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Bee Products Properties, Applications, and Apitherapy


Edited by

Avshalom Mizrahi The Israeli College of Complementary Medicine Tel Aviv, Israel

and

Yaacov Lensky Triwaks Bee Research Center The Hebrew University Rehovot, Israel

Springer Science+Business Media, LLC

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Proceedings of an International Conference on Bee Products: Properties, Applications, and Apitherapy, held May 2 6 - 3 0 , 1996, in Tel Aviv, Israel

ISBN 978-1-4757-9373-4 DOI 10.1007/978-1-4757-9371-0

ISBN 978-1-4757-9371-0 (eBook)

© Springer Science+Business Media New York 1997 Originally published by Plenum Press, New York in 1997 Softcover reprint of the hardcover 1st edition 1997 http://www.plenum.com All rights reserved 109 8 7 6 5 4 3 2 1 No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

PREFACE

The nature .and diversity of presentations at the conference on: "Bee Products: Properties, Applications and Apitherapy" held at Tel-Aviv on May 26--30, 1996, emphasize the increasing interest of physicians, practitioners, scientists, herbalists, dieticians, cosmeticians, microbiologists, and beekeepers in different facets of bee products. This volume consists of a selection of 31 contributions presented at the conference and which provide information on the present status of our knowledge in this area. In spite of their diversity, they reflect the mainstream of the conference, namely: "Imported" Products (honey, pollen and propolis), Exocrine Secretions of Workers (venom, royal jelly). Toxicity and Contaminants, Quality Control, Marketing, Apitherapy, Cosmetics, etc. Since antiquity, honey as well as other bee products were used as food, as a cure for ailments of humans and animals, and as cosmetics. We hope that this volume will contribute to interdisciplinary studies on chemical composition, pharmacological effects, nutrition, and other aspects of bee products. Critical and unbiased experimental research may unravel the yet unknown composition and mode of action of bee products and elucidate many unanswered questions. The noteworthy features of this conference were the participants from all parts of the world and of different cultural backgrounds, who shared their keen interest and curiosity regarding honey bees and their products. We thank all of them for their personal contribution to the success of this conference. Avshalom Mizrahi Yaacov Lensky Editors

v

THE CONFERENCE ON BEE PRODUCTS

The Conference was organized by: The Israeli Honey Production and Marketing Board and The Israeli Beekeepers' Associations and in informal alliances with: • • • • • • •

American Apitherapy Society Apimondia - The International Federation of Beekeeping Association Asian Apicultural Association International Bee Research Association Israeli Dietetic Association Ministry of Agriculture, State ofIsrael Ministry of Tourism, State of Israel

Local Organizing Committee Avshalom Mizrahi, Ph.D. (Chairman) Yaacov Lensky, Ph.D. (Vice Chairman) Moshe Almaliah, M.Sc. Tsila Dvir, M.Sc. Abraham Hefez, Ph.D. Anatol Karakowsky, M.D. Yanay Sachs David Sadeh Yeshayahu Stem, M.Sc. Boris Yakobson, D.V.M. International Advisory Committee Stefan Bogdanov, Ph.D. (Switzerland) Raymond Borneck, President, Apimondia (France) Kate Chatot (U.S.A.) Theodore Cherbuliez, M.D., President AAS (U.S.A.) Zhibin Lin, M.D. (China) Charles Mraz (U.S.A.) Tetsuo Sakai, Ph.D., President, AAA (Japan) Mira Spitzer-Adir (Croatia) Artur Stojko, Ph.D. (Poland) Bradford S. Weeks, M.D. (U.S.A.) Siriwat Wangsiri, Ph.D. (Thailand)

vii

CONTENTS

1. The Past and Present Importance of Bee Products to Man Eya Crane 2. Bee Products: Chemical Composition and Application . . . . . . . . . . . . . . . . . . . . . Justin o. Schmidt

15

3. Honey as an Antimicrobial Agent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. C. Molan

27

4. Non-Peroxide Antibacterial Activity of Honey Stefan Bogdanov

39

5. Antioxidant Properties of Honey Produced by Bees Fed with Medical Plant Extracts ..................................................... Gennady Rosenblat, Stephane Angonnet, Alexandr Goroshit, Mina Tabak, and Ishak Neeman 6. Speeding Up the Healing of Burns with Honey: An Experimental Study with Histological Assessment of Wound Biopsies ........................ Th. J. Postmes, M. M. C. Bosch, R. Dutrieux, J. van Baare, and M. J. Hoekstra

49

57

7. The Effect of Honey on Human Tooth Enamel and Oral Bacteria S. R. Grobler and N. 1. Basson

65

8. Honey Contact with Teeth in Situ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Gedalia, S. R. Grobler, I. Grizim D. Steinberg, L. Shapira, I. Lewinstein, and Mo. Sela

73

9. Medicinal Herbs as a Potential Source of High-Quality Honeys. . . . . . . . . . . . . . Zohara Yaniv and Michal Rudich

77

10. The Unique Properties of Honey as Related to Its Application in Food Processing Tsila Dvir

83

11. Honey as a Clarifying and Anti-browning Agent in Food Processing and a New Method of Mead Production. .. .. . . .. .. . . .. . . . . . . . .. . .. . . . . . . . . . . ChangY. Lee

89

ix

x

Contents

12. Bee-Pollen: Composition, Properties, and Applications M. G. Campos, A. Cunha, and K. R. Markham

93

13. Clinical Evaluation ofa New HypoaUergic Formula of Pro polis in Dressings. . . W. Fierro Morales and 1. Lopez Garbarino

101

14. Present State of Basic Studies on Propolis in Japan. . . . . . . . . . . . . . . . . . . . . . . . Tsuguo Yamamoto

107

15. The Usage and Composition of Propolis Added Cosmetics in Korea Park Jong-Sung and Woo Kun-Suk

121

16. Eucalyptus Propolis Beverages with Their Composition and Effects Woo Kun-Suk and Park Jong-Sung

125

17. An Inhibitory Effect of Propolis on Germination and Cell Division in the Root Tips of Wheat Seedlings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K. Sorkun, S. Bozcuk, A. N. Gomiirgen, and F. Tekin

129

18. The Exocrine Glands of the Honey Bees: Their Structure and Secretory Products Pierre Cassier and Yaacov Lensky

137

19. Alarm Pheromones of the Queen and Worker Honey Bees (Apis mellifera L.) Yaacov Lensky and Pierre Cassier

151

20. Protein Traffic between Body Compartments of the Female Honey Bee (Apis melli/era L.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yoseph Rakover and Yaacov Lensky

161

21. Effects of Feeding, Age of the Larvae, and Queenlessness on the Production of Royal Jelly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nuray Sahinler and Osman Kaftanoglu

173

22. The Use of Royal Jelly during Treatment of Childhood Malignancies Osman Kaftanoglu and Atilla Tanyeli

179

23. The Role of Hymenopterous Venoms in Nature Eli Zlotkin .

185

24. Effect of Apamin and Melittin on Ion Channels and Intracellular Calcium of Heart Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. G. Bkaily, M. Simaan, D. Jaalouk, and P. Pothier

203

25. Bee Venom in Treatment of Chronic Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . .. Th. Cherbuliez

213

26. Apitherapy in Orthopaedic Diseases Franco Feraboli

221

27. The Monitoring of Possible Biological and Chemical Contaminants in Bee Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Boris A. Yakobson

227

Contents

28. Heavy Metals in Propolis: Practical and Simple Procedures to Reduce the Lead Level in the Brazilian Propolis ................................... Nivia Macedo Freire Alcici

xi

231

29. Acaricide Residues in Beeswax and Honey. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. Bogdanov, V. Ki1chenmann, and A. Imdorf

239

30. Judging the Quality of Honey by Sensory Analysis. . . . . . . . . . . . . . . . . . . . . . .. Michel Gonnet

247

31. Methods for the Characterization of the Botanical and Geographical Origin of Some Bee Products and for Their Quality Control . . . . . . . . . . . . . . . . . . .. Giancarlo Ricciardelli D'Albore Index

253 263


1

THE PAST AND PRESENT IMPORTANCE OF BEE PRODUCTS TO MAN Eva Crane International Bee Research Association Woodside House, Woodside Hill, Gerrards Cross Bucks SL9 9TE United Kingdom

1. THE BEES FROM WHICH PRODUCTS ARE HARVESTED At this Conference we are considering the products of social bees, which beekeepers harvest from them. Candidate bees (Table I, Figure I) are: first, all the honey bees: Apis melli/era from Europe, eastern Mediterranean lands and Africa; Apis cerana the hive bee in Asia, and Apis dorsata, Apis jlorea and related species in the tropics of Asia. Second, in the tropics of all continents there are stingless bees (Meliponinae), some 500 species in all. In addition, honey-but not wax-is produced by colonies of honey wasps (Vespidae) and honey ants (Formicinae) and is harvested from them. The wasps live in parts oftropical South America, and the ants in some dry areas of Australia and North America. What we now think of as bee products were essential to the bees for their survival and development during and after the evolutionary period: this was and is their function. Stingless bees and honey bees evolved roughly 100 million and 50 million years ago, respectively, whereas man has existed to use the products for only I or 2 million years-a tiny fraction as long as social bees. The earliest records of man's harvesting from bees' nests are in the Mesolithic rock art of Europe and Asia, painted not more than 8000 years ago (Figures 2 and 3). There are also rock paintings in Australia showing stingless bee nests. Man used bee products in many ways: beeswax in various technologies, and honey as food and also in medicine and as offerings to the gods he worshipped. Man also had ideas about the origins of the various bee products, and attributed certain properties to them. But their true origins were not known until a few centuries ago, and their detailed chemical compositions were determined only in the late 1900s. I shall say most about honey, and then deal with other products: beeswax, propolis, rollen, bee brood, bee venom and royal jelly. Finally, I shall discuss changes in the importance of the various bee products during the period when man has been harvesting them.

E. Crane

2

Table 1. Substances collected or produced by certain social insects Insect

Where native

Honey

Wax

Prop.

Pollen

xx xx xx xx xx x x

xx xx xx xx xx

xx

xx x x x x

Brood Venom

Rj

Honey bees (Apis) A. melli/era

Old World Europe & E. Mediterranean; Africa A. cerana Asia Asia, tropics A. dorsata A.jlorea Asia, tropics Stingless bees (Meliponinae) tropics S. America, tropics Honey wasps Parts of Australia & N. Honey ants America

x x x

xx x x x x x x

xx x x x

xx x x x

x Collected or produced by the insects. xx Known to be commercially harvested and marketed by man.

2. HONEY The earliest known written records' of the use of honey by man relate to religious sacrifices in various regions; indeed honey may well have been one of the earliest nonanimal sacrifices. It was sometimes offered together with milk, or butter or ghee, oil, or incense. According to inscriptions on clay cylinders from Sumer in Mesopotamia, when the foundations of a new temple for the god Ningirsu were laid about 2500 Be, Gudea the ruler of Lagash made offerings of honey and butter. Then, when the image of the god was finally erected, he offered honey with other foods. The use of honey as an offering probably had a still older origin, because other inscriptions show that it was already customary by Gudea's time. In Ancient Egypt much honey was sacrificed in religious ceremonies, and when Israelites later presented the first harvest of their produce to God, this included honey. For instance in Jerusalem at the time of Hezekiah in the late 700s Be, 'they gave generously from the first fruits of their corn and new wine, oil and honey, .. .' (II Chronicles 31.5). Honey had, however, been forbidden as a burnt offering around 1300 Be: 'You shall not burn any leaven or any honey as a food-offering to the Lord' (Leviticus 2.11). What may be the earliest recorded medical prescription that includes honey is also from Sumer, dated to about 2000 Be. Oil was to be spread over a preparation of river dust kneaded with honey, water, and other ingredients. This was presumably for external application, and many Ancient peoples used honey in this way2. In the Ebers papyrus compiled in Egypt about 1550 Be, I found honey in 147 prescriptions for external use, and in 102 for internal use, both out of a total of several hundred. For internal use, honey was sometimes included because of its own properties, sometimes as a binder, and often to disguise the taste of other, unpalatable ingredients. The Roman poet Lucretius (c. 99-55 BC) referred to this use of honey: Physician-like, who when a bitter draught Of wormwood is disgusted by a child To cheat his taste, he brims the nauseous cup With the sweet lure of honey. • Historial records cited will be detailed in a forthcoming book on the history of man's use of bees, to be published by Duckworth in London.

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Figure 2. Mesolithic rock painting from around 6000 Be, showing honey collection from Apis mellifera, La Arana shelter, Bicorp, eastern Spain (copy by E. Hernandez-Pacheco who found the painting in 1924).

Past and Present Importance of Bee Products to Man

5

Figure 3. Post-Mesolithic rock painting showing honey collection from Api" dorsa/a, Rajat Prapat, Central India (drawing: Y. Mathpal, 1984).

Honey was also used as a preservative, notably for the body of a king or general killed in battle, so that this could be taken home for burial. The custom is known for instance from Babylon, and from Ancient Greece. But according to Plutarch, when Agesilaus King of Sparta died in 360 BC, his body was preserved in beeswax 'since they had no honey'. The Hebrew scriptures refer many times to honey as plentiful in Canaan, and four passages indicate the source of the honey or how it was obtained. The first (Deuteronomy 32.13) referred to Jacob, in a period about 1700 BC: the Lord ' satisfied him with honey from the crags' . This was referred to again in Psalms 81.16, written around 1000 BC: the God of Jacob 'satisfied him with honey from the rocks'. So honey was obtained from bees nesting in rocks, as is usual in dry country; there is no mention of nests in trees. The other two passages are intermediate in date. In Judges 14.8, Samson 'turned aside to look at the carcass of the lion, and he saw a swarm of bees in it, and honey. He scraped the honey into his hands and went on, eating as he went'. Then in I Samuel 14.25- 27: 'There was honey comb in the countryside ', and Jonathan ' stretched out the stick that was in his hand, dipped the end of it in the honey comb. put it to his mouth and was refreshed.' These passages describe common methods of harvesting honey from bees' nests or from traditional hives, used all over the world. Of course the fact that man used honey or other bee products does not show that he was a beekeeper. By keeping bees in hives, however, man could harvest honey in larger amounts, and more easily. Hives are known to have been used in Egypt from about 2500 BC, and Figure 4 shows a more complete later pictorial record . In Mesopotamia hives are known from the 700s BC. But the earliest references I found relating to Israel are in the Babylonian Talmud, compiled about AD 500. Already in Ancient civilizations, honeys from different plants, and in different regions, were differentiated. In dry Mediterranean regions much honey came from aromatic

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plants such as thyme, and Roman writers praised this honey, for instance from Mount Hymettus in Greece and Mount Hybla in Sicily. They disliked honey from Spanish broom (Spartium junceum). Export-import trade in honey also started early. Although Ancient Egypt produced much honey, some was imported from several places in Asia Minor, and from Syria, Rhodes and Greece. Until about 200 years ago it was believed that honey had its origin in the heavens, and this idea contributed to its status as a sacred substance in pre-religious beliefs and in religions. In the 300s BC, Aristotle said that 'honey falls from the air, principally at the rising of the stars, and when the rainbow rests upon the earth'. Pliny (AD 23- 79) questioned 'whether it is that this liquid is the sweet of the heavens, or whether a saliva emanating from the stars, or a juice exuding from the air while purifying itself ... it comes to us pure, limpid and genuine' . As late as 1609 in England the Reverend Charles Butler, who was very knowledgeable about beekeeping, in England wrote: 'The greatest plenty of purest nectar cometh from above; which Almighty God doth miraculously distil out of the air ... which thence doth descend into the earth in a dew or small drizzling rain.' Vaillant in France has been quoted as the first to state-in 1717-that the nectar the bees collect is produced by nectaries in the flowers. But the belief that it fell from heaven persisted into the 1800s. We now know that honey is made mainly from the nectar of flowers by bees and a few other insects-which evaporate water from the nectar and, while doing so, secrete into it enzymes invertase and glucose oxidase. The bees thereby produce a highly supersaturated solution of certain sugars which has antimicrobial properties essential for its storage in the nest safe from spoilage. Some of the tropical social bees--especially the cavity-nesting stingless bees--evolved in an environment where maintenance of nest hygiene was very difficult, with no cold winters, and often a hot and humid atmosphere which favoured the growth of pathogenic micro-organisms. And the entrance to a nest of stingless bees must be very small (Figure 5), as a protection against the many enemies, so the nest is poorly ventilated. Perhaps as a result, stingless bee honeys differ from Apis

Figure 5. Nest of Melipona interrupta grandis'.

8

E. Crane

mellifera honeys in several ways: for instance, they contain more acids, their enzyme composition is somewhat different, and they have greater antimicrobial activity. Although their water content is higher, they are stored safe from spoilage 4 , although we still do not fully understand the reasons.

3. BEESWAX Beeswax is a very inert substance, which is not digested by mammals (including humans), and any eaten is excreted. Until the 1700s it was believed that beeswax was collected by worker bees from flowers, but various observers then found wax scales on the underside of the bee's abdomen, and in 1793 Fran"ois Huber in Switzerland finally established that the wax is secreted by the bees themselves. The importance of beeswax to bees-and to man-is due to its inertness and its physical properties. The wax is plastic at temperatures from 32° upwards, so the bees can manipulate it in the hive. The wax from anyone species of bee has a composition which is remarkably uniform, although it is very complex. Beeswax was used by early civilizations to cast copper-and later other metals-from an original wax model into superb objects of art, by the lost-wax process. The earliest such casting known, dated to between 3500 and 3000 BC, was found in a cave in the Judean desert. Other notable centres of early lost-wax casting were in the Yellow River basin of China, the Red River basin in Vietnam, and Benin in West Africa. In Mesoamerica wax from stingless bees was used to cast gold into splendid ornaments and jewellery, but most of them were looted by the Spanish in the 1500s, and many destroyed. Beeswax candles were used for lighting in Ancient Egypt, Crete, Greece and Rome, and more widely in later centuries. By the AD 400s, beeswax was the mandatory material for lights in the Christian churches. Saint Augustine said that 'the wax of the candle produced by the virgin bees from the flowers of the earth is a symbol of the Redeemer born of a Virgin Mother'. This created a huge demand for beeswax by churches and monasteries in Christian countries, until after the Reformation of the Roman Catholic Church in the 1500s, when reformed churches prohibited the use of candles. Beeswax was an important component of ointments and cosmetics during Ancient times and later. It was also used in making incendiary weapons. For instance during the siege of Jerusalem in 1099, the Muslim defenders threw at the advancing Christians incendiary devices which contained pitch, beeswax, sulphur and tow. They used rather similar mixtures in 1097 and 1147.

4. PROPOLIS Propolis is the name given to various sticky substances which bees collect; some of these are plant secretions, and others are plant exudates from wounds. It is quite hard when cold, but becomes viscous when warmed, and Apis mellifera uses it in various building operations in the nest or hive. The Asian hive bee Apis cerana does not collect or use propolis, but stingless bees use it extensively. The composition of all propolis is very complex, and varies according to the plant of origin. Anyone sample may contain a hundred different substances, including about forty flavonoids which are the main source of its antimicrobial action. Bees may mix wax with propolis, but I do not know that they otherwise change propolis in any way.


9

Propolis has been used by man since early times, for various purposes: as an adhesive and to seal cracks; to protect wooden and other surfaces; and especially in medicine because of its antimicrobial properties. We do not know what methods were used for harvesting it in the Ancient World, although writers in Greece and Rome were familiar with it. The Greek Historia animalium referred to a substance mitys which was probably propolis, as 'a cure for bruises and suppurating sores'. According to Varro in Rome, propolis was used 'by physicians in making poultices, and for this reason it brings even a higher price than honey on the Via Sacra'. A number of early records mention substances which mayor may not have been propolis: Asis 6 believed that 'black wax' referred to in the Egyptian Ebers papyrus (c. 1550 BC) may have been propolis. He also considered that Hebrew tzori was an early word for propolis. This occurs six times in the Hebrew scriptures, and was usually translated as balm or balsam. In Genesis (c. 1700 BC), tzori was taken to Egypt, once with honey; in Ezekiel it is mentioned together with honey, and its healing properties are noted three times in Jeremiah. Twice, tzori came from Gilead, but it was not balm of Gilead which is produced from a tree, Commiphora opobalsamum. As far as I know, propolis was first produced commercially in the 1950s. It was harvested by fitting-at the periphery of a hive-a grid or grids, with holes about 2 mm; the bees closed these up with propolis, which could be removed by shattering, after cooling the grid in a deep-freeze. In 1984 exports included 55 tonnes from China and smaller quantities from Argentina, Canada, Chile and Uruguay, with unknown amounts from at least eleven other countries.

5. POLLEN Bees evolved during the same period as the flowering plants from which they collect nectar and pollen, and they pollinate plants by transferring pollen from one flower to another, although this was not understood until Arthur Dobbs in Ireland established it in 1750. Bees also collect pollen and store it in their combs; it supplies them with protein and essential minerals and vitamins. Hunter-gatherer peoples doubtless ate pollen along with the honey combs they harvested from bees' nests. But if early man deliberately collected pollen, it would probably have been shaken or blown from wind-pollinated flowers. There are few past references to uses of pollen. The earliest T know to its application in medicine are in books by Arab and Jewish physicians in Islamic Spain. Maimonides (1135-1204), a Jew in Cordoba who was a physician to the Sultan of Egypt, recommended it as an astringent and sedative tonic. In the early 1200s Ibn el-Beithar described pollen as an aphrodisiac, also beneficial for the stomach, bowels and heart; it reduced the 'fervour' of the blood, and cured swellings produced by eating certain foods 7 • The device beekeepers use today for harvesting pollen removes it from the bees' legs as they enter the hive. It dates only from 1941, when the first pollen traps were produced in both Germany and the USA; a hive pollen dispenser, to coat outgoing bees with hard-collected pollen, had been used 9 years earlier. The possibility of marketing bee-collected pollen for dietary purposes was explored in the 1950s, after some of the difficulties of commercial royal jelly production were realized. Pollen collection was best done in dry regions of the world where handling and storing were much easier than in humid areas. By the late 1980s, bee-collected pollen was produced commercially in at least 18 countries, and Western Australia alone produced between 60 and 130 tonnes a year.

10

E. Crane

Harvested pollen is sold as a dietary supplement and for treating certain diseases, and it is essential for some types of work on crop pollination and plant breeding.

6. BEE BROOD Aristotle said that bees collected their young from flowers, but in 1586 Luiz Mendez de Torres in Spain established that new bees are produced from 'seed' placed by the female queen in cells of the comb, and that she is the mother of all the other bees. Bee brood was the main protein food that man harvested from bees' nests or hives. To many peoples in the tropics, insects--especially immature stages-were a main source of protein; in fact, bee brood was their food from bees' nests, and honey was a seasonal treat and a medicine. On the other hand peoples of European origin did not eat insects; honey was their food from bees, and in the western world brood was rarely listed among bee products. In 1951 Bodenheimer8 said that 'the aversion to insect food in Western civilization is ... not based on hereditary instinct. It is established by custom and prejudice.' He pointed out that animal rearing and crop growing, which developed earlier in Mediterranean regions and Europe than in the tropics, provided people with an adequate diet without the need to hunt such tiny game as insects, so Europeans came to despise food insects, and the peoples who ate them. The eating of certain insects was forbidden by religions of eastern Mediterranean peoples. Laws attributed to Moses in about 1300 Be (Leviticus 11.21) allowed insects such as locusts to be eaten, whereas others-which would include bees-seem to have been proscribed as unclean. According to Allegro 9 , eating bee brood was forbidden in Zadokite fragments of the Dead Sea scrolls: 'Let no man defile his soul with any living being or creeping thing by eating of them, from the larvae of bees [in honey] to all the living things that creep in water'. Muslims have explained to me that they do not eat the digestive system of an animal (it is unclean), and since this cannot be removed from the bee, bees were not to be eaten. Buddhism proscribed the killing or eating of any animals. In some Asian countries bee brood is a well known and important product, but I do not know amounts produced, exported or imported.

7. BEE VENOM It was known in Ancient Greece that, when a bee stings, she cannot retract her sting from human skin, and dies as a result. According to Broadman 1o , bee venom was referred to as early as the 400s Be by the physician Hippocrates, and by other writers in Antiquity. But the composition of bee venom was not established until the late 1900s. I found more documentation from past centuries on the use of stinging bees for military purposes than in medicine. In the Ancient World, besieged people sometimes released bees (among other animals) into tunnels which had been excavated and occupied by the enemy below their defended position. Tacitus described such action in about 357 Be, and Appian's Roman history recorded how in 72 Be the Roman army under Lucullus suffered a reverse in this way in Pontus, south of the Black Sea. In the Middle Ages the military tactics were different: hives of bees were dropped, thrown or projected at the enemy. There are various unsubstantiated records, but two of


11

the more reliable relate to incidents in 908 in England, and in 1191 at Akko (Acre) 100 km north of Tel-Aviv. An English manuscript from 1326 now in Christ Church, Oxford, included a design for a machine to hurl skeps of bees at a besieged castle. Several reports of the military use of skeps survive from the Thirty Years' War in Europe (1618-1648). During the fighting in tropical Africa in the 1914-1918 World War, trip-wires were tied to hives hidden in trees, where passing enemy troops would activate them and cause the bees to sting. For use in medicine, an injectable solution of bee venom is prepared, so that doses can be quantified. As far as I know this was not attempted until the late 1880s, and the first person to succeed was J. Langer at the University of Prague, in 1897/99. From 1930 the firm Mack at Illertissen in south Germany produced bee venom solution commercially!!. Dr Bodog Beck in the USA was one of the pioneers in the use of bee venom, and his 1935 book!2 gives much information. A method developed later, which I saw as a commercial operation in Czechoslovakia in 1960, used a framework covered with a very thin membrane, fixed in front of a hive entrance. Bare wires were stretched across the membrane so that bees leaving the hive received an electric shock and stung into the membrane, which was so thin that a bee could retract her sting, and she could sting again. Drops of venom crystallized on the underside of the membrane, and were scraped off. Since 1973, bee venom is known to have been used in medicine in at least 12 countries in Europe, 3 in Asia, and 3 in the Americas.

8. ROYAL JELLY Young worker bees provide food for larvae (from glands in the head), and the food is richer for larvae in queen cells (royal jelly) than for larvae in worker or drone cells. Huber in Switzerland, in 1793, was the first to distinguish between worker brood food (gelee) and queen brood food (gelee royale), and the prestigious name royal jelly has been used for the queen food ever since. The first person to get a chemical analysis of royal jelly done was probably Langstroth in the USA, in 1852, but an effective analysis was not possible until the 1940s. The only reference I have found to a specific traditional use of royal jelly is Raymond Borneck's 1976 observation in certain areas of Ivory Coast in West Africa l3. If honey hunters there found royal jelly in queen cells, it was given to the old people. The harvesting of royal jelly involves much labour-intensive work, done to a strict timetable. Colonies of bees are organized so that they rear a large number of female larvae as queens, providing them with royal jelly. When the larvae are 3 days old the royal jelly is removed from the cell with a vacuum pump, and the larvae are discarded. Royal jelly was sold as a commercial product in the early 1950s in France, which produced l.5 tonnes of it in 1958. In 1984 world production included 400 tonnes in China and 234 tonnes in Taiwan, much of which was exported. Most royal jelly is sold for medicinal and dietary purposes.

9. HOW THE IMPORTANCE OF BEE PRODUCTS TO MAN CHANGED THROUGH TIME I believe that man ate the contents of bees' nests from his first existence as a species. Early peoples used the entire contents of a nest for one or more purposes: as food,

E. Crane

12

medicine, preservative, adhesive, or military weapon; for modelling or casting metals; for purposes of magic or as a religious symbol. Bees, honey and beeswax were held in especially high regard, and in some religions they were sacred. Until about 1600, there was very little alteration in beliefs about bee products, or in uses of them, but every century since then has brought one or more fundamental change. In the 1600s honey bees (Apis mellifera) native to Europe were introduced by settlers to North America in the New World, and flourished and spread there. During the 1700s the true origins of most of the bee products were established for the first time. Around 1800 the Industrial Revolution started, and many new materials were then manufactured. Some of these competed with bee products, and could be sold much more cheaply. For instance in England in 1400 sugar cost 20 times as much as honey. Soon after 1800 the two cost about the same, and by 1900 sugar cost only a fifth as much as honey. The place of honey in diets of various peoples up to the 1800s has been discussed in a recent paperl4. Paraffin wax was manufactured from the 1850s, and later competed successfully with beeswax for many purposes. In the 1800s also, European honey bees were introduced to Australia and New Zealand, and to some parts of non-tropical Asia where they became much more cost-effective producers than the native Apis cerana. And in 1853 Langstroth in the USA described his movable-frame hive, which has become the basis of present world beekeeping. Throughout the 1900s the world's trade in the main bee products, honey and beeswax, has been dominated by production from temperate-zone Apis mellifera in movableframe hives, and latterly from parts of the world where this bee is not native. Also, after low-cost alternatives to honey and beeswax became available, most purchasers of them were in affluent societies. By 1950, honey-the main product of movable-frame beekeeping-was difficult to sell, and I well remember how interested and excited beekeepers became at the idea of obtaining income by harvesting other bee products. These new products were more expensive to produce than honey or beeswax, but they commanded much higher prices. Advanced analytical methods were developed, and these provided a more detailed knowledge of their composition, and it became possible to explore new uses of them, especially in medicine. In addition, it was established that honeys from certain plants contained specific substances with potentially useful properties. Each bee product varies somewhat according to the species of bee from which it is harvested. Honey, wax, brood, venom and royal jelly vary because of the physiology of the bees themselves; honey, pollen and propolis vary because of the different plants in the regions where the different bee species live. Almost all the papers at forthcoming Sessions relate to products harvested from one bee, European Apis mellifera native to the temperate zone. It is less easy to harvest the products from other bee species, but products of tropical bees are likely to have somewhat different compositions, properties and uses, and I think that more knowledge about them might well lead to further possibilities for diversification.

REFERENCES I. Crane E. Bees and Beekeeping: Science, Practice and World Resources. Heinemann Newnes, Oxford, 1990 2. Manjo G. The Healing Hand: Man and Wound in the Ancient World. Harvard University Press, Cambridge, MA, USA, 1975 3. Davies N. de. The Tomb of Rekhmire at Thebes. Ayer Co., Salem, NH, USA, 1944


13

4. Bruijn L. L. M. de (1996) Composition and Properties of Honeys of Stingless Bees (Apidae, Meliponinae). In press 5. Camargo J. M. F. de (1970) Ninhos e Biologia de algumas Especies de Meliponideos (Hymenoptera: Apidae) de Regiiio de Porto Velho, Territ6rio de Rondonia, Brasil. Rev. BioI. trop. 16(2),207-239 6. Asis M. Propoleo: el Oro purpura de las Abejas. CIDA. Havana, 1989 7. Monferrer J. P. (1991) La Miel en la Espana musulmana (al-Andalus). Vida apic. (46), 64-68; (47), 24-28 8. Bodenheimer, F. S. Insects as Human Food: A Chapter in the Ecology of Man. W. Junk, The Hague, 1951 9. Allegro J. M. (1956) Personal communication 10. Broadman J. Bee Venom: The Natural Curative for Arthritis and Rheumatism. G. P. Putnam's Sons, New York,1962 II. Forster H. (1985) Personal communication 12. Beck B. F. Bee Venom Therapy. Appleton Century. New York, 1935 13. Bomeck R. (1976) L'Apiculture en Cote d'Ivoire. Rev. fro Apic. (344), 334-335, 338-339 14. Allsop K. A., Miller J. B. (1996) Honey revisited: A Reappraisal of Honey in pre-industrial Diets. Brit. J. Nutrition 75(4),513-520

2

BEE PRODUCTS Chemical Composition and Application

Justin O. Schmidt' Carl Hayden Bee Research Center USDA-ARS 2000 E. Allen Road, Tucson, Arizona 85719

Honey bees are master chemists and chemical engineers. Their success in the animal kingdom is largely because of the chemistry and the application of their products: honey, beeswax, venom, propolis, pollen, and royal jelly. Three of these products, beeswax, venom, and royal jelly, are chemically synthesized by the bees themselves. The other three are derived from plants and are modified and engineered by the bees for their own use. The use of these products explains the amazing honey bee success: honey is used as a stable, reliable food source that serves during times of shortages, enables the bees to warm their nest during cold weather, and has allowed them to become perennial species that can exploit virtually all habitats in the world; beeswax is used as a pliable, stable and moisture-proof material with which to construct their nest, to store honey safely, and to rear their brood; venom gives honey bees the advantage of a formidable defense that is capable of stopping or deterring all but the most determined and capable of predators; propolis is an outstandingly good caulking for use in sealing the nest cavity and is also one of the best antimicrobial agents known; pollen is a nutrient-rich food that, like honey, can be stored in the hive indefinitely to seJ;ve as a reserve during times or seasons of shortages; and royal jelly is a balanced food source that does not spoil readily and is used to feed bee larvae. Without these unique products honey bees likely would have evolved to be !ittle different from their ancestors-solitary bees in which each female bee during a brief season provisions a few cells with pollen and nectar for the next generation. The usefulness of honey bee products for mankind is based on the same properties that make these products useful for the bees themselves. In the case of propolis these properties extend back beyond the bees to the plants themselves which produce the original resins that bees collected to become propolis. Quite simply, honey is an excellent, stable sweetener and energy source for humans, just as it is for bees; beeswax is a malleable • Address correspondence to: Dr. Justin O. Schmidt, Carl Hayden Bee Research Ctr., 2000 E. Allen Road, Tucson, AZ 85719. Tel: 602 1l70--6380; e 109 Fax: 602 670--6493.

15

16

J. O. Schmidt

plastic material, that in addition be being an excellent material for molding, bums cleanly; venom is useful because it causes pain and possesses a host of pharmacological activities; propolis is anti-microbial toward bacteria, viruses, fungi, molds, and possesses a multitude of other pharmacological activities; pollen is a phenomenonally nutritious and well-balanced food that can be consumed by people and domestic animals; and royal jelly has a variety of moisturizing, emulsifying and stabilizing properties that make it useful to people. The goal of this chapter is to examine the chemistry of honey bee products, to use this information to explain the application of the products, and to predict their usefulness.

1. POLLEN Pollen as trapped by beekeepers from honey bee colonies is a product collected from many, often dozens, of species of plants visited by the bees. This feature enhances the nutritional balance of the pollen, but also means that bee pollen is not a uniform product, rather it varies somewhat from sample to sample. This variability complicates the analysis of pollen chemistry and requires that statements vis-a-vis pollen be given as averages or as values for a specific species of pollen. All chemical and nutritional analyses here will be given as means derived from large numbers of literature reports that appear reliable. Table I is a listing of the chemical composition of pollen and a comparison of pollen nutrient density with that established for Recommended Daily Allowance (RDA) or Estimated Daily Intake (EDI = Estimated Safe and Adequate Daily Dietary Intakes) for human dietary needs (I). In general, compared to many standard human foods, pollen is rich in protein, low in fat, and possesses a wealth of minerals and vitamins. No obvious human nutritional deficiencies are present in pollen with the possible exceptions of vitamin BI2 and the fat soluble vitaminsD and K. In the case ofB l2 , the vitamin is not usually in shortage because the body usually retains a multi-year reserve. Shortage only occurs in cases of defective body recycling (pernicious anemia) and is particularly needed for pregnant women who have metabolic deficiencies, or are strict vegetarians. Vitamin D is somewhat of a misnomer, as it is not truly a vitamin. Humans can synthesize the vitamin from 7-dehydrocholesterol if they are exposed to sunlight. Vitamin K is a minor vitamin whose sole role is to aid in blood clotting and which is produced naturally by intestinal bacteria. Evidence of the digestibility of pollen is provided by Bell et al. (2) and Schmidt et al. (3) and a testament to its overall balance is demonstrated by mice that survived well for over a year on a diet containing only pollen (4). Pollen has not been analyzed in detail for some of the trace elements such as boron, chromium, molybdenum, iodine, fluoride, and selenium, but it would not be surprising if it also contained adequate quantities of these elements. One means to evaluate the nutritional content of pollen is to compare the levels of dietary nutrients in good wholesome food to those in pollen. In Table 2 the quantities of 11 well established and measured nutrients for two vegetables, one fruit, two meats, and two staples are compared to pollen. Pollen ranks number I in quantity for four of the nutrients, number 2 for another four, and ranked lower only for vitamin C, sodium, and fat. Overall, pollen has a higher ranking than any of the compared foods, even tomatoes and cabbage which are considered to be classic examples of the most nutritious foods available. In terms of protein, pollen ranked number 2, and above beef. The overall conclusion is that pollen is a food source par excellence that is probably not exceeded by any other food. The one caveat is that pollen is much too expensive to be considered a primary food and, indeed, consumption of large quantities can cause adverse effects (4). However, this

Bee Products: Chemical Composition and Application

17

Table 1. Average chemical composition and nutritional value of bee pollen Chemical

Composition'

Energy Protein Carbohydrates Lipids Cholesterol Phosphorus Potassium Calcium Magnesium Sodium Iron Manganese Zinc Copper Nickel Boron Chromium Molybdenum Iodine Fluoride Selenium Thiamin Niacin Riboflavin Pyridoxine Pantothenate Folic acid Biotin Vit.B 11 VitaminC Vitamin A Carotenes Vitamin D Vitamin E Vitamin K

2.46 23.7 27 4.8

kcal/g' % % %

-0 .53 % .58 % .225 % .148% .044% 140 ppm ppm 100 ppm 78 14 ppm 4.5 ppm trace

420 83 59 d _Od

590 190 250 470 27d 830 2500 580 560

? ? ? ? ?

? ? ? ? ? 9.4 157 18.6 9 28 5.2 .32 0 350 0 95 0 14 0

% of RDA/EDI b

ppm ppm ppm ppm ppm ppm ppm ppm

760 940 1300 500 450 2600 440 0 520 Of

ppm ppm

-900 f 0 160 0

'Data for pollen from (II). bCalculations based on RDA (Recommended Daily Allowance) and ED! (Estimated Daily Intake = Estimated Safe and Adequate Daily Dietary Intakes) using U.S. women aged 25-50 with a 2200 kcal intake (\). 'Calculated on the basis of 4 kcal/g for protein and carbohydrates. and 9 kcal/g for fat (28). dThese values are recommended maximum intakes for nutrients generally accepted as harmful to health in excess (I). eNutritional requirements in the human diet not established. fPollen contains no preformed ViI. A. but carotenes can be can-verted to ViI. A equivalents based on 6 Ilg ~-carotene = I ViI. A equivalent and assuming half pollen carotenes are ~-caratene.

96.3 50.0 54.1 152.8 40.1 3.4 43.2 59.4

Pollen Tomato Cabbage Chicken Beans Apple Bread Beef

19.5 8.8 8.3 35.9 6.5 10.3 12.3 82.7

Fat (g)

.7

1.1

2.4 11.0 2.4 2.0 1.7 1.9

Potassium (g) 915 588 2037 60 443 122 407 26

Calcium (mg) 179 138 835 484 3800 19 2200 145

Sodium (mg) 57 22 16 8.9 15 5.3 12 7.5

Iron (mg) 14500 41000 5410 484 1070 1560 trace 143

ViI. A (Inl. U) 3.82 2.75 2.11 .28 .65 .53 1.06 .17

Thiamin (mg) 7.56 1.88 2.75 1.29 .25 .34 .49 .46

Riboflavin (mg)

63.8 31.2 12.8 57.7 4.9 1.9 11.5 12.2

Niacin (mg)

142 1050 1950 0 16 68 trace 0

Vil.C (mg)

62 60 58 31 30 27 23 17

Rank score'

'All values are based on amount in the quantity of food that provides 1000 kcal of energy; data for the foods from (29) and for pollen calculated based on the values in Table I. 'Fresh raw tomato, cabbage, apple; fried chicken leg and breast; baked beans; whole wheat bread; broiled sirloin beer steak. 'Each food is ranked from 7 (highest nutrient content) to 0 (lowest nutrient content) for each of the II nutrient categories. Because increased dietary levels of fat and sodium are considered detrimental to health, the rankings are reversed (7=lowest; O=highest) for these two nutrients. Rank score is the total of the II rankings for each food (lowest possible score=O; highest=77)

Protein (g)

Food'

Table 2. Nutritional comparison of pollen and typical nutritious foods a

P

~

9

CO'

1{'

!-

QO

-


19

does not preclude pollen from being an excellent food supplement which can enhance the health and well-being of individuals, especially those who otherwise might have an unbalanced diet. Pollen or pollen products have been shown to have several beneficial applications for human use. Pollen has been successfully used for treatment of some cases of benign prostatitis (5,6,7,8,9) and for oral desensitization of children who have pollen allergy (10). Pollen has been shown to be an excellent dietary component in diets for specialty or valuable animals (see (11) for more discussion).

2. HONEY Honey is a supersaturated solution of sugars, mainly fructose, glucose, and maltoselike sugars, with traces of sucrose, glucose oxidase, hydrogen peroxide, phenolics, flavonoids, terpenes, etc. (12). The sugars make honey hygroscopic (moisture absorbing) and viscous, and the sugar concentration plus other factors including low pH, hydrogen peroxide, and the flavonoids, phenolics and terpenes make honey antimicrobial or prevent microbial growth (13). The main use of honey is as a flavorful sweetener and energy source which is eaten with and as a component of a wide variety of foods. The sweetness is from the sugars, particularly fructose, and flavor is created by a wide variety of trace essences derived from plant esters, alcohols, aldehydes, and other compounds (12). Secondary, but important, uses of honey are for the promotion of health and well being. Some of these uses include aiding in the healing of wounds, healing of serious skin burns, and healing gastric ulcers. The basis for the wound and burn healing properties of honey is its antimicrobial, moisturizinglfluid removal, and oxygen barrier properties. By keeping a wound or burn clean and moist, and free from bacteria and the damaging effects of oxygen, the wound can heal much more quickly than if left unaided. Modern creams and antibiotics may help heal these types of wounds, but they often have the disadvantages of killing tissue and causing heavy scabs and scars. The healing properties of honey were clearly demonstrated in a study comparing honey treatment to that of silver sulfadiazine, the standard treatment, for burn victims. The results of the study (Table 3) clearly showed that honey treatments resulted in a much greater sterility of the wounds, a faster rate of healing, and a faster onset of healing (14). Similar results have been shown by T. Postmes in tests with burned pigs. In these experiments, honey was shown not only to be better than standard treatments, but also better than artificial honey made from the sugars, but omitting the glucose oxidase, hydrogen peroxide, flavonoids, and other minor components of honey (T. Postmes, personal communication). For many years, advocates have claimed that honey can help treat gastric ulcers. With recent discoveries, an understanding of how this can occur has emerged. Until recently the bacterial origin of gastric ulcers was unknown. Now, the culprit is known to be the bacterium Helicobacter pylori. Some honey has been shown to inhibit H. pylori (P.C. Table 3. Honey and silver sulfadiazine (Std) treatments for burns a % Sterile in 7

Treatment (n=52)

days

Honey Silver sulfadiazine

91 7

"Data from (14)

Mean days for granulation 7.4 13.4

Healing in 15 days (%) 87 10

20

J. O. Schmidt

Molan, pers. communication) and the flavonoid content and low pH of honey likely aid in stimulating growth and healing.

3. PROPOLIS Propolis is plant resin collected by bees for use in and around the hive. In plants it is usually the sticky coating around buds that serves to protect them from the elements of weather plus from attack by bacteria, fungi, molds and viruses. These are properties that are useful to the bees and are enhanced by the sticky properties of the propolis. Like pollen, propolis is a bee product that cannot be clearly defined and varies from sample to sample. This is a natural outcome of the collection process---propolis collecting bees will use resins from a large variety of tree and other plant species, and these naturally will differ in their qualitative and quantitative chemical composition. Nevertheless, different propolis samples do share considerable similarity in their physical and overall general chemical nature, thereby enabling a general discussion ofthe properties of pro polis. Much work has been conducted on the chemistry and properties of propolis. Hundreds of chemical compounds have been identified from propolis. The main chemical classes present in propolis are flavonoids, phenolics, and various aromatic compounds (Figure 1). These compounds are poorly soluble in water, usually are soluble in alcohols,

o

OH

HO

QUERCETIN

H 0 - ) 0 > - CH=CHCOOH

HO CAFFEIC ACID

o

HO PINOCEMBRIN Figure 1. Typical flavonoids and phenolics present in propolis.


21

Table 4. Compounds in propolis that possess known pharmacological activities Chemical Quercetin

Pinocembrin

Caffeic acid

Caffeic acid phenethyl ester Acacetin Pinostrobin

Activities Anti-viral Antihistamine Ulcer healing Capillary strengthening Anti-bacterial Anti-fungal Anti-mold Local anesthesia Anti-bacterial Anti-fungal Anti-viral Anti-inflammatory Tumor cytotoxicity Anti-inflammatory Local anesthesia

References

30 31 32 33 34 35 32 33 30 36 37 36 35

and are often poorly soluble in hydrocarbon solvents. Propolis also contains some volatile oils, terpenes, and beeswax, but these compounds are not believed to contribute as significantly to the chemical properties and effects of propolis. Flavonoids are well-known plant compounds that have antioxidant, anti-bacterial, anti-fungal, anti-viral, and anti-inflammatory properties. Other properties of propolis include acting as a local anesthetic, reducing spasms, healing gastric ulcers, and strengthening capillaries. Compounds responsible for these activities are listed in Table 4.

4. BEE VENOM Venom is synthesized by honey bees for only one purpose: as a defensive agent against predators, primarily large mammalian and other vertebrate predators. In order to be of defensive value the venom must induce pain, cause damage, or have some other pharmacological or sensory activity in the potential predator (15). Bee venom, unlike many other insect allomones, or chemical defenses. is water soluble, not fat soluble, and must be injected or applied to moist tissues to be active. This water solubility is an advantage as it allows a whole new suite of highly active defensive compounds to be used. Bee venom is composed of a diversity of proteins, peptides, active amines, and other compounds which possess a variety of activities (16). The major chemical components and their primary activities are listed in Table 5. The main pain-inducing and lethal component appears to be melittin (17) and this component might be responsible for much of the activity of bee venom in apitherapy use. Mankind has used bee venom primarily for apitherapy to treat a variety of autoimmune diseases, with recent usage for immunotherapy of bee sting allergic patients. The immunotherapy use will not be considered further (see (18) for further discussion). Apitherapy has been particularly successful with individuals suffering from rheumatoid arthritis, gout, and multiple sclerosis, but a variety of other immune disorders including scleroderma and asthma have been treated (T. Cherbuliez, pers. communication). The benefit of apitherapy for treatment of arthritis has received some research attention by the

22

J. O. Schmidt

Table 5. Components in honey bee venom and some of their activities a Component (% venom) Melittin (3G-50) Phospholipase A2 (10-20) Apamin (3) Hyaluronidase (2) Mast cell degranulating peptide (2) Histamine

«1)

Chemical nature Small, highly basic 26 amino acid polypeptide of2840 Mol. Wt. Basic, stable protein of 15,800 Mol. Wt. Highly basic 18 amino acid polypeptide Protein of 35,000 Mol. WI. Highly basic 22 amino acid polypeptide Small, unstable biogenic amine of III MoI.Wt.

Activity/pharmacology Pain, cardiotoxin, hemolysin, membrane activity Membrane and phospholipid disruptant, toxic, lungs are target Neurotoxin, causes tremors Promotes spreading of other components, no other activities Releases histamine, etc. from mast cells, pain, anti-inflammatory Bum-itch, redness, immediate local skin effects

'Data from references (16) and (38).

medical establishment. Cohen et al. (19) demonstrated in controlled experiments that bee venom and local pain-inducing agents significantly improved the symptoms of rheumatoid arthritis patients. Steigerwaldt et al. (20) reported moderate improvement in 66% of bee venom treated patients versus only 27% improvement in the controls. Vick et al (21) using severely arthritic dogs reported significant improvement in mobility and activity in their cages of bee venom treated animals compared with controls. Some of the problems in demonstrating efficacy of bee venom treatments for immune diseases stem from the very nature of immune disorders. Immune disorders are characterized by "flare ups" and remissions that occur unpredictably. In addition, immune disorders are particularly susceptible to treatment placebo effects. These two factors combine to make clinical research trials on immune diseases very difficult and often inconclusive. These same problems also plague medical research concerned with evaluating established treatments. In the cases of arthritis and multiple sclerosis, modern medicine has no cures, it simply treats to suppress symptoms. The established treatments include use of steroids, strong anti-inflammatory drugs, antibiotics, antimalarials, and gold salts--drugs with serious side effects, and that often fail to deliver relief. This frustrating situation led one researcher to comment "rheumatoid arthritis rarely kills the patient; corticosteroids often do" (22). These problems lead this writer to observe that apitherapy has never killed anyone and has negligible side effects. Thus, what valid criticisms can be raised against apitherapy for rheumatoid arthritis and multiple sclerosis? The question arises: how does bee venom work? The answer is not clear, but we have some hints. Bee venom has anti-inflammatory effects, it might well "shock" the immune system which somehow might correct imbalances, it causes pain, and it might stimulate the nervous system which, in turn, can exert influence on the immune system. Bee venom possesses chemical components responsible for these activities: anti-inflammatory action - mast cell degranulating peptide, apamin; "shocks" immune system-phospholipase A2 , hyaluronidase; pain-melittin; stimulates nervous system-melittin, apamin, mast cell degranulating peptide. Overall, bee venom appears to have the chemical properties to affect the immune system and immune disorders, and apitherapy has been shown to work in many cases-so all that is needed is a clearer understanding of how apitherapy works and to convince mainstream practitioners to use apitherapy.


23

5. ROYAL JELLY Royal jelly is a creamy product secreted by young nurse worker bees for feeding to the queen, queen larvae, and other young larvae. It is totally synthesized by the bees in the hypopharyngeal and mandibular glands and is derived from the proteins and nutrients in the pollen ingested by the secreting bees. Royal jelly consists of an emulsion of proteins, sugars, and lipids in a water base (Table 6). The proteins have no particularly unusual properties (23) and have the main presumed function of providing the growing larva or the queen a readily digested source of protein. The remainder of the composition, except the lipids, also appears to be oriented toward providing a balance of nutrients for the consuming individuals. The lipids are unusual because they lack the normal triglycerides and diglycerides that are composed of fatty acids having carbon chains of even numbers from 14 to 20 that are typical of insect fats. Instead royal jelly lipids are composed mostly of short chained 8-10 carbon hydroxy fatty acids or diacids. These compounds have active functionalities at both ends of the molecule, are more soluble in water than usual fatty acids, are highly acidic, and act as good detergents and antimicrobial agents. It is this latter property, antimicrobial activity, that appears to be the main function of the lipids in royal jelly. For humans, royal jelly possesses the appealing properties of being a creamy emulsion that is strongly antibacterial (24,25). These make it an ideal component of cosmetics and skin care products. Internal uses of royal jelly are less promising, as all the antibacterial activities disappear when the pH is raised to above 6 by the natural buffering systems in the body (which maintain a pH of about 7.4) (24). In fact, no clear evidence from controlled experiments exists to support claims of internal usefulness of royal jelly; that in conjunction with the lack of a theoretical chemical basis for activity leads to the conclusion that there is little future promise for pharmaceutical use of royal jelly. Royal jelly is a

Table 6. Chemistry of royal jelly" Component

Quantity

Water Proteins

67.0% 12.5%

Sugars Fructose Glucose Others Fats (£)-10-Hydroxydec-2-enoic acid

11.0% 6.0% 4.2% .8% 5.0% 31.8% of fat

10-Hydroxydecanoic acid Other hydroxy fatty acids Dicarboxylic acids Gluconic acid Others Minerals Vitamins

21.6% offat 9.5% offat 4.5% offat 24.0% offat 8.6% of fat <1.0% <0.1%

Sterols Undetermined pH 'See (11) for references and (23) for proteins.

<.01% 3.5% 3.8

Comments 18+ major pep tides/proteins of 14-94 kd with digestibility greater than beef Similar in composition to honey

Mostly hydroxy fatty acids, diacids, simple acids

K, Mg, Na, Ca, Zn, Fe, Cu, Mn Thiamin, riboflavin, pyridoxine, niacin, pantothenic acid, inositol, biotin, folic acid, Vito C 24-Methylene cholesterol, !3-stigmasterol, etc.

24

J. O. Schmidt

highly nutritious material. However, its cost precludes its use for any but the most specialized food products for people or animals and its benefits are questionable. Recently, royal jelly has been shown to cause serious reactions, including death, in some individuals who ingest it (26). This indicates that both more research into the causes of the adverse reactions, and caution in ingesting royal jelly are needed.

6. BEESWAX Beeswax is synthesized de novo by honey bees in four pairs of glands located on the ventral side of the abdomen. Bees use the wax as their primary building material for making combs for rearing their brood and for storage of honey and pollen. Beeswax is composed of a variety of monoesters, diesters, hydroxylated esters, hydrocarbons, and free fatty acids (Table 7). This composition distinguishes the material as a wax rather than a fat because it is composed mostly of esters and long chained hydrocarbons, classic wax components. Triglycerides and diglycerides, typical of fats, are missing. The chemistry of the beeswax components is ideal for the uses of it by both bees and man. These components, and the wax itself, are not soluble in water (or honey), repel water soluble materials, remain strong to temperatures of 50° C, are reasonably flexible, and are not readily degraded or decomposed by moisture or microorganisms. The strength, flexibility, and waterproofing qualities of beeswax have made it an excellent material for polishes, finishes, and waxes that preserve, add shine, and generally enhance products coated with it. Beeswax stability also makes it an excellent wax for addition to cosmetics and skin products. Historically, beeswax was an excellent material for making molds for castings; indeed, even today we have artifacts over 3000 years old that were produced by the lost-wax process (27). Beeswax also bums with a clean flame and produces a pleasant odor. This, plus the resistance of beeswax to degradation, has made it ideal for use as candles. The stable, flexible, and preserving properties of beeswax are good for use as waxes for musical instrument strings, skis, archery, and a variety of other specialty uses. Finally, the flexibility, safety, stability, and ability to accept colors of beeswax has made it a prime material for modem crafts and hobbies for both children and adults.

7. CONCLUSIONS Although honey bees and humans are dramatically different, they share two fundamental features-both are social animals, and both live in highly complex societies. These Table 7. Gross chemical composition of beeswax a Chemical class Monoesters Diesters Triesters Hydroxy esters & polyesters Acid esters & polyesters Long-chained hydrocarbons Long-chained fatty acids 'Data from (39)

Quantity (%) 35 14

3 12 3 14

12


25

features cause both species to maintain more or less permanent residences, to have developed specialized behaviors, to engage in a diversity of activities, and to need for a multitude of materials. Material properties and uses are governed by their chemistry and vice versa. Honey bees need a stable food supply for long-term energy and growth; people likewise need a stable food supply. Honey bees need structural materials such beeswax and propolis to construct their nest; people likewise have housing needs. Honey bees need materials such as propolis and venom to defend against diseases and predators; people have similar needs. Is it any wonder then, that since antiquity, human beings have gone to honey bees as a chemical warehouse of materials and foods. Honey and pollen are the foods that promote health and well being in honey bees. They have served the same function for people. Bees use wax to build their combs and people have taken advantage of the wonderful chemical properties of beeswax to make objects for their homes and daily lives and to coat and preserve materials. Bees use propolis and venom to defend against microorganisms and enemies. People also use propolis, sometimes in conjunction with honey, for its antimicrobial properties. People use the same properties in bee venom that drive off predators of bees to enhance human health by fighting off some of their bodies' own internal enemies that cause autoimmune diseases. Overall, much of the human application of bee products can be explained on the basis of the chemistry of the bee products. This is not to say that bee products should not be used for purposes for which we have no chemical understanding; indeed, the process has usually operated in reverse-first, people discovered uses for bee products, then later came the chemical understanding of how and why the bee products were useful. Perhaps the message from this is that we should look to traditional uses of bee products to guide us in our investigations and to use research to discover how best to use bee products and their components to improve human life. But for this process to operate, individuals concerned with bee products must be fair and honest in representing the legitimate uses and benefits of the bee products.

8. REFERENCES I. Recommended Dietary Allowances, 10th Edition. National Academy Press, Washington, DC. 1989. 2. Bell, R.R., E.J. Thornber, J.L.L. Seet, M.T. Groves, N.R. Ho and D.T. Bell. (1983) Composition and Protein Quality of Honeybee-Collected Pollen of Eucalyptus calophylla. J. Nutr. 113,2479--2484. 3. Schmidt, PJ., J.O. Schmidt and c.w. Weber. (1984) Mesquite Pollen as a Dietary Protein Source for Mice. Nutr. Reports IntI. 30, 513-22. 4. Liebelt, R.A., D. Lyle and J.Walker. (1994) Effects of a Bee Pollen Diet on Survival and Growth of Inbred Strains of Mice. Am. Bee J. 134,615-620. 5. Denis, L.J. (1966). Chronic Prostatitis. Acta Urol. Belg. 34,49-55. 6. Ask-Upmark, E. (1967). Prostatitis and its Treatment. Acta Med. Scand, 181,355-57. 7. Hayashi, A.U., J. Mitsui. H. Yamakawa et al. (1986). Clinical Evaluation ofCemilton in Benign Prostatic Hypertrophy. Hinyokika Kiyo 32, 135-41. 8. Samochowiec, L., T. Dutkiewicz, J. Wojcicki and J. Gieldanowski. (1992) The Influence of Pollen Extracts (Cemitin GBX and Cernitin T60) on Allergic Reactions. Phytother. Res. 6, 314--317. 9. Rugendorff, E.W., W. Weidner, L. Ebeling and A.C. Buck. (1993) Results of Treatment with Pollen Extract (Cernilton N) in Chronic Prostatitis and Prostatodynia. Brit. J. Urology 7l, 433-438. 10. Wortmann, F. Oral Immunotherapy. in: Clinical Immunology and Allergology. (Steffen, C. and H. Ludwig Editors) ElsevierlNorth-Holland, Amsterdam. 1981. pp. 389-398. II. Schmidt, J.O. and S.L. Buchmann. Other Products of the Hive. in: The Hive and the Honey Bee (Graham, J.M., Editor) Dadant & Sons, Hamilton, IL. 1992. pp. 927-988. 12. White, J.W.Jr. Composition of Honey. in: Honey A Comprehensive Survey (Crane, E. Editor). Heinemann, London. 1975. pp. 157-206. 13. Molan, P.c. (1992) The Antibacterial Activity of Honey I. The Nature of the Antibacterial Activity. Bee World 73, 5-28.

J. O. Schmidt

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14. Subrachmanyam, M. (1991) Topical Application of Honey in Treatment of Burns. Brit. J. Surg. 78, 497-498. 15. Schmidt, J.~. Hymenopteran Venoms: Striving Toward the Ultimate Defense Against Vertebrates. in: Insect Defenses Adaptive Mechanisms and Strategies of Prey and Predators (Evans, D.L. and J.~., Schmidt Editors), State Univ. New York Press, Albany, NY. 1990. pp. 387-419. 16. Banks, B.E.C. and R.A. Shipolini. Chemistry and Pharmacology of Honey-bee Venom. in: Venoms of the Hymenoptera (Piek T., Editor). Academic Press, London. 1986 pp. 330-416. 17. Schmidt, J.O. (1995) Toxinology of Venoms from the Honeybee Genus Apis. Toxicon 33, 917-927. 18. Schmidt, J.O. Allergy to Venomous Insects. in: The Hive and the Honey Bee (Graham, J.M., Editor) Dadant & Sons, Hamilton, IL. 1992. pp. 1209-1269. 19. Cohen, A., J.B. Pearah, A.w. Dubbs and CJ. Best. (1942) Bee Venom in the Treatment of Chronic Arthritis: A Comparative Study. Trans Med. Soc. State Pennsylvania 45, 957-959. 20. Steigerwaldt, F., H. Mathies and F. Damrau. (1966) Standardized Bee Venom (SBV) Therapy of Arthritis. Indust. Med. Surg. 35, 1045--1049. 21. Vick, J.A., G.B. Warren and R.B. Brooks. (1975) The Effect of Treatment with Whole Bee Venom on Daily Cage Activity and Plasma Cortisol Levels in the Arthritic Dog. Am. Bee J. 115,52-53,58. 22. Calin, A. Diagnosis and Management of Rheumatoid Arthritis. Addison-Wesley, Menlo Park, CA. 1983. 23. Thien, F.C.K., R. Leung, B.A. Adldo, J.A. Weiner, R. Plomley and D. Czarny. (1996) Asthma and Anaphylaxis Induced by Royal Jelly. Clin. Exp. Allerg. 26, 216--222. 24. Blum, M.S., A. F. Novak and S. Taber, III. (1959) IO-Hydroxy-L\2decenoic Acid, an Antibiotic Found in Royal Jelly. Science 130,452-453. 25. Yatsunami, K. and T. Echigo. (1985) Antibacterial Action of Royal Jelly. Bull. Fac. Agr. Tamagawa Univ. No. 25, 13-22. 26. Bullock, RJ., A. Rohan and J-A. Straatmans. (1994) Fatal Royal Jelly-Induced Asthma. Med. J. Australia

160,44. 27. Crane, E. The Archaeology of Beekeeping. Duckworth, London. 1983. 28. Atwater, W.O. (1910) Principles of Nutrition and Nutritive Values of Food. U.S. Dept. Agric. Bull. 142 (Second review). 29. Adams, C.F. (1975). Nutritive Value of American foods in Common Units. USDA Agric. Handb. No. 456. Washington DC: Government Printing Office. 30. Konig, B. and J.H. Dustmann. (1985) Fortschritte der celler Untersuchungen zur Antivirotischen Aktivitiit von Propolis. Apidologie 16, 228--230. 31. Budavari, S. (Editor). The Merck Index. Merck & Co, Rahway, NJ 1989. 32. Vilanueva, Y.R., M. Barbier, M. Gonnet and P. Lavie. (1970) Les Flavonoides de la Propolis Isolement d'une Nouvelle Substance Bacteriostatique: la Pinocembrine {dihydroxy-5,7-flavone} Ann. Inst. Pasteur. Paris llB, 84--87. 33. Metzner, J., E.-M.Schneidewind and E. Friedrich. (I 977} Zur Wirkung von Propolis und Pinocembrin auf Sprosspilze. Pharmazie 32, 730. 34. Miyakado, M., T. Kato, N. Ohno and TJ Mabry. (1976) Pinocembrin and (+)-I3-endesmol from Hymenoclea monog)Jra and Baccharis glutinosa. Phytochemistry 15, 846. 35. Paintz, M. and J. Metzner. (1979) Zur lokalaniisthetischen Wirkung von Propolis und einigen Inhaltsstoffen. Pharmazie 34, 839-841. 36. Bankova, Y.S, S.S. Popov and N.L. Marekov. (1983) A Study of Flavonoids of Propolis. J. Natural Prod. 46,471-474. 37. Grunberger, D., R. Ganerjee, K. Eisinger, E.M. Oltz, L. Efros, M. Caldwell, V. Estevez and K. Nakanishi. (1980) Preferential Cytotoxicity on Tumor Cells by Caffeic Acid Phenethyl Ester Isolated from Propolis. Experientia 44, 230--232. 38. Schumacher, M.J., J.O. Schmidt, N.B. Egen and J.E. Lowry. (1990) Quantity, Analysis and Lethality of European and Africanized Honey Bee Venoms. Amer. J. Trop. Med. Hyg. 43, 79-86. 39. Tulloch, A.P. {I 980) Beeswax--Composition and Analysis. Bee World 61, 47--62.

3

HONEY AS AN ANTIMICROBIAL AGENT P. C. Molan Honey Research Unit Department of Biological Sciences University ofWaikato Hamilton, New Zealand

1. INTRODUCTION That honey has antibacterial properties has been known for more than a centuryl. Although it has been used as a medicine since ancient times in many cultures 2 .3 , in its ancient usage there was no recognition of its antibacterial properties - it was just known to be an effective remedy. This is not surprising considering that it is only since the latter part of the last century that it has become known that many ailments are the result of infection by microorganisms. Now it can be seen that the effectiveness of honey in many of its medical uses is probably due to its antibacterial activity. It is well established that honey inhibits a broad spectrum of bacterial species. There are many reports of bactericidal as well as bacteriostatic activity. There have also been reports of honey having antifungal activity. These numerous reports of the antimicrobial activity of honey have been comprehensively reviewed4 ; the collation of data shows that honey is active against a wide range of bacterial and fungal species, many of which cause infections. However, there are ailments which may be treated with honey which have not had the infectious agents tested for their sensitivity to the antimicrobial activity of honey. Also, there has not been much distinction made in the different types of antimicrobial activity in honey to which the various microbial species are sensitive. For serious consideration to be given to the use of honey as a therapeutic agent it is necessary that these aspects be further investigated.

2. ANTIMICROBIAL PROPERTIES OF HONEY The numerous reports of investigations which have established the nature of the antimicrobial factors in honey are cited in a comprehensive review of this subject 4 ,s. A brief summary of what has been established is given here.

2.1. Explanation of Antibacterial Activity 2.1.1. Osmotic Effect. Honey is a saturated or super-saturated solution of sugars, 84% being a mixture of fructose and glucose. The water content is usually only l5~21% 27

28

P.c. Molan

by weight. The strong interaction of these sugar molecules with water molecules leaves very few of the water molecules available for microorganisms. This "free" water is what is measured as the water activity (a w ): mean values for honey have been reported from 0.562 to 0.62. Although some yeasts can live in honeys that have a high water content, causing spoilage of the honey, the aw of ripened honey is too low to support the growth of any species, no fermentation occurring if the water content is below 17.1 %. Many species of bacteria have their growth completely inhibited if the a w is in the range 0.94-0.99. These values correspond to solutions of a typical honey (a w of 0.6 undiluted) of concentrations from 12% down to 2% (v/v). On the other hand, some species have their maximum rate of growth when the aw is 0.99, so inhibition by the osmotic (water-withdrawing) effect of dilute solutions of honey obviously depends on the species of bacteria. 2.1.2. Acidity. Honey is characteristically quite acidic, its pH being between 3.2 and 4.5, which is low enough to be inhibitory to many animal pathogens. The optimum pH for growth of these species normally falls between 7.2 and 7.4. The minimum pH values for growth of some common wound-infecting species is: Escherichia coli, 4.3; Salmonella sp., 4.0; Pseudomonas aeruginosa, 4.4; Streptococcus pyogenes, 4.5. Thus in undiluted honey the acidity is a significant antibacterial factor. But if honey is diluted, especially by body fluids which are well buffered, the pH will not be so low and the acidity of honey may not be an effective inhibitor of many species of bacteria. 2.1.3. Hydrogen Peroxide. The major antibacterial activity in honey has been found to be due to hydrogen peroxide produced enzymically in the honey. The glucose oxidase enzyme is secreted from the hypopharyngeal gland of the bee into the nectar to assist in the formation of honey from the nectar. The hydrogen peroxide and acidity produced by the reaction:

serve to preserve the honey. The hydrogen peroxide produced would be of effect as a sterilising agent only during the ripening of honey. Full-strength honey has a negligible level of hydrogen peroxide because this substance is short-lived in the presence of the transition metal ions and ascorbic acid in honey which catalyse its decomposition to oxygen and water. The enzyme has been found to be practically inactive in full-strength honey, it giving rise to hydrogen peroxide only when the honey is diluted. This is because the acidity produced in the action of the enzyme drops the pH to a point which is too low for the enzyme to work any more. On dilution of honey the activity increases by a factor of 2 500 50 000, thus giving a "slow-release" antiseptic at a level which is antibacterial but not tissue-damaging. 2.1.4. Phytochemical Factors. The evidence for the existence of other antibacterial factors is mainly that the peroxide-generating system does not account for all of the observed antibacterial activity, but there have also been some reports of isolation of antibacterial substances from honey that are not hydrogen peroxide. Furthermore, it has ben found that heating honey, which inactivates the glucose oxidase, causes loss of activity against some species whilst it is retained against others. Although the stability of the enzyme varies in different honeys, there have been reports of honeys with stability well in

Honey as an Antimicrobial Agent

29

excess of this variation, showing that there must be an additional antibacterial factor involved. The most direct evidence for the existence of non-peroxide antibacterial factors in honey is seen in the reports of activity persisting in honeys treated with catalase to remove the hydrogen peroxide activity. Several chemicals with antibacterial activity have been identified in honey by various researchers: pinocembrin, terpenes, benzyl alcohol, 3,5-dimethoxy-4-hydroxybenzoic acid (syringic acid), methyl 3,5-dimethoxy-4-hydroxybenzoate (methyl syringate), 3,4,5trimethoxybenzoic acid, 2-hydroxy-3-phenylpropionic acid, 2-hydroxybenzoic acid and 1,4-dihydroxybenzene. However, the quantities of these present were far too low to account for any significant amount of activity.

2.2. Variation in Antibacterial Activity In almost all reports on the medical use of honey as an antibacterial agent no consideration is given to the selection of type of honey for therapeutic use. Aristotle, c.350 B.C. 6, and Dioscorides, c.50 A.D. 7, recommended that honey collected in specific regions and seasons (and therefore presumably from different floral sources) be used for the treatment of particular ailments, but in modem medicine clinical practitioners have not heeded these views nor the laboratory findings of large differences in the antibacterial potency of different honeys. It was recognised more than 40 years ago that there are differences in the antibacterial activity of different honeys, and a method was devised to determine the "inhibine number" of honeys as a measure of their antibacterial activity. The "inhibine number" is the degree of dilution to which a honey will retain its antibacterial activity, representing sequential dilutions of honey in steps of 5% from 25% to 5%. Studies measuring the "inhibine number" of honeys report activity to range over the five-fold difference in concentration in the dilution series, and studies using a wider range of dilutions report the minimum inhibitory concentrations of the honeys tested to range from 25 to 0.25%, >50 to 1.5%, 2O--D.6%, and 50-1.5%. The data showed activities to be fairly well spread over these ranges. A study of 345 samples of New Zealand honeys8 found a large number with low activity (36% of the samples had activity near or below the level of detection), the rest having almost a Gaussian distribution over a twenty-fold range of activity. The major variations seen in overall antibacterial activity are due to variation in the level of hydrogen peroxide that arises in honey, and in some cases to the level of non-peroxide factors. Hydrogen peroxide can be destroyed by components of honey: it can be degraded by reaction with ascorbic acid and metal ions, and by the action of the enzyme catalase which comes from the pollen and nectar of certain plants, more from the nectar. Also, very large differences have been found between honeys from different floral sources in the thermal stability of their glucose oxidase content, and in the sensitivity of this hydrogen peroxide-producing enzyme to denaturation by light because of a photosensitizing component that comes from some floral sources. Although it appears that the honey from certain plants has better antibacterial activity than that from others, there is not enough evidence for such definite conclusions to be justified because the data are from small numbers of samples. However, honeys from some sources have been been studied in large enough numbers or have been included in enough different studies for some trends to be noted. Honeydew honey from the conifer forests of the mountainous regions of central Europe has been found to have particularly high antibacterial activity. Also honey from manuka (Leptospermum scoparium) in New

30

P.C. Molan

Zealand has been found to have a high activity, about half of this type of honey having an exceptionally high level of non-peroxide activity 9. Thus it is important that when honey is to be used as an antimicrobial agent it is selected from honeys that have been assayed in the laboratory for antimicrobial activity. It is also important that honey for use as an antimicrobial agent be stored at low temperature and not exposed to light, so that none of the glucose oxidase activity is lost. Although all honey will stop the growth of bacteria because of its high sugar content, when the sugars are diluted by body fluids this antibacterial action is lost. The additional antibacterial components then become important.

3. POTENTIAL USES OF HONEY AS AN ANTIMICROBIAL AGENT 3.1. Limitations to Usage The popular literature on health and self-treatment of ailments gives the impression that honey can be taken to cure almost anything, but a rational consideration would suggest that the antimicrobial activity would be insignificant when an oral dose of honey becomes diluted after absorption from the gut into the many litres of fluid in the circulation and tissues of the body. Realistically, the potential for honey as an antimicrobial agent in medicine is in topical application rather than as a systemic agent, although there are some situations such as gastrointestinal infections or mastitis where the honey could remain localised and thus not become too dilute to be effectively antibacterial.

3.2. Honey as an Antiseptic Dressing 3.2.1 Established Usage of Honey as a Dressing. Honey has a well established usage as a wound dressing in ancient and traditional medicine lo . In recent times this has been rediscovered, and honey is in fairly widespread use as a topical antibacterial agent for the treatment of wounds, burns and skin ulcers, there being many reports of its effectiveness ll - 23 . The observations recorded are that inflammation, swelling and pain are quickly reduced, unpleasant odours cease, sloughing of necrotic tissue occurs without the need for debridement, dressings can be removed painlessly and without causing damage to regrowing tissue, and healing occurs rapidly with minimal scarring, grafting being unnecessary. In many of the cases honey was used on infected lesions not responding to standard antibiotic and antiseptic therapy. It was found in almost all of the cases to be very effective in rapidly clearing up infection and promoting healing. 3.2.2. Importance ofAntibacterial Activity. Much of the effectiveness of honey as a dressing appears to be due to its antimicrobial properties. The healing process will not occur unless infection is cleared from a lesion: swabbing of wounds dressed with honey has shown that the infecting bacteria are rapidly cleared 13,16,18,20.24. In this respect honey is superior to the expensive modern hydrocolloid wound dressings as a moist dressing. Although tissue re-growth in the healing process is enhanced by a moist environment, and deformity is prevented if the re-growth is not forced down by a dry seab forming on the surface, moist conditions favour the growth of infecting bacteria. Antibiotics are ineffective in this situation, and antiseptics cause tissue damage, so slow the healing process 25 . Honey is reported to cause no tissue damage, and appears to actually promote the healing process.


31

There are also numerous reports of sugar being used as a wound dressing, this also being found to be effective 26-31. Antibacterial activity is attributed by several authors to the high osmolarity of the sugar or honeyll. 17.22.27, it not being generally recognised that some honeys can have additional antibacterial activity considerably greater than that due to the osmolarity. This additional activity would be of particular significance in situations where the dressing becomes diluted by body fluids, and in regions of a lesion that are not in direct contact with the dressing. Staphylococcus aureus is exceptionally osmotolerant: for complete inhibition of its growth the a w has to be lowered below 0.86, which would be a typical honey at 29% (v/v). In the reports of sucrose syrup or paste being used as a wound dressing it is noted that infection with Staphylococcus aureus is hard to clear. Measurements that have been reported27 of the dilution occurring from the uptake of water from surrounding tissues when an abdominal wound was packed with sugar reveal that a saturated sucrose syrup containing undissolved granules becomes diluted in 7.5 hours to a concentration that is 30% of that of a saturated solution. Although the aw of this solution is low enough to prevent the growth of most human pathogens, it is not low enough to seriously restrict the growth of Staphylococcus aureus, a species which has developed resistance to many antibiotics and has become the predominant agent of wound sepsis in hospitals 32 . But Staphylococcus aureus is one of the species most sensitive to the antibacterial activity of honey. There have been many reports of complete inhibition of Staphylococcus aureus by honeys diluted to much lower concentrations 4 , showing the importance of the other antibacterial factors in selected honeys. To know for certain the clinical significance of the additional antibacterial activity in honey, a clinical trial will need to be conducted to compare dressings of sugar and selected honeys. The little comparative work reported to date indicates that more rapid healing is achieved with honey than with sugar I2 . IS • Since infection is one of the most common impediments to wound healing 33 , then such results would be expected if the sugar dressing were not able to fully suppress the growth of bacteria as the sugar became diluted. The additional antibacterial activity of honey could be the reason for the remarkable rates of healing reported when honey has been used as a dressing 1 I. 13. 14. 3.2.3. Effectiveness against Wound-Infecting Species of Bacteria. The seven species of bacteria most commonly involved in wound infection have been tested for their sensitivity to the antibacterial activity of honei 4 . The two major forms of antibacterial activity were examined separately: a honey with an average level of activity due to hydrogen peroxide and no detectable non-peroxide activity was used; also a manuka honey with an average level of non-peroxide activity, with catalase added to remove any hydrogen peroxide. The results of this study are summarised in Table 1. Overall there was little difference between the two types of antibacterial activity in their effectiveness, although Table 1. The minimum concentration of honey (%, v/v) in the growth medium needed to completely inhibit the growth of various specics of wound-infecting bacteria Species

Escherichia coli Proteus mirabilis Pseudomonas aeruginosa Salmonella typhimurium Serratia marcescens Staphylococcus aureus Streptococcus pyogenes

Manuka honey

Other honey

3.7 7.3 10.8 6.0 6.3 1.8 3.6

7.1 3.3 6.8 4.1 4.7 4.9 2.6

32

P. C. Molan

some species were more sensitive to the action of one type of honey than they were to the other. The results thus showed that these honeys, with an average level of activity, could be diluted nearly ten-fold yet still completely inhibit the growth of all the major wound-infecting species of bacteria. It is notable that the manuka honey, with an average level of activity, could be diluted with 54 times its volume of fluid yet still completely inhibit the growth of Staphylococcus aureus, the major wound-infecting species, and a species notorious for its development of resistance to antibiotics. There are frequent reports of hospital wards being closed because of the presence of strains of methicillin-resistant Staphylococcus aureus (MRSA). Because these strains are resistant to all of the antibiotics in common use it is necessary to protect patients with impaired immunity from exposure to them in case they contract infections which will not respond to treatment. The collection of strains of MRSA at Waikato Hospital have been tested for sensitivity to the two honeys described above J5 . All of the strains were found to be completely inhibited by both honeys at 10% (v/v) in the growth medium, and many of the strains by the honeys at 5% (v/v).

3.2.4. Microbiological Safety. The use of honey as a wound dressing has been argued against because of the risk of it possibly causing wound botulism 36 . Clostridia are widely distributed in nature, but there is a very low incidence of wound botulism. However, honey sometimes contains spores of Clostridium botulinum J7 , so there is a definite risk of introducing the spores into wounds if honey is used as a dressing. Ifhoney could be sterilized for use as a wound dressing this would remove the risk. The glucose oxidase activity which generates the hydrogen peroxide is very labile and would not withstand autoclaving 5. The non-peroxide activity of man uk a honey is stable to much more heating 38 , but there is some loss on autoclaving J9, and any hydrogen peroxide activity present in addition to the non-peroxide activity would be completely lost. Honey is too viscous for sterilization by filtration through microporous membranes, but sterilization by gamma-irradiation is a possibility. However, there have been no reports on whether or not the antibacterial factors in honey withstand this sterilizing treatment. Therefore a study was recently undertaken to determine the effect of gamma-irradiation on the antibacterial activity of hone/D. Honey samples were selected for their antibacterial activity, some manuka honeys with a high level of non-peroxide activity and a low level of hydrogen peroxide activity, others honeys with a high level of hydrogen peroxide activity only. They were put through a commercial sterilising plant which subjected all items processed to the standard 25 kGy of gamma-irradiation used for sterilising medical materials. The results of this study, summarised in Table 2, showed that there was no significant loss of either type of antibacterial activity when the honey samples were gamma-irradiated. A control honey very heavily seeded with Clostridial spores had no viable spores present after the same irradiation treatment.

3.3. Honey for the Treatment of Mastitis in Dairy Animals One type of infection in which a localised high concentration of honey could be achieved is mastitis in dairy cows and goats. This can be an expensive and difficult condition to treat. The standard treatment is the introduction of antibiotics into the teat canal of the infected udder, but milk has to be withheld from use until clear of antibiotic residues. Honey could possibly be suitable for the treatment of mastitis if inserted into the infected udder via the teat canal as it is harmless to tissues and would leave no undesirable residues in milk.


33

Table 2. Comparison of the antibacterial activity of various samples of honey before and after sterilization by gamma-irradiation. The activity is shown as the diameter (mm), ± S.D. (n=16), of the clear zone obtained in an agar well diffusion assay using plates seeded with Staphylococcus aureus. Total activity (i.e. 25% w/v honey in water) and non-peroxide activity (i.e. 25% w/v honey in catalase solution) are shown

Untreated, no catalase Irradiated, no catalase Untreated, with catalase Irradiated, with catalase

Honey 1

Honey 2

Honey 3

Honey 4

Honey 5

20.8±1.1 21.3±1.0 O.O±O.O O.O±O.O

16.0±0.8 14.9±0.6 O.O±O.O O.O±O.O

12.7±0.5 13.1±0.7 12.8±0.4 13.2±0.4

13.6±0.5 14.6±0.5 12.1±O.6 13.5±0.5

15.4±0.6 16.3±0.6 16.3±0.5 16.5±0.6

As a first step in evaluating this possibility, the seven species of bacteria that most commonly cause mastitis in dairy cattle were tested for their sensitivity to the antibacterial activity of honey. Cultures of these were spread on nutrient agar plates containing various concentrations of two types of natural honey and an artificial honey, and the growth of the bacteria was assessed to find the concentration of honey that was necessary to prevent growth of the bacteria. The natural honeys used were a rewarewa honey with an average level of activity due to hydrogen peroxide and no detectable non-peroxide activity, and a manuka honey with an average level of non-peroxide activity and no detectable non-peroxide activity. The artificial honey was used to assess the sensitivity of the bacteria to the osmotic action and acidity of honey. The results of this studl 1 are summarised in Table 3. It can be seen that the growth of all seven species was completely inhibited by a I in 10 dilution of the natural honeys, in some cases a I in 20 dilution being sufficient. The manuka honey was noticeably more effective. Since only one species was inhibited by the artificial honey at a I in 10 dilution, it can be seen that the other antibacterial factors in natural honeys are important, and honeys should be selected for a high level of these if they are to be subjected to trial in the treatment of clinical mastitis.

3.4. Honey for the Treatment of Peptic Ulcers Honey is a traditional remedy for dyspepsia and peptic ulcers42, but there has been no rational basis for its use. The finding that Helicohacter pylori is probably the causative agent in many cases of dyspepsia and peptic ulcers raised the possibility that the antibacterial properties may be responsible for its therapeutic action. Consequently, the sensitivity of Helicobacter pylori to honey was tested42 , using isolates of Helicobacter pylori from biTable 3. Minimum inhibitory concentration of honeys (% v/v in nutrient agar) for cultures of various mastitis-causing bacteria streaked on the agar plates Bacterial species

Actinomyces pyogenes Klebsiella pneumoniae Nocardia asteroides Staphylococcus aureus Streptococcus agalactiae Streptococcus dysgalactiae Streptococcus uberis

Manuka honey 1-5% 5--10% 1-5% 1-5% 1-5% 1-5% 1-5%

Rewarewa honey 1-5% 5--10% 5--10% 1-5% 5--10% 5--10% 5--10%

Artificial honey 5--10% >10% >10% >10% >10% >10% >10%

34

P. C. Molan

opsies of gastric ulcers. All five isolates tested were found to be sensitive in an agar well diffusion assay to a 20% (v/v) solution of a manuka honey with an average level of nonperoxide activity, but none showed sensitivity to a 50% (v/v) solution of a honey in which the antibacterial activity was due primarily to its content of hydrogen peroxide. Assessment of the minimum inhibitory concentration by inclusion of manuka honey in the agar showed that the growth of all of a further seven isolates tested was completely inhibited over the incubation period of 72 h by the presence of 5% (v/v) honey.

3.5. Honey for the Treatment of Gastroenteritis Honey has been found to be effective in treating bacterial gastroenteritis in infants 43 . Used in place of glucose in an oral re-hydration fluid, it was found to be as effective as glucose in achieving re-hydration, whilst the antibacterial activity cleared the infection in bacterial diarrhoea. However, there is little information available on the sensitivity of the gastroenteritis-causing species of bacteria to the antibacterial activity of honey, and on which of the antibacterial factors in honey is most effective against them. Therefore honey was tested for its relative antibacterial potency against all the bacterial species that commonly cause gastroenteritis, comparing manuka honey and a honey with the usual hydrogen peroxide activity, also an artificial honey to assess how much of the antibacterial activity was due simply to the acidity and the osmotic effect of the sugar in hone/ 4 , With some of the species of bacteria the assessment was repeated with additional strains obtained from clinical isolates supplied by medical and animal health laboratories to see if there was any variation in sensitivity between different strains of a species. Cultures of the bacteria were streaked on nutrient agar plates containing various concentrations of the honeys, and the growth of the bacteria was assessed to find the concentration of honey that was necessary to prevent growth of the bacteria. The honeys used were a mixed pasture honey with an average level of activity due to hydrogen peroxide and no detectable non-peroxide activity, and a man uk a honey with an average level of non-peroxide activity. Honey concentrations were in a 5% (v/v) step dilution series initially and then with I % dilution steps, the honey being diluted with either sterile distilled water (for the pasture honey and artificial honey) or a sterile solution of 0.2% catalase (for the manuka honey). Plates where inhibition of growth was observed were swabbed with a loopful of sterile water and streaked onto freshly prepared nutrient agar plates which did not contain honey. The plates were then incubated to find any surviving bacteria growing into visible colonies if the initial inhibition had been due to prevention of growth (bateriostasis) rather than killing the bacteria (bactericidal activity). The results, summarised in Table 4, showed that honey with an average level of hydrogen peroxide activity is bacteriostatic at 4-8% (v/v) and bactericidal at 5-10% (v/v). The non-peroxide activity of an average manuka honey is bacteriostatic at 5-11 % (v/v) and bactericidal at 8-15% (v/v). Activity (just bacteriostatic) was not seen with artificial honey unless it was at 20-30% (v/v), clearly showing the importance of factors other than sugar and acidity,

3.5. Honey for the Treatment of Tineas Honey has been reported to have antifungal activity, but not many species of fungi have been tested. An important group of fungi which regularly infect humans are the dermatophytes (Deuteromycotina). Cutaneous or superficial mycoses, caused through host infection by these fungi, are one of the most common diseases of humans. Only a small


35

Table 4. Minimum inhibitory concentration of honeys in nutrient agar plates (% v/v) giving partial inhibition (PI), bacteriostatic activity (BS) and bactericidal activity(BC) against various strains of bacteria which cause gastroenteritis Manuka honey with catalase PI

Bacterial strain Escherichia coli 916 Escherichia coli ex AHL Escherichia coli K88+ Salmonella enteritis 3484 Salmonella hadar 326 Salmonella infantis 93 Salmonella typhimurium 298 Salmonella typhimurium 1739 Salmonella typhimurium ex WH Shigella boydii 2616 Shigella jlexneri 983 Shigella sonnei 86 Shigella sonnei ex WH Vibrio cholorae Vibrio paraheamolyticus Yersinia enterocolitica

6% 6% 6% 7% 6% 7% 6% 6% 6% 6% 6% 5% 5% 5% 10%

BS

7% 7% 7% 8% 7% 8% 7% 7% 5% 7% 7% 7% 6% 7% 6% 11%

Pasture honey

BC

10% 10% 10% 10% 8% 10% 8% 9% 10% 10% 10% 10% 10% 10% 10% 15%

BS

PI

5% 4% 6%

6% 7%

6% 6% 6% 5% 6% 7% 6% 6% 5% 5% 6% 5% 6% 7% 4% 8%

BC

6% 6% 6% 6% 6% 10% 8% 7% 10% 6% 6% 5% 10% 10% 6% 9%

number of species of these, from the genera Epidermophyton , Microsporum and Trichophyton, regularly infect humans 45 • Superficial fungal infections are amongst the most difficult diseases to successfully treat, antibiotics which successfully combat bacterial diseases being largely ineffective against fungi. A common predisposition to some fungal infections is poor host immunity, thus bacterial infections may also be present quite often. So a treatment which has both antifungal and antibacterial activities would be most beneficial. Therefore the effectiveness of honey against the dermatophyte species which most frequently cause superficial mycoses (tineas such as ringworm and athletes foot) was invest igated46 • For this investigation two sorts of natural honey were used: a mixed pasture honey with an average level of antibacterial activity due to hydrogen peroxide production, and a manuka honey with an average level of non-peroxide antibacterial activity. An artificial honey was also used, to assess how much of the antibacterial activity was due simply to the acidity and the osmotic effect. The honeys were tested against clinical isolates of seven species of dermatophytes. An agar well diffusion assay was used, the contents of the wells being replaced with freshly prepared honey solutions at 24 hour intervals over the 3 - 4 days of incubation. The honeys were diluted with either sterile distilled water or a sterile solution of 0.2% catalase, a 5% (v/v) step dilution series being used for testing. The results are summarised in Table 5. No inhibitory activity was detected with any of the seven species with the pasture honey at any concentration up to the highest tested, 50% (v/v), when catalase was present, nor with the artificial honey even at 100%. This showed that it was the the hydrogen peroxide in the pasture honey, and the non-peroxide activity in the manuka honey, that were inhibiting the growth of the fungi. Although the concentrations of honey needed to inhibit some of the dermatophytes are higher than needed to inhibit bacteria, less dilution of the honey is likely with a tinea than with infected wounds, bums and ulcers where there would be serum exudation. It could be that

36

P. C. Molan

Table 5. Minimum inhibitory concentration of honeys in agar wells (% v/v) giving a clear zone around the wells in an agar well diffusion assay against seven species of fungi which cause tineas Species

Epidermophyton jloccosum Microsporum canis Microsporum gypseum Trichophyton rubrum Trichophyton tonsurans T. mentagrophytes var. interdigitate T. mentagrophytes var. mentagrophytes

Pasture honey

Manuka honey

Manuka honey with catalase

5-10% 10-15% 15-20% 2.5-5% 15-20% 10-15% 10-15%

5-10% 20-25% 45-50% 5-10 20-25% 20-25% 15-20%

20-25% 20-25% 50-55% 15-20% 20-25% 40-45% 20-25%

manuka honey may be more effective, even though the dermatophytes are less sensitive to its activity than they are to hydrogen peroxide, if there is insufficient dilution of honey on tineas for the enzymic production of hydrogen peroxide to be activated. Which type of honey is most effective, and the practical usefulness of honey as a topical antifungal salve, will only be known if comparative clinical trials are conducted.

4. REFERENCES I. Dustmann J H. (I 979) Antibacterial Effect of Honey. Apiacta 14, 7-11. 2. Majno G: The Healing Hand. Man and Wound in the Ancient World. Harvard University Press Cambridge, Massachusetts. 1975. 3. Ransome H M: The Sacred Bee in Ancient Times and Folklore. George Allen and Unwin London. 1937. 4. Molan PC. (1992) The Antibacterial Activity of Honey. I. The Nature of the Antibacterial Activity. Bee World 73, 5-28. 5. Molan PC. (\992) The Antibacterial Activity of Honey. 2. Variation in the Potency of the Antibacterial Activity. Bee World 73,59-76. 6. Aristotle (350 B.C.). Translated by Thompson D'A W. Historia Animalium in: The Works of Aristotle (Smith J A, Ross W D editors) Oxford University Press Oxford 1910 Volume IV. 7. Gunther R T: The Greek Herbal of Dioscorides (Translated by Goodyear J, 1655). Hafner N. Y. 1934, reprinted 1959. 8. Allen K L, Molan PC, Reid G M. (\ 991) A Survey of the Antibacterial Activity of Some New Zealand Honeys. J. Pharm. Pharmacol. 43, 817-822. 9. Allen K L, Molan P C, Reid G M. (1991) The Variability of the Aantibacterial Activity of Honey. Apiacta 26, 114--121. 10. Zumla A, Lulat A. (\ 989) Honey - a Remedy Rediscovered. J. Royal Soc. Med. 82, 384--385. II. Bulman M W. (\ 955) Honey as a Surgical Dressing. Middlesex Hosp. J. 55, 188-189. 12. Hutton D J. (1966) Treatment of Pressure Sores. Nurs. Times 62,1533-1534. 13. Cavanagh D, Beazley J, Ostapowicz F. (1970) Radical Operation for Carcinoma of the Vulva. A New Approach to Wound Healing. J. Obstet. Gynaecol. Br. Cmwlth. 77. 1037-1040. 14. BIomfield R. (1973) Honey for Decubitus Ulcers. J. Am. Med. Assoc. 224, 905. 15. Burlando F. (1978) Sull'azione Terapeutica del Miele nelle Ustioni. Minerva Dermat.1l3, 699-706. 16. Armon P J. (1980) The Use of Honey in the Treatment ofInfected Wounds. Trop. Doct. 10,91. 17. Bose B. (1982) Honey or Sugar in Treatment ofInfected Wounds? Lancet i, 963. 18. Dumronglert E. (1983) A Follow-up Study of Chronic Wound Healing Dressing with Pure Natural Honey. J. Natl Res. Counc. Thail. 15,39-66. 19. Kandil A. Elbanby M, Abd-Elwahed K, Abou Sehly G, Ezzat N. (1987) Healing Effect of True Floral and False Nonfloral Honey on Medical Wounds. J. Drug Res. (Cairo) 17, 71-75. 20. Effem SEE. (1988) Clinical Observations on the Wound Healing Properties of Honey. Br. J. Surg. 75, 679-681. 21. Farouk A, Hassan T, KashifH, Khalid S A, Mutawali I, Wadi M. (1988) Studies on Sudanese Bee Honey: Laboratory and Clinical Evaluation. Int. J. Crude Drug Res. 26, 161-168.


22. 23. 24. 25. 26. 27.

28. 29. 30. 31.

32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.

37

Green A E.(l988) Wound Healing Properties of Honey. Br. 1. Surg. 75, 1278. Mcinerney R J F. (1990) Honey - a Remedy Rediscovered. 1. Royal Soc. Med. 83, 127. Braniki F J. (1981) Surgery in Western Kenya. Ann. Royal CoIl. Surg. Engl. 63, 348-352. Branemark P-I, Ekholm R, Albrektsson B, Lindstrom J, Lundborg G, Lundskog J. (1967) Tissue Injury Caused by Wound Disinfectants. 1. Bone Joint Surg. Am. Vol. 49, 48-62. Knutson R A, Merbit L A, Creekmore M A, Snipes H G. (1981) Use of Sugar and Povidone-iodine to Eenhance Wound Healing: Five Years Experience. South. Med. J. 74. 1329-1335. Chi rife J, Herszage L, Joseph A, Koh E S. (1983) In Vitro study of Bacterial Growth Inhibition in Concentrated Sugar Solutions: Microbiological Basis for the Use of Sugar in Treating Infected Wounds. Antimicrab. Agents Chemother. 23, 766--773. Rahal F, Mimica I M, Pereira V, Athie E. (1984) Sugar in the Treatment aflnfected Surgical Wounds. Internal. Surg. 69, 308. Middleton K, Seal 0 V. (1985) Sugar as an Aid to Wound Healing. Pharm. J. 235, 757-758. Trouillet J L, Fagon J Y, Domart Y, Chastre J, Pierre J, Gibert C. (1985) Use of Granulated Sugar in Treatment of Open Mediastinitis after Cardiac Surgery. Lancet ii, 180··184. Shimamoto Y, Shimamoto H, Fujihata H, Nakamura H, Matsuura Y. (1986) Topical Application of Sugar and Povidone-iodine in the Management of Decubitus Ulcers in Aged Patients. Hiroshima J. Med. Sci. 35, 167-169. Lowbury E J L, Ayliffe G A J: Drug Resistance in Antimicrobial Therapy. Thomas Springfield, Illinois. 1974. Smith M, Enquist I F. (1967) A Quantitative Study of Impaired Healing Resulting from Infection. Surg. Gynecol. Obstet. 125,965--973. Willix 0 J, Molan P C, Harfoot C 1. (J 992) A Comparison of the Sensitivity of Wound-infecting Species of Bacteria to the Antibacterial Activity of Manuka Honey and Other Honey. 1. Appl. Bacteriol. 73,388-394. Hancock B M: Microbiology Dept., Waikato Hospital, Hamilton, New Zealand: unpublished findings. Mossel 0 A A. (1980) Honey for Necrotic Breast Ulcers. Lancet ii, 1091. Huhtanen C N, Knox 0, Shimanuki H. (1981) Incidence of Clostridium botulinum Spores in Honey. J. Food Prot. 44, 812-814. Molan PC, Russell K M. (1988) Non-peroxide Antibacterial Activity in Some New Zealand Honeys. 1. Apic. Res. 27, 62-67. Allen K L: University ofWaikato, Hamilton, New Zealand: unpublished findings. Molan PC, Allen K L. (1996) The Effect of Gamma-irradiation on the Antibacterial Activity of Honey. J. Pharm. Pharmacol. In press. Allen K L, Molan pc. The Sensitivity of Mastitis-causing Bacteria to the Antibacterial Activity of Honey. Submitted for publication. Al Somai N, Coley K E, Molan PC, Hancock BM. (1994) Susceptibility of Helicobacter pylori to the Antibacterial Activity of Manuka Honey. J. Royal Soc. Med. 87, 9-12. Haffejee I E, Moosa A. (1985) Honey in the Treatment of Infantile Gastroenteritis. Br. Med. 1. 290, 1866--1867. Brady N F, Molan P C. The Sensitivity of Enteropathogenic Bacteria to the Antibacterial Activity of Honey. Paper in preparation. Rademaker M. (1993). Superficial Dermatophyte Infections. N. Z. Med. J. 106, 14--16. Brady N F, Molan P C, Harfoot C G. The sensitivity of dermatophytes to the antimicrobial activity of honey. Submitted for publication.

4

NON-PEROXIDE ANTIBACTERIAL ACTIVITY OF HONEY Stefan Bogdanov Federal Dairy Research Institute Bee Department 3097 Liebefeld, Switzerland

1. INTRODUCTION Honey acts as an antibacterial agent against many bacteria (1). There are two sorts of antibacterial agents or so called "inhibines." One of them is heat- and light-sensitive and has its origin in the HP2 ' produced by honey glucose oxidase (2,3,4). Some workers believe that hydrogen peroxide is the main antibacterial agent (2,5,6). Other authors find that the non-peroxide activity is the more important one (7,8,9). The H 20 2 amount in honey is very small and it can be produced only after aerobic incubation of diluted honey solutions, which might mean that it is not very important for the antibacterial action of honey (10). The argumentations of the pro and contra peroxide side are based on the results with the specific antibacterial test used. However, a certain antibacterial test might be sensitive only to certain types of antibacterian substances. In a previous study from dur laboratory it was found that while in an agar disc diffusion test only the peroxide activity was measured, in a liquid medium test only the non-peroxide substances were active( 10).

1.1. Antibacterial Substances The main honey substances are sugars, which by their osmotic effect exert an antibacterial action (1). The antimicrobial tests used in different studies are mostly carried out at concentrations, where the sugars are not osmotically active. Many different antibacterial substances, which are more or less heat- and light-stable have been characterised. It has bee claimed, that honey contains lysozyme, a well known antibacterial agent (8). However, in an other study it was found that no lysozyme activity was present in honey (10). Honey contains a number of flavonoids (11,12), many of which are known to have an antibacterial action (13). The profile and the concentration of the different flavonoids have been correlated to the floral origin of honey (11,12) , but not to its antibacterial activity (14). From New Zealand honeys, mainly manuka and viper's bugloss honey, several aromatic acidic substances with antibacterial activity have 39

S.Bogdanov

40

been isolated (1). These substances were proved to have a floral origin. Only in viper's bugloss honey the antibacterial activity measured could be explained by the amount of the antibacterial substance present. Another investigation claimed, that the low honey pH was responsible for the antibacterial activity (15). Some workers have isolated volatile substances with antibacterial activity (17,18), but their contribution to the antibacterial action was not examined. Other antibacterial substances have also been chemically characterized, mainly in New Zealand honey (1). Also, other workers found non-peroxide activity of honey, extractable by organic solvents, but were not able to identify the chemical nature of the substances (18,19).

1.2. Origin of the Antibacterial Substances Several clearly identified substances were shown to have a floral origin (1). In one study it was found, that the non-peroxide activity in blossom and honeydew honeys was not significantly different (20). Another study claimed, that the non-peroxide, partly volatile antibacterial activity of honey, has bee origin (21). Summarising the results of the previous studies, it seems that only in very rare cases the antimicrobial activity was quantitatively correlated to the amounts of the antimicrobial substances present. Also, in most studies it was not clear, whether the activity had a bee or a plant origin. This is surely due to the heterogenous nature of non-peroxide activity. In our study we looked for answers to following questions: I. Which different honey substance groups are responsible for the antimicrobial activity? 2. Does the activity have a plant or a bee origin? 3. What is the effect of heat and storage of honey on the non-peroxide activity? Here we summarise the work done in our laboratory, dealing with the non-peroxide honey activity, using an antibacterial test, which was shown to reflect only the non-peroxide part of antimicrobial activity (10)

2. MATERIALS AND METHODS 2.1. Materials and Honey Samples Turbidity Test. For the turbidity test we used the following liquid medium: I % pepton, 1% Lab-Lemco (both Oxoid) and 0.1 % glucose Test strains: Staphylococcus aureus and Sarcina lutea were both used for inocculation of bacteria growth tests as suspensions with 0.2 absorption units at 520 nm The spectrometer for measuring the turbidity of the bacterial suspensions was a Spectronic 20, where 20 mi test tubes could be read directly at 520 nm Thermostatable shaking incubator 20 mi sterile test tubes A "honey-sugar" standard was a solution of 40% fructose, 35% glucose, 7% maltose, 0,2 gil 00 g KCI and mM lactic acid. Honey Fractionation. The columns used for honey fractionation were:

• C-18 1000 mg SPE (Solid Phase Extraction) Baker 7020 disposable columns

Non-peroxide Antibacterial Activity of Honey

41

• 2 cm 3 /column 50 mesh (Dowex 50 W x 8) strong acidic cation exchanger • 2 cm 3 /column 50 mesh (Dowex I x 8) strong basic anion exchanger The SPE columns were mounted on Baker-IO SPE extraction manifold with vacuum. Biorad polypropylene disposable columns Nr.73l-l550 were used for the ion exchange fractionation, without the use of vacuum. Reagents and Honey Samples. All reagents were of analytical purity grade. Destilled water was used for all dilution steps. The honey samples anlysed in this study were either market samples (of Swiss and foreign origin) or they were taken at different parts of Switzerland for the purpose of the present study.

2.2. Methods 2.2.1. Honey Analysis. The honey humidity, acidity, invertase- and diastase (Phadebas method) activities were all determined according to the Swiss Food Manual (22). The free acidity is expressed in maeq/kg, the invertase in Hadorn units (invertase number) and the diastase in Schade units (diastase number). HP2 production was determined as described (10). 2.2.2. Antibacterial Growth Test. • Mix 10 mlliquid growth medium with 10 ml20g/100g honey solution. Add one drop of bacteria suspension and mix well. • Incubate in the shaking water bath at a constant shake-speed for a maximal bacterial grwoth: 12 hours for Staphylococcus and 36 hours for Sarcina. Whole honeys were tsted only against Staph.aureus was tested, while honey fractions were tested against both strains • Read turbidity at 520 nm Control incubation: 10 ml liquid growth medium were mixed with 10 ml 20gll00g honey sugar standard. It was shown earlier that due to osmosis, this sugar concentration has an inhibitory effect of about 10--20 %, compared to the growth in the medium without the "honey sugar" standard. 2.2.3. Honey Fractions. 50 gllOO ml honey water solutions were used for all fractionation steps. The initial pH of each honey solution was measured. The antimicrobial tests were carried out with the initial honey solutions and with the honey solutions after the removal of the fraction by each fractionation step. Before the antibacterial test the honey concentration of the solution after each fractionation step was adjusted to 20 gil 00 ml and the pH of the solution was set at the pH ofthe honey solution before the fractionation. 2.3.3.1. Removal of Volatile Substances. 50 gllOO ml honey solution was heated at 60° C under vacuum (15 mg Hg) for 2 hours in order to remove all volatile substances. 2.3.3.2. Removal of Non-Polar Non-Volatile Substances. The Baker C-IS columns were activated with one volume of ethanol, followed by one volume of water. The honey solution was then passed through under constant vacuum. The honey filtrate was used for the antimicrobial test.

S.Bogdanov

42 • TEST INITIAL ANTIMICROBIAL ACTIVITY OF WHOLE HONEY

Honey Solution • FRACTIONATION ./ Destilation 1. Removal of volatile substances (2 h. 60°C under vacuum)

~

honey without volatiles

1

Columns Removal of 2. Non-polar, non-volatiles (C-18) 3. Acids (anion exchange) 4. Bases (cation exchange)

• TEST LOSS OF ANTIMICROBIAL ACTIVITY OF HONEY SOLUTIONS AFTER REMOVAL OF THE DIFFERENT FRACTIONS AND COMPARE TO INITIAL ANTIBACTERIAL ACTIVITY Figure 1. Scheme of the fractionation and testing of the different antimicrobial fractions.

2.3.3.3. Removal of Bases (Relatively Polar). The cation exchange column was converted into the H-form by passing 2 ml of 2 M Hel. Then it was washed with water until the eluate was neutral. The honey solution, where the bases at the acidic pH of honey are in protonized form, is passed through. The flitrate is then set at the pH of the initial honey solution. 2.3.3.3. Removal of Acids (Relativly Polar). The anion exchange column was converted into the OH-form by passing of2 ml of2 M NaOH. Then it was washed with water until the eluate was neutral. The honey solution is then set at pH 11. The honey solution, where the acids at this high pH are in their dissociated form, is passed through. The flitrate is then set at the pH of the initial honey solution. 2.2.4. Expression ofAntimicrobial Inhibition. 2.2.4.1. Whole Honey Solutions. Results were calculated by the turbidities of the incubation mixtures at the end of the bacterial test. They were expressed in % inhibition compared to the absorbance of the control (control = 0 % inhibition or 100 % growth).

2.2.4.2 Honey Fractions. The increased bacterial growth after the removal of a certain honey fraction (see below) was attributed to the removal of antibacterial substances by this fractionation step.


43

Example: A honey has an initial bacterial inhibition of 90 %. The inhibition after removal of: volatiles: 80%, non-polars: 65%, bases: 40% and acids: 20%. The initial inhibition of 90% was set as 100 parts to represent the whole antibacterial capacity of this honey. The partial inhibition after the loss of the different fractions in relation to the antibacterial capacitys of the whole honey was calculated as: 80/90x I 00, 65/90x I 00 etc.= 89, 72, 44, 22. The inhibition capacity of each absorbed fraction is then: 100-89; 100-72 etc = 11,28, 56 and 78. Then the sum of the inhibition capacities of the fractions was set as 100 %, e.g. for the above example:

11+28+56+78 = 173 = 100 %. In table 2 the % inhibition, attributed to each fraction, is a relative inhibition number, e.g. the relative inhibition of the volatile fraction is 1lI173xl 00 = 6%. The average sum of the inhibition parts of each fraction of the ten honeys in table 2 was 119 for Staph.aureus and 223 for Sarcina lutea. If all antibacterial substances were fractionated by our procedure and if their action is additive a sum of 100 should be expected in the above example. The significantly higher percentage than 100 for the fractional inhibition of Sarcina might be due to interactions of the different antibacterial fractions, when they act as a whole.

3. RESULTS AND DISCUSSION 3.1. Relative Distribution of Antimicrobial Activity among Different Honey Fractions We fractionated honey in 4 basic substance groups: volatile, non-volatile and nonpolar, acidic and basic substances.

Table 1. Relative distribution of antibacterial activity in different honey fractions The sum of the antibacterial activities against Staph. aureus und Sarcina /utea, attributed to each fraction is set as 100 % (see Methods) % antibacterial activity in different fractions Acidic Honey

St.

Manuca N.Z. Sunflower It Rape CH Lavender Fr Mountain CH Blossom S. America Honeydew CH Honeydew CH Honeydew CH Honeydew Europe

100 58 25 25 24 62 45 32 43 43

Average Minimum Maximum

46 24 100

Non-polar

Basic

Say.

51.

Say.

St.

Say.

75 46 40 25 73 46 31 26 32

0 13 7 34 60 13 26 37 22 25

10 15 33 30 25 20 15 31 26 31

0 16 63 23 8 9 26 19 19 26

5 25 22 29 25 7 15 31 26 37

42 25 75

24 0 60

24 10

21 0 63

22 5 37

27

33

Volatile

St.

Sar.

0 13

10 15

5 18 8 16 2 12 15 6

14 24 0 24 6 23 0

10 0 18

12 0 24

44

S.Bogdanov

Table 2. Correlation between free acidity, diastase and invertase activity and bacterial inhibition of Slaph.aureus. r-correlation coefficient, p---probability level at 95 %. For units see Methods

p n

Free acidity vs. inhibition

Diastase number vs. inhibiton

Invertase number vs. inhibition

0.35 0.001 81

0.65 0.0005 37

0.58 0.001 37

In table I the relative distribution of antibacterial activity in these fractions against Staph.aureus and Sarcina lutea is summarised. The acidic fraction has the greatest inhibitory power, while the volatiles are the weakest bacterial inhibitors. The relative distribution of the antibacterial activity in the different fractions is the same against both bacterial strains tested. On the average, for both strains the following relative distribution of antibacterial activity was observed: acids: 44%, bases: 24%; non-polars: 21 % and 11: volatiles. If the differences between the distribution of activity among the different groups were tested by a t-test, only the difference between the volatile activity on one side and the activity in the acidic (p=O.OOO) and the basic fraction (p=0.05) proved to be significantly different. This is due to the variation of the distribution among the fractions of the different honey types. In the manuca honey 90% of the activity was found in the acidic fraction, in the rape honey the greatest part of the activity was in the non-polar fraction and in one Swiss blossom honey the basic fraction had the highest activity.

3.2. Origin of the Non-peroxide Bacterial Inhibition 3.2.1. Plant Origin. In fig .2 the bacterial inhibition of 9 unifloral and 2 mixed (different blossom and different honeydew origins) are shown. Rhododendron honey had the

80

70 60 c .250

-

::cs 40 c

.- 30 ~

20 10

o

o

a:::

ro

o

o

:c

ro

a:::

Figure 2. Non-peroxide activity of unifloral honey against Staph.aureus: The values (averages) are for: Rh Rhododendron, n=3 ; Eucalyptus, n=4; Orange, n=3 ; Chestnut, n=7, Blossom, n=30; Acacia, n=7; Sunflower, n=4; Lavender, Dandelion, n=2; Honeydew, n=IO; Rape, n=7.


45

lowest, while rape honey had the highest inhibitory power. However, there is a considerable variation in each honey type. In order to prove for significant differences between the honey types, a greater number of honeys should be analysed. Together with the fact, that the relative distribution of the antibacterial activity is different in the various honey types, the data shown here suggest, that some of the non-peroxide activity is of floral origin. 3.2.2 Bee Origin. Honey acidity, distase and and invertase are known to have a bee origin. There is a highly significant correlation between the free acidity, the diastase and the invertase activity on one hand and the bacterial inhibition on the other (table 2). These results show, that a part of the non-peroxide antibacterial activity has a bee origin.

3.3. Influence of Heat and Storage The experiments were carried out with light blossom- and dark honeydew honeys. Heating of both honey types at 70° C for 15 minutes had no or very little effect on the non-peroxide actitivity (table 3.). Under the same conditions the peroxide accumulation capacity of blossom honeys is severely damaged (10). In a next experiment glass pots with blossom or honeydew honeys were stored in the light (day-light) or in the dark at room temperature (about 20-25° C). The results are summarised in fig. 3. After 5 months of storage the activity remains the same, while after 15 months there is a small drop of activity of about 20 %. The results were the same both for both types of honeys stored in the light or in the dark. Under the same storage conditions the peroxide accumulating capacity of honey is strongly reduced, especially when blossom honeys are stored in the light (10).

4. CONCLUSIONS 1. There are four different honey fractions, which account for the non-peroxide antibacterial activity. The antibacterial activity of these fractions is: acids> bases"" non- polars > volatiles. 2. There is evidence for the floral- and for the botanical origin of the non-peroxide antibacterial activity 3. The non-peroxide activity is only slightly affected by heat and by storage for 15 months in the light or in the dark.

Table 3. Effect of heat on non-peroxide activity Fresh honeys of floral or honeydew origin were heated for 15 minutes at 70° C. Values are mcans ± SEM and are expressed in % of the initial inhibition Honey

n

Bacterial inhibiton % of initial

Blossom (light) Honeydew (dark)

3 4

94 ± I

86 ±4

S.Bogdanov

46

120

.."'

100

~

.:; u

80

"'iii 60

~

c

0

~

0

40 20 0 blossom, light

blossom, dark

honeydew, light

honeydew, dark

3, 5 and 15 months Figure 3. Effect of storage on the non-peroxide activity The non-peroxide activity of 7 mixed blossom honeys and 5 honeydew honeys, stored in glass pots in the light and in the dark, was tested. The values are averages for the above honeys.

SUMMARY In honey there are two sorts of antibacterial agents or so called inhibines. One of them is heat- and light sensitive and has its origin in HP2 . The other consists of thermostable substances. In our study we tested the antibacterial activity by a turbidity test with 20% honey solutions with Staphylococcus aureus and Sarcina lutea strains. Under these conditions destroying all the HP2 with catalase had no effect on the antibacterial activity. Thus with this test only the non-peroxide antibacterial activity is measured. We used this test to measure the non-peroxide antibacterial activity of whole honeys and of different honey fractions. The results can be summarised as follows: I. By fractionation in different substance classes the non-peroxid antibacterial activity is distributed among 4 fractions with different chemical characteristics: acidic; basic (both relatively polar); non-volatile and non-polar; volatile. 2. The antibacterial activity of the different fractions , tested in 10 different honeys was: acids> bases

= non- polar, non-volatiles> volatiles.

This order was the same for both Staph. au reus and Sarcina lutea as test strains 3. The distribution of activity was, however, dependent on the honey type : In manuca honey almost the whole activity was found in the acidic fraction, in rape honey the greatest activity was in the non-polar fraction and in one Swiss mountain honey the basic fraction had the highest inhibitory power.


47

4. There were differences between the antibacterial actIVIties of different honey types: rhododendron, eucalyptus and orange honeys had a relatively low, lavender, dandelion, honeydew and rape honeys had a relatively higher activity. This result, together with point 3. suggests, that some of the non-peroxide activity has a plant orign. 5. There is also a significant correlation between the acidity, diastase- and invertase activity, all of bee origin, on one hand, and the non-peroxide activity, on the other. Thus, a substantial part of the non-peroxide activity has also a bee origin. 6. The non-peroxide activity is not or only slightly affected by heat (15 minutes 70 C and by storage for 15 months at room temperature. 0

REFERENCES I. Molan. P. (1992) The antimicrobial activity of honey I. The nature of antibacterial activity. Bee world, 73. 5-28 2. White, J.w., Subers, M.H. and Schepartz A.I., (1963), The identification ofinhibine, the antibacterial factor in honey, as hydrogen peroxide and its origin in honey glucose-oxidase system. Biochim. Biophys. Acta n 57-70 3. White, 1. W. and Subers, M.H. (1964), Studies of honey inhibine, 3. The effect of heat. J.Apic.Res. 3 454-450 4. Dustmann, J.H. (1972) Ueber den Einfluss des Lichtes auf den Peroxid-Wert des Honigs. L.Lebensm.Unters.Forsch. 148,263-268 5. Dustmann.J.H. (1979) Antibacterial effect of honey. Apiacta 14, 7-11 6. Morse, R.A. (1986) The antibiotic properties of honey. Pan-Pacific Entomologist 62, 370-371 7. Gonnet, M. and Lavie, P. (1960) Influence du chaufage sur Ie facteur antibiotic du miel. Annales de I' Abeille (Paris) 3, 349-364 8. Mohrig, W. and Messner, R. (1968), Lysozym als antibacterielles Agens im Honig und Bienengift. Acta Biologica Medica Germanica 21, 85-95 9. Radwan S.S. EI-Essawy A.A. and Sarhan, M.M. (1984) Experimental evidence for thc occurrence in honey of specific substances active against micro-organisms. Zentralblalt Mikrobio!. 139,249-255 10. Bogdanov, S. (1984) Characterisation of antibacterial substances in honey. Lebensm. Wiss. Techno!., 17, 74-76 II. Sabaticr, S., Amiot, M.1., Tachini, M and Aubert. S. (1992) Identification offlavonoids in sunflower honey. I.Food Sci. 57, 773-774 12. Tomas Barberan, FA., Ferreres, F Garcia-Viguera, C. and Tomas-Lorente, F Flavonoids in honey of different geographical origin. (1993) Z.Lebensm.Untersuch.Forsch. 196,38-44 13. Metzner 1., Bekemeier, H .• Paintz, M and Scheidewand E. and (1975) Zur antimikrobiellen Wirksamkeit von Propolis und Propolisinhaltsstoffen. Pharmazie, 34, 97-102 14. Bogdanov S. (1989) Determination of pinocembrin in honey using HPLC. J.Apic.Res. 28, 55-57 15. Yatsunami, K. and Echigo, T. (1984) Antibacterial activity of honey and royal jelly. Honeybee Science 5, 125-130 16. Lavie, P. (1963) Sur I'identification des substances antibacteriennes presentes dans Ie mie!. C.R.Seanc.Acad.Sci. Paris. 256, 1858-1960 17. Toth, G., Lemberkovics, E. and Kutasi-Szabo (1987) The volatile components of some Hungarian honeys and their antimicrobial effects. 127,496-497 18. Roth, L.A., Kwan, S. and Sporns P. (1986) Use ofa disc assay to detect oxytetracycline residues of honey. I.Food Prot. 49, 436-441 19. Schuler, R and Vogel, R. (1956) Wirkstoffe des Bienenhonigs. Arzneimittel Forsch. 6, 661-663 20. Bogdanov, S. , Rieder, K. and Ruegg, M. Neue Qualitatskriterien bei Honiguntersuchungen. Apidologie, 18,267-278 21. Lavie, P. Proprietes antibactcriennes et action physiologique des produits de la ruche et des abeilles in: Traite de Biologie de l'Abeille (R.Chauvin, editor) Masson & Cie pp.2-115 22. Swiss Food Manual, Chapter 23 A, Honey, Bern. Eidgen6ssische Druck und Materialzentralle, 1995

5

ANTIOXIDANT PROPERTIES OF HONEY PRODUCED BY BEES FED WITH MEDICAL PLANT EXTRACTS Gennady Rosenblat,1 Stephane Angonnet,2 Alexandr Goroshit,3 Mina Tabak, I and Ishak Neeman l IDepartment of Food Engineering and Biotechnology Technion-Israel Institute of Technology Haifa 32000, Israel 2ENSIA Paris 91305, France 3Tzuf Laboratori es Ltd. P.O.B. 408, Kiryat Shmona, Israel

ABSTRACT Honey is known to exert beneficial effect on many pathological conditions. The full gamut of its biological activity has yet to be elucidated. In this study five type of honey (regular commercial honey, Chinese honey and three other honeys, namely, Laryngomel, Bronchomel, and Dermomel) were studied for their antioxidant effect. Unlike regular honey, three last honey species were a product of bees which have been fed on a mixture of several medico-herbal water extracts with regular honey. All the assessed honeys demonstrated prevention of j3-carotene degradation in linoleic acid emulsion and obviation of superoxide radical generation by xanthine/xanthine oxidase system. Laryngomel and Bronchomel were particularly effective in reactive oxygen species scavenging at a concentration range 7-50 J.lg/ml. The relationship between the antioxidant properties of honey and its physiological activity is discussed.

INTRODUCTION One of the common properties of many plant natural products is the considerable degree of protection afforded against oxidative attack. The spontaneous reaction of oxygen or oxygen containing radicals with organic compounds leads to cell and tissue damage. Radicals and the radical-generating process are normally neutralized by antioxidative defense mechanisms [1], but in certain situation such as aging, inflammation, etc., radical 49

50

G. Rosenblat et al.

generation may increase with a consequent acceleration of accumulating damage [2,3]. In such situation the use of antioxidants in medication or of plant extracts with antioxidant properties in food has been advocated to minimize any oxidative degradation of the target molecules [4,5]. The antioxidant potential of honey, which is a widely used natural product containing mostly floral compounds, has not been sufficiently evaluated. Most studies on the biological properties of honey have thus far focused on its antibacterial activity [6]. Yet, in " folk medicine" honey also is believed to exert a beneficial effect on ulcers, the nervous system, the heart, the liver and the digestion [7]. The full spectrum of honey biological activities is still unc1arified but evidently much extend beyond were antibacterial activity. The present investigation attempted to fill the gap by studying the antioxidant effect of several types of honey.

MATERIALS AND METHODS All solvents were obtained either from Frutarom (Haifa, Israel) or Biolab (Jerusalem, Israel). Specific compounds were all purchased from Sigma (St. Louis, USA) excepting linoleic acid (from Fluka Chemie, Buchs, Swizerland) and OPA (from Zymed, USA)

Honey Venue and Production Commercial Chinese honey was received from Cho Yung International, Israel. Regular commercial honey was produced by kibbutz Ayelet HASHAHAR (Israel). The commercial honeys Bronchomel, Laryngomel and Dermomel were produced by Tzuf Laboratories Ltd., Kiriat Shmona, Israel; unlike regular honey, these three honey species were a product of bees which have been fed on a mixture of several medico-herbal water extracts with regular honey. The taxonomical data on the medical herbs used in the mentioned extracts are given in Table 1. Glucose-fructose syrup, which was used in part of the experiments as a model solution, was prepared by adding 35 g of each sugar to 100 ml of hot bidistil1ed water. ~-carotene

Degradation Test

Antioxidant activity was assessed via emulsion with p-carotene and linoleic acid, whose degradation was determined by the method of Marco [8] with modifications. Briefly, 0.4 mg of p-carotene, 100 !-II of linoleic acid and 200 !-II of Tween 40 were dissolved in 20 ml of chloroform, which was then evaporated by nitrogen. The model emulsion was now prepared by adding 30 ml of water. One gram of honey was next dissolved in 2 ml of water (or of appropriate buffer) and mixed with 3 ml of the emulsion; 2 ml of 95 % ethanol was added to the mixture, which was finally incubated at 50°C for several time. The antioxidant activity of I g of honey in 7 ml of reaction solution was compared with that of 1 !-Ig/ml (5.5 10-3 mM) and 5 !-Ig/ml (2.8 10.2 mM) ofBHA. The degradation of honey protein in the different tests was achieved by autoc1aving for 30 min 2 ml of water (or buffer) containing 19 of honey at I atm. Citric buffer, 0.1 M, pH=2, or citric-phosphate buffer, 0.1 M, pH=4.5 or phosphate buffer, 0.1 M, pH=7 were used for the pH-change in the different tests.

Antioxidant Properties of Honey and Medical Plant Extracts

51

Table 1. Medicinal herbal are used in forming of honey Laryngomel , Bronchomel , and Dermomel and medical-biological characteristic of the honey Type of honey Laryngomel

Bronchomel

Dermomel

Family of the herbalLauraceae Asteraceae Apiaceae Labiatae Betulaceae Chenopodiaceae Rutaceae Liliaceae Mirtaceae Lamiaceae Lauraceae Pinaceae Apiaceae Cruciferaceae Rosaceae Umbelliferae Juglandaceae Labiatae Salicaceae

Medical- biological characteristic active against laringitis,tracheitis, glossitis

active against inflamation of the upper respiratory tracts

active against suppurative wounds and chronic ulcer

*All plants are used in conventional or alternative medicine

Inhibition of O 2- Production by Xanthine/Xanthine Oxidase System (X/XO) The production of superoxide anion was evaluated by chemoluminescence. Reaction was initiated at room temperature by adding 250 f.ll solution of xantine oxidase solution (0.65 units/ml) to 2-ml of Hanks buffer (pH=8.3) containing 100 f.lM of xanthine and 60 f.lM of lucigenin. The mix was put in a luminometer (model 597B, Technion Physics Department, Haifa, Israel). The light production remined constant throughout the 2 - 5 minutes following onset of the reaction, during which period 25 f.ll of the differently diluted (in water) honey was added dropwise. The chemoluminiescence was now measured directly in triplicate samples.

Xanthine Oxidase Activity Activity of xanthine oxidase was determined by uric acid production. Uric acid generation was monitored by absorption spectroscopy, at 290 nm, under the same condition as in experiment with superoxide anion determination.

RESULTS Effect of Honey on the Degradation of ~-Carotene in Emulsion with Linoleic Acid The oxidation of ~-carotene in emulsion with linoleic acid in the presence of various honey species is shown on Fig. 1. A change in the absorbency at 470 nm (expressed in

52

G. Rosenblat et al. 120

100

QJ

u

to

...'0"

,.Q rJl

E c:

'~ci " ..."

,.Q

0

•.r::

•

.2 0 -~ ""'0 0~

60

40

...

.

20

o w............ o 50

'

I ",

I I

I

100 150 200 250 300 350

Time [min] Figure 1. Effect of honey on p-carotene degradation in emulsion with linoleic acid.

percent of initial absorbency) is characteristic of 0-carotene degradation in course of the oxidation. As can be seen from the data the antioxidant activities of all the honey solutions in distillated water (142,8 mg/ml) at normal for honey pH (about 4.0 ) are comparable with that of BHA (which is a potent antioxidant employed in the food and chemical industries [9]) in a concentration of I /-!g/ml or 5 /-!g/ml, respectively . The antioxidant effect of honey in emulsion with linoleic acid was found to be pH dependent. The antioxidant effect of all species of honey is maximal at physiological pH (7) but reduces with decrease in the pH, so that at pH of human stomach (pH=2) the tested honey types do not show antioxidant activity excepting Laryngomel and Bronchomel which demonstrate a negligible antioxidant effect. The capacity of honey to protect against 0-carotene oxidation was not altered by heating of the honey solution in the protein- cleaving state, thus supporting our contention regarding the non-proteine nature of the honey antioxidant moiety (data not shown). Nevertheless, the possibility in this case that thermal inactivation of antioxidant protein is compensated for by products of Maillard reaction cannot be entirely excluded, and it has been observed repeatedly that various Maillard reaction products formed through the interaction of protein and carbohydrates during the heat-processing of food do exert antioxidant activity [1OJ-

Inhibition of 02- Production by Xanthine / Xanthine Oxidase Xanthine / xanthine oxidase system is one of the two natural system of superoxide anion generation . Xanthine oxidase-derived superoxide anion has been linked to postischemic tissue injury and the generation of neutrophil chemotaxis [II] . Hence curtailment of 0 2' generation by this enzymatic pathway would be beneficial in the case of ischemia. Honey demonstrates a significant inhibitory effect on radical generation by xanthine/xanthine oxidase system. Indeed extintion of the chemoluminescence was observed in the presence of the regular honey (159-900 /-!g/ml), while an even greater effect was shown


53

12° ~1

100

o

20

o

o

675

1450

2 25

3000

Concentration, IJ.gfml Figure 2. Effect of honey on superoxide anion production by a xanthine/xanthine oxidase enzymatic system.

by Dermomel and Chinese honey in the same concentration range. Strong effect on superoxide amount was found for Bronchomel and Laryngomel at a concentration of 7- 50 Jlg/ml (Fig 2. but data for Bronchomel is not shown). This effect was not abnogated by heating the honey samples at 100 °C and I atm for 30 min , thus supporting the supporting nonproteine nature of the inhibiting compounds. No significant inhibitory effect on the superoxide anion generation by xanthine/xanthine oxidase was displayed by glucose-fructose syrup even in markedly concentrations. To justify whether this activity was due to an inhibitory effect on the enzyme itself, a control experiment was carried out, in which production of uric acid was measured by UV spectroscopy at 290 nm. Under the same experimental condition it was not demostrated an inhibition of uric acid production by xanthine oxidase even in presence of markedly concentrations of honey.

DISCUSSION Honey contains various minerals and organic compounds but is comprised mainly of sugars (about 80 %) and water (about 20%) [12]. In addition, honey is known to contain a number of enzymes such as diastase, invertase, saccharase, catalase, glucose oxidase. Depending on the source from which bees obtain the material for honey production, the honey will acqure its specific aroma, color and test. Apparently , the medicinal and the nutritional properties of honey depend in part, also from the chemical composition of the flowers from which bees collect nectar. For this regard, there is growing interest in the honey created by bees that are nourished on medical herbs for the properties of the final product are enhanced during processing of the natural nectar in the bee's body. Using various plant extract that are well known in alternative and conventional medicine, it have been able to develope honey substitutes for easing cough and bronchitis (Bronchomel),

G. Rosenblat et al.

54

sore and inflamed throat (Laryngomel ), or for treatment of wounds (Dermomel). This and natural honey studied by us have all demonstrated antioxidant activity, particularly an inhibitory effect on superoxide radical production by xanthine Ixanthine oxidase. The inhibition of 02- production is manly due to scavenger properties since the tested honey are not xanthine oxidase inhibitors. Regular honey was found to be a weaker than the other samples tested by us. Presumably the protective properties of manufactured honey against reactive oxygen species reside mainly in the substances extracted from the medicinal herbs. Although butulated hydroxyanisole was used by us as the standard for assesing the antioxidant properties of honey it would be incorrect to compare BHA and honey antioxidant activity quantitatively, for obviously honey contains the biologically active ingradients in very low concentration. Nevertheless a simple calculation (using data from the ~-carotene degradation test) shows that active dose of natural honey which is recommended for medicinal use (one teaspoonful, which is about 7 g ) contains an amount of antioxidant which is equal to 50-250 Ilg of the potent antioxidant BHA. Turning now to the mechanism undrelying honey biological activity, accumulating evidence supports the assumption that the protective effect of honey against inflammation of whatever sourse is associated with its antioxidant moiety. Indeed, it has been shown that synthesis of inflammation mediators like leukotrienes and prostaglandins is involves the formation of arachidonic acid lipoperoxides [13]. Furthermore, in the wake of many types injury, including trauma, cells are known to rupture and release their contents, which include transition metal ions that can rapidly catalyze radical-mediated transformation and tissue injury [2]. Macrophage activation and the disruption of mitochondrial function may also result in the formation of excess reactive oxygen species [14,15]. Human phagocytes destroy bacteria or virus-infected cells throughout an oxidative burst of nitric acid NO", hypochlorite CIO' and superoxide 02- , but unfortunately the damage produced by radicals liberated from the phagocytic cells can sometime extend beyond the intended target and injure also surrounding tissue [16 ]. Uniquely vulnerable to such oxidative damage are epithelial cells lining the respiratory airways [17]. A part from potential oxidant exposure owing to normal cellular metabolism, the respiratory epithelium is exposed to relatively high oxygen tension and often also exposed to air pollutants, phagocytes, catalase-negative bacteria, and reactive xenobiotic-drug metabolites. From all the above, we can conjecture that the significant protective effect of honey (particularly Bronchomel and Laryngomel) against respiratory airway inflammation is explanable, in part, by its antioxidant properties. In conclusion, the in vitro antioxidant effect of honey revealed by the present study seems to comprise an is important attribute of honey, especially for such as was produced by bees nourished on mediI' ina I herbs.

REFERENCES I. Reiter R.J. (1995). Oxidative processes and antioxidative defense mechanisms in the aging brain. FASEB J.

9,526-533 2. Kehrer J.P., Smith c.v. Free radicals in biology: sourse, reactivities, and roles in the etiology of human diseases in: Natural antioxidants in human health and disease (Balz Fri Editor.), Academic Press, Foreword, 1994, pp.25--62, 3. Harman D. (1993). Free radical involvement in aging. Pathophysiology and therapeutic implication. Drags Aging. 3, 60-80 4. Bermond P. Biological effects of food antioxidants in: Food antioxidant, (Hudson BJ.F. Editor), Elsevier Science Publishing Co., Inc. N.Y. 1990, pp. 193-251 5. Sies H., (1993). Strategies of antioxidant defense. Eur. J. Biochemistry. 215, 213-219 6. Molan P.c. (1992). The antibacterial activity of honey. Bee World. 73,5- 26


55

7. Loyrish N: Bees and people. Mir Publishers, Moskow. 1977 8. Marco, G.J. ( 1968). A rapid method for evaluation of antioxidants. JAm Oil Chem.Soc .. 45, 594-598 9. Kukagawa K., Kunugi A., Kurechi T. Chemistry and implications of degradation of phenolic antioxidants in Food antioxidant. (Hudson B.1.F, Editor), Elsevier Science Publishing Co. Inc. N.V. 1990 10. Eichner, K. (1981). Antioxidative effects of Maillard reaction intermediates. Prog.Food Nut/:Sci .. 5, 441-451 11. Cotelle, N., bernier, J.L., Henichart, J.P., Catteau, J.P., Gaydou, E.. Wallet, 1. C. (1992). Scavenger and antioxidant properties of ten synthetic flavones. Free Rad.Biol.Med .. 13,211-219 12. White, I.w., Jr. (1978). Honey. Adv. Food Res., 24, 288--374 13. Borgeat,P., Samuelson, B. (1979). Proc. Natl.A cad. Sci. USA. 76.3213-3217 14. Kehrer, J.P.(l993). Free radicals as mediators of tissue injury and disease. Crit.Rev. Toxicol .. 23,21-48 15. Rosen, G.M., Pou S., Ramos c.L., Cohen M.S., Britigan B.E.(1995). Free radicals and phagocytic cells, FASEB J. 9. 200--209 16. Wright D.T., Cohn L.A., Li H., Fisher B., Li C.M., Adler K.B. (1994). Interaction of oxygen radicals with airway epithelium, Environ. Health Penpect. 102 (Suppl 10),85--90 17. Meyer A.S., Isaksen A. (1995). Application of enzymes as food antioxidants. Trend. FoodSci. Technol. 6, 300304

6

SPEEDING UP THE HEALING OF BURNS WITH HONEY An Experimental Study with Histological Assessment of Wound Biopsies

Th. 1. Postmes 1: M. M. C. Bosch 2 , R. Dutrieux 3, 1. van Baare 4 , and M. 1. Hoekstra 4 IDepartment ofInternal Medicine Academic Hospital Maastricht 2Leiden Cytology and Pathology Laboratory 3Dcpartment of histopathology Academic Hospital, Utrecht 4Burns Unit Red Cross Hospital Beverwijk, The Netherlands.

ABSTRACT In a pilot-study deep dermal burns, identical in depth and extent, were made at each flank of Yorkshire pigs. Wound healing characteristics and measurements of dermal thickness were investigated by comparing biopsies histologically in pairs. Wounds were treated with either honey of a defined antibacterial activity, or sugar, or silver sulfadiazine (SSD). Biopsies were taken on post burn days 7,14,21,28,35 and 42. Wounds treated with SSD were fully epithelialized after 28-35 days, whereas those treated with honey and sugar were closed within 21 days. In 5 out of 6 wounds the neodermis of the sugar treated burns was thicker than the neodermis of those treated with honey. In all honey experiments, on day 21, wounds were best microscopically characterized by (i) a quiet granulation tissue, (ii) an inconspicuous inflammation and (iii) a decrease of actine staining of myofibroblasts. In contrast, sugar treated wounds appeared ditTerent especially on day 21 and later. Furthermore, we observed in various sections signs of inflammation which were linkcd to pcrivascular infiltrates and well-stained myofibroblasts. These results suggest a difference between sugar and honey treatment. If antibacterial activity and anti-inflammatory activity count in wound treatment, then honey has to be prefered above sugar. * Correspondence should be addressed to: Dr. Th.Postmes, St.Servaasklooster 22; 6211 TE Maastrcht.

57

S8

Th. J. Postmes et al.

INTRODUCTION Epidermal regeneration of a wound is a complex process in which residual epithelial cells proliferate in an integrated manner into intact epidermis. In deep second degree burns re-epithelialisation primarily takes place from the wound margins; apparently most epithelial cells from hair follicles in the thermal injured area are not viable anymore. Granulation tissue is formed on the base of vessels and fibroblasts in the residual dermis and from the fatty tissue septa. Since the choreography of wound healing factors is still in the dark, an evaluation of the efficacy of current wound dressings remains worthwhile. Topical application of honey to open wounds has been recognized for centuries to be effective in controlling infection and producing a clean granulating wound bed. Both honey and sugar have been highly praised and recommended as proper treatments for traumatic wounds, burns, decubitis and ulcera l - 5 • Though honey is a complex medium it is safe and non-toxic 3- s. Moreover it is able to disinfect infected burn wounds, but it can also prevent colonization of non-infected burn wounds by bacteria until complete epithelization3 • According to Condon (1993) honey acts mainly as a hyperosmolar medium that precludes bacterial growth and as such does not differ from simple sugar6. Thus the real explanation of the antimicrobial effect of honey lies in its physical properties, not in its chemical composition 6 • However, the antibacterial effect after dilution up to 4% proves that honey must have intrinsic antibacterial properties quite distinct from sugar 7,R. Moreover the presence of e.g. traces of zinc, vitamins, amino acids and a large number of organic substances may contribute to and support the nutrition of the injured tissue. In spite of the numerous publications on honey and sugar as topicals for wound treatment, not a single clinical study comparing both substances has been published. In this pilot experiment, which is part of a larger evaluation on topical treatments, we wish to report some observations on the healing of deep second degree burns after treatment with honey, sugar or silver sulfadiazine.

MATERIALS AND METHODS A standard burn wound model, published by Hoekstra et al. (1993)., was strictly followed as described 9 • In brief:

Animals Experiments were performed in accordance with the Dutch Law on Animal Experimentation and the experimental protocol employed was approved by the Animal Welfare Committee of the University of Amsterdam. Three Yorkshire pigs of approximately 14 weeks (with a body weight of 25-35 kg) were thermally injured with a brass block of 6.7 x 6.7 cm, weighing 450 g. Twelve areas of 7 x 7 cm ( six on each flank) were marked in a symmetrical way, using the processus spinosi as an anatomical landmark. The block was heated up to 170°C and applied during 20 s without exerting pressure. The epidermal remnants of the burned skin were removed shortly after thermal injury. The total burned surface area amounted to not more than 10 % of the total body surface area.

Speeding Up the Healing of Burns with Honey

59

The details of the control of the animal's wellbeing, housing, food, general anaesthesia, daily wound treatment and covering were all similar to Hoekstra et aI., 1993.

Topical Agents Experiment (a), pig no 120, Silver sulfadiazine 1% cream vs honey. SSD ( Flammazine R , Duphar, Weesp, The Netherlands), as a topical agent and the most commonly used ointment for treating burns, was compared to unprocessed honey with a known antibacterial activity. The total inhibine (i) score of this particular lime honey was 15 as described previouslylo. Experiment (b), pig no 64. Honey vs honey. The honey was the same as in exp.(a). Experiment (c), pig 137. Honey vs sugar (artificial honey). The honey was the same as in expo (a). The osmolarity of the sugar paste containing 40% glucose, 40% fructose, 10% saccharose and 10% water was approximately that of honey.

Biopsy and Histology Each week (on post burn days 7, 14, 21,28, 35 and 42) biopsies were taken symmetrically from wounds on the left and right flanks. Biopsies included the wound bed as well as the healthy skin of the wound margins. Tissue fixing and staining were as described previousl/. An additional staining was included to detect myofibroblasts by an anti-Alpha smooth muscle actin stain.

RESULTS In this pilot study the wound healing pattern of the silver sulfadiazine treated flank of pig study no 120 was similar to the pattern reported earlier9 . Macroscopically it is almost impossible to assess the grade of epithelialization due to the presence of the crust. The time of a hundred percent epithelialization and the measurements of the dermal thickness of experiments a, band c, as found microscopically, are summarized in table I. An uninterrupted epidermis was found on day 21 for both honey (experiments a,b and c) and sugar (experiment c). Placed side by side the histology of the biopsies turned Table 1. Some data related to deep second degree burns treated with silver sulfadiazine (SSD), honey and sugar (artificial honey) Exp. no

Pig

a.

120

b.

64

c.

137

Thickness of the dermis I after day:

Treatment

100% epithelia1ised

14

21

28

35

42

honey vs SSD honey vs honey sugar vs honey

21-28 days' 28-35 days 21 days 21 days 21 days 21 days

1.7 1.0 3.3 2.5 1.8 1.3

3.3 1.1 1.5 3.3 3.5 2.5

2.7 2.7 2.0 1.8 2.7 1.5

2.9 3.3 2.0 1.5 2.3 1.3

3.0 2.g 1.7 1.7 2.2 2.4

'The thickness of the dermis is measured in the middle of:he biopsy. All data are given as a ratio of thickness of the burn wound versus that of normal skin. The pig's skin is normally 3 to 4 mm thick and in many ways it resembles the human skin. 'On day 21 the wound is almost fully epitheiialised, the next biopsy was on day 28, so the 100% point lies somewhere between day 21 and 28.

60

Th. J. Postmes et a!.

.'

:

,

.. .

,<

.

" ,

'",;

•

or

-

,

,

..

•

Figure I. On day 21 , in sugar treated burns, deeper layers still show perivascular infiltration (Papanicoulaou staining, x25 ).

out to be quite different. All sections of honey treated burns were, more or less, similar. On post burn day 21 the general picture was primarily a quiet granulation tissue and a light degree of inflammation was seen. In contrast, the sugar treated burns showed more inflammation as revealed by the many perivascular infiltrations. According to the stained sections of the biopsies on day 28 the dermal thickness of the honey treated pig wounds was 5.1 mm, whereas the neodermal layer sugar of the treated wounds of the contralateral side was much thicker (7 .6 mm), see fig 2. On days 14, 21, 28 and 35 the honey treated burns showed a variation in dermal thickness between 4.0 mm and 9.5 mm while healed bum wounds in the contralateral sugar side varied between 5.5 and 10.5 mm. Part of the swelling of the derm is can be put down to local oedema in the first week of wound healing. In the honey treated wounds the typical actin staining in myofibroblasts appeared to be weak or diminished whereas sugar treated burns still showed a positive actin stain in many myofibroblasts. The honey treated burns were covered with a translucent layer of possibly non absorbed honey. This layer, however, could not be observed in the sugar experiments. In both honey and sugar treated wounds a few isolated bacterial colonies were found. These were less frequently seen in the honey than in the sugar wounds. A few micro-pustules were found in the neo-epidermis of the sugar treated wounds and less in the contralateral control side treated with honey. Bacteria were seen in the eschars of the sugar and the honey treated bums, though less in the latter and none in those of the SSD treated burns.


61

Figure 2. (a): Normal pig's skin with subdermal fat and muscle tissue in the depth. (Papanicoulaou staining, x 12.5). (b). Pig number 137: honey treated side on day 28, the epidermis is uninterrupted and thus the wound is full y epilheliali sed. Clearly the dermis is thicker than normal. Staining and enlargement as in (a). (c): Pig number 137: sugar treated contralateral side on day 28. Staining and enlargement as in (a). Note: the dermis is 2.5 times thicker than normal.

DISCUSSION Honey and sugar paste, in comparable hyperosmolar concentrations, are non-toxic substances. The outgrowth of epithelial cells of the hair follicles is most likely inhibited by the uptake of either silver or SSDII. For the speeding up of wound healing by honey and sugar there are at least two explanations. First, in deep second degree burns, there are still a number of epithelial cells which might survive. Some belong to hair follicles , and others to sweat glands. Second, simple sugars in honey and sugar create a moist environment, which, as we know today, is a conditio sine qua non for quicker wound healing. Our study confirms the clinical study with honey vs SSD of Subrahmanyan (1991). In his study, honey turned out to be much better than SSD, which was proved by a significant reduction of the number of days spent in hospital 5 Today, in terms of re-epithelialisation, honey scores better than SSD (Flammazine R) and also seems to be better than sugar. In fig. 2 the most striking difference between sugar and honey is the thickness of the dermis. At cellular level the latter coincides mainly with a quantitative difference in inflammatory reaction and myofibroblast expression, both being less in the honey treated wounds. During the process of wound healing myofibroblasts are supposed to playa role in wound contraction. However, when contraction stops and the wound is fully epithelialized, these myofibroblasts which contain alpha myofibroblast smooth muscle actin disappear. The scar classically becomes less cellular probably as a result of apoptosis. It is then composed of typical fibroblasts with well-developed rough endoplasmatic reticulum but with no more actin filaments. In hypertrophic scars, on the other hand, the expression of alpha-smooth muscle actin in myofibroblasts persists l2 . Actually, in our study a persisting actin staining was seen in the sugar treated bums of pig 137 (table I).

62

Th. J. Postmes et al.

Hence, the "early" disappearance of myofibroblasts in the honey treated bums suggests a more advanced phase of wound healing than that of the sugar treated wound on the contralateral side. Granulation tissue, as required for the formation of the neodermis, is clearly suppressed initially by SSD. The effect of sugar on dermal thickness, as illustrated in fig. 2, is an interesting observation. Apparently, the large variation in dermal thickness in experiment b, where honey was applied on both flanks, implies that more data are needed to reach a final conclusion in the honey vs sugar inquiry. Unprocessed honey, unlike sugar, generates hydrogen peroxide when it becomes diluted. Hydrogen peroxide has been used for decades as an antibacterial agent with great success. Mainly due to the so-called Fenton reaction it can easily produce free hydroxyl (OB) radicals which are very bactericidal indeed. In vitro, hydrogen peroxide has also a biphasic effect on fibroblasts. Within a concentration of 10-8 and 10--{i molliiter it stimulates cell proliferation, whereas at higher concentrations an inhibiting effect becomes prominent I 3. In vivo, data are lacking. Nevertheless there is no reason to assume that fibroblasts in situ would react otherwise. The hydrogen peroxide concentration provided by honey in an open wound may have a growth inhibiting effect on the fibroblast. According to White et al. (1963), a 14 % honey solution may generate in one hour 0.0-2.12 mmollliter 7• Of the 90 samples investigated the one hour mean value was 0.47 mmolll ± 0.55 (s.d.Y4. In bleeding wounds or in the presence of traces of catalase, hydrogen peroxide breaks down almost instantly. This and other micro-environmental conditions make it almost impossible to predict the concentration of hydrogen peroxide at the interface of honey and wound bed.

CONCLUSION Honey is an ideal topical therapeutic agent, for it does not adhere to the wound surface. In comparison to silver sulfadiazine it is definitely superior because of its quick reepithelialization and its absence of a sustained inflammatory reaction, which in SSD is seen, even long after a complete epithilialization. Honey is also better than sugar paste, for it has a natural antibacterial activity, which is lacking in sugar paste of similar osmolarity. In addition it provides and maintains an environment in which healing can take place at an optimal rate. Honey treatment of burns is certainly cost effective, because it shortens the duration of treatment with ca. 25%, as shown in this study, and it certainly reduces, beyond all question, the duration of hospitalizations.

ACKNOWLEDGMENT We wish to thank the Dutch Burns Foundation for its financial and personal support of this study.

REFERENCES I. Knutson, R.A., Merbitz, L.A., Creekmore, M.A. and Snipes HG. (1981). Use of sugar and povidone -iodine to enhance wound healing:five years' experience. Southern Medical Journal 74, 1329--1335.


63

2. Orouet, N. (1983) Utilisation du sucre et du miel dans Ie traitement des plaies infectees. Nouv Press Med 12,2355-2356. 3. Efem, S.E. (1988). Clinical observations on the wound healing properties of honey. British Journal of Surgery 75, 679--{i81. 4. Efem, S.E. (1993) Recent advances in the management of Fournier's gangrene: Preliminary observations. Surgery 113, 200 -204 5. Subrahmanyam, M. (1991). Topical application of honey in treatment of bums. British Journal of Surgery 78,497-498. 6. Condon, R.E. (1993). Curious interactions of bugs and bees. Surgery 113, 234-235. 7. White). W., Subers, M.H. and Shepartz AI. (1963). The identification of inhibine, the antibacterial factor in honey,as hydrogen peroxide and its origin in a honey glucose-oxidase system. Biochemica Biophysica Acta 73,57-70. 8. Molan, P.c. and Russel, K.M. (1988). Non-peroxide antibacterial activity in some New Zealand honeys. Journal of Apicultural Research 27, 62 -67. 9. Hoekstra, MJ, Hupkens, P., Outrieux, R.P., Bosch, M.M.C and Kreis, R.W. (1993). A comparative burn wound model in the New Yorkshire pig for the histo- pathological evaluation of local therapeutic regimens: silver sulfadiazine cream as a standard. British Journal of Plastic Surgery 46,585--589. 10. Postmes, L Bogaard van den A.E. and Hazen, M. (1993). Honey for wounds, ulcers, and skin graft preservation. Lancet 341,756--757. II. Teepe, R.G.C., Koebrugge, E.l, L6wik, C.W.G.M., Petit, P.L.c.P., Bosboom, R.W., Twiss, I.M., Boxma, H., Vermeer, BJ. and Ponec, M. (1993). Cytotoxic effects of topical antimicrobial and antseptic agents of human kerationocytes in vitro. The Journal of Trauma 35, 8-19. 12. Schmitt-Graff., Oesmouliere A. and Gabbiani G. (1994) Heterogeneity of myofibro blastic cell plasticity. Virchows Archive 425, 3--24. 13. Schmidt RJ., Chung, L.Y., Andrews, A.M. and Turner, T.O. (1992). Hydrogen is a murine (L 929) fibroblast cell proliferant at micro- and nanomolar concentrations. In:Second European Conference in advances in wound management. Proc. Int. Conf.Center Harrogate Oct. 20th-23th p. 117-121. 14. White, lW. and Subers, M.H. (1963). Studies on honey inhibine. 2. A chemical assay. Journal of Apicultural Research 2, 93-100.

7

THE EFFECT OF HONEY ON HUMAN TOOTH ENAMEL AND ORAL BACTERIA S. R. Grobler' and N. J. Basson Oral and Dental Research Institute Faculty of Dentistry University of Stellenbosch Private Bag Xl 7505 Tygerberg, South Africa.

SUMMARY Various fruit juices with relatively low pH values are known to have erosive effects on human tooth enamel in a reasonably short time (Grobler et al.1989! Clin Prev Dent, 11:23-28; 12:5--8). Honey, however, with a relatively low pH, could do the same. The honey sample consisted mainly of nectar gathered from the blossoms of Eucalyptus trees. The honey used did not contain any artificial preservatives or dilutents, neither had it been heated by any artificial method. Sixteen human incisor crowns were ultimately ground wet using 1200-grade silicon carbide paper. Each surface was divided into five segments and each segment exposed to pure honey and diluted honey as well as to artificial honey for different periods of time. The enamel segments were then investigated for their hardness as well as for any etch pattern, by scanning electron microscopy. Scanning electron microscopy showed no erosion of enamel by natural honey over a period of thirty minutes neither did Knoop microhardness tests show any deterioration of the enamel structure even in a 4 times diluted honey solution. However, the theoretical solubility and ion product values can be linked to the results obtained by the SEM study for the undiluted as well as for the four times diluted artificial honey sample. The absence of any effect by pure honey could only be partially attributed to the normal building blocks of enamel, namely calcium, phosphorus and fluoride levels. Seven different oral Streptococcus species, a Candida albicans strain and a Staphylococcus aureus strain were tested for antibacterial sensitivity towards the honey. Minimal inhibitory concentrations (MIC) were determined with a broth dilution method. The MIC was the lowest concentration of the honey which yielded no growth. * Correspondence address: S.R. Grobler, Oral and Dental Research Institute, University of Stellenbosch, Private Bag XI, Tygerberg, 7505, South Africa.

65

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S. R. Grobler and N. J. Basson

The oral streptococci as well as the C. albicans strain were relatively resistant to Bluegum honey. However, the two species Streptococcus anginosus and Streptococcus oratis were inhibited at 17% and 12% respectively.

INTRODUCTION Honey has been used by man for many centuries. Written records date back to 3000 BC, which describe the practice of loading beehives onto small boats and moving them up and down the Nile River, in Egypt, depending on the season, to enable the bees to collect nectar where it was most abundant. Honey was also used by the Assyrians about 4000 years ago to embalm the bodies of their dead. Today honey is used extensively as a sweetening agent and is also considered as a health food, for example, it is used as a sweetener in cough mixtures. In a recent study in the British Journal of Surgery (1991)2, it was reported that honey not only renders burn wounds sterile within 7 days. but that 87% of burn wounds also healed within 15 days, against 10% in the control group. Large expansion of fruit farming practices took place in such a way that additional pollination became a necessity for flowering crops and orchards. Here hived honey-bees are employed to help with the pollination process. These small hard worker-bees are perfect pollinators which beekeepers can manipulate to supply in large quantities at appointed times. Two sub-species of the true honey-bee are found in South Africa. namely, the striped African honey-bee and the Cape honey-bee l . The pH of honey produced by these bees is slightly low, with an average value of about 3.9 for South Africa. It is interesting to note that the average value reported is the same as that for the USA 4. The demineralizing effects of excessive intake of fruit drinks, especially of citrus fruits and other acidic beverages, are well documented because of their low pH values s- 7 • Furthermore, it was reported that the highest dental caries incidence was found in farm workers in a citrus producing group in comparison to a control group. Published results 7 on different soft drinks showed the following degree of enamel demineralization: Pepsi Cola = orange juice> apple juice> Diet Pepsi Cola. Therefore, owing to the low pH values reported for honey one could presume that it might have a potential to cause erosion when it comes into contact with enamel. At the same time acid produced from dietary sugars through the metabolism of oral bacteria are also responsible for enamel erosion and dental caries 8 • Several workers 9- 1J investigated the antibacterial properties of honey and found it effective against organisms such as Staphylococcus aureus, Salmonella typhi, Shigella species, Escherichia coli and other pathogenic species. However, little information is available with regard to the antimicrobial effect of honey on the oral bacteria. Therefore, the purpose of this study was to determine the erosive effect of honey on human tooth enamel and to evaluate honey for its antimicrobial activity on certain oral bacteria.

MATERIAL AND METHODS The honey samples used in this experiment were harvested in the Western Cape of South Africa and consisted mainly of nectar gathered from the blossoms of Bluegum (Eucalyptus) trees. In order to be 100% certain that the honey sample collected were not

Human Tooth Enamel, Honey, and Bacteria

67

diameter

~

3 mm

Figure 1. The division of the enamel surface into four segments as used in subsequent phases of the study.

contaminated it was harvested by the researchers and was not heated by any artificial method. Sixteen human incisor crowns were polished up to 1200 grit fineness with silicon carbide paper before being exposed to the different honey concentrations for different periods of time. The surfaces were ground until an area of enamel approximately 3 m in diameter at the mid-central region had been smoothed. This was necessary to obtain a very smooth enamel surface which would highlight the slightest signs of enamel erosion, should any take place. The polished areas were washed and covered with a circular plastic adhesive tape (Fig. 1) and the tape then cut with a surgical scalpel into four segments.

Phase 1 Seven crowns were used in the first phase. The first specimen was prepared by removing the tape from one of the four segments from a crown. Honey was poured into a Petri dish and the crown with the one exposed segment immersed in the honey and continuously agitated for 10 minutes. A second outer segment of tape was then removed and the specimen again agitated in the honey for 10 minutes. Similarly, the third segment was removed and agitated for 10 minutes. The fourth segment was not exposed to honey and served as the control. In this way the four segments were exposed for 30, 20, 10 and 0 minutes. The effect of the honey treatments of these specimens were evaluated by scanning electron microscopy.

Phase 2 In this phase three crowns were exposed to a solution of 50% honey to 50% distilled water by volume, while three were exposed to a solution of 25% honey to 75% water. Again the exposure periods were 0, 10, 20, and 30 minutes per segment.

Phase 3 Six crowns were also divided into segments as explained above. In this investigation the surfaces were exposed to artificial undiluted honey (3 crowns) and to a solution containing 25% artificial honey and 75% distilled water (3 crowns). In this phase the immersion times were 0, 0, 30 and 30 minutes per segment. The artificial honey contained: 38%

68

S. R. Grobler and N. J. Hasson

Table 1. The minimal inhibitory concentration of honey and ofa carbohydrate control for different oral Streptococcus species (% vol/vol) 50% Organism (NCTC) Streptococcus mutans (10449) Streptococcus salivarius (8618) Streptococcus sanguis (7864) Streptococcus anginosus (10708) Streptococcus gordonii (3165) Streptococcus oralis (11427) Streptococcus sobrinus (10921) Candida albicans (NCPF 3118) Staphylococcus aureus (8530)

H

C

30%

25%

21%

H

H

H C

C

+ +

C

+ +

+ + + + + + +

+ + + + + + + + +

17% H

C

+ + + + + + + + + - + + + + + + +

12% H

+ + + + +

C

+ + + + + + + + + + + +

6%

H

C

+ + + + + + + + + + + + + + + + + +

3% H

C

+ + + + + + + + + + + + + + + + + +

H-honey, C~ontrol, + growth, - no growth NCPF-National Collection of Pathogenic Fungi (London)

D-fructose, 31 % D-glucose, 7% maltose, 1.5% sucrose, 100 mg Call, 308 mg PII, 0.7mg FII and the pH adjusted to that of the natural honey sample with HCI 4,14. All the segments in all the phases were evaluated for erosion by SEM. The honey was tested for its antimicrobial activity against a control solution with a sugar content similar to that of natural honey namely the abovementioned artificial honey. Eight different oral microbial species, obtained from the National Collection of Type Cultures (NCTC) (London), were used in the study (Table I). A strain of Staphylococcus aureus was included as a reference organism. The cultures were maintained on Brain Heart Infusion (BRI) agar (Oxoid) and subcultured weekly. The Minimal Inhibitory Concentration (MIC) of the honey (expressed as a vollvol percentage) were determined with the broth dilution method described by Ericsson and Sherris 15 • In short, two-fold dilutions of natural honey and the control solution were aseptically prepared in double strength sterile BHI broth (Oxoid) to give final volumes of 5 ml in each tube. The inocula used were prepared from overnight growth cultures in BHI broth. One drop of a 1I 100th dilution of a culture was used to inoculate 5 ml of the test solution. The tubes were incubated at 37°C for 24 hours and examined for growth. The MIC was noted as the lowest concentration of honey which yielded no growth.

RESULTS Fig I gives a diagrammatical outlay of the shape of the circular plastic adhesive tape which covered the smoothed enamel surface. The circular area was divided into 4 different segments by cutting it with a scalpel. All the segments in phase 1 and 2 showed no sign of enamel erosion under the scanning electron microscope (5l00x magnification) as a result of exposure to the different honey concentration for 0, 10, 20 and 30 minutes. Figure 2 gives a scanning electron photomicrograph (5100x magnification) of a typical polished enamel surface which was not exposed to any honey solution. In phase 3, Figure 3 (5100x) shows a high degree of enamel erosion when the enamel was exposed for 30 minutes to the 4 times diluted honey sample (25% artificial honey and 75% water). A lower degree of enamel erosion was also observed when the enamel was exposed for 30 minutes to undiluted artificial honey.


69

Figure 2. Scanning electron micrograph of the control enamel section, which was not exposed to honey. x5100 magnification.

Figure 3. Scanning electron micrograph of enamel section exposed to 4 times diluted artificial honey for 30 min. x5100 magnification.

70

S. R. Grobler and N. J. Basson

The MIC of the natural honey and of the control solution is shown in Table I. Except for the yeast species, all the organisms tested failed to grow at honey and sugar concentrations higher than 21 %. The two oral strains Streptococcus anginosus and Streptococcus oralis were more sensitive to the antimicrobial activity of honey than the S. aureus strain and failed to grow at concentrations of 17% and 12% respectively. The oral yeast Candida albicans was more resistant to honey than the bacteria and was able to grow at concentrations up to 30%.

DISCUSSION The natural honey was analysed l4 and the artificial honey prepared to contain the same amounts of different carbohydrates (namely fructose, glucose, maltose, sucrose), calcium, phoshorous and fluoride. This was done to investigate the effect of these species (which form the building blocks of enamel) on the solubility of enamel in honey, BECAUSE we soon observed that enamel does not dissolve in pure honey even when diluted up to 4 times. However, a low degree of erosion was already observed for the undiluted artificial honey in contrast to pure honey. while the 4x diluted artificial honey illustrated a higher degree of enamel erosion, again in contrast to 4 x diluted pure honey (30 min. 5100x enlargement). The fact that enamel dissolves in artificial honey and not in pure honey can be attributed to factors other than the concentrations of the elements that are normally associated with the solubility of enamel (calcium, phosphorous and fluoride). In order to establish why enamel dissolves more in diluted artificial honey than in undiluted artificial honey we have to consider the composition of pure honey and artificial honey at different dilutions. We looked mainly at the concentrations of the elements which are also found in the apatite crystal because these elements will have an affect on the solubility and the solubility product of enamel. What was very interesting to note is that honey also contains F (this sample 0.7 ppm). The question arose as to whether any correlation exists between concentrations of fluoride, calcium and phosphorous in the honey and the water used in its processing. Bees use water extensively, in the making of honey and to cool their hives, especially in very hot weather4 • The rapid movement of their wings produces draughts which cause evaporation, thus cooling the hives as well as condensing the honey. However, in an extensive study by Grobler et aI 16 ., on the analysis of honey samples from various areas and of the water collected from each source used by the bees for their water supply, it was concluded that the elemental composition of water does not contribute substantially towards the levels of calcium, phosphorous or fluoride of honey. On the other hand, it is possible that the relatively high concentrations of the above mentioned elements in honey relative to that in water could mask such a possible relationship. The pH of the different honey solutions is similar, namely 4.24 regardless of the dilution. Under a normal situation this pH is low enough to dissolve enamel quite rapidlyl7. This is due to the fact that the buffering capacity of honey is quite high-4x higher than that of saliva. This means that a small amount of saliva will not decrease the acidicity of honey to a harmless level of about 6.8. The joint effect can be summarised by calculating the ionic products of the honey solutions for hydroxyapatite (HAP) and fluoroapatite (F AP) as well as the solubility products of hydroxy-apatite and fluoro-apatite I4 . From these values it can be seen that the ionic product value for artificial honey with respect to HAP (63.2) is higher than the solubility


71

product value for HAP (54.6), which means that the 100% honey solution is under saturated with respect to HAP and HAP will dissolve but not FAP. On the other hand the ionic product of honey with respect to FAP (57.9) is lower than the solubility product (59.6) with respect to F AP and the honey solution is supersaturated with respect to F AP and FAP will not dissolve in the 100% honey. This explains why enamel which consists of both HAP and FAP will dissolve only slightly in 100% artificial honey, but will dissolve more readily in the 4x diluted honey solution as was found in this study. It has been shown convincingly that honey contain antibacterial activity and that this activity can be ascribed to much more than just the high sugar content of honei 8 • However, it is obvious from our results that the high sugar content can mask the real antibacterial activity of honey at honey concentrations of 25% and higher. Our results also show that except for S. anginosus and S. oratis, the oral streptococci are relatively resistant to the true antimicrobial activity of honey.

REFERENCES I. Grobler S.R .• Senekal P.J.c. and Van Wyk Kotze T.1. (1989) The degree of enamel erosion by five different kinds of fruit. c/in. Prev. Dent. 11,23-28. 2. Subrahmanyam M. (1991) Topical application of honey in treatment of burns. Br. 1. Surg. 78,497-498. 3. Anderson R.H., Buys B. and Johannsmeier M.F. (1983) Beekeeping in South AJrica. 2nd edn, Bulletin No. 394. p. 144. Department of Agriculture. 4. Root AI. (1983) ABC and XYZ oj Bee Culture. A.1. Root Pub!. Co. Medina, Ohio. 5. Grobler S.R. and Van der Horst G. (1982) Biochemical analysis of various cool drinks with regard to enamel erosion, de- and remineralization. 1. Dent. Assoc. South Afi-ica 37, 681-684. 6. Grobler S.R., Jenkins G.N. and Kotze D. (1985) The effects of the composition and method of drinking of soft drinks on plaque pH. & Dent. 1. 158,293-296. 7. Grabler S.R., Senekal P.J.c. and Laubser J.A. (1990) In vitro demineralization by orange juice, apple juice, Pepsi Cola and Diet Pepsi Cola. Clin. Prevo Dent. 12. 5--8. 8. Silverstone L.M., Johnson N.W., Hardie 1.M. and Williams R.A.D. (1981) Dental caries: Aetiology, pathology and prevention. MacMillan Press Ltd. London. 9. Cavanagh, D, Beazley, J & Ostapowicz. F (I 970) Radical operation for carcinoma of the vulva: a new approach to wound healing. Journal o{ Ohstetrics and Gynaecologv o{ the British Common Wealth, 77, 1037-1040. 10. Dold, H, Du, DH & Dziao, ST (1937) Nachweis antibakterieller, hitzc-und lichtempfindlicher hemmungsstoffe (inhibine) im naturhonig (Bliitenhonig). Zeitschrifi (iir Hygiene und Infektions-Krankheiten, 120,155-167. 11. Ibrahim A.S.( 1981). Antibacterial action of honey. Proceedings of the First International Conference on Islamic Medicine. Kuwait: Minister of Health, 363- 365. 12. Jeddcr A.,Kharsany A.,Ramsaroop U.G., Bhamjee A., Haffejee I.E. and Moosa A. (1985). The antibacterial action of honey. An in vitro study. S. Aji: Med. 1. 67. 257-258. 13. Zumla A. and Lulat A. (1989). Honey a remedy rediscovered. 1. Roy. Soc. Med. 82,384-385. 14. Grobler S.R., Du Toit 1.1. and Basson N.J. (J 994) The etlect of honey on human tooth enamel in vitro observed by electron microscopy and microhardness measurements. Arch. Oral Bioi. 39. 147-153. 15. Ericsson H.M. and Sherris J.e. (1971) Antibiotic sensitivity testing. Report of an international collaborative study. Acta Path Microbial Scand 79B, Suppl 217, 1-82. 16. Du Toit 1.1., Grobler S.R., Van Wyk Kotze TJ. and Basson N.J. (1995) Fluoride, calcium and phosphorus levels in bee honey and water. S. AJr. 1. Sci. 91, 391-392. 17. Driessens F.e.M. (1982) Mineral aspects oj dentistry (Edited by Meyers H.M.) Vol. 10, p. 117. S. Karger, London. 18. Molan P.c. (1992) The antibacterial activity of honey. 1. The nature of the antibacterial activity. Bee Wold 73,5-28.

8

HONEY CONTACT WITH TEETH IN SITU 1. Gedalia: S. R. Grobler, 1. Grizim D. Steinberg, L. Shapira, I. Lewinstein, and Mo. Sela

Hebrew University-Hadassah School of Dental Medicine Jerusalem, Israel; and Faculty of Dentistry University of Stellenbosch Tygerberg, South Africa.

SUMMARY Honey is a sweetening agent affecting dental caries like sucrose. It contains also a solubility-reducing agent, an organic phosphorus ester that is degradable by salivary emzymes. In the experimental design the changes of microhardness in prepared enamel surfaces from extracted human teeth were monitored by measurements of the tooth enamel microhardness at baseline and after intra-oral exposure, during a certain time period, to honey. Normal and salivary' flow deficient subjects volunteered for the study. pH measurements of saliva were carried out at baseline, during and after exposure of the enamel specimens in the mouth to the honey. The pH of the saliva (close to 7.0 at start) mixed up with that of the honey (3.9), decreased from about 6 to 4 in the saliva-honey mixture. After swallowing the mixture the pH returned to the baseline value. The microhardness of the surface enamel did not change in subjects with almost complete lack of saliva flow (dry-mouth subjects), as opposed in the subjects with a regular flow of saliva.

INTRODUCTION It was observed in rats that tooth destruction increased at a high rate with the addition of honey to their basal bread did. In vitro tests showed that pure honey with a relatively low pH (3.9) does not exert an erosive effect on human tooth enamel 2 • An organic phosphorus ester was suggested as the solubility reducing agent in honey, that is degradable by salivary enzymes 3 * Correspondence to: Prof. I. Gedalia, Oral Biology-Dental Research, Hebrew University-Hadassah School of Dental Medicine, Jerusalem, Israel.

73

74

I. Gedalia et al.

The effect of honey consumption on human dental enamel was investigated in drymouth subjects.

MATERIALS AND METHODS Honey samples harvested in the southern part of Israel (Yad Mordechai) during autumn, consisting of nectar from the blossoms of wild flowers, were used. Ca, P and F were analyzed according to previous examinations in honey 4-7 . Changes of microhardness in prepared tooth enamel surfaces were monitored by measurements of the tooth enamel micro hardness at baseline and after intraoral exposure to honey, during a similar time period. Normal and salivary flow deficient subjects (xerostomic) volunteered for the study g.9. pH measurements were taken of the saliva at baseline, during and after exposure of the enamel specimens in the mouth to the honey-saliva mixture by means of indicator paper. Significance of the differences in microhardness between the baselines of the tested groups was determined by the Student t-test. Paired t-test was used to determine the significance of changes in microhardness before and after honey consumption within each subject group .

RESULTS The results of the Ca, P and F composition of the honey are: 166 ppm Ca ± SD 5, 410 ppm P ± SD 15 and 0.05 ppm F ± SD 0.01. The salivary pH levels and the microhardness levels are presented in Figs. 1 and 2. The pH of the honey-saliva mixture decreased from about 6 to 4 in the normal and the salivary-flow deficient subject groups (p < 0.05), returning to the baseline pH after the mixture was swallowed (p < 0.05), Fig. 1. The initial microhardenss of the surface of the enamel specimens decreased significantly (p < 0.000 I) after the honey consumption in the subjects with a regular flow of saliva, whereas in the dry-mouth subjects no enamel microhardness decrease took place (Fig.2).

8,----------------------,

o

7

•

Normal Irradiated

I

0.6

1\1

.~

iij 5

en

4

3~--------------------~

Figure I. Mean saliva pH values (±SE) at baseline, in the mouth, during consumption of the honey and at completion of swallowing the honey-saliva mixture.

7S

Honey Contact with Teeth in Situ

Z 300

:r:

>

-; 250

=

Before

=

After

,--!-

Ul

III C

"E

III

r--

200

:r: Qi 150 E III

Figure 2. Mean microhardness (±SE) expressed in Vickers hardness numbers before and after exposure to honey in the mouth of human enamel specimens.

C UJ

100

DISCUSSION A pH below 5.5 at the tooth enamel surface leads to mineral loss 10. 11 • The results of the present study indicate that tooth enamel decalcification occurred during consumption of the acidic honey in subjects with normal saliva secretion (Fig. 2). It is known that even trace amounts, 0.01 ppm of F in solution, decrease the rate of enamel solubility 12 • The F concentration was very low in the honey used in our study , 0.05 ppm, which probably explains why it did not suppress bacterial fermentation or exert a remineralizing effect (return oflost Ca and P to the tooth enamel surface). No erosive defects on in vitro exposed enamel surface to pure honey at a pH 4.24 was observed 2• The difference between the in vitro2 and the present in situ results regarding erosive effects on tooth enamel from honey, may be in part explained by the suggested solubility-reducing agent in honey, the phosphate ester, that is degradable by salivary enzymes 3 . In the dry-mouth subjects, the expected solubility-reducing agent in the honey was probably active protecting the tooth enamel when the honey-saliva mixture was below the pH level of 5.5 10• 11 , Fig. I.

A-CKNOWLEDGMENT The authors are indebted to Prof. I Roman, Dept. of Applied Science and Applied Physics, Hebrew University, Jerusalem, for his first-rate cooperation in this study. Thanks to Mrs. Shula Konig for her secretarial assistance.

REFERENCES I. Koenig K.G. ( 1967) Caries Induced in Laboratory Rats. Br. Dent. J. 123,585- 589. 2. Grobler R.S .. Du Toit I.J., Basson N.J. (1994) The Effect of Honey on Human Tooth Enamel In Vitro Observed by Electron Microscopy and Microhardness Measurements. Arch. Oral BioI. 39. 147- 153. 3. Edgar W.M .• Jenkins G.N. (1974) Solubility-Reducing Agents in Honey and Partly-Refined Crystalline Sugar. Br. Dent. J. 136. 7- 14. 4. Chakrabarti C .. L. ( 198 1) Progress in Analytical Atomic Spectroscopy. 2, 207. 5. Havezov I., Russeva E., Jordanov N . (1979) Fl ameless Atomic Absorption Determination of Phosphorus Using ZrC Coated Graphite Atomizer Tubes. Fresenius Z. Anal. Chern. 296, 125- 127. 6. Nicholson K. , DuffE.J. (1981) Fluoride Determination in Water. Anal. Lett. 14, 493- 517.

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I. Gedalia et al. 7. du Toit I., Grobler S.S., v Kotze W.TJ., Basson, NJ. (1996) Fluoride, Calcium and Phosphorus Levels in Bee Honey and Water. S.Afr. J. of Science 91,391-392. 8. Gedalia I., Davidov I., Lewinstein I., Shapira L. (1992) Effect of Hard Cheese Exposure, With and Without Fluoride Prerinse, on the Rehardening of Softened Human Enamel. Caries Res. 26,290-292. 9. Sela Mo., Gedalia I., Shah L., Skobe Z., Kashket S., Lewinstein I. (1994) Enamel Rehardening with Cheese in Irradiated Patients. Amer. 1. Dent. 7, 134-136. 10. Grobler R.S., Jenkins G.N., Kotze D. (1985) The Effect of the Composition and Method of Drinking of Soft Drinks on Plaque pH. Br. Dent. J. 158,293--296. II. Gedalia I., Dakuar A., Shapira L., Lewinstein I., Goultschin J., Rahamim E. (1991) Enamel Softening with Coca-Cola & Rehardening with Milk or Saliva. Am. J. Dent. 4, 120-122. 12. White OJ. (1987) Reactivity of Fluoride Dentifrices with Artificial Caries. I. Effects of Early Lesions: F Uptake, Surface Hardening and Remineralization. Caries Res. 21, 126--140.

9

MEDICINAL HERBS AS A POTENTIAL SOURCE OF HIGH-QUALITY HONEYS Zohara Yaniv and Michal Rudich ARO The Volcani Center Bet Dagan, Israel

The use of plants for therapeutic purposes dates back to the beginning of time. Ancient civilizations, such as Egypt, China, India and the Incas of the Americas knew well the medicinal properties of herbs and used them in the prevention and treatment of diseases. Through the writings of great physicians, such as Hippocrates and Dioscorides, much of this ancient art had survived to our times. Nowadays we are witnessing the revival of the herbal tradition. There is a great interest in using this tradition in order to develop and define new, natural drugs for human use. Honey, too, has its important place in human nutrition since prehistoric time. It was considered not only as a sweetener but also as a high-quality food, with curative properties. The appreciation of honey is expressed in old manuscripts and documents, legends and mythologies. Honeybees feed on plants nectar, and honey is produced. Its qualities reflect the source of nectar which was used,so that the properties of the nectar and its chemical content are of great importance. In fact, the quality of the honey is directly related to the source of nectar. Therefore, there is a very great potential for using medicinal plants and nectars with very specific chemical compositions to produce honeys in the future with curative properties above and beyond those known hitherto. The taste, aroma and color of the honey is directly affected by the source of the plant nectar. (Table I). Examples include eucalyptus honey, which is dark, aromatic and spicy; thyme honey, light and scented; carrot honey, reddish and spicy, and so on. These examples indicate a direct transfer of components from the nectar to the honey. The use of honeys produced from diverse sources of nectar for specific therapeutical purposes is widely practised in France. Table 2 illustrates some examples of honeys, their source of nectar and their special medicinal indications. It is clear that the honeys and their source of nectar are used for identical medicinal treatments. Examples include lavender honey for diseases of the respiratory system, tilia honey, for spasms, insomnia and as a tranquilizer; acacia honey as an intestinal regulator, etc. (Table 2). It should be noted that this practice falls within the framework of folk medicine. 77

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Z. Yaniv and M. Rudich Table 1. Colors and aromas of honeys Source of nectar Eucalyptus Thistle Carrot Onion Red Clover Cotton Sage Rosmary Thyme Savory

Color

Aroma and taste

dark dark green tone reddish brown bright yellow light light light light light

spicy spicy spicy onion flavor delicate flavor non·scented delicate flavor delicate flavor delicate scent delicate flavor

Scientific evidence for a transfer of active metabolites from the nectar to the honey produced is provided by studies performed in several laboratories. Some important topics are summarized as follows:

THE EFFECT OF PLANT ORIGIN ON THE ANTIBACTERIAL PROPERTIES OF HONEYS The antibacterial properties of honey have been known for a long time (Molan et ai, 1988). Scientists in various parts of the world have noticed that the intensity of these antibacterial properties depends on the plant source of the honey. In Brazil, Cortopassi-Laurino and Gelli showed that in Apis-bee honeys, the strongest antibacterial properties were found in honeys produced from mimosa and eucalyptus pollen. Mimosa was found to be also the best source of antibacterial honey produced by Melipona subnitida and Plebeiasp bees. (Cortopassi-Laurino and Gelli, 1991). Indeed, the use of mimosa and eucalyptus is known in phytotherapy and aromotherapy as antiseptic and antiflammatory agents. A study was performed in Poland recently on the antibacterial properties of honeys againsts everal strains of bacteria. In this study, too, it was found that various types of

Table 2. Honeys: sources and curative properties (folk medicine in France) Source of nectar

Curative properties

Acacia Erica

Intestinal regulator. Antiseptic (urinary tract), Diuretic.

Chestnut, forest flowers, sunflower

Stimulates blood circulation.

Lavender

Antiseptic, Anti-inflammatory (respiratory system), Anti-spasmodic. Anti-anemic,Antiseptic Anti-inflammatory (respiratory system),Diuretic. Anti spasmodic, Tranquilizer

Oak sap, fir tree

Tilia

Particular indications Good for intestinal stasis in infants. Urinary tract infections and kidney insufficiency. Improves blood circulation in general and varicose veins in particular. Diseases of the respiratory system. Diseases of the arteries. Certain types of anemia

Spasms of diverse origin, restlessness, insomnia and epilepsy.

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honey differ substantially in their antibacterial activity, depending on the plant source. The most active ones were honeydew and lime honey (Leszczynska Fik and Fik, 1993). This difference could be due to the compositions or amounts of essential oils and other components which were transferred from the plant to the honey. The direct effect of feeding medicinal plant extracts to honeybee colonies, on the antimicrobial activity of the honey produced, was studied in Egypt (Mishref et ai, 1989). Extracts from geranium, chamomile and majoram were fed to honeybee colonies once during an 8-week period. At the end of this period, honey in the combs was extracted and its antimicrobial activity was determined. Results showed that the honeys from colonies fed with medicinal plant extracts showed greater antibacterial activities than honey from control colonies, in the order chamomile> geranium> majoram. These findings correlate with the actual use of geranium, majoram and chamomile, as antiseptic and antimicrobial agents in folk medicine. The essential oils of geranium and majoram are widely used as antiseptic components of aromatherapic mixtures.

THE PRESENCE OF IDENTICAL FLAVONOIDS, CAROTENOIDS AND GLUCOSIDES IN BOTH HONEY AND THE NECTAR OF ITS HONEYBEES The following examples demonstrate the possibility that diverse secondary metabolites, besides essential oils, can be transferred by the honeybee, from the nectar to the honey, thus creating a honey with a specific chemical composition: 1. Sunflower honey was shown to be a very rich source of flavonoids. (Sabatier et ai, 1992; Sabatier et ai, 1988).The flavonoids were identified and their structures could provide an index of floral origin. 2. Analysis of many experimental and commercial citrus honey samples revealed the presence of the flavanone hesperetin in all samples. This flavanone was not detected in any non-citrus honey samples. The analysis of the flavonoids present in orange nectar revealed that the flavanone hesperidin was the major flavonoid detected. Hesperetin is probably produced by hydrolysis of hesperidin by the bee enzymes present in honey (Ferreres et ai, 1993). 3. Of a similar nature is the study of Czeczuga (Czeczuga, 1985), showing the presence of identical carotenoids in working bees and in the flowers being visited by the bees. The author suggested that some of the flower carotenoids are converted to more highly oxygenated forms in the honeybee. The total content and composition of carotenoids in foraging bees was related to the flower species visited. 4. The presence of the glucoside, arbutin in both bitter honey and in the nectar of the strawberry tree, Arbustus unedo, confirmed this tree to be the main source of the honey. (Floris and Prota, 1989). In Sardinia, this bitter honey is considered as having therapeutic value.

THE DANGER IN TOXIC HONEY We should be aware of the possibility of the presence of plant toxins in honey. Nectars from plants such as azalea, andromeda and rhododendrom (Ericaceae) are known to

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contain gayanotoxins. Cases of intoxication, following the ingestion of such honey have been reported. Symptoms are similar to aconitine intoxication: progressive paralysis from the extremities to the diaphragm. (Alcaraz and Rios, 1991). The more recent report of the presence ofpyrrolizidine alkaloids in honey, may indicate a new health hazard (Deinzer et ai, 1977): these authors found that the hepatotoxic alkaloids known to occur in tansy ragwort (Senecio jacobaea L.) are also present in honey produced from the nectar of this species. These alkaloids (six were identified), are potentially carcinogenic, mutagenic and teratogenic, and may pose health hazards to the human consumer. However, due to the fact that ragwort honey is very bitter in taste and off-color, it is not likely that the individual consumer would consume enough honey to suffer acute effects.

FUTURE PRODUCTION OF HIGH QUALITY, THERAPEUTICAL HONEY Since the source of plant nectar can affect the composition and properties of the honey, future production of therapeutical honeys can be envisaged. Nectar-rich medicinal plants with desired therapeutical properties could be used as the source of nectar. Table 3 contains a list of some medicinal plants which could be used as a source of active compounds. Some of these plants have nectar-rich flowers, visited by the honeybee. Examples include aromatic plants from the Labiatae family, known for their high content of essential oils. Salvia ojJicinalis and S. Fruticosa; Coridothymus capitatus and Majorana syriaca. All three species are known for the treatment of colds, indigestion and external inflammations. Crataegus oxycantha is prescribed as a cardiac depressant and as a hypotensive. Relama raelam, a desert plant, is used for rheumatic pain and external wounds and has a potential application in chemotherapy against cancer. Echinacea angustifolia stimulates and strengthens the immune system and Cassia senna is a very popular natural laxative. Plants could also be used by feeding the bees with a sweetened extract of plant parts, such as leaves. roots, stems, on flowers. These plant parts should be selected on the basis of a high content of active compounds. The sweet extracts would then serve as a rich source for the production of medicinal honey. We hope that in the future the list of curative and medicinal honeys will be unlimited. This direction will offer a new avenue into natural medicine.

Table 3. Potential medicinal qualities of honeys Plant origin Salvia officinalis (Sage) Coridothymus capitatus (thyme) Majorana syriaca (Majoram) Crataegus oxycantha (Hawthorn) Retama raetam (White Broom) Echinacea angustifolia Cassia senna

Medicinal potential Stimulates bile secretion. wound and acne treatment, regulates menstruation. Antiseptic, expectorant and anti-spasmodic Expectorant, toothache and gum infection, indigestion, heart problems. Vasodilator, regulates blood pressure. Relieves abdominal and rheumatic pain, and wound remedial. Anti-carcinogenic activity. Anti-inflammatory, strengthens and stimulates the immune system. Laxative.

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REFERENCES Alcaraz, M.J. and Rios, J.L (1991). Pharmacology ofDiterpenoids. in: Ecological Chemistry and Biochemistry of Plant Terpenoids. (Harborne, 1.8. and Tomas-Barberan, F.A. eds.) p 230-263. Clarendon Press Oxford. Cortopassi-Laurino, M. and Gelli, D.S. (1991). Pollen analysis, physico-chemical properties and antibacterial action of Brazilian honeys from Africanized honeybees (Apis mellifera ) and stingless bees. Apidologie. 22. 61-73. Czeczuga, B. 1985. Investigations on carotenoids in insects. VII. Contents of carotenoids in worker bees feeding on flowers of different plants. Zoologica Poloniae 32. 183--190. Deinzer, M.L. Thomson, P.A Burgett, D.M. and Isaacson, D.L. 1977. Pyrro1izidine alkaloids: their occurrence in honey from tansy ragwort (Seneciojacobaea L.) Science, 195497-499. Ferreres, F. Garcia-Viguera, C. Tomas-Lorents, F. and Tomas-Barberan, F.A. 1993. Hesperetin: a marker of the floral origin of citrus honey. J. Sci. Food. Agric. Sussex. 61. 121-123. Floris I. and"Prota, R. 1989. The bitter honey of Sardinia. Apicoitore-Moderno. 80. 55--{)7. Leszczynska Fik, A. and Fik, M. (1993). Antibacterial properties of various types of honey and the effect of honey heating on antibacterial activity. Medycyna- Weterynaryjna. 49.415-419. Mishref, A. Magda, S.A. and Ghazi, I.M. 1989. The effect of feeding medicinal plant extracts to honeybee colonies on the antibacterial activity of the honey produced. Proceedings o[the Fourth International Conference on Apiculture in Tropical Climates cairo, Egypt. 80-87. Molan, P.C Smith, I.M. and Reid, G.M. 1988 . A comparison of the antibacterial activities of some New Zealand honeys. J. of Apicultural Research. 27. 252-256. Sabatier, S. Amiot, M.J.Aubert, S.Tacchini, M.and Gonnet, M. 1988. Importance of flavonoids in sunflower honeys. BuUetin-Technique-Apicole. 15. 171-178. Sabatier, S.Amiot. M.J. Tacchini, M. and Aubert, S. 1992. Identification of flavonoids in sunflower honey. J. Food. Sci. off. Publ. Ins!. Food. Techno!. Chicago. ILL. The Institute. 57. 773--774.

10

THE UNIQUE PROPERTIES OF HONEY AS RELATED TO ITS APPLICATION IN FOOD PROCESSING Tsila Dvir Human Nutrition Unit Ministry of Agriculture 12, Aranya St., Tel Aviv, Israel

1. ABSTRACT Honey has a wide range of characteristics which differentiate it from sugar and other sweeteners. Some of the unique features of honey are the following: • Honey is a natural ingredient. Nowadays, when natural foods are so valued, this is very significant. • As opposed to the common belief, honey is not particularly calorie rich. • Honey contains many important nutritional values, like minerals and vitamins. • Honey has an important role in natural health care. It has potential in prevention and treatment of certain diseases. • Honey can serve many functions in cooking like browning (especially with microwave). It is an excellent bonding material and an ideal base for sauces. A strategy aimed at increasing the consumption of honey by the private consumers must emphasis these qualities and highlight honey's advantages over "competing" ingredients. Furthermore, these features should be attractive to food manufacturers because of their appeal to the consumer.. Such strategy can include the following components: • Bold and detailed labeling of products that contain honey. The label should list the nutritional values of the honey. • Presentation of honey and honey products in separate sections and on many shelves that are devoted to specific categories, like health food, honey products, spreads, sauces, cake decorations etc. • Marketing honey in new forms which are more convenient to use, like spread instead of liquid honey. • Unique and unusual containers that emphasis the natural aspects of honey. 83

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2. HONEY CONSUMPTION-A BRIEF HISTORY In ancient times honey was the common and unquestionable sweetener, and also a symbol to good life and wealth. All this was completely changed with the industrial revolution, the massive growth of sugar cane in the Americas and the knowledge to cheaply extract sugar from cane and sugar beets. This was an unavoidable development; there was no way by which honey could possibly supply the rapidly growing population which coincided with the rise of the standard of living; among other things this meant an enormous demand for sweeteners. The unfortunate thing that happened alongside this development was that gradually the wonderful food called bee-honey lost its glory: people regarded it as not much different from jam or cane sugar. In the last few decades the honey suffered two additional blows. First, the western world has become more calorie aware; and with the massive campaign against overweight all the natural sweeteners were among the first to be blamed as "the enemy", "the danger". Needless to say, the consumption of honey has suffered greatly. As a "natural" result, people started to shift to artificial sweeteners. This forum is not the place to analyze the consequences of this shift to artificial sweeteners, or to put it more boldly-the health problems which directly result from the exaggerated use of these non-natural substitutes. However, I feel that this matter is of critical importance to the honey future and therefore I will take the liberty to give at least a few clues: • Aspartame sugar substitutes cause symptoms from memory loss to brain tumors. It is sought to be one of the most dangerous substance [1]. • Saccharin, found in many "diet" drinks is considered a possible cancer hazard [2]. • The real amount of sugar substitute consumed in out diet exceeds by far the daily amounts approved as "safe" by USA FDA [1]. • It appears that many of the health problems that the US soldiers contracted in the Golf war are a result of the huge quantities of diet drink that they have consumed [3]. As if the damage caused by artificial sweeteners substitutes was not enough, another problem evolved. the public, and many professionals, got the idea the actually honey is almost the same as sugar. Consequently, housewives, cooks, institute mangers and industrialists said: if so, why bother with honey since sugar is cheaper and readily available? I am not sure how much of this basic misconception was due of somebody claiming that "honey equals sugar" or simply that there was nobody to clearly emphasize the differences between them. Whatever the reason-the damage is very real.

3. HONEY VS. SUGAR To show the full scope of the difference we should compare honey with sugar, item by item. To sum it up: honey is very different-and clearly superior-to sugar, when compared by almost all criteria.

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Table 1. The differences between honey and sugar [4,5] Property Source Health Qualities

Honey

A natural, unprocessed product Mild antibiotic Curing sore throat Avoiding constipation Curing diarrhea in babies Nutritional Properties: Water Content 20% Calories 304 cal per 100 gr. 13 kinds of sugars (glucose 33%, fructose Sugar Kinds 40%, other II kinds 5%) Vitamins (mg per 100 gr.) 0.004-0.006 Thaine B1: B6 Pyridoxine 0.008--0.032 0.1l..{J.36 Niacin 2.2-2.4 VitaminC mg per 100 gr. Minerals 0.4-3 Calcium 0.1-3.4 Iron Potassium 1.0-47.0 Also: Iodine, Copper, Magnesium, and traces of other Gourmet Qualities Rich taste Rich aroma Rich color Translucence Smooth texture Syrupy quality Used "as is" Cooking and Baking Qualities Adds color Browning Binding Preserves freshness & moisture Adds market value Symbolized tradition and "roots" Special Qualities Relates to the "back to nature" trends

Refined Sugar Industrially processed None

1% 375 cal per 100 gr only sucrose None

None

None

None

None

These two misconceptions--that honey, like sugar, is Calorie rich, and that sugar is an equivalent substitute to honey, have discouraged many potential honey consumers. The potential lose is estimated by perhaps hundreds of millions of consumers that would have used considerable amounts of honey in there daily diet. So we must find ways to reverse the trend-that is, to re-introduce wide sectors of the public and food industry with honey and its properties. Most of the data listed above is probably known to you. Furthermore, I will tell you a secret. Just a few days ago I went over note of a lecture I have delivered some 8 years ago. Surprise--it was almost all there. Now, the fact that we all knew the benefits of honey and the misconceptions about it is not the important issue. The real problem is that the users--I mean the public and the food industry as well as the professionals-are either ignorant of the facts or, as in the industry case, simply prefer to ignore them because of economical and other practical reasons.

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T.Dvir

4. A STRATEGY TO INCREASE HONEY CONSUMPTION The honey industry has to form a strategy to face this problem. I would like to mention some of my thoughts on the subject.

4.1. General Finding Let me start with some general findings relating to the issue: • A "Back to Nature" fad is growing in Israel • The amount of health groups has doubled since 1987 • Confusion exists as to the difference between health foods and dietetic/low Calorie foods. • Women pay attention more than men to the food they eat. • Men think that health food/dietetic food is for sick people

4.2. Honey Drawbacks I then ask myself why honey is consumed less than other sweet spreads: • • • •

Availability-not always available Price--more expensive Variety-less then other Authenticity-how to tell real honey apart from imitations?

4.3. Honey vs. Sugar We should start a campaign to show that honey is not sugar-All facts listed in Table 1. We should emphasize nutritional value of honey-Again, see table 1.

4.4. Honey Is Much Safer We must convey the fact that honey is much safer then the artificial sweeteners. To do so we may have to point out what is reported about the dangers: Aspartame sugar substitutes cause worrying symptoms from memory loss to brain tumours. But despite USA FDA approval as a "safe" food additive, aspartame is one of the most dangerous substance ever to be foisted upon an unsuspecting public (Nexus Magazine, ref. 1).

4.5. Honey Uses Then we should talk about honey uses • • • • •

as a natural alternative to refined sugar barbecue sauce and dips in hot beverages, such as tea in cold beverages, such as ice tea, ice coffee, ice water+lemon and honey in manufactured foods: breads, cereals, sauces, bottled and canned beverages, fruit and carbonated drinks, milk products and candies

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4.6. Honey in Cooking and Baking Honey can and should be inserted into many home cooking and baking recipes. Honey can be used as food softener, for browning (good idea for micro-oven users), as a binding element, and as sweetener. It would serve well in many recipes, like: • • • • • • • • •

salads & vegetables spreads & butter breads, muffins and rolls marmalades and sauces cakes (traditional cakes, cheese cakes, oriental cakes, fruit cakes) cookies deserts and pies candies & snacks main entree

4.7. Honey Promotion And finally, promotion of honey consumption in all sectors is important. For example, honey should be placed in different sectors throughout supermarkets. Such sectors are spreads, cake decorating, sauces & salad dressings, Chinese food, natural and health foods [6,7]. Marketing honey in new forms which are more convenient to use, like spread instead ofliquid honey. Unique and unusual containers that emphasis the natural aspects of honey.

5. CONCLUSIONS Ifwe take these actions, I believe that 4 years from today, in 2000 Honey congress, the chairman or chairwoman will probably rise and point to a graph titled "Honey consumption 199Cr2000". It will show a dramatic increase.

REFERENCES I. 2. 3. 4. 5. 6. 7.

Gold M. D. (95), The Bitter Truth about Artificial Sweeteners, Nexus magazine, Vol. 2 # 28 Ames and Gold (I 987),Ranking Possibly Carcinogenic Hazards, Science pp. 236-271 Woodrow C M, Aspartame, Methanol & Public Health in: Journal of Applied Nutrition, 36 (I), pp. 42-53 Crane E., Honey, Heinemann London 1976, p. 264 Gebhards S. and Mathews R. Nutritive Values Foods, USA department of Agriculture, 1991 Hayes G. W. (1985), Lets Promote Honey, American Bee Journal, Feb 1985 Thomas and Payne (1988), A Leak at Japanese Market, American Bee Journal, June 1988

11

HONEY AS A CLARIFYING AND ANTI-BROWNING AGENT IN FOOD PROCESSING AND A NEW METHOD OF MEAD PRODUCTION ChangY Lee Department of Food Science and Technology Cornell University Geneva, New York 14456

Today's consumers demand foods more natural with less food additives. We found honey to have very useful characteristics in food processing such as a fruit juice clarifying agent, an anti-browning agent among others. Honey can replace some of the conventional food additives in fruit juice or wine production and it can also be processed into a wide range of new beverage products. The following describes some of our research on honey that has been carried out for over ten years in our laboratory. Honey consists primarily of carbohydrates with an average concentration of about 80%. Water is the second major component at around 17%. The other important component, although its concentration is relatively low, is protein. The protein content was reported to have a wide range of 58-786 mgllOOg of honey with a mean value of 169 mgll OOg (1). In spite of low concentrations, honey protein is an important constituent because it influences many properties of honey and honey products (2). One of the unique properties of honey protein is that it readily interacts with phenolic polymers, including tannins, and forms macromolecule complexes (3). It is known that the reaction between proteins and tannins produces hazes in most natural fruit juices, and that sediments of fruit juices consist largely of phenolic materials mixed with protein (4). When a protein such as gelatin is added to a hazy juice, it entraps the particles and coagulates them by hydrogen bonds between the hydroxy groups of polyphenolic tannins and the carbonyl groups of the proteins (5,6). In a model solution of honey protein with tannic acid, it was found that the maximum rate of interaction occurred at the ratio of 1:2-3, tannic acid:honey protein (3). We employed this characteristic of honey protein in apple juice processing and found that the honey protein acts as gelatin-like in that it entraps the hazy particles and clarifies the juice. The optimum clarification conditions were found to be 4-5% honey concentration by weight at pH 3--4 at room temperature. Clarification oc-

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ChangY. Lee

curred over a wide temperature range with the rate increasing rapidly as the temperature was raised (7). This process has been used commercially for several years. In order to find the derivation of this unique honey protein, honey samples from various floral sources, including citrus, dandelion, locust, basswood, clover, goldenrod, and honey from sucrose-fed bees were collected from various regions and compared for their juice clarification characteristics and electrophoretic patterns. There are reports that the honey protein could be originated in either the plant nectarines or the bees and that honey protein consisted of 4-7 components (8,9). We found that all honey samples we studied, regardless of their origin, contained the specific protein fraction that is responsible for clarifying apple juice and that is originated from honey bees (Apis melli/era) (10). In order to compare the protein component among honeys from different bee species. honey from A. melli/era bees from Cornell University, honey from A. laboriosa bees from Chhomrong, Nepal at the altitude of about 2,000 m and honey from A. cerana, common Indian honey bees from Kapre Chhap, Nepal at about 1,200 m from sea level were analyzed for their protein component. The electrophoretic pattern showed that protein fractions from the three diverse bee species were different and only Apis meli!fera and A. cerana bees produced the protein fraction with the apple juice clarifying activity, but A. laboriosa bees did not (11). Honey also has an inhibitory activity on polyphenol oxidase which is the major cause of enzymatic browning in fruit and vegetable products. In model solutions of polyphenols and polyphenol oxidase, added honey prevented browning reactions (12). We found this inhibitory effect of honey on Drowning in preparation of several fruit juices and wines and dehydrated fruit products. Grape juice prepared with added honey exhibited a similar color to commercial juice that was treated with ascorbic acid and enzyme. Table 1 shows effect of honey added to Niagara grape juice on color. Juice prepared with no treatment (control) was dark-brown with a higher absorbency at 420 nm and lower Hunter "L" value, but juices treated with honey showed no brown color with a low absorbency and higher Hunter "L" value. As early as in 1935, honey was added to fruit juices to make fermented products as a supplementary sweetening agent (13). We added honey to apple cider to bring soluble solids to 20° Brix and fermentation was carried out in the normal procedure at room temperature, 18-22°C for 30 days. The final products were analyzed for sensory quality and we found that apple wine treated with mild honey (orange blossom or clover-locust) was much better than apple wine made with added sugar (14). We also used honey in white grape wine production with no added sulfur dioxide. Several white grape cultivars were made into wines by conventional and new honey-treated (ameliorated with 22% honey solution) methods. We observed that wines made by the honey treatment with no S02 were very close to the conventionally prepared wines (commercial) in overall sensory quality. It appeared that honey acted as an anti-browning agent similar to S02.

Table 1. Application of honey in Niagara grape juice production Juice preparation

pH

Absorbency (420 nm)

Hunter"L"

Control (no treatment) Commercial" Honey treated#

3.78 3.11 3.11

0.71 0.07 0.07

47.30 58.41 58.36

*Treated with ascorbic acid and enzyme. #Added honey (3% by weight).

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In order to apply honey as an anti-browning agent in minimal processing of fruits, apple slices were treated by dipping them into a 3-5 % honey solution, packaged under the modified atmosphere conditions and then stored for 2-3 week at 3-5°C. We found that apple slices treated with honey were lighter in color and firmer in texture compared to those of apple slices prepared with commercial anti-browning acidulants. We also applied honey to various fruit slices for dehydrated products. Sensory quality of the dehydrated fruits (apple and pear slices, raisin) treated with honey was much better than those prepared by a conventional method with added S02' Although mead (honey wine) has been produced for many years, it has never been as popular as grape wines in the U. S. One of several reasons is that conventional mead lacks the overall quality of a typical alcoholic beverage. Due to the hazing problem in mead caused by honey protein, traditional mead-making requires boiling a honey solution for 30-{:)0 minutes before fermentation. This heat process is so excessive that it develops undesirable flavors, such as harshness and bitterness. To mask these undesirable flavors, a high residual sugar content and a long aging process are required. In order to overcome these problems and improve the mead quality, honey was passed through an ultra-filtration (UF) membrane with various molecular weight cut-offs (l0-50K) to remove haze forming proteins and then fermented in the usual manner. A complete fermentation and bottling process were accomplished within 3 weeks so there was no need for a long clarification and stabilization period. Sensory quality and stability of the UF-treated mead are superior to mead made by the conventional method. Several commercial firms are producing products by using this new method. This UF -treatment of honey opened the door for a wide range of honey application in food processing and there are already many new products being produced commercially using UF-treated honey.

REFERENCES 1. White, J. w.; Rudyj, O. N. (1978) The protein content of honey. J. Apic. Res. 17.234-238. 2. Paine, H. S.; Gertler, S. I.; Lothrop. R. E. (1934) Colloidal constituents of honey. Ind. Engng. Chem. Analyt. Edn. 26, 73-81. 3. Lee, C. Y. (1984) Interaction of honey protein and tannic acid. J. Apic. Res. 23, 106-109. 4. Johnson, G.; Donelly, B. J.; Johnson, D. K. (1968) The chemical nature and precursors of clarified apple juice sediment. 1. Food Sci. 33, 254-257. 5. Calderon, P.; Van Buren, J.; Robinson, W. B. (1968) Factors influencing the formation of precipitates and hazes by gelatine and condensed and hydrolyzable tannins. J. Agric. Food Chem. 16,479-482. 6. Gustavson, K. H. (1954) Interaction of vegetable tannins with polyamides as proof of the dominant function of the peptide bond of collagen for its binding of tannins. J. Polymer Sci. 12,317-324. 7. Lee, C. Y.; Kime, R. W. (1984) The use of honey for clarifying apple juice. J. Apic. Res. 23, 45-49. 8. White, J. W.; Kushnir, I. (1967) Composition of honey. VII. Proteins. 1. Apic. Res. 6, 163-178. 9. Bergner, K. G.; Diemair, S. (1975) Protein in honey. II. Gel-chromatography, enzymatic activity and origin of honey proteins. Z. Lebensm. Forsch. 157.7-13. 10. Lee, C. Y.;Smith, N. L.; Kime, R. w.; Morse, R. A. (1985) Source ofthe honey protein responsible for apple juice clarification. J. Apic. Res. 24, 190-194. 11. Lee, C. Y.; Smith, N. L.; Underwood, B. A.; Morse, R. A. (1990) Honey protein from different bee species in relation to apple juice clarification activity. Amer. Bee 1. 130,478-479. 12. Oszmianski, J.; Lee, C. Y. (1990) Inhibition of polyphenol oxidase activity and browning by honey. J. Agric. Food Chem. 38, 1892-1895. 13. Fabian, F. W. (1935) The use of honey in making fermented drinks. The Fruit Prod. J.I4, 363-365. 14. Kime, R. W.; Lee, C. Y. (1987) The use of honey in apple wine making. Amer. Bee J. 127,270-271.

12

BEE-POLLEN Composition, Properties, and Applications

M. G. Campos,] A. Cunha,l and K. R. Markham 2 ILaborat6rio de Farmacognosia da Faculdade de Farmacia Universidade de Coimbra 3000 Coimbra, Portugal 2New Zealand Institute for Industrial Research and Development POBox 31310, Lower Hutt, New Zealand.

1. INTRODUCTION During ancient times, people throughout the world commonly used pollen, praising it for its goodness and medicinal properties. Some of the reasons the ancients used beepollen are why wc use it today. To date no scientific evidence has been cited to disprove the claimed properties of bee-pollen. One claim, attributes to bee-pollen the ability to reduce the rate of ageing. It is said that this product has a special factor that can improve health, and increase vital energy. The basis for this claim is the fact that the queen bee, and only the queen bee, can live five or six ycars on a diet of royal jelly, that the bees do with bee-pollen. Other bees only eat this diet for the first two days of their life and after that, they eat honey. They live for only a few weeks. An undeniable fact is that, this product can't be synthesised in the laboratory, can't be easy adulterated and has been taken by people over thousands of years, without manipulation or recorded side effects. It is fortunate that a number of scientists, world wide are earring out research in many different areas, out of interest, to test and prove the nutritive and curative properties claimed for the bee-pollen that we "steal" from the hive.

2. NUTRITIVE VALUE Concerning the value of bee pollen as a natural health food for people, it is well known that bees collect pollen because of its high content of protein (average 35%). Approximately half of the "protein" fraction is in the form of free amino acids which can be assimilated immediately by the body. 93

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It has been established by Peris (1984)1, that in a daily dose of 15 g (about a soup spoon) of bee-pollen provides the minimum amount of amino acids that the human body needs. Further, at least 40% of the content is made up of various forms of sugar. Huidobro et al. (1986,1987)2,3 reported a value of 61%, but they included in this fraction not only carbohydrates like reducing sugars (fructose, glucose), maltose, sucrose and polysaccharides, but also starch and other polysaccharides that can't be absorbed such as cellulose, hemicellulose and lignin, esporopolenin, etc" We have also verified that about 50% (Campos et ai, 1994)4 of the weight of the bee-pollen corresponds to a material that can't be extracted with organic or aqueous solvents. Lipids and minerals (carriers of calcium, phosphorus, magnesium, iron, copper and manganese, etc.) represent 5 and 3% respectively. Most (60%) of the fatty acids aTe in the free form. Bound fatty acids, which reflect the compositional profile of pollen, were characterised by a high level of a-linolenic acid (70%) and by small amounts of linoleic and oleic acid. Palmitic acid is the most abundant saturated fatty acid (Seppanen et al.,l989f Vitamins in bee-pollen include not only vitamin B complex and ascorbic acid, but also vitamins A, D and E. The levels vary between the pollen species and with season. For example, Herbert et aI., (1987)6 established that the seasonal thiamine (vitamin B 1) levels in bee-collected pollen varied greatly depending on the floral source and the time of year. It is important not to forget that in biological samples, vitamin B 1 activity is due not only to thiamine but also to the mono, di and triphosphate derivatives of thiamine. Thiamine and its mono and diphosphate forms are normally the most prevalent. Pollen apparently supplies the honey bees requirement for this vitamin since there is no real evidence that it can be synthesised by insects. Thiamine is relatively stable in acid pH, and pollen provides an acid environment. In stored pollen the vitamin has been shown to exist for up to 4 years (Hagedorn and Burger, 1968l Xie et al. (1994), in China, have studied the effect of bee pollen on maternal nutrition and foetal growth. They state that plant pollen collected by the honeybee is a natural nutrient. They studied the effects of the bee pollen from Brassica campestres L. on maternal nutrition and foetal growth using pregnant Sprague-Dawley rats. Pollen-fed dams had greater body weight and higher levels of haemoglobin, total protein, serum iron and albumin while the foetuses of pollen-fed dams had greater body weight and lower death rate. No gross external, visceral or skeletal malformation was observed in the foetuses. These results were interpreted as indicating that bee-pollen could improve maternal nutrition without affecting normal foetal development. Bee pollen was therefore considered a practical and effective nutrient during pregnancy.s

3. ANTIBIOTIC ACTIVITY Gillian et al., 1989 isolated one-hundred and forty-eight moulds from the following samples of almond, Prunus du/cis. pollen: floral pollen collected by hand; corbicular pollen from pollen traps placed in colonies of honeybees, Apis mellifera, in the almond orchard; and bee bread stored in comb cells for one, three and six weeks. They verified that corbicular pollen (pelletized bee pollen) contained 99,8% almond pollen when collected from pollen traps placed in bee colonies in an almond orchard. If micro-organisms are responsible for the fermentation and accompanying chemical changes in pollen stored in comb cells by honeybees, the moulds maybe a component of a required microbial content. It was considered, for example that they could produce antibiotics, organic acids and enzymes, products for which there are uses in industry. These compounds may limit the

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growth of deleterious micro-organisms in stored pollen and provide enzymes for utilisation of nutrients. If so, pollen moulds must produce enzymes involved in protein, lipid, and carbohydrate metabolism. Indeed, most moulds from all pollen sources were shown to produce caprylate esterase-lipase, leucine aminopeptidase, acid phosphatase, phosphoamidase, ~-glucosidase and N-acetyl-~-glucosamidase. A high percent of the isolates (50 %) from all sources also gave positive reactions for alkaline phosphatase. The majority of moulds identified were Aspergilli (17%), Mucorales (21%) and Penicillium (32%). We have also found a predominance of Penicillium in samples of Eucalyptus corbicular pollen collected in Portugal9 • In general, the number of isolates decreased in pollen following collection and storage by the bees. Moulds that may have been introduced by bees during collection and storage of pollen include Aureobasidium pullulans, Penicillium corylophilum, Penicillium crustosum and Rhizopus nigricans.. Mucor sp., the dominant moulds in floral pollen, were not found in corbicular pollen and bee bread. Thus, as with yeast and Bacillus ssp, the mould flora found in corbicular pollen and bee bread may be the result of microbial inoculations by bees. Chemical changes in pollen therefore may result both from additions by bees of secretions of glands during regurgitation of honey sac contents and from microbial fermentation. Such modifications may allow some species to survive but not others. Even though moulds were more numerous than yeast or Bacillus ssp in the samples, pollen was rarely overgrown by moulds. Potential microbial spoilage of stored pollen thus may be controlled by antibiotic substances produced by the normal microflora of bees or by those naturally present in pollen and/or honey. These results may assist in the understanding of the antibiotic power of bee pollen. We must emphasis that this is our interpretation of the research work referred to. The antibiotic power of flavonoids that has already been proved for propolis activity (Houghton et aI., 1995)\0 must also be considered a factor. 39 Chauvin and Lavie (1956)11 in a study of the antibiotic activity of bee-pollen, found a "factor" with activity against Spullorum. S. gallinarum. S. Dublin, E. coli, Proteus vulgaris, S. subtilis Caron and B. pyocyaneus. We have followed the extraction procedure that they used to extract this factor and it seems that this factor could be the flavonoids. We are currently checking the activity of flavonoids from extracts of bee-pollens from different floral origins. Chauvin and Lavie also verified that different floral sources of bee pollen gave different levels of activity. In our research we have found that flavonoid type varies according to the family of pollen plant source (Campos et aI., 1996).12

4. ANTIATHEROSCLEROTIC ACTIVITY Another property accredited to well known species of bee-pollen when consumed as a medicine is the lipid-lowering effect on serum. Literature relating to bee pollen however was not found in our literature search. We therefore quote below, research carried out on pollen generally which may help to provide an understanding of this activity. Some groups have revealed that extracts of pollen have beneficial properties, lowering serum lipid levels (Samochowiec et Wojcicki, 1981; Wojcicki et Samochowiec, 1984)13., reducing atherosclerosis plaque intensity (Wojcicki et aI., 1986)1\ and decreasing platelet aggregation both in vitro (Kosmider et al. 1983)15 and in vivo (Wojcicki et Samochowiec, 1984)16. These findings have been confirmed in humans (Wojcicki et aI., 1983)17. Additionally, studies in humans suggest that a diet supplemented with polyunsaturated fatty acids (as found in pollen extracts) decreases whole blood viscosity and re-

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duces triglyceride and cholesterol levels in patients with cardiovascular disease (Saynor et al. 1984)18. With these studies in mind Seppanen et al. (1989),19 analysed the fatty acid composition of the fat-soluble pollen extract (Cernitin GBX) by gas chromatography in order to account for the anti-atherosclerotic activity. The analyses revealed that most (more than 60%) of the fatty acids were in the free form, characterised by a high content oflinolenic acid (18:3n-3, a-LLA) (70%) If fatty acids are involved in the beneficial effects referred to, the role of a-linolenic acid as a precursor of eicosapentenoic acid (20: 5n-3, EPA) is significant, since EPA is considered to be responsible for reduced platelet aggregation. EPA in vivo is incorporated into platelet phospholipids, to some extent replacing arachidonic acid and exerting an antithrombotic effect either by competing with remaining arachidinic acid for cyclo-oxygenase and lip oxygenase or by being converted to less proagreggatory PGH 3 and TXA 3 (Moncada et Vane, 1984).20

5. ANTI-NEOPLASTIC ACTIVITY The same extract of pollen from AB Cernelle, Vegeholm, Sweden (Cernitin GBX), was analysed by Zhang et aI., 1995 21 , who isolated and characterised a cyclic hydroxamic acid [2,4-dihydroxy-2H-I,4-Benzoxazin-3(4H)-one] from a pollen extract, which inhibits cancerous cell growth in vitro. This hydroxamic acid is the active compound in the pollen extract which might be responsible for the symtomatic relief in patients with benign prostate hyperplasia. Seventy-nine patients with this disease, aged from 62 to 89 years, were treated with pollen extract and showed a mild beneficial effect on prostate volume and on urination (Habib et aI, 1995; Yasumoto et aI., 1995). Interestingly, one of the major beneficial qualities attributed to bee-pollen by the ancients was its usefulness in the treatment of prostatitis.

6. POLLEN PHENOLICS AND ANTI-OXIDANT AND FREE RADICAL SCAVENGING ACTIVITY As evident from the above, many chemical, biochemical and microbiological studies have been carried out with a wide variety of compounds from pollen, but only recently have scientists focused on a special group, the phenolic compounds. In fact, these exist in low quantities in bee pollen and other plant parts (Campos et aI., 1990)22 (Markham, 1982)23, (Markham and Campos, 1996)24, as is common for non-nutrient compounds, which are important for life. Because in pollen we can find that many groups of compounds differ during the year or from one species to another, we have developed a method that can identify the corbicular pollen species with precision using the phenolic compounds. This method offers reproducible conditions that produce a consistent profile (Campos et aI., 1996)13. We have verified that in bee pollen, and in pollen in general these compounds are reliable species indicators when. mature pollen is used. A special type of flavonoid glycoside was commonly found in bee-pollen. After comparison with the literature we verified that these glycosides often had the same glycosidic linkage. Because these pollens attract insects these glycosides may have a special taste which honeybees recognize as indicating a suitable food, as been observed with other

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insects (Harborne, 1994)25. Certain types of flavonoids are known to have a distinctive taste that insects are attracted to. The profile of phenolic compounds in pollen is different from one species to another thus providing a species-specific character for pollen identification. In our studies we have verified that in the bee-pollen collected from the Central Coast of Portugal, the floral species were Eucalyptus globulus, Ranunculus sardous, Salix atrocinerea, Taraxacum sp and Ulex europaeus. Between the Northern and Southern regions we found the Cistus ladanifer and in the Central Interior Erica australis was the major species collected for the bees. Salix atrocinerea and Rhaphanus raphanistrum were minor species in the samples examined by us. New Zealand samples were found to contain the same species as those from the Central Portuguese Coast and the Central Interior. This may be due to the climate and because the samples were also collected close to the coast. Only the Eucalyptus was not found in these samples. In contrast the Eucalyptus bee-pollen was commonly found in samples of bee pollen from Australia. The results of Godinho et Nansen, 1995 26 suggest, as do ours, that weeds are preferred by the bee, effectively competing with the cultivated plants. After analysis of the free-radical and anti-oxidant activity of bee-pollens, it was evident that only the phenolic compounds are active. The derivatives of cinnamic acid are the most effective phenolics, as evidenced by the loss of much activity when they are absent. This activity is relevant to the claim that bee-pollen has regenerative properties for the body and long lives are often attained by bee-pollen users. The literature records much research work that proves that flavonoids, and in general, phenolic compounds, have an antioxidant / radical scavenging effect in the human body and that they can be given to prevent and cure some diseases (Pathak et aI., 1991)27. It is probable that active free radicals, together with other factors are responsible for cellular ageing and can cause biological imbalance that in extreme conditions can be responsible for premature death. Ageing is a concern in all stories of human life and the story of the search for the elixir o/youth, continues to this day. There are many theories presented in literature to explain the ageing process. These include the "Genetic Theory"-that ageing and long life are genetically programmed ("Programmed Ageing") (Hayflick, 1965 28 ; Warner et aI., 1987 29 ; Holliday et al. 1985 3°). The Stochastic Theory-this theory claims that ageing is the result of a series of destructive events that affect all levels cellular organisation ("Random ageing"), e. g. Error catastrophe theory (Orgel, 1963)31 and Free radical theory (Harman, 1956)32. All these events cumulatively induce ageing and cellular death. A free radical is a neutral or charged structure that possesses an unpaired electron and is represented by the symbol R·. Our body produces active forms of oxygen and oxygen free radicals in the course of normal metabolism. These radical species are very reactive, produce secondary free radicals, inter and intramolecular bridges, oxidation, halogenations and molecular fragmentation. Cell membrane lipids are vulnerable to free radicals. "Peroxidation" of polyunsaturated fatty acids gives many derivatives that are indicative of the intensity of the phenomena of cellular oxidation: for example, lipid hydroperoxides, aldehydes (malonylaldehyde-MDA and 4-0H nonenal), conjugated dialdehydes, hydrocarbons and fluorescent conjugates (lipofuscins). Some of these derivatives have biological activities (chemostatic action, cellular division effect). Other deleterious effects of free radicals result from their action on polysaccharides (hialuronic acid de-polymerization), proteins (chemical modification of the crucial aminoacids for the enzymatic functions, fragmentation of the peptide chain), and nucleic acids (chromosome bridges).

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The free radical theory of ageing became more credible after the discovery that active free radicals are involved in cellular degradation process, such as cardiovascular diseases, arthritis, cancer, diabetes, etc .. They have also been implicated in Parkinson disease and Alzheimer disease (Feher et al., 1986)33,34. Bee-pollen has been used for centuries to protect the body from diseases and especially to slow the process of ageing. In studying this, Dudov et Starodub (1994)35, fed rats with bee pollen for one month and studied the resulting state of the erythrocyte redox system. It was established that the content of glutathione, total SH-groups, as well as activities of glutathione peroxidase and glutathione reductase in these animals were increased in comparision with the control group. Simultaneously a decrease of malondialdehyde and dienic conjugates in erythrocytes was demonstrated. It was concluded that the antioxidative system is non-specifically activated and oxidative processes blocked in erythrocytes of rats fed on bee pollen. In another study, primary and secondary humoral immune response (the level of specific IgM and IgG) as well as the intensity of delayed-type hypersensitivity of sheep erythrocytes were investigated in rabbits fed with bee pollen for one month. It was shown that bee pollen, acts as an immunomodulator in that it stimulated humoral immune response and changed the reaction of delayed-type hypersensitivity (Dudov et al.,1994).36 The free radical scavenging activity of different floral species of bee pollens were recently studied by us. The results show a big difference between the species containing derivatives of phenolic acids (which seem to be the more effective scavengers) and those containing only flavonoids (Campos et aI, 1994)37 (Campos et al., 1996 b)38. Returning to the story of life and ageing, lipid oxidation in the presence of oxygen, causes rancidity, a problem recognised since ancient times in relation to the preservation of oils and fats. Oxidation of vegetable oils and animal fats, produces organoleptic changes (colour, smell and taste), as well as changes in density, viscosity and solubility. Surprisingly, it was not until 1950 that scientists started to recognize the significance of lipid oxidation in Biology and Medicine (Dinis, 1995).39 Other evidence of the benefits of bee-pollen comes from Olympic athletes that eat this product as a supplement. The coach concluded that bee pollen increased the athletes crucial recovery time after stressed performance and enabled them to actually improve their performance the second time around (Fischer, 1986)40. This may be explained by the anti-oxidant theory. Physical exercise with aerobic characteristics Clln impose on the organism a supplementary consumption of oxygen that in humans can amount to ten times the level of basal metabolism. The concept of "oxidative stress" implies a disruption of the precarious equilibrium between the production and quenching of oxygen free radicals. With physical exercise this "stress" is brought about by excessive production of such radicals. Paradoxically, people involved in sport regularly live in a situation of "permanent oxidative stress" with the consequent biological cost. Today scientists know that fitness training, like an adaptive mechanism, increases the anti-oxidant defences. Thus more oxygen is consumed without negative consequences. Nevertheless, in studies carried out at "Faculty of Sport Sciences and at the Centre of Experimental Cytology of University of Porto", by Silva (1993)41, it has been verified that a fit athlete when exercised to exhaustion, suffers dramatically high oxidative injury. These results suggest that anti-oxidant defences could be insufficient in an extreme situation like this one. It was also shown in this study that those athletes with a high level of oxidative injury, who recovered quickest (within one hour after the exercise) produced high plasma levels of vit.E. This suggests that there is a systemic anti-oxidant response to the physical exercise.

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It is tempting to speculate about the anti-oxidant protection provided by bee pollen in these cases, because of the amount of phenolic compounds in the product given to the Olympic athletes. It is known today, for example, that some flavonoids can protect vit C, because when taken together the flavonoids are oxidized first. On the basis of this and other factors discussed above we can rationalize the benefits of bee pollen in the diet (Bors et aI., 1995)42. Diet has long been linked to Medicine and good health, and the use of bee products, including bee pollen, is frequently recorded. For example, about 487-380 b.c. (before Christ), Herodicos de Selymbrie, master of gymnastics, inaugurated a diet method, the "Grand Arte". This formed the basis of the Medicine, that Hippocrate de Cos (460-377 a.c.) established in his Diet Treatise, The Food and the Man Nature (Debry, 1991).43 Even then, in an empirical way, they knew that the utilisation of a correct diet could have a prophylactic effect in delaying or preventing the onset of some diseases. Research of the biological activity of compounds isolated from natural sources and used in folk medicine has established a relationship between chemical composition and pharmacological activity of many such natural drugs. With bee pollen, the results ofinvestigations of its therapeutic activity, add rationale to the "magic" properties attributed to it in the past. Bee pollen it seems, can be more than a prophylatic and has a place in treatment alongside other natural or synthetic products. More scientists are now beginning to give credence to the potential of folk remedies, and bee-pollen is one of them. "We need to ponder the present, to survive in the future, while learning from the past."

REFERENCES I. Peris J. (1984), "Produccion y comercio de los produtos apicolas en Espana". El Campo del Banco de Bilbao. Apicultura 93. Bilbao. 2. Huidobro J.F., Simal J., Muniategui S. (1986) "El polen: Determinacion del contenido en agua" Offarm 5 (3), 73-77. 3. Huidobro J.F., Simal J., Muniategui S. (1987) "El polen apicola: Determinacion del contenido em glucidos" Offarm 6 (5) 57-71. 4. Campos M.G., Cunha A. Rauter A. (1994) "Portuguese bee-pollen as a source of flavonoids" Acta Horticulturae,429-432. 5. Sepp"nen T., Laakso 1., Wojcicki J., Samochowiec L., (1989). An Analytical study on fatty acids in pollen extract. Phytotheraphy Research, 3 (3) 115-116. 6. Herbert E. W. Jr., Vanderslice J.T., Meis-Hsia Huang, Higgs D. J. (1987) Levels of thiamine and its esters in bee collected pollen using liquid chromatography and robotics. Apidologie 18 (2), 129-136. 7. Hagedorn H. H. and Burger M. (1967) Effect of the age of pollen used in pollen supplements on their nutritive value for the honeybee. II. Effect of vitamin content on pollens. J. Apic. Res .• 7,97-101. 8. Xie Y., Wan B., Li W. (1994) Effect of bee-pollen on maternal nutrition and foetal growth. Hua-Hsi-I-KoTa-Hsueh-Hsueh-Pao. 25 (4) 434-437. 9. Results not published 10. Houghton P., Woldemariam T., Basar A., Lau C. (1995) Quantification of pinocembrin content of propolis by densitometry and high performance liquid chromatography. Phytochemical Analysis, 6, 207-2\0. II. Chauvin R., Lavie P. (1956) Recherches sur la substance antibiotique du pollen. Annales de I'Institute Pasteur 90 (4) 523-527. 12. Campos M.G., Markham K., Mitchell K., Cunha A. (1996). An approach to the characterisation of bee pollens via their flavonoid/phenolic profiles. Phytochemical Analysis to be submited 13. Samochowiec L., Wojcicki J., (l981) Effect of pollen on serum and liver lipids in rats fed on a high-lipid diet. Herba Polon. 27, 33 Wojcicki J., Samochowiec L., (1984) Further studies in Cernitins: screening of the hypolipidemic activity in rats. Herba Pol on. 30, 115.

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14. W6jcicki J., Samochowiec L., Bartlomowicz B., Hinek A., laworska M., Gawronska-Szklarz B., (1986). Effect of atherosclerosis in rabbits. Atherosclerosis 62, 39. IS. Kosmider K., W6jcicki 1., Samochowiec L.. Woyke M., G6rnik w., (1983). Effect ofCernilton on platelet aggregation in vivo. Herba Polon. 29, 237. 16. W6jcicki 1., Samochowiec L (1984). Further studies on Cernitins: screening of the hypolipidemic activity in rats. Herba Polon. 30, lIS. 17. Wojcicki J., Kosmider K., Samochowiec L., Woyke M. (1983) Clinical evaluation of Cernilton as lipidlowering agent. Herba Polon. 29, 55. 18. 19. Sepp"nen T., Laakso I., Wojcicki J., Samochowiec L., (1989). An Analytical study on fatty acids in pollen extract. Phytotheraphy Research, 3 (3) 115--116. 20. Moncada S., Vane J. R., (1984) Prostacyclin and its clinical applications. An. Clin. Res. 16,241. 21. Zhang-X., Habib F.K., Ross M., Burger U., Lewenstein A., Rose K.. Jaton 1.e. (1995) Isolation and characterisation of a cyclic hydroxamic acid from a pollen extract, which inhibits cancerous cell growth in vitro. 1. Med. Chern. 38 (4) 735--738. 22. Campos M.G., Sabatier S., Amiot M., Aubert S. (1990) Characterisation of flavonoids in three hive products: Bee-pollen, propolis and honey. Planta Medica 56 (7) 580--581. 23. Markham K. R. Techniques of Flavonoid Identification. London, Academic Press. 1982, 24. Markham K., Campos (1996) 7- and 8-0-methylherbacetin-3-0-sophorosides from bee-pollens and some structure/activity observations. Phytochemistry in press 25. Harborne J. in The Flavonoids: advances in research since 1986. (1. Harborn Editor) Chapman & Hall. 1994 26. Godinho J. et Nansen e. (1995) Estrategia alimentar da abelha domestica (Apis melliferaj como polinizador da cultura da meloa (Cucumis melo) em estufa. 0 Apicultor 3 (8) 25--29. 27. Pathak D., Pathak K., Singla A. (1991). Flavonoids as medicinal agents--recent advances. Fitoterapia 28. Hayflick (1965) The limit in vitro lifetime of human diploid strains. Exp. Cell Res. 37, 614-636. 29. Warner H.T., Butler R.N., Sprott R.L., Schneider E.L.. Modem theories of ageing. Ageing vol. 31 (Raven Press N. Y.) 1987 30. Holliday R.. Kirkwood T.B.L., Cuzin F. (l985}--EMBO Workshop on "Oncogenes, immortalization and cellular Ageing" Grignon, 3-7 Setembro. 31. Orgel L.E .. (1963) The maintenance of the accuracy of protein synthesis and its relevance 5' to ageing. Prod. Natl. Acad. Sci., 49, 517-521. 32. Harman D. (I 956}--Thefree radical theory of ageing in "Free Radicals in Biology, (A. Pryor Editor) Academic Press, 1984,5,255--275. 33. Feher J., Csmos, Vereckci A. (1986) Clinical importance of free radical reactions and their role in the pathogenesis of various human diseases. in Free Radical Reactions in Medicine, 48-147. 34. Various Comunications--IQ Congresso de Radicais Livres em Quimica, Biologia e Medicina. Instituto Superior Tecnico, Lisboa. Portugal 21-23 de Junho de 1993. 35. Dudov LA., Starodub N.F., (1994). Antioxidant system of rat erythrocytes under conditions of prolonged intake honeybee{lower pollen load. Ukr. Biokhim. Zh.66 (6) 94-96. 36. Dudov LA., Morenets A.A., Artiukh v.P.. Starodub N.F., (1994). 1mmunomodulatory effect of honeybee flower pol/en load. Ukr. Biokhim. Zh. 66 (6) 91-93. 37. Campos M.G., Cunha A., Navarro M.e., Utrilla M.P. (1994). Free radical scavenging activity of bee pollen. Bull. Group Polyphenols. 17,415-416. 38. Campos M. Markham K., Mitchell K., Veiga J. Cunha A., Paredes F., Frazao L. (1996 b) Therapeutic activity of bee pollen-Preliminary essays. International Conference on: Bee-products: properties. applications and apitherapy. May 26--30. Tel-Aviv, Israel. 39. Dinis, T. (1995). Peroxida,iio /ipidica membranar. Actividade antioxidante de farmacos fen6licos (acetaminofeno, salicilato e 5-aminosalicilato). Disserta9ao de doutoramento apresentada it Faculdade de Farmacia da Universidade de Coimbra. pp 13-14. 40. Fischer W. L. How to fight cancer & win. 1986 41. Silva P. C. 1993 Radicais livres de oxigenio em Medicina Desportiva, I Q Congresso de Radicais Iivres em Quimica, Biologia e Medicina 42. Bors w., Michel e., Schikora S., (1995) interaction offlavonoids with ascorbate and determination of their univalent redox potentials: A pulse radiolysis study. Free Radic. BioI. Med. 19 (I) 45--52. 43. Debry G. (1991). Evolution des concepts en nutrition humain. Cah. Nutr. Diet. 26 (6),435-442.

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CLINICAL EVALUATION OF A NEW HYPOALLERGIC FORMULA OF PROPOLIS IN DRESSINGS w. Fierro Morales and 1. Lopez Garbarino Department of Surgery--Outpatient Service Hospital "G. Saint Bois" Montevideo, Uruguay

ABSTRACT A new hypoallergic formula of propolis in dressings was evaluated against the standard formula. Patients (229) with wounds of diferent types that required ambulatory care were included. The new formula presented the same therapeutic action than the standard one with a notorious disminution of signs oflocal intolerance (l ,8% vs.18%) The therapeutics properties of pro polis were demostrated : anti-intlamatory, anti-microbian and stimulant of wound healing by a faster grow and initiation of granulation tissue. Other properties comprobated were the analgesic effect mainly in bums, the easier handling of the ambulatory patient and the posibility of avoid hurting the granulation tissue. The wounds and bum were completed healed in an average of I I days, the septic wounds en 17,5 days, and a complete reparation in the 67% of the ulcers in 36 days. In this paper a methodology for the use of this dressing is described, pointing the importance of acomplishing it by the medical personnel.

INTRODUCTION The complexity of the composition of propolis and the sinergic action among its diferent componcnts was well established by researchers of the Oxford University in 1990 (1,2).

At the local level a group of actions are important: anti-microbian, anti-inflamatory (by inhibition of the hydrofolate reductase(3), minimizing the prostaglandin production) and anti-oxidant(4) (neutralizing the nocive effects of the free radicals at the local level). These properties complemented each other, explain the therapeutic benefits obtained. Other benefit obtained is the analgesic effect at the locallevel(5). 101

W. Fierro Morales and J. Lopez Garbarino

102

The utilization of propolis in the treatment of cutaneous lesions of diferent nature such as burns, wounds and ulcers is positive in the reparation process, shortening the healing of wounds and reducing the risk ofinfections(6), but in some cases it is observed allergic dermititis by contact. In 1990 it was published en Acta Chir. Plast.(7) a paper studying the beneficial effects of propolis in the treatment of burns against other products. The treatment of burns is an old issue not exent of contradictions(8). The utilization of topic anti-microbians it has become generalizated in spite of the lack of alleatory research. In the cutaneous burns it is increased the local production of prostaglandins and free radicals. The liberation of proteolitic enzimes and the aditional production of oxigen radicals contributes to the production of aedema, controlling these factors limits the convertion of the burns of partial thickness in complete burns(9). These phenomenae also are presents in the phisiophatology of the wouds. wich in the case of a terrain with vascular insufficiency or metabolic anomalies. are lessening or stopping the repairing process. Carefully pathologic studies were done in rats and explain the anti-inflamatory and healing properties of propolis : minimizing the acute inflamatory exudate (by inhibition of the degranulation of basofiles), estimulating the macrophagic activity, promoting the colagen production and stimulating the epitelization ( incremente of the number of mitosis of the basal layer and favoring the queratinization) Diaz, P.Prof of Histology, School of Medical Sciences of La Habana (oral comunication); (10,11,12).

OBJECTIVES To evaluate the tolerance and efficacy of the new hypo allergic formula of propolis in dressings against the standard formula.

MATERIAL AND METHODS The present research was done in the period from Sept./91 to Aug.l93 at the Emergency - Dept.of Surgery- Hosp. "G.Saint Bois" Hospital (Montevideo) with ambulatory patients. In the initial months were progressively evaluated 4 formulae named A, B, C, and S, and the best results were obtained with the type C (or NF ); 229 patients were in-

Table 1. Population of study and days of treatment. Propo Ii s type No.of cases

Bums Wounds Infected Wounds Ulcers

Days oftreatm.

Age average

NF(C),

B'

NF(C)

B

61 29 53 22

15 10 30 9

25 41 48 63

35 29 46 70

'Propolis C=Ncw Fonnula. 2% of pro polis in hydrosoluble cream. 'Propolis B=Standard formula. 8% of propolis in hydrosoluble cream.

NF(C)

B

II

16,5

11 17,5 36

11,2

16

New Hypoallergic Formula of Propolis in Dressings

103

c1uded, 115 females and 114 males, from I year old to 92 years old, with varied pathology classified as follow: a) bums, b) wounds, c) infected wounds, and d) ulcers.

METHODOLOGY OF TREATMENT • Wounds. Washing with SF an Benzalconiun c1orure, applying the propolis dressing and closing over it with simple cotton dressing (oclusive treatment)(l3,14). Each other 48 hs.- 72 maximum - washing and replacement of the propolis dressing (burns excepted). • Burns. Flictenae resection, washing with SF, drying with warm air, applying the propolis dressing that remains there until complete granulation when fall by itself. Clinical controls each 48-72 hs., allowing moisture the dressing with propolis solution. • Infected wounds, ahcesses and ulcers. After bacteriological study it was made a surgical cleaning and/or drainage of the abcess, washing with SF and benzalconiun c1orure. Applying the propolis dressing and simple cotton dressing. In some cases it was used propolis lotion in order to get a better penetration. In 13 cases the area was drained with dressing. Clinical control and replacement each 48-72 hs. or whenever is indicated. In patients with ulcers were indicated to remain in bed with an elevated leg to favour the venous pressure.

RESULTS AND DISCUSSION 1. Among the 165 patients treated with propolis NF it were observed 3 cases

(1,8%) presenting local signs of intolerance and occurring in an average of 16 days after the treatment was initiated. The signs were prurite, eriteme and exudation and they promtly disappeared after the treatment was suspended. Unlikeness, the group treated with the propolis B registered a 18% of local intolerance and appearing at an average of the 9th. day of the initiated the treatment. 2. Only 4 patients had antecedents of local intolerance against the propolis B but they did not registered any intolerance with treatment of propolis NF. 3. After this initial period it was confirmed the good tolerance of propolis NF, so from that moment the study continued only with propolis NF. 4. There were included 61 patients with burns according the following age categorization: less of 10 years 19 cases; from II to 40 years 25 cases; from 40 and more years 17 cases. It was obtained good reparation and analgesic in an average of 11 days in the 3 groups, but we had the clinical impression that the younger group got a faster evolution. In 36% of the cases it was indicated ATB. The bums included were only those that did not required an inpatient treatment, most of them of 2nd. degree, with a few hours of evolution, and produced by agents as water, foods, bitumen and some cases by a direct contact with fire or hot metals. Those bums with several hours of evolution and signs of infection were included in the group of infected wounds and treated as such. It was observed, the same as other authors( 15), the propolis has the following beneficial effects: Easy handling of the ambulatory patient; less nursing care by replacing

104

W. Fierro Morales and J. Lopez Garbarino

fewer times the dressing, analgesic effect, minimized pain with no replacing and only moisture the dressing, it has a revitalizing power, because the non replacement it has no trauma effect in the granulation tissue. 5. A group of 83 patients with infected wounds and abcesses was formed(ages from 3 to 84 years). In 18 of them it was found the following germs : estaf.aureus (5 cases) estrepto B hemoliticcus (2 cases), B.piocianic (2 cases) and only a case of nuemococcus, proteus and serratia. In 6 patients no pathogenic germ was isolated. In 85% of cases it was indicated a simultaneous treatment of ATB and propolis. In all but one case was observed good tolerance to propolis. A cure average of 17,5 days was obtained. 6. Non infected wounds with a few hours of evolution formed a group of 39 patients. Only in 41 % of the cases were indicated ATB. In 3 cases the wounds were drained with a dressing. In 28 cases the treatement with propolis NF was effective and it was obtained the cure in II days average. 7. The repairing process also was evaluated in 31 patients with ulcers, 22 were treated with propolis NF (age X = 65 r=I7-92 y.), 12 cases older than 62 years. Localization all of them in legs: 8 maleol.intern., 7 maleol.extern., 2 bi-maleol.; 16 had a terrain of veinstasis, 8 had arteriolar compromise, 5 post-trauma ulcers, and 2 diabetic patients. In 10 cases a pathogen germ was isolated: estafil.aureus in 4 cases, estreptoc. in 2, piocianic en 3, and proteus in I. ATB was indicated in 62% of the cases. All the skin layers were compromised by the wound and in 2 cases reached the aponeurotic level. The size of the ulcers was somewhere among 2 and 5 cms. A patient 17 years old with a postthrauma ulcer of 10 cms.of 45 days of evolution and already including the aponeurotic layer was healed in 28 days. The cicatrization was obtained in 68% of the cases in an average of 36 days. Similar results were obtained by Rodriguez in Cuba(16) testing the propolis in 80 patients. In those cases treated with propolis B, the treatment was suspended due to allergic contact dermatitis. The venous estasis subjacent was responsible for the slow evolution and the lack of cicatrizal response in most of them (17,18). The anti-inflamatory and regenerative effect of propolis was shown in the cicatrization of those wounds with a previous history of non tendency to the cicatrization. 8. In 16 cases of wounds drained with the propolis dressing, it was shown the fast regression of the inflamatory process and the supuration (anti-bacterian action). We must stress that the dressing was putted after a surgical cleaning and that the same was performed as many times as necessary (19). In all cases the cicatrization was obtained in an average of 22 days.

CONCLUSIONS I) 229 patient with wounds of varied nature were evaluated and it was shown the good tolerance to the new formula of propolis dressing with only a 1,8% of the cases with signs of local intolerance. There is no quantitative research in this field in Uruguay, according to our previous experiences the results obtained with the treatment of propolis NF in the present research, as a measure of the allergic reaction to the propolis, were significantly lower than those observed with the propolis standard formula. 2) A very satisfactory evolution and cicatrization was obtained in c~ses of wounds with and without

New Hypoallergic Formula of Propolis in Dressings

105

infection and burns. A fast cure, shorter treatment period and less septic complications were obtained. In 67% of the cases with ulcers (trophics and non trophics) the cicatrization was obtained. The anti-inflamatory property was demostrated by the fast reduction of aedema of the limits of the wound. The cicatrizing action was evident by the early formation of the granulation tissue(between 4th. and 5th.day) being more notorius in those with a torpid evolution. The anti-microbian capacity was evident by a fast regression of the septic component of the supurated wounds. 3) Other positive elements to be quoted are: the importance of carefully follow the methodology, to be a natural product, easy application, cheap, and produced in this country by standard pharmaceutical procedures.

REFERENCES 1. Villanueva V. Barbier N. Gonnet M, Lavie P. Les flavonoids de la propolis isolement de une nouvelle substance bacteriostatique: Ie pinocembrine(dihidroxy-5,7flavonona). Annales del, Institute Pasteur 1970; 118( 1):84--7. 2) Greenaway W, Scaysbrook T and Whatley FR. The composition and plant origins of propolis: A report of work at Oxford Bee World 1990;71 (3): I 07-18. 3. Strehl E. Vol pert R, Eistner E, Biochemical Activities of propolis extracts. Inhibition of Dihydrofolate Reductase. Zeitschrift Fur Naturforschung C-A Journal of Biosciences 1994; 49 (1-2):39--43. 4. Pascual C, Torricella RG, Gonzalez R. Scavengig action of propolis extract against oxigen radicals. J. Ethnopharmacology 1994;41 (1-2):9-13. 5. Silvestre N, Stranieri G. y Bezerque P. Anestesia troncular de propolcos comparado con lidocaina. International Dental Research 1984. 6. Ponce de Leon R. y Benitez P. Estudio morfologico comparativo del efecto de la propolina, el alcohol y el balsamo de Shostakovski como agentes cicatrizantes. Investigaciones Cubanas sobre el propoleo. I Simposio sobre Propoleos. Varadero-Cuba 1988:269-71. 7. Troshev K. and others. Regulation of traumatic skin wound healing by influencing the process in the regnbouring bone of wound. Acta Chir. Plast.1990;32(3): 152-163. 8. Glastrup H, Knudsen L. y Mazanti C. Tratamiento de las quemaduras, nuevos enfoques de accidentes y catastrofes. Hezagono 1980;7(8): 1-10 9. Muller MJ, y Hernodon ON. Cirugia. EI reto de las quemaduras. The Lancet 1994;24(6):360-364. 10. Magro F. Application of propolis to dental sockets and skin wounds. Univ. Sch. Dent. 1990;32( I): 4--13 11. Bunta S. Efecto anti-inflamatorio de las pomadas con propoleos I1ISimposio Intemacional de Apiterapia. APIMONDIA 1978:94-7. 12. Neychev H. and others. Inmunomodulatory action of pro polis. Acta Microbiol. Bulg. 1988;23:58--62 13. Boswick J. Quemados. Clin. Quinirg. Norteamerica. 1987; I. 14. Lepore G. y Giuria H. Quemaduras menores, tratamiento de urgencia y seguimiento ambulatorio. Catedra de Cirugia Plastica y Quemados. ProfJ. Hornblas. 15. Ramirez M.Propoleos en el tratamiento de quemados. I' Jornadas Nacionales de asistencia integral del nino quemado. 1989. 16. Rodriguez A. y col. Resultados de la aplicacion del propoleos en ulceras y gangrenas de las extremidades inferiores. Investigaciones Cubanas sobre el propoleos. I Simposio sobre Propoleos. Varader-Cuba 1988:171-74 17. Mescon H.y col. Dermatitis por estasis 0 eczema varicoso. In: Robbins. Patologia estructural y funciona!. Interamericana Mexico. 1975: 1331-2. 18. Apositos para las ulceras de las piemas. Drug and Therapeutics Bulletin. 1094;24(3): 172-5. 19. Temesio P. Nuevo metodo del tratamiento local con prop6leos. Investigacion clinica. Policlinica de diabetes. Hosp.Maciel 1983.

14

PRESENT STATE OF BASIC STUDIES ON PROPOLIS IN JAPAN Tsuguo Yamamoto Nihon Natural Foods Co., Ltd. 6-26-12 Nishishinjuku, Shinjuku-ku, Tokyo, Japan

I. INTRODUCTION In October 1985, the 30th International Apicultural Congress held in Nagoya first introduced propolis into Japan, when the latest reports on basic studies on propolis and the clinical application of propolis to the intractable diseases were presented by the researchers and the medical doctors from various countries abroad. Together with the introduction of a crude propolis by the Brasilian beekeepers, the name of 'propolis' became known instantly with the possibility of future promising substance. However during first five years since 1985 propolis was regarded only as health foods and folk remedies and was not paid much attention to by scientists. Propolis studies made public in 1987 related only to general findings, introduction of overseas literatures, and distribution of flavonoid components I). In 1991 at the 50th Japan Cancer Society Meeting, Matsuno of the National Institute of Health reported three compounds with tumor killing activities in propolis from his experience curing terminal-stage uterine cancer with propolis2) With this as momentum, many of pharmaceutical companies and research institutes have started studying propolis with the prospect of its use more promising. Among them Hayashibara Biochemical Laboratories Inc. in the course of his interferon research, took an interest in the BRM (biological response modifiers)-like effects in propolis, such as the antivirus effect 3 ) and immune activating effect 4 ) and has been studying propolis these ten years, and published a fairly number of remarkable research results. This paper describes representative studies on the antimicrobial effect and cytotoxic effect of propolis from 1985 up to now in Japan.

II. ANTIMICROBIAL ACTIVITY OF PRO POLIS The strong antimicrobial activity of Propolis is often compared to a "natural antibiotic". Its antimicrobial characteristic against various microbes has recently been studied throughout the world. 107

108

T. Yamamoto

In Japan, Aga et a1. 5), 6) Matsun0 71, ltoh et a1. 8), and Nakano et a1. 9 ) have studied and reported the effects of propolis and antimicrobial substances isolated from propolis on various types of fungi, yeast's and bacteria, as well as on specific pathogenic microbes, such as Helicobacter pylori, and Methicillin-resistant Staphylococcus aureus (MRS A) between 1992 and 1995.

1. Antimicrobial Activity of Brazilian Propolis 5) In 1992, Aga et al. of Hayashibara Biochemical Laboratories Inc. published their research results on the antimicrobial activity of an ethanol aqueous solution extract of Brazilian propolis against 8 strains of fungi, 4 strains of yeast, and 42 strains of bacteria, including Enterobacter and Actinomyces. In their study, the propolis extract showed strong antimicrobial activity against Micrococcus lysodeikticus (MIC: 15.6 /-tg/ml), Bacillus cereus, Enterobacter aerogenes (MIC: each 31.3 /-tg/ml), Corynebacterium equi, Mycobacterium phlei, Thermoactinomyces intermedius, Arthroderma benhamiae & Microsporum gypseum (MIC: each 62.5 /-tg/ml), but showed very weak antimicrobial activity against Enterobacter such as Bifidobacterium, Lactobacillus, Eubacterium, and Bacteroides. This antimicrobial spectrum agreed with the results of German propolis studied by 1. Metzner et al. 10) and of American propolis studied by L. A. Lindenfelser lll in which antimicrobial activity was recognized against 7 strains of bacteria such as Micrococcus lysodeikticus & Bacillus cereus, and Arthroderma benhamiae. The MIC values of Brazilian propolis show about 3 times more antimicrobial activity against Bacillus subtilis, Staphylococcus aureus and Arthroderma benhamiae than that shown by German propolis. This result suggests a difference in antimicrobial substance contents and the presence of stronger antimicrobial substances. Accordingly, it is expected that isolation and identification of these substances and further elucidation of the mechanism of the antimicrobial action will be conducted.

2. Isolation and Identification of Antimicrobial Compounds in Propolis 6) In a later study, Aga et a1. isolated and identified three antimicrobial compounds from Brazilian propolis. As shown in Fig.l, they identified these compounds as 3,5diprenyl-4-hydroxy cinnamic acid (as Compound I), 3-prenyl-4-dihydro cinnamoloxy cinnamic acid (as Compound 2), and 2,2-dimethyl-6-carboxy ethenyl-2H-l-benzopyran (as Compound 3). The results of this study were published in 1994. Table I gives the antimicrobial activity (MIC values) of these compounds against three types of microbes selected as specimens to be studied. As shown in Table 2, compared with the original propolis extract, 3,5-diprenyl-4-hydroxycinnamic acid (Artepillin C) had stronger antimicrobial activity against cutaneous fungi (ex. Microsporum, Arthroderma), putrefying bacteria (ex. Bacillus), Corynebacterium, and pyogenic bacteria (ex. Pseudomonas). It has long been said that propolis is effective for the treatment of dermal disorder and burns. This characteristic of propolis suggests that Artepillin C plays a leading role in the antimicrobial and anti-inflammatory activities of pro polis. Later, Kimoto et a1. disclosed that Artepillin C played a principal role not only antimicrobial activity, but also in "anticancer action" of Brazilian propolis to be introduced below. 121

Present State of Basic Studies on Pro polis in Japan

109

o OH

R'=H, R 2 =CH 2CH=C(CH 3 )2 Compound 1 (3,5-diprenyl-4-hydroxycinnamic acid) (ArLepillin C) R'=CO(CH 2 )2 Ph , R2=H Compound 2 (3-prenyl-4-dihydrocinnamoloxycinnamic acid)

o OH Compound 3 (2,2-dimeLhyl-6-carboxyeLhenyl-2H-l-benzopyran)

Figure 1.

3. Anti-Helicobacter pylori Substances in Propolis

8)

Itoh et a1. of the Zenyaku Kogyo Co., Research Institute examined the antimicrobial activity of Chinese, Argentine and Brazilian propolis against Heliocobacter pylori, whose connection to gastritis and gastric ulcer was suspected. Their research results were published in 1994. According to these results, Argentine propolis showed the highest antimicrobial activity with an MIC value of 50 f..\g/ml, followed by Chinese propolis at 100 Ilg/ml, and Brazilian propolis at 200 Ilg/m1. They reported that each propolis showed an anti-Heliocobacter pylori effect.

Table 1. Antimicrobial activity of isolated compounds 1_3 6 )

MIC ( '-' g/ml)

Composilion'

B.cereus

Compound

5.296

15.6

31. 3

compound 2

2.396

31. 3

62.5

compound 3

0.896

Crude propolis

125 31. 3

E.aerogenes

125 31. 3

A.benhamiae 15.6 ,250 62.5 125

, as a percenlage relalive lo lhe dry solid crude propolis

T. Yamamoto

110

Table 2. Antimicrobial activity of artepillin C6) MlC Slrains

g/ml) Propolis

Microsporum gypseum (lFO 8231) Arlhroderma benhamiae (JCM 1885)

(/I.

Arlepillin C 7.8

62. S

15.6

62. S

Bacillus cereus (lFO 3466)

15.6

31. 3

Bacillus sublilis (ATCC 6633)

31. 3

31. 3

Corynebaclerium equi (lFO 3730)

31. 3

62. S

Micrococcus lysodeiklicus (lFO 3333)

31. 3

Pseudomonas aeruginosa (lFO 3453)

31. 3

Enlerobacler aerogenes (lFO 3321)

31. 3

Mycobaclerium smegmalis (JCM 5866 T )

31. 3

Mycobaclerium phlei (JCM 5865 T )

62.5

Slaphylococcus aureus (ATCC 6538P)

62.5

2S0

Slaphylococcus epidermidis (ATCC 12228)

62.5

SOO

Thermoaclinomyces inlermedius (JCM 3312 T )

62.S

lS.6 12S 31. 3

SOO 62. S

62.5

Micrococcus luleus (lAM 1099)

125

2S0

Propionibaclerium acnes (JCM 6425 T )

12S

SOO

Flavobaclerium meningoseplicum (lFO 12535)

2S0

2S0

Kloeckera apiculala (JCM 5947)

SOO

SOO

Saccharomyces cerevisiae (lFO 0214)

SOO

2S0

HO

HO OH 0

OH 0

pinocembrin

compound

galangin (R=OH), chrysin (R=H)

MIC

( /.1

g/ml)

pinocembrin

12.5

galangin

25

chrysin

25

Figure 2.

111

Present State of Basic Studies on Propolis in Japan

They further isolated fractions that had anti-Heliocobacter pylori activity from propolis by column chromatography, and identified these fractions as pinocembrin (MIC: 12.5 Ilg/mi), galangin and chrysin (MIC: each 25 Ilg/ml). The anti-Heliocobacter pylori activity of pinocembrin showed an antimicrobial activity equal to that of Lansoprazole, which was used as a control (Fig.2). However, pinocembrin showed a lower antimicrobial activity against another microbes other than Heliocobacter pylori (MIC: 50 ->200 Ilg/ml). This suggested that one of the factors contributing to propolis's anti-ulcer effect was specific anti-Heliocobacter pylori activity due to flavonoids such as pinocembrin.

4. Anti-MRSA Compound Isolated from Brazilian Propolis9 ) In 1995, Nakano et al. of Hayashibara Biochemical Laboratories Inc. studied active substances in Brazilian propolis to determine whether Brazilian propolis exhibits antiMRSA activity. This substance was 3-prenyl-4-dihydrocinnamoloxycinnamic acid having a chemical structure shown in Fig.3. As illustrated in Fig.3, only 2 Ilg/ml of this substance evaluated by the MIC method had an anti-MRSA activity 100 to 400 times higher than that of each component of the propolis ethanol extract and propolis. In 1994, this substance had been isolated from Brazilian propolis by Aga et a1. 6) at Hayashibara Biochemical Laboratories as an antimicrobial substance together with Artepillin C. At the time, however, no study was made on anti-MRSA activity. Regarding studies of anti-MRS A activity of propolis made so far, Grange et al. 13 ) reported on the relatively strong anti-MRS A activity of the extract of French propolis in 1990. Activity of the same degree as that in this report was recognized in Brazilian propolis. The anti-MRSA activity of propolis is considered due to the synergetic effect of complex components in propolis. During their work of isolation, the fraction containing this 3-prenyl-4-dihydro-cinnamoloxycinnamic acid was the most active, so Nakano et al.

o

o

~O

OH

3-prenyl-4-dihydrocinnamoloxycinnamic acid

co.pound

IIIC( JL9.m1)

compound

IIIC( JL 9.111)

200

9a1an9in

)aoo

caffeic acid

)800

Kaemferol

>800

coumarie acid

)800

ArLepi11in C

cinnallic acid

)800

anLi-KRSA compound

propolis exLracL

Figure 3.

200 2

112

T. Yamamoto

estimated that this substance was the main component of the anti-MRSA activity of Brazilian propolis. Findings on the anti-microbial activity of propolis and isolated substances against specific microbes in Japan have been described here in. It is interesting that, in addition to the known effect of flavonoids, both Artepillin C reported by Aga et al. and New Clerodane Diterpenoid founded by Matsuno as an anti-tumor substance have strong anti-microbial activities. We expect that new substances with antimicrobial activity against specific pathogenic microbes will be discovered from propolis, and through basic research, application studies for clinical use will be soon to follow.

III. IMMUNE ACTIVATING EFFECT AND CYTOTOXIC EFFECT OFPROPOLIS Commercially available books and European papers l4) describe that propolis and its components are effective in inhibiting human malignant tumors and cancer cells. However, there had been no papers objectively evaluating the mechanism or degree of their antitumor effects before 1990. In Japan, Matsun0 2 )?) of the National Institute of Health, Arai et al. 15 ). Kimoto et al. 12 ) of Hayashibara Biochemical Laboratories Inc., and Suzuki et al. 16 ) of the Suzuka College of Technology published results of studies on propolis, and on some of its isolated or fractionated active components between 1991 and 1996.

1. Cytotoxic Effect of New Clerodane Diterpenoid Isolated from

Brazilian Pro polis 7)

In 1990, Dr. Matsuno of the National Institute of Health found that the ethanol extract of Brazilian propolis transformed human hepatic carcinoma cells and uterine carcinoma cells cultured in vitro, and that it inhibited their growth. Afterwards, he orally administered a large quantity of a propolis drink to one of his relatives, who had cancer of uterine cervix and was unable to undergo surgery because of her poor physical strength. He also continuously applied the propolis ethanol extract directly to the affected part. As a result, the lesion became a burn-like scar several weeks later, and her cancer of uterine cervix disappeared2 ). Since then, he started to study the purification and isolation of the antitumor active substances contained in propolis, and observed the cytotoxic effect of fractionated substances on human hepatocellular carcinoma, HuHI3. As a result, he found this effect in the following three substances that is, the known substances of quercetin, and caffeic acid phenethylester, and a new compound belonging to clerodane diterpenoid. He reported his findings at the 50th Japanese Cancer Association Congress in 1991. Clerodane diterpenoid, in particular, was very active in destroying tumor cells, especially human cervical carcinoma cells (HeLa cells) and Burkitt's Lymphoma cells in addition to HuHI3. His findings suggested that this substance showed selective toxicity to tumor cells, stopped the cell growth cycle in the gene synthesis phase (phase S), changed the properties of the cell membranes, and killed cells by disturbing their ionic permeability.


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Although the details of the mechanism are still being analyzed, this substance acts on tumor cells in phase S, when tumor cells are growing more actively than normal cells and synthesizing genes rapidly. Therefore, Dr. Matsuno assumes that tumor cells are ultimately destroyed because they are growing at a different speed. The effects of clerodane diterpenoid on HuH 13 and two normal cells ( untransformed primary rabbit kidney cells and human diploid fibroblast cells) were examined. As a result, as is shown in FigA, a large difference in the effective concentrations of this substance was found, and it was proved that an appropriate concentration of this substance could kill tumor cells without affecting normal cells. These results emphasized the possibility that a new treatment, which could kill tumor cells exclusively without damaging normal cells, could be developed by determining an appropriate concentration and administration method for this substance. Dr. Matsuno confirmed its treatment effects on cancer patients, and made public the details of his experience at the 51 st Japanese Cancer Association Congress.

2. Biological Effects of Propolis on Macrophage Function and Tumor Metastasis 15) 17) Dr. Arai et al. of Hayashibara Biochemical Laboratories Inc. has begun to study the biological activity of Brazilian propolis from various perspectives since 1990. In order to make the propolis easy to use, they powdered the propolis extract using anhydrous maltose, and tried to confirm the effectiveness and mechanism of a BRM-like substance, aiming at isolating and identifying this substance. This propolis powder was dispersive in water, was free of endotoxin that macrophage activates, and contained 13.8% propolis-derived solids, so that concentrations during the experiment were all expressed in terms of propolis powder. They discovered a macrophage activation phenomenon related to the immune function of living organisms, then in 1993 published the results of their minute studies on the effects of propolis on macrophage spreading, phagocytosis, motility, and cytokine production!7). In 1994, they made public its inhibitory effect on lung metastasis in mice!5). After adding a culture medium containing propolis powder solution to the abdominal macrophage obtained from the BALB/c mouse, stretching as shown in Fig.5 was observed. This phenomenon correlated to propolis concentration and time, and a dose reaction was recognized.

114

T. Yamamoto

Figure 5.

Similarly, the effects of this propolis on fowl phagocytosis and motility, TNF production by LPS (lipopolysaccharide; pyrogen) coexistence. cytotoxic factor NO (nitrogen oxides) production inhibition were studied. As a result, it was revealed in vitro that its effects depended on concentration and time, or the presence or absence of an LPS stimulus. Moreover, in vivo, though TNF production in mouse blood was increased by an LPS stimulus, by administering 0.2 mg and 2.0 mg propolis Imouse 3 hours before an LPS stimulus, ten times more TNF could be produced. Propolis as one of BRM (biological response modifiers)-like substance activated macrophages by, Propolis by itself did not produce cytokine in vivo; however cytokine production was sharply accelerated by an LPS stimulus. These results suggest the activation effect of immune cells which produce cytokines. Prior to a test on the inhibition of tumor metastasis by this propolis, its effects on the growth of mouse colon carcinoma cell Colon 26 were studied. As shown in Fig.6, this propolis inhibited the growth of the cells in a concentration- dependent way, and had a direct effect. Next, 40 f.lg of this propolis was administered to BALB/c mice (7-week-old females), on to which Colon 26 cells were then transplanted. Various concentrations of this propolis were continuously administered for 6 days, and the metastatic lesions in the lung were counted 14 days later. As shown in Table 3, the dose of propolis was optimum. Lung metastases were reduced to 80% in the group receiving 0.1 mg propolis powder, 57% in the 0.2 mg group, and 70% in the 0.4 mg group. Based on these results, they assumed the following . The dose of propolis administered to the mice was too small to directly inhibit cell growth in vivo. Administration of propolis activated immune cells, mainly macrophages, and inhibited and removed the implantation of metastatic tumor cells as foreign matter to lung tissues, leading to a reduction in the number of metastatic lesions.


115

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3. Cytotoxic Effect of Artepillin C Isolated from Brazilian Propolis t2 ) Three substances isolated and identified from Brazilian propolis by Aga et aI., especially Artepillin C, have been shown to have a stronger antimicrobial activity than the crude propolis61. The above-mentioned study on the antitumor effect of propolis made by Dr. Arai et al. suggested the presence of a substance in propolis that was effective in killing tumor cells. This study also suggested that c1erodane diterpenoid, an antitumor substance in

Table 3. Inhibition lung metastasis of mouse colon carcinoma (colon 26) by propolis l51 dose Group Propolis powder

Anhydrous maltose Physiological saline

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Colonies numbers

Average ± SE

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13

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13

66. P±

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13

81. 0 ± 13.3

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13

134.0 ±

O.2ml

13

115.9 ± 15.4

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propolis discovered by Dr. Matsuno, also had relatively strong antimicrobial activity 7). Stimulated by these facts, Kimoto et al. examined the antitumor effect of these three antimicrobial substances, and found that only Artepillin C had a strong cytotoxic effect on various cultured tumor cells and transplanted carcinoma cells. They published their results in 1995 12). Artepillin C showed a noticeable inhibitory effect on the growth of the 18 types of cultured tumor cells tested, with a dose of only 1{}-100 I!g/ml. Its cytotoxic effect was especially eminent in rapidly growing cells. Fig.7 shows the effects of Artepillin C on human gastric carcinoma cells (HOC), human lung cancer cells (BLC), and mouse colon carcinoma cells (Colon 26), when 100 I!g/ml each of Artepillin C was added and cultured. Fig.7 also presents the effects of Artepillin C at different concentrations on malignant melanoma cultured cells (0-361, IHARA and B-16). Artepillin C demonstrated a conspicuous effect in inhibiting the growth of these cells. Fig.8 sets out the effects of 100 I!g/ml of Artepillin C on HLC (Fig.8-a and -b) and HTSA (Fig.8-c and -d). Fig.8-a and-c are microscopic photos showing cells cultured without treatment. Fig.8-b and-d show cells given Artepillin C and cultured for 24 - 48 hours. Severe cell injury (Fig.8-b) and cell necrosis (Fig.8-d) can be observed. Though Artepillin C is insoluble in water, newly developed water-soluble [Artepillin C]-Na was used to examine its antitumor effect. The results of this examination are shown in Fig.9 and Fig.IO. [Artepillin C]-Na was more effective than Artepillin C in its anticancer effect, resulting from DNA synthesis inhibition of human leukemia cells (HL-60 and THP-I) and malignant lymphoma (U937) (Fig.9). Also [Artepillin C]-Na was more effective than Artepillin C in antitumor effects causing cell death and growth inhibition of hepatocellular

117


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carcinoma cells (rat-derived; HTSA and RL-34, human-derived; PLC /PRFIS), and human larynx carcinoma cells (KB) (Fig .! 0). Next, in vivo, 500 f.!g of Artepillin C were intravenously administered for 4 weeks every other day to nude mice transplanted with HLC, HOC, and PLC /PRFIS as xenografts, and Colon 26 and HTSA as allografts. Fig.!! shows the growth inhibition of the transplanted carcinoma cells, and the histopathologic findings. As a result, nuclear degeneration (caryolysis and pycnosis) and apotosis-Iike population death were observed in these carcinoma cells (Fig.ll-c and -d). Small-population physalis turned to large-population degeneration and necrosis, which inhibited cell growth (Fig. I!-e and -f). Afterwards, collagen, macrophages, and helper cells increased around carcinoma cells which resembled solitary islands (Fig. I I-f). Conversely, in Fig.ll-a and -b, HLC in the mouse given Artepillin C separated into two small tumors, and no further swelling was observed. These results suggest that the cytotoxicity of Artepillin C to many carcinoma cells inhibits cancer cell growth by inhibiting the DNA synthesis of carcinoma cells (Fig.9) and by damaging the respiratory enzyme system of intracytoplasm glomerular intima (Fig. I 0). In particular, Artepillin C has a strong antitumor effect on leukemia cells, and its future application as an adjuvant drug for intravenous injection chemotherapy is expected. Several research reports in Japan on the antitumor effects of propolis have been described here. However, there is no complete agreement between propolis and the components isolated from propolis in terms of the effect on cancer cells and the mechanism. This fact suggests that new antitumor substances and cytotoxic substances (antimicrobial substances) may be discovered in propolis in the future.


119

Figure 11.

In concluding this paper, we would like to say that we expect Japanese researchers to discover new substances which are more effective in dealing with tumor cells without damaging normal cells in another phases than the gene synthesis period (S period); for instance, in the mitotic period (M period).

ACKNOWLEDGMENT I wish to thank Prof. Avshalon Mizrahi of the Israel College of Complementary Medicine and Prof. Mitsuo Matsuka of Tamagawa Univ. for this opportunity to introduce "the present status of propolis in Japan", and Mr. Satoshi Iritani of Hayashibara for his valuable comments and support to complete this paper.

T. Yamamoto

120

REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Characterization and Actions of Propolis; Fragrance Journal, 83, 1987 Matsuno T.; Hammingbird, 1, 14, 1991 Tatefuji T. et a1.; Shoyakugaku Zasshi. 47(1), 60, 1993 Moriyasu 1. et a1.; Biotherapy, 7(3), 364. 1993 Aga H. et a1.; Medicine and Biology, 124 (5), 205, 1992 Aga H. et a1.; Biosci. Biotech. Biochem., 58 (5), 945, 1994 Matsuno T.; Honeybee Science, 13 (2), 49, 1992 Itoh K. et a1.; Honeybee Science. 15(4), 171, 1994 Nakano M. et a1.; Honeybee Science, 16 (4),175,1995 Metzner 1. et a1.; Pharmazie, 34, 97, 1979 Lindenfelser L.A.: Amer Bee 1., 107 (3), 90, 1967 Kimoto T. et a1.: Nihon Iji Shinpou, 3726. 43. 1995 Grange 1. M. et a!.; J. Royal Soc. Med., 83, 159, 1990. Grunberger D. et a1.: Experientia, 44, 23, 1988 Arai S. and Kurimoto M.; Honeybee Science. 15 (4),155.1994 Suzuki l. et a1.; Honeybee Science, 17 (1), I, 1996 Moriyasu J. et a1.: Biotherapy 8(3). 346, 1993

15

THE USAGE AND COMPOSITION OF PROPOLIS ADDED COSMETICS IN KOREA Park Jong-Sung and Woo Kun-Suk lNHB International Co. Ltd. Janghakhoegwan Bldg. 602-ho, Oaechi-dong 945-15, Kangnam-gu, 135-280, Seoul, Korea 20epartment of Agricultural Biology, College of Agriculture and Life Sciences Seoul National University 44 1-744, Suwon, Korea

1. ABSTRACT Propolis cosmetics commercialized in Korea are the liquid foundation (GAC 4927), Propoleo cream (GAC 4914), and the eye cream (GAC 4922). Liquid foundation has 3% of propolis with 20 other ingredients. The propoleo cream includes 2% of propolis. The cream is specially beneficial for oily or pimply face. The eye cream with I % of propolis helps to absorb nutrients and to prevent excessive moisturizing. PropoJis in cosmetic shows a great deal of advantages in antibacterial activity, moisturization, revitalization, and elasticity of skin. Additionally, Amiga Lotion (G 153- 1122) and Amiga Skin (G 1531123) are also products with propolis. To see the consumer's satisfaction, questionnaires were distributed, and the results are shown 8 I % on the liquid foundation and Propoleo cream, and 83% on the eye cream as table 1,2,3.

2. INTRODUCTION The demand of consumers for natural ingredient in the cosmetology is increasing recent decade. To meet the desire, Taepyengyang trade Co. founded in 1988 and imported cosmetic raw materials at the beginning. With changing name to Korea Antuco in 1990 and Amiga Co. in 1994, company developed 5 natural cosmetics in 1990. Founding the marketing company, NHB International Co. Ltd. the company has developed 12 propolis added cosmetics in 1990. Propolis cosmetics initially commercialized and attracted many people's attention in Korea. These products are foundation (GAC 4927), Propoleo cream (GAC 4914), and eye cream (GAC 4922). Additionally, Amiga Lotion, cleansing water, 121

122

Park Jong-Sung and Woo Kun-Suk

Brice massage cream, revital pack, royal jelly cream, and essence lotion are also products with propolis. Propolis imported from a satellite town in Chile, because Korean propolis may have an allergy problem on the skin due to a lacquer tree. Propolis, what is called bee glue, is resinous materials collected by honey bees from the bark and buds of various trees (1). The composition generally consists of terpenes, resins, volatile oils, flavonoids and other compounds. Though about 130 chemicals identified from propolis, all propolis do not have same composition (1). The physical characters is 65.5°C melting point and brittle in cold room temperature (3). The solubility are partial in water, but rather completely in ether or chloroform. The color ranges from yellow to dark reddish brown (3). , The new brand name Brice is combined word from English brilliant and italian Dolce, which means bright and beautiful grace. All the ingredients of products came from the natural source. The marketing system is multi-level marketing and organized by members. The current members are about 20 thousands and the number of member is increasing rapidly since consumers have begun to realize the benefits of natural ingredients and functional cosmetics. Because of extraordinary variability of propolis in the area and origin of plant, it is not reasonable to declare that propolis shows a great effect on skin and in health of everybody, but there is no doubt it is effective on inflammations and other skin troubles, according to the literatures (2,4) and empirical data showed in this study.

3. MATERIALS AND METHODS To evaluate the benefits of Brice, questionnaires were distributed among 1,600 members during marketing seminar and the empirical data were collected in 1996. The members are divided into 4 groups after questioned in according to their general impression on the cosmetics, so A group is the most favorable persons and D group is the least. Questionnaires were asked on the effects of individual cosmetics.

4. RESULTS 4.1. Liquid Foundation Liquid foundation has 3% of pro polis with 20 other ingredients. It helps balance pH of your skin, blocks the aging UV rays as sunscreen, and affinity enables to stay longer on the skin. Propolis works as antibacterial agent on the skin. Moisturizing effect is excellent and long-lasting. The satisfaction of consumers varies as shown table 1, and the average is 81 %.

4.2. Propoleo cream The Propoleo Cream includes 2% of propolis. The cream is specially beneficial for oily or acne. It also has characteristic in soothing your vulnerable skin and improving troubles. Propoleo cream also shows 81 % in consumer's satisfaction as in table 2.

4.3. Eye cream Eye cream with 1% of propolis helps to absorb nutrients and to prevent excessive moisturizing. This also increases the firmness and elasticity of skin. Lines and wrinkles

Usage and Composition of Propolis Added Cosmetics in Korea

123

Table 1. Satisfaction rates on an liquid foundation (unit: percent) Group Characters

A

B

C

D

Average

pH balance UV blocking Bacteriostasis Moisturizing Affinity Elasticity

90 85 95 90 90 90

90 85 80 90 85 85

85 80 70 85 80 80

80 70 60 70 60 75

86 80 76 84 79 82

are softened and smoothed. With the help of eye cream, blood circulation around eyes improved to velvety and smoothy skin. Users massage the cream around eyes in the morning and evening. The average effect is 83% as shown in table 3.

4.4. Other Cosmetics Moisture shampoo prevents scurf, acne, and depilation. Approximately 48 kinds natural ingredient exist in the shampoo. The essence lotion makes healthy hairs by providing nutrients and coating the hair for protection. This lotion including propolis has 50 different nutrients. Brice massage cream is able to cleans dust, exudate, and sebum on the skin, even in the skin pore. The major compositions of massage cream are propolis and rose hip oil, which makes silky skin. Brice royal-jelly cream inhibits the reduction of collagen, which is cause of wrinkle and fine lines. Plus it protects skin from UV and oxidant with balancing moisture and nutrients.

5. DISCUSSION As a result of questionnaires, the liquid foundation needs improvement on bacteriostasis and affinity, which shows lower satisfaction on the characteristics. Propoleo cream generally shows very good characters except skin pore care and revitalization of skin. Eye cream also proved to have excellent effects on the skin care, but satisfaction on wrinkle care need some improvement.

Table 2. Satisfaction on a Propoleo cream (unit: percent) Group Characters

A

B

C

D

Average

pH balance Bacteriostasis Moisturizing Affinity Elasticity Acne and scar care Skin pore care Revitalization

90 95 90 90 95 98 85 85

90 85 90 85 90 80 80 80

85 80 80 75 75 70 70 75

80 70 80 75 70 65 70 65

86 82 85 81 82 78 76 76

124

Park Jong-Sung and Woo Kun-Suk

Table 3. Satisfaction on an eye cream (unit: percent) Group Characters

A

B

C

D

Average

Puffiness reducing Blood circulation Wrinkle care Elasticity

95 90 85 90

90 90 80 85

85 80 75 85

80 75 70 70

88 84 78 83

At this moment only the propolis from Chile is manufactured in the process, but natural propolis in Korea can be purchased and used commercially as long as the restriction is made on the origin of plant like the green house environment. Propolis in cosmetic shows a great deal of advantages in antibacterial activity, moisturizing, revitalization, and elasticity on skin. The products can be used for everybody regardless gender and sex and age. The great benefits proved on skin trouble, scar, and acne. Body cares, soaps, two-way cakes, and lipsticks will be developed.

6. REFERENCES I. 2. 3. 4.

Bonney, R. (1995) Propolis. Bee Culture. II, 630-634. Maeda G. The miracle of propolis (translated into Korean). Young-woo Publisher, Seoul. 1994.115pp. Root, A. I. ABC and XYZ of Bee Culture. A.1. Root Company, Medina, Ohio, U.S.A. 1954. pp 538-541. Toth, G. (1988) Cosmetic use of hive products: facts and prospect. American Bee Journal. 6, 431---434.

16

EUCALYPTUS PROPOLIS BEVERAGES WITH THEIR COMPOSITION AND EFFECTS Woo Kun-Suk and Park Jong-Sung lDepartment of Agricultural Biology, College of Agriculture and Life Sciences Seoul National University 441-744, Suwon, Korea 2Amiga Food Co. Ltd. Janghakhoegwan Bldg. 602-ho, Daechi-dong 945-15 Kangnam-gu, 135-280, Seoul, Korea

1. ABSTRACT The Combi Propol and the Pro Rapa are propolis beverages made from Eucalyptus. The brand-new Combi Propol is a foaming agent in two types, foaming tablets and foaming granules. Both have 20% of propolis. The Pro Rapa is concentrated beverage and 85% of the component without water is propolis. Water is added to 1-5 ml of Pro Rapa for drinking. The procedure for making the Pro Rapa is the following: wax extracts matured for 50-60 days in ethanol, and then the extract mixed and filtered at 25-30°C. After 24 hours, filtrate was mixed with honey, wax extract, and peppermint.

2. INTRODUCTION Bees use propolis to build hives with wax, to polish hive walls (1, 5). They also polish combs before larva rearing, which results in black and smaller combs by successive use. They also embalm dead animals which are too big to take out from hive. They stuff the cracks with propolis for water-proof (2). The propolis has the bactericide effect in the hive. Propolis is able to inhibit the germination of some seeds and a half of hemp seed inhibited with 111»20 dilution. The lack of air-borne flora around apiary may have something to do with the antibiotic effect of propolis (4). Propolis is hard and pliable at 15°C. Melting point is 60-69 DC. The color of propolis is variable from yellow to dark brown and it is thermostable and partially hydrosoluble. Alcohol or chloroform readily resolves propolis. Main components of propolis is flavonoids (3, 9). Flavonoids works in the body in enhancing the exchange between blood and tissues, which provide nutrients and remove 125

126

Woo Kun-Suk and Park Jong-Sung

residues (2). Flavonoids components contain glucides, protides, and lipids, which could explain the possibility of its integration with physiological metabolisms (2). Flavonoids undergo several cleavages under the intestinal flora environment and some flavonoids are removed by urine and some by feces, and only I % of the administrated dose is absorbed with the rest being removed unchanged (2). Vitamin P or citrine, some called this as bioflavonoids or flavonoid derivates, works synergically with vitamin C (2). Propolis has curative bactericidal properties, but they are very variable depending on the bacteria studied (4). Propolis with different origins do not always have a constant antibiotic value. Bacteriostatic effect of propolis in vitro was tested on cultures of E. coli, bacillus, entrococci, acidolactic bacteria, which are found in the large intestine or animals as well as on staphylococcus cultures and acidophilous bacteria (6). Propolis beverages made from Eucalyptus were produced in Korea. The Combi Propol and Pro Rapa is the beverages having propolis as a major component. The Combi Propol is tablet-type and the Pro Rapa is liquid-type, which needs to blend with water before drinking.

3. MATERIALS AND METHODS Questionnaires were distributed among 1,600 members during marketing seminar in 1996. Most of users have been administrated the Pro Rapa for 6-7 years. To see consumers' satisfaction rates on effects of products, we distributed questions on characteristics of the Combi Propol and the Pro Rapa.

4. RESULTS 4.1. The Combi Propol Two kinds of the Combi Propol is selling, one is for children with chocolate flavor and the other is for adults with propolis natural flavor. The types ofthe Combi Propol are foaming agents, foaming tablets and foaming granules. Propolis is about 20 percent and other ingredients are cellulose, foaming agent, ketonic acid, and ~-carotene. Questionnaires were asked about the effects on antibiotics and cell revitalization, and so forth (table 1).

4.2. The Pro Rapa The composition of the Pro Rapa is 85% of propolis, 0.5% of peppermint, and 10% of honey. The procedure for making the Pro Rapa is extracting and maturing propolis for Table 1. Effects of the Combi Propoi (unit: percent) Characteristics Antibiotics Immune and anticancer activity Cell revitalization and restoration Strengthening veins Propolis flavor Consumers' satisfaction

Excellent

Very good

97.8 98.1 98.3 96.9 87.8 93.8

Good

Not bad

127

Eucalyptus Propolis Beverages with Their Composition and Effects Table 2. Effects of the Pro Rapa (unit: percent) Characteristics Circulatory disease Gastrointestinal disease Respiratory disease Arthritis Dermatitis Anti-oxidant Anti-intlammation

Excellent

Very good

Good

Not bad

90.6 91.2 94.4 86.6 96.1 84.6 99.0

5(}-{)0 days in ethanol, and then the extract mixed and filtered at 25-30 0c. After 24 hours, filtrate was mixed with honey, wax extract, and peppermint. The products were filled in the bottles and were inspected for distribution. The Pro Rapa is liquid product with propolis natural flavor and is administrated with water. The effects of the Pro Rapa were arranged in table 2.

5. DISCUSSION Propolis has used as a mean of anaesthetizing the pains in the operations on the abdomen in domestic animals without side effects (7). It also used as a antioxidant in stabilizing the quality of frozen fish (8). According to Laszt (2), a successful treatment of circulatory disease, it is necessary that the troubles should be detected in their incipient stage. Propolis shows a great deal of advantages in curing and preventing disease (5), but because of propolis variability and not knowing exact way of metabolism of propolis, the benefits need to have scientific background in further studies.

6. REFERENCES I. Bonney, R. (1995) Propolis. Bee Culture. II, 630-634. 2. Derevici, A. Contribution to the study of pro polis in: A remarkable hive product propolis. Apimondia Publishing House. Bucharest. 1978. pp.78-95 3. Derevici, A and A. Popescu and N. Popescu. Considerations on the characteristics of alcohol propolis extract in: A remarkable hive product propolis. Apimondia Publishing House. Bucharest. 1978. pp.74-78. 4. Lavie, P. The antibiotic from propolis in: A remarkable hive product propolis. Apimondia Publishing House. Bucharest. 1978. pp.41-48. 5. Maeda G. The miracle ofpropolis( translated into Korean). Young-woo Publisher, Seoul. 1994. 115pp. 6. Palmbakha, S. E. Study of the antimicrobial effects of propolis on the gastro-intestinal microtlora in: A remarkable hive product propolis. Apimondia Publishing House. Bucharest. 1978. pp.49-51. 7. Tsakoff, T. Study of the local anaesthetic characteristics of propolis and their effect in operations on sheep and dogs in: A remarkable hive product propolis. Apimondia Publishing House. Bucharest. 1978. pp.67-71. 8. Ushkalova, V. N. Antioxidant value of pro polis in: A remarkable hive product propolis. Apimondia Publishing House. Bucharest. 1978. pp.71-73. 9. Graham, J. E. The hive and the honeybee. Dadant & Sons Inc. 1992. pp.943-952.

17

AN INHIBITORY EFFECT OF PROPOLIS ON GERMINATION AND CELL DIVISION IN THE ROOT TIPS OF WHEAT SEEDLINGS K. Sorkun, S. Bozcuk, A. N. Gi:imiirgen, and F. Tekin Hacettepe University Science Faculty Department of Biology Beytepe-Ankara Tiirkiye

ABSTRACT In the present work, the effect of five different propolis samples collected from different regions of Turkey «:ankiri, Aksaray, Milas-Kalemli, Giimii~hane-Kaleta~) in 1994 and from Giimii~hane-Kaleta~ in 1995 on germination percentage and cell division in the root tips of wheat seedlings (Triticum durum Desf. cv.Kunduru) were studied. For this reason, the ethanolic extracts of propolis (EEP) were prepared in four different concentrations. Our findings show that, propolis solutions (EEP) depending on the concentration, significantly inhibited the percentage of germination in comparison with distilled water and their self alcohol controls. In the root tip cells of EEP-treated seedlings, the mitotic cell division was also inhibited significantly with respect to distilled water controL Propolis samples from Milas-Kalemli and (:ankiri regions inhibited the mitotic index significantly as compared with their alcohol controls. The maximum inhibitions in both germination and mitotic index were obtained with the propolis collected from (:ankiri. There was a significant difference between the 1994-1995 Giimii~hane-Kaleta~ propolis collections, on germination.

INTRODUCTION Propolis is a kind of bee product, collected by bees from the buds and leaves of various plants and trees 1. Its chemical composition is very complex and has therefore attracted the attention of various researchers 2.3,4. It was shown that the chemical composition of propolis changed depending on climate, locations and years 5.6. In order to determine the plant sources and the composition of propolis samples another research was done 7. There are also several works showing that propolis can be used for medical pur129

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K. Sorkun et al.

poses 8,9. Keskin et al. (1995) studied the antibacterial effect and Arkan et al. (1995) reported the antifungal effect of propolis 10, 11, Besides the use of propolis in the medical and cosmetic industries 12, there are also various studies that show the effect of propolis on plant diseases 13, 14,15, In the literature, it is reported that propolis has an inhibitory effect on seed germination as well as on plant growth and development 16, 17,18 , Abdou and Omar (1988) investigated the effect of propolis on mitotic cell division in the root tip cells of broad beans, barley, onion and garlic 19 , They have shown that propolis has an inhibitory effect on mitotic division in the root tips of the plants mentioned, Recently Sorkun and Bozcuk (1994) showed that propolis delayed and/or inhibited the rate of germination in the seeds of some culture plants 20 , In another research, the inhibitive effect of propolis on cell division of human lymphocyte cells was also shown 21 , The aim of this study is to investigate the effects of propolis collected from different regions of Turkey on germination and mitotic cell division in the root tip cells of wheat seedlings,

2. MATERIALS AND METHOD 2.1. Research Materials Propolis samples obtained from hives in four different regions in Turkey were used , The propolis samples were collected from the <;ankiri, Aksaray, Milas-Kalemli and Gumu~hane-Kaleta~ regions (Figure 1) in 1994 and only from the Gumu~hane-Kaleta~ re-

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Inhibitory Effect of Pro polis on Germination

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gion in 1995. The samples were obtained by scraping the walls, frames and other hive parts. On the other hand the wheat seeds (Triticum durum Desf. cv. Kunduru) were supplied by the Ministry of Agriculture'S Research Center in Ankara.

2.2. Preparation of Ethanolic Extracts of Propolis (EEP) Propolis samples were kept in a deep freezer at -20"C for a few days. Then the hardened propolis was ground by a grinder 22 and 30 g of ground propolis was dissolved in 50 ml ethanol (96%). This mixture was preserved for a couple of days in a bottle corked tightly and kept in the incubator at 30"C. After dissolving, it was filtered twice with Whatman No:4 and No: I filter papers. The amount of alcohol which had evaporated during the extraction process was completed up to 50 ml by adding ethanol. This solution was called the ethanolic extracts of propolis (EEP). From this solution 30 ml was taken and completed up to I It with distilled water. This solution was then named the 30 EEP stock solution 23 and kept in a refrigerator at 4"C until needed. Later on other propolis solutions at 4 different concentrations (3.75, 7.5, 15 and 20 EEP) were prepared from the 30 EEP stock solution by adding the proper amounts of distilled water. For the control group, 30 ml ethanol (96%) was taken and completed up to I It with distilled water. This solution was named the alcohol stock solution and it was diluted properly with distilled water for alcohol controls.

2.3. Germination Procedure Several groups each having 100 wheat seeds of a medium size were selected. After taking the dry weights of the seeds each group was soaked in one of the test solutions mentioned earlier. At the end of 24 hours soaking time, the test solutions were drained and surface of the seeds were gently dried with filter paper and then reweighed. The difference between the two weights was accepted as the amount of imbibition water. The experiment was performed using three groups. After the imbibition period the seeds were taken to the plastic germinating boxes, 24xl4xl6 cm in size. Beforehand, a thin layer of cotton and two sheets of filter paper were placed at the bottom of the boxes and each box was moisturized with 25 ml of the proper test solutions . Then the imbibed seeds were placed between the two sheets of the filter paper. The germinating boxes with lids closed were kept in a growth chamber at 25"C, in the dark for 5 days. The total percentage of germination was determined for the five day period. For mitotic index studies, approximately 1-1.5 cm pieces were cut from the root tips of two day-old seedlings and fixed in the Farmer's fixative (alcohol and glacial acetic acid, 3: 1)) and kept in a refrigerator until needed. From the root tips, the squash preparations were made in 1% acetocarmine. For each test solution 10 root tips were prepared using the squash method and in 10 different grid areas, dividing and non dividing cells were counted for each preparation. The mitotic index was determined as a percentage of the number of mitotic cells in relation to the number of counted cells. The means, standard errors, ArcSinv'P transformations and analysis of variance were calculated with an IBM computer using the Systat 5 package program. The differences among the means were compared with LSD at a 5% level 24.

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3. RESULTS Effects on seed germination of 5 propolis samples collected from different locations and each of four different concentrations are shown in Fig. 2. As can be seen clearly all propolis samples studied, inhibited germination significantly in comparison to both distilled water and their self alcohol controls (P<0.05). Among the propolis samples; <;ankiri was the most effective one, reducing the germination percentage most at 7.5, 15 and 20 EEP. Indeed the inhibitory effects of 15 and 20 EEP were so great that no germination had taken place. On the other hand, Giimih;;hane-Kaleta~ 95 and Milas-Kalemli samples had their maximum inhibitory effects at 3.75 and 20 EEP respectively. Figure 3 represents the amount of water imbibed by the seeds during the 24 hoursoaking period. It was shown that all propolis samples induced the entrance of imbibition

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water significantly at 3.75 EEP in relation to the self alcohol control (P
4. DISCUSSION In the present study, it was shown that propolis had a phytoinhibitory effect on seed germination depending on the concentration used, location and the time collected. The inhibitory effect of propolis on seed germination was also shown earlier by other workers. For example, ethanol extracts of propolis, was shown to be highly inhibitory on seed germination of Cannabis sativa (Derevici et al. 1964), Triticum vulgare and Zea mays (Sorkun and Bozcuk, 1994) and this effect was highly dependent on propolis concentration 16.20. According to Gonnet (1968) the seedling growth of rice and lettuce was also inhibited by ethanol extracts of propolis 18 . Furthermore, Albore (1979), Barberan et al. (1993) reported that propolis collected from different locations at different times would have different chemical compositions and therefore different phytoinhibitory effects 7. 5. All these findings support our observations. On the other hand, Zimonjic et al. (1989) have found that propolis at low concentration (0.3 mg/ml) inhibited the mitotic activity of human lymphocyte cells and reduced the mitotic index by approximately 40% 2 1. Similarly Abdou and Omar (1988) have shown in

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the root tips of Vicia faba, Hordeum vulgare, Allium cepa and Allium sativum that the mitotic cell division is adversely affected with propolis concentrations 19. All these studies again support our findings to a great extent. H.owever, our results in Figure 2 and 4 indicate that there is a great similarity between the propolis effects upon seed germination and the mitotic index. This could mean that, propolis inhibits the seed germination through the mitotic division of the embryonic cells. On the other hand, according to our findings, propolis does not inhibit, on the contrary it can even increase the entrance of imbibition water (Figure 3) which is crucial at the beginning of germination. So, the scarcity of water within the seed can not be the reason for the inhibited germination. Therefore, it is possible that the propolis molecules that enter into the seeds at the beginning of germination either playa role as an inhibitor or cause the synthesis of some inhibitory substances within the seeds so that mitotic activity and also germination can not take place. Further work is needed to elucidate these possibilities.

REFERENCES I. Root, A.I., "The ABC and XYZ of Bee Culture", Root Company. Medina, Ohio, U.S.A., 1972, pp.538 -539. 2. Garcia, c., Viguera, F., Ferreres, F., Barberan, T., Study of Canadian propolis by GC- MS and HPLC Propolis Rcsearch, North Yorkshire YO 139 HT., 1995, pp. 12-16. 3. Bankova, v., Christov, R., Stoev, G., Popov, S., (1992) Determination of phenolics from propolis by capillary gas Chromotography, Journal of Chromatography, 607: I, 150-153 4. Christov, R., Bankova, R., (1992) Gas chromatographic analysis using an electron capture detector, Journal of Chromatography, 623: I, 182-185 5. Barberan, T., Garcia, c., Olivier, P., Ferreres F., (1993) Phytochemical Evidence for the Botanical Origin of Tropical Propolis from Venezuela., Phytochemistry, 34--1, 191-196. 6. Omar, M. (1989) Some characteristics of pro polis from Upper Egypt. Proceedings of the Forth Int. Conference on Apiculture in Tropical Climates, Cairo, Egypt., 6-10 Nov. 1988, 1989, pp 8&--92. 7. Albore, G.R., (1979) I 'origine Geographique de la Propolis. Apidologie, I 0(3),241-267. 8. Grunberger, D., Banerjee, R., Eisinger, E.M., Oltz, L., Efros, M., Coldwell, v., Nakanishi, K., Preferential Cytoxicity on Tumor Cells by Caffeic Asid Phenethyl Ester Isolated from Propolis. Propolis Research, North YorkshireYO 138 HT., 1995, pp.79-86. 9. Gafar, M., Sacalus, A. M., Treatment of Common Chronic Recurring Aphthae with Propolis, A Remarkable hive product propolis, Apimondia Publishing House-Bucharest- Romcnia, 1978, pp. 133-138. 10. Keskin, N., Hazir, S., Sorkun K., Dogan, c., (1995) Antibacterial activity of Propolis extracts, 34. Congress Apimondia Lausenne- Switzerland. II. Arkan 0., Sorkun, K., Dogan, c., GuIer, P., (1995) Mycelial forms of Morchella conica Pers. on the Nutrition with pollen and propolis, 34. Congress Apimondia Lausenne-Switzerland. 12. Moise, A., Hooper, T., Encyclopedia of Beekeeping, Butler and Tenner Ltd. U.K., 1985, pp. 308--309. 13. Saeed, F.A., Mohamed, S.H., (1992) The Influence of Propolis Extracts on Soy-bean and Sunflower Wild Disease, Assiut-lournal of Agricultured Science, 23-2, 190 14. Fahmy, F.G., Omar, M.O.M., (1989) Potential use of Pro polis to control white rot disease of onion, AssiutJournal of Agricultural Sciences, 20: 1,265-275. 15. Fahmy, F.G., Omar, M.O.M., (1988) Effect of Propolis extracts on certain potato viruses., Fourth International Congress on Agriculture in Tropical Climates, Cairo, Egypt, 6-10, November. 16. Derevici, A., Popesco, A., Popesco, N., (1964) Research on Certain Biological Properties of Pro polis., Annals Abeille, 7 (3), 191-200. 17. Ghisalberti, E.L., (1979) Propolis, A Review International Bee Research Assosiation, Thirtieth Annual General Meeting, U.K. pp.75. 18. Gonnet, M., (1968) Proprietes phytoinhibitrices de quelques substances extraites de la colonie d' abeilles (Apis mellifica L.) I. Action sur la crosissance de Lactuca sativa. AnnIs Abeille 11(1):41--47. 19. Abdou, R.F., Omar, M., (1988) Effect of the bee product propolis on the mitotic cells of Vicia faba, Hordeum vulgare, Allium cepa and Allium sativum, Assiut-Journal of Agricultural Sciences. 19:2, 159-170.

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20. Sorkun, K., Bozcuk, S., Investigation of the effect of Propolis on Seed Germination of some Culture Plants., XII. Ulusal Biyoloji Kongresi 6--8 Temmuz, Edirne- TURK lYE, 1994, pp.54--59. 21. Zimonjic, D., Giga, D., Popeskovi<;: D., (1989) Propolis Induces Decrease of Mitotic Activity but Does not Effect SCE Frequency in Cultured Human Lymphocytes, Acta- Veterinaria, 39 (1),11-18. 22. Bianchi, E.M., (1995) The Preparation of the Tincture, The soft Extract, The Oinment, The Soap and Other Propolis-Based products, Apiacta 3--4, 121-127. 23. Krol, w., Scheller, S., Shabi, 1., Pietsz, G., Czuba, Z., (1993) Synergistic Effect of Ethanolic Extract of Propolis and Antibiotics on the Growth of Staphylococcus aureus. Arzneim.-Forsch.l Drug Res. 43 (I), 607--60 24. Snedecor, G.W. and Cochran, w.G. (1979) Statistical Methods, Iowa State Univ" Ames Iowa, U.S.A.

18

THE EXOCRINE GLANDS OF THE HONEY BEES Their Structure and Secretory Products

Pierre Cassier' and Yaacov Lensky2 'Universite Pierre et Marie Curie Laboratoire d'Evolution des Etres organises 105 boulevard Raspail, 75006 Paris, France 2The Hebrew University of Jerusalem, Faculty of Agriculture Triwaks Bee Research Center 76100 Rehovot, Israel

The insect societies are stable structures submitted to various qualitative and quantitative regulations funded on the perception of mechanical, acoustic, visual and mainly chemical stimuli. The chemical informations provided by the biotic environment are named allomones if they trigger a defensive behaviour, kairomone if they allow an attractive one to the emitor. The pheromones secreted by conspecific individuals, belonging or not to the same group or society, act immediatiy on the behaviour (primer) or after a lag time on the physiology (releaser) of the receptive insect. The blend or pheromonal bouquet is truly the identity card of each insect. The honey bee (Apis mellifera L.) possess numerous exocrine glands (Fig. 1) at the level of the buccal apparatus (mandibular, hypopharyngeal, genal, and labial glands), of the sting apparatus (Koschewnikow glands, sheaths of the sting, setaceous membrane), of the legs (tarsal glands), of the abdominal tergites (Nassanof or Renner glands) or of the abdominal stemites (wax glands). Their pheromonal and volatile secretions are always mixed with nonvolatile, proteinic or glycoproteinic ones. They act separatly, or synergistically according to multiple schemes, in the relations between the members of the society, in the regulation of various functions : reproduction, breeding, wax building, thermoregulation, territorial defence, swarming, alarm, foraging activities, caste differentiation, recruitment, ... and in the location and the optimal working of each nutritive source. So, the society of the honey bee is frequently compared with an homeostatic ally regulated superorganism. The exocrine glands of the queen, drones and workers of the Honey bee belong to types I and II as defined by Noirot and Quennedey (1). They have already been described with some details (2, 3, 4). The structural, cytological parameters as well as the ontogenetic and analytical ones allow to suggest the level of the activity, the interactions and, to a certain extend, the nature of the secretory products.

137

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P. Cassier and Y. Lensky

Figure 1. The exocrine g lands of the honey bee. External and internal views. I... VIII : tergites I to VII ; Abd : abdomen ; Ant: antenna; DG : Dufour gland; H : head; HG : h ypopharyngeal gland; Md : mandible ; MG : m andibular gland; NG: Nassanofgland; 0: ocelli ; Prb: probosci s; SG : salivary gland: St : sting; TG : tergal glands ; Th : thorax : WI , W2 : wings I and 2; WG . wax gland .

I. THE SECRETORY EXOCRINE GLANDS OF THE TYPE I In the type I (Fig. 2), the epidermal and glandular cells are directly apposed to a porous cuticle; enlarged pore canals and epicuticular pores allow secretory products to move to the exterior throught the cuticular barrier. There is no intermediate cell. The secretory product is mainly a non-proteinic one. 1. 1. The tarsal glands or glands of Arnhart (5) are located into the fifth tarsomere of each leg of the queen, the drones and the workers (2) but the level of activity varies according to the sex and the physiological state of the bee (Table I). They are lined by a thick palisade-like epithelium (50--80 J..lm) and by a thin cuticular intima of which only the inner endocuticular layers (2-5 J..lm) are associated with the apical part of the glandular cells. The outer layers edge the central reservoir. The cytoplasm contains numerous smooth and coated vesicles, mitochondria , lipid droplets, a well developed smooth endoplasmic reticulum, some cisternae of rough endoplasmic reticulum (6 , 7). The oily, colourless, alcohol soluble secretory product is extruded through an articular slit located between the 5th tarsomere and the arolium and forms a trail (foot-print) behind moving bees. The study of the foot-print substance using gas chromatography and mass spectrometer (GC-MS) procedures allows to detect at least 120 fractions (alkanes, alkenes, alcohols, organic acids, esters, aldehydes, ... ) and to identified 27 compounds (C S-C 3S ; MW : 86 - 492). Eleven fractions are specific of queens, 10 of workers and 2 of drones . The others compounds are also detected in the secretion of various exocrine glands of the honey bee (8, 9). The role of the foot-print secretions is only partly known. In the queen bees, the secretions of the tarsal and of the mandibular glands act synergistically and inhibit the construction of swarming cups and cells (10). In the workers bees, the tarsal secretions are not directly involved in the orientation of the foragers to the hive entrance or to the food sources; their frequently emphazised attractive power is linked to contamination by components of the flowers, by others bees, or by nectar regurgitation. Also, they are not involved in the attraction of flying drones, in the acceptance of grafted female larvae in

Exocrine Glands of the Honey Bees

139

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queen cups, etc ... (6) . Likely, the «adhesive» tarsal secretion acts synergistically as «slow release substance» trapping pheromones produced by various exocrine glands (mandibular glands, Nassanof glands, Koschewnikow glands, tergal glands).

1. 2. The proximal parts of the sting sheaths of the honey bee workers have all the characteristics of very primitive exocrine glands: porous cuticle, enlarged pore canals and epicuticular pores, hypertrophied epithelial cells secreting an electron dense material. The non-volatile part of the secretory product embedded the setae of the sheaths.The intensity of alarm behaviour triggered by the volatile components of the Koschewnikow glands was much lower than that elicited by sting sheaths. The volalile compounds released by both the sting sheaths and Koschewnikow glands launch a recruiting activity and hence may have a synergistic effect. The chemical identity of the volatile components released at the level of the sting sheaths is always unknown (11,12 , 13). 1. 3. Of all the organs tested the setaceous membrane (tergum IX) volatiles (isoamyl acetate, isoamyl alcohol, hexyl acetate, nonanol, benzyl acetate, benzyl alcohol) elicited the strongest reactions of the guards (II, 14). Attraction : setaceous membrane > sting sheaths > quadrate plates and Koschewnikow glands > venom glands and venom sac > Koschewnikow glands > Dufour gland and pure venom.

Table I. Secretory activity of the tarsal glands. Quantitative data. (Lensky et coil., 1984; Hyams, 1988) Categories 3-day-old queens 6-month-old queens 18-24-month-old queens Workers Drones

Number n of insects 5 8 8 70 70

Mg / hour / bee x ± SD 0.2180 ± 0.6500 ± 0.4660 ± 0.0718 ± 0.0630 ±

0.009 0.051 0.038 0.020 0.015

140


Alarm: sting sheaths> setaceous membrane> venom gland and venom sac. Stinging: setaceous membrane> sting sheaths> quadrate plates and Koschewnikow glands> venom sacs. The setaceous membrane is directly connected to the sting sheaths and is disposed all around the base of the sting. The fine structure of the epithelium of the setaceous membrane of workers and queens is similar and does not show any characteristic of an exocrine gland. The flexible, untanned cuticle (4--5 !lm) rests on a flattened, inactive epithelium (3.5-4.5 !lm). It is abundantly covered with cuticular infoldings and multiforked bristles which, in the vicinity of the sting sheaths are embedded in an electron densc substance as on the sting sheaths. These cuticular expansions increase the surface of the evaporation area; so, the outer surface of the setaceous membrane is ideally suited for the discharge of pheromonal secretions from the sting sheaths and the Koschewnikow glands. The function of the setaceous membrane is to serve as a platform for the discharge of alarm pheromones that are secreted elsewhere and accumulate on its surface. I. 4. The wax gland complex (15, 16) of the honey bee workers consists of three cell types: epithelial cel1s, oenocytes and adipocytes, which act synergistical1y to secrete wax, a complex mixture of hydrocarbons, fatty acids and proteins (lipophorins). The hydrocarbons coming from the oenocytes and the proteins (17) from the haemolymph transit across the epithelium via the large smooth endoplasmic reticulum cisternae connected to extracellular spaces and through the mirror cuticular plates along a wel1 developed extracellular and pore canal filamentous system linked to wax canal filaments of the epicuticle. There is no excretory ducts or intermediate cel1s. The height of the epithelial cells is agedependent; in freshly emerged workers « one week old) the epidermis is a flat epithelium. At the height of wax secretion (2 week-old) the lengthened cells have a fibrillated appearance. In fact, the secretion of wax is a constant process even in overwintering workers. During the ageing of the workers, the fat body cells and the oenocytes show the volumetric variations similar to the epithelial cells. The wax may act as a slow release substance for pheromones secreted by other exocrine glands. The volatile and odoriferous part of the wax secretion may also act as a pheromonal component implies in the regulation of the wax secretion or building?

II. THE SECRETORY EXOCRINE GLANDS OF THE TYPE III In the type III (Fig. 3) exocrine glands (2, 3), each glandular cell shows a central reservoir lined by the invaginated plasma membrane; this membrane forms a10 () numerous cristae and microvilli; the content of the secretory vesicles is poured between them. The glandular cell is caped by a tubular cell named the duct cell because it secrete, in the central cavity, a thin duct edged by an epicuticular wall. In the reservoir of the glandular cell, the proximal part of the duct (end-apparatus) has a porous structure permeable to the secretory products. So, each glandular cell and its associated duct cell form a glandular unit. The ducts of the glandular units are connected either directly to the external cuticle (ex. : Nassanov, Renner or Koschewnikow glands), or, in the most developed conditions, to a general excretory canal (ex. : mandibular glands). Glandular cells involved in the secretion of non-proteinic pheromones contain numerous mitochondria and a well developed endoplasmic. During the secretory process mitochondria undergo maturation (swelling, disappearance of cristae and inner membrane, sticking to endoplasmic membranes, accumulation of macromolecular substances), and generate secretory vesicles. Rough en-


141

Figure 3. Type III exocrine gland. BL : basal laminae; EC : epithelial cell; Ep : epicuticle; D :desmosome: DC : duct cell : GC : glandular cell ; Mt : microtubule; MVB : multi vesicular body; N :nucleus; Pc : procuticle; RER : rough endoplasmic reticulum: SD : spetate desmosome ; Tr : tracheole.

doplasmic reticulum and Golgi apparatus are implied in the synthesis of the proteinic part of the secretory products. II. I. The Nassanov gland or abdominal scent gland of the workers of the honey bee (18, 19) is a complex structure where the glandular units are associated with oenocytes and fat body cells; the respective part of these cells to the secretory activity of the Nassanof gland is to be elucidated using labelled precursors of the pheromonal or protein components (20). The Nassanov scent is used for orientation, particularly at the nest entrance, in swarm clustering, at water collection sites and possibly at flowers. The proteins may act as pheromone carriers, enzymes for pheromone degradation, slow release substances, caste or sex specific modulators of pheromone activity; they enhance the attractiveness of the volatile fraction. They may also form part or all of the surface cuticular proteinic repertoire . The pheromones produced by the Nassanov gland have been chemically identified; they are composed of the following terpenoids : geraniol, nerolic acid, geranic acid, (E)citral, (Z)-citral, (E-E)-famesol, nero!. If the geraniol is the major compound, the most attractive and efficient components are (E)-citral and geranic acid (18). II. 2. The Renner's glands or tergal glands (Figs. 4-10) of the queen located near the rear of tergites III, IV and V, (2, 21 , 22, 23, 24). The secretion of the tergal glands attracted drones to queens on their mating flights from distances of less than 30 cm and increased their mating activities. Queens with paraffin-coated tergites were about 16 % less attractive than queens with no coating (25). As the mandibular gland secretion, the tergal gland products are also attractive to workers at distances of several centimeters; so, in the queen's retinue the honey bee workers lick her with their tongues or antennates her abdomen. This behaviour stabilizes the court once it has been formed and likely participate in the inhibition of the ovaries of the workers. The general organization of the Renner gland of the queen bee is similar to the Nassanov glands of the workers although they are located in different segments. So, the type III glandular units are intimately associated with fat body cells and oenocytes. The coexistence of rough and smooth endoplasmic reticulum in the glandular cells suggests that they are able to synthesize two kinds of products: the non-volatile macromolecular protein components and the volatile, small molecular, phero-

142


Figure 4. Tergal glands of the queen bee. Scanning electron microscopy. Dorsal view of a tergite (T) and of an articular membrane (AM) showing glandular pores (frame).

monal components. Although, Renner and Baumann (1964) and Sanford (1977) suggested that the gland is active throughout a queen's lifetime, there is no convincing evidence concerning the age-dependence of tergal gland secretion. The various analysis of the tergal gland secretions are only preliminary ones. De Hazan et al. (26), after wiping of the tergal gland areas, have identified by GC-MS 14 compounds (Table II). Six compounds are specific for tergal gland secretion: benzoic acid, 9-hexade-

Figure 5. Tergal glands of the queen bee. Scanning electron microscopy. Detail of the articular membrane showing cuticular scales (Sc) and ovoid or ci rcul ar glandular pores (arrows).


143

Figure 6. Tergal glands of the queen bee. Scanning electron microscopy. Inner view of the cuticular membrane with glandular cell (GIC), ducts (d) connected to cuticular holes (arrow). Oe : oenocytes.

cenoic acid, 9, 12-octadecadienoic acid, 2-dodecanol, 5-methyl-cyclo-hexanal, 2-decanal. The others compounds are also present in other exocrine glands as follows: 1- mandibular glands : diethyl-I ,2-benzene dicarboxylate, 3-methyl-2,6-dioxo-4-hexenoic acid, hexadecanoic acid, tetradecanoic acid, tetratetracontane and 1,2-dodecadiene (27, 28); 2- tarsal glands: hexadecanoic acid and 3-methyl-2,6-dioxo-4-hexenoic acid (8); 3- Koschewnikow glands of 4-dayold queens: tetradecanoic acid and hexadecanoic acid (29). These results are not consistent with those of Espelie et al.. (30) who prepared the extracts for GC-MS analysis from dissected tergites of 1- to 4-day-old italian queen bees.

Figure 7. Tergal glands of the queen bee. Scanning electron microscopy. Detail of a group of glandular cells (GlC) with their duct (d). Tergal glands of the queen bee. The glandular unit.

Figure 8. Tergal gland of Ihe queen bee. Eleclron microscopy. General view of a glandular cell (GlC) under Ihe cUlicular inlima (CUI) of Ihe arlicular membrane. BL : basal laminae; M : milochondria; N :nucleus; n : nucleolus; R : reservoir; SV : secrelory vesicle .

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Figure 9. Tergal gland of the queen bee. Electron microscopy. Detail of the central part of the reservoir with the tenninal part or end apparatus of the duct showing the perforated inner epicuticle and the reticular outer epicuticle (arrow). m : microvilli.

II . 3.The Koschewnikow glands are associated with the sting apparatus of the majority of Apidae, Vespidae and primitive Formicidae (29, 31 , 32). In the workers and queen of the honey bee they show the same localization and structure but glandular products and ontogeny related to aging are different. The Koschewnikow glands are present in the 7th abdominal segment and appear as a wide cellular mass (workers: 450 x 200 x 60 ~m; queens: 600 x 330 x 60 ~m) organized along the intersegmental membrane joining the quadrate plate and the spiracular plate. They are composed of about 600 type III secretory units each including a large glandular cell and a narrow duct cell. The secretions extruded by thin ducts, emitted on the intersegmental membrane can flow into the sting chamber and reach the setaceous membrane. In an old, egg-laying queen the thickness of the gland decreases to 30 ~m or less as a result of degeneration (33). The secretory cells contain dense proteinic and lipidic granules in workers, glycoproteinic granules in queens . II. 3. 1. In the workers, the Koschewnikow glands secrete an alarm pheromone. When guard worker bee are disturbed at the entrance of the hive, they extrude their stings, expose their setaceous membrane while fanning and release alarm pheromone (34, 35)

146


Figure 10. Tergal gland of the queen bee. Electron microscopy. Peripheral part of a glandular cell (GIC) with numerous mitochondria (M) and well developed smooth and rough endoplasmic reticulum (arrows). BL : basal laminae.

Table II. Chemical composition of the tergal glands secretions of 1- to 4-day-old queens I.

Benzoic acid

C7H,O,

2.

2-decanal

C,oH 18 O

3.

3-methyl-2.6-dioxo-4-hexenoic acid

C 7H,04

4.

1,2-dodecadiene

C"H 2O

5.

Cyclododecane

C"H24

6.

2-dodecanol

7.

S-methyl-cyclohexanal

C"H 2O C,oH,oO

8.

Diethyl-I ,2-benzene dicarboxylate

9.

Tetradecanoic acid

C"H'404 C14 Hn02

10.

9-hexadecenoic acid

C'6 H,OO,

II.

Hexadecanoic acid

C"H 32 O,

12.

9, 12-octadecadienoic acid

13.

9-(Z)-octadecenoic acid

14.

Tetratetracontane

C'8 HJ2 0 , C 18 H'40, C 44 H9


147

rich in isoamyl acetate,but containing also isoamyl alcohol, hexyl acetate, nonanol, benzyl acetate, benzyl alcohol, etc ... (36). II. 3. 2. The queens secrete a « stress pheromone» (Lensky et coll., 1991) which may elicit: a.- an aggressive behaviour between two queens, following mutual detection, which in general results in the death of one of them;b.- The balling behaviour of worker bees towards either an introduced or one of multiple queens in the nest, except during swarming season. Topical treatment of worker bees (<< pseudoqueens) with ethanolic extracts of queen Koschewnikow glands induced typical queen balling behaviour in workers of a bee colony. It seems assume the maintenance of a monogynous status of each bee colony. Twenty-eight compounds including acids, alcohols, alkanes and alkenes (C SHI6 C43 HRS ) were characterized by GC-MS in queen extract. None of them is present in worker alarm pheromone which is secreted from worker Koschewnikow gland (29). II. 4.The mandibular glands of the queen (25, 37) and workers (3S) of the honey bee are paired organs connected to the basis of the mandibles where the secretory products can flow along a grove to the spatular end. In the two castes the mandibular glands show the same general organization: they consist of an axial cavity (2S0 x 90 11m) lined by a thin cuticular intima (4 11m) elaborated by a flat epithelium and of a secretory epithelium exclusively composed of type III glandular units. Endoplasmic reticulum and mitochondria are preponderent organelles. The maturation of these last organelles generates an oily secretory product (3S). In the drones, the reduced mandibular glands are exclusively composed by the reservoir lined by a flat epithelum; there is no glandular units (39). n. 4. I. The pheromonal activity of the mandibular glands secretion of workers has been partially explored. A « queen substance-like» compound, the I 0-hydroxy-trans-2decenoic acid has been isolated (40), as well as a ketone, 2-heptanone which, according to several authors, acts synergistically with isopentyl acetate from Koschewnikow glands as an alarm pheromone (3). During the ageing of the workers, the size of the gland and the amount of 2-heptanone per headspace sample progressively increase from 0.1 III at emergence to 7 III in foraging bees. This increasing quantity of 2-heptanone si correlated to the increase of hemolymphatic juvenile hormone ITT (41). Similar age-dependent changes of 2-heptanone were found in mandibular glands of workers from docile and aggressive colonies and the amount of 2-heptanone in the two groups was alike. Neither crushed glands nor 2-heptanone showed any direct effect as an alarm pheromone on guardian bees at hive entrance. 2-heptanone had either an attractive or a repUlsive effect on gards, according to the season. It showed a repulsive effect when added to sucrose solution which was visited by foragers. 2-heptanone had a temporary repulsive effect on the visitation of flowers by foraging bees, hence it seems to act as a « foraging-marking pheromone» (3S). II. 4. 2. The pheromonal secretions of the honey bee queen's mandibular glands play an important role in the control of social activities and in communication among the colony members. The gland secretion is a complex mixture of different compounds some of which remain to be chemically identified (2, 3, IS). The problem is a very complex one because the compounds are progressively modified during the ageing of the queen bee (25, 2S, 37). The most important compound, the queen substance, the 9-oxo-trans-2-decenoic acid, is known since 1960. The pheromonal secretions concern: attraction of workers to the queen, attraction of the drones during the nuptial flights, court formation, inhibition of ovaries, level of foraging, inhibition of the construction of swarming cups and cells, cohesion of the swarming group (IS). So, the queen substance is a sexual and social pheromone and an anti-gonadotropic hormone. It acts as a releaser or as a primer. The highest activity occurs in the 6- to IS-month-old queens; in IS- to 24-month-old

148


queens the reduction of the secretory and pheromonal activities are associated with the reduction of the mitochondrial population as well as with the accumulation of lipid droplets and various lytic structures. II. 4. 3. The nature of the pheromonal secretion of the mandibular glands of the drones is unknown but its acts synergistically with the queen substance as recruitment pheromone during the mating flights. The secretory product appears in the mandibular glands of 3-, 4-day-old drones. In the 7 -, 9-day-old drones the involution of the mandibular glands begins; in the Il-day-old drones the glands are reduced to the cuticular intima (39).

CONCLUSION Owing to the diversity of the exocrine glands and of their pheromonal blend, the honey bees are provided with a tremendous chemical repertoire adapted to each sex, caste, period or physiological state, function. The chemical diversity contrasts with the relative uniformity of the glandular structures suggesting endogenous and exogenous regulations; so, in spite of the same general organisation the secretions of the mandibular glands of the workers and of the queens are quite different. Moreover, if it is difficult to appreciate the exact function of each compound of the pheromonal blends, it is necessary to take also account of the temporarily variable chemical messages emitted by the flowers, the stored products (nectar, honey, wax, propolis). These informations have according to time and physiological state of the bees synergistic or antagonistic effects. The pheromones are only a part of the social communication of bees; the language or dances of the bees is an achieved mean of communication involving mechanical, chemical, chemical stimuli.

REFERENCES 1. Noirot Ch. and Quennedey A. (1974) Fine structure of insect epidermal glands. Ann. Rev. Entomol.. 19, 61-80. 2. Cassier P. and Lensky Y. (1992) Structure et role social de quelques glandes exocrines it secretion pheromonale chez I' Abeille domestique Apis melli/era L. (Hymenoptera: Apidae). 1.- Glandes tarsales, glandes tergales et glandes de Koschewnikow. Annee BioI., 31, 61-78. 3. Cassier P. and Lcnsky Y. (1992) Structure et role social de quelques glandes exocrines it secretion pheromonale chez I' Abeille domestique Apis melli/era L. (Hymenoptera: Apidae). 11.- Glandes mandibulaires. Annee BioI., 31, 78-95. 4. Lensky Y. and Cassier P. (1995) The alarm pheromones of queen and worker honey bees. Bee World, 73, 3, 119-129. 5. Arnhart L. (1923) Das Krallenglied der Honigbiene. Arch. Bienenk., 5, 37-86. 6. Lenky Y., Cassier P., Finkel A., Teeshbee A., Schlesinger R., Delorme-loulie C. and and Levinsohn M. (1984) Les glandes tarsales de l'Abeille mellifique (A pis melli/era L.) reines, ouvrieres et faux-bourdons (Hymenoptera, Apidae). 11.- Role biologique. Ann. Sci. Nat., Zool., 18eme serie, 6, 167-175. 7. Lensky Y., Cassier P., Finkel A., Delorme-joulie C. and Levinsohn M. (l985a) The fine structure of the tarsal gland of the honeybee Apis melli/era L. (Hymenoptera). Cell Tissue Res., 240,153-158. 8. Finkel A. (1983) Biological effects and chemical characterization of the footprint substance of the honeybee (Apis melli/era). M. Sc. Thesis, Hebrew University of Jerusalem, 46 pp. 9. Lensky Y., Finkel A., Cassier P., Teeshbee A. and Schlesinger R. (1887) The tarsal glands of the honeybee (Apis melli/era L.) queens, workers and drones. Chemical characterisation of foot-print secretions. Honeybee Science, 8, 97-102. 10. Lensky Y. and Siabezki Y (1981) The inhibiting effect of the queen bee (Apis mellifera L.) foot-print pheromone on the construction of swarming queen cups. 1. Insect Physiol., 27, 313-323.


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II. Cassier P., Tel-Zur D. and Lensky, A. (1994) The sting sheaths of honey bee workers (Apis mellifera L.) : structure and alarm pheromone secretion. 1. Insect Physiol., 40, 23-32. 12. Tel-Zur D. (1993). Alarm pheromones of the sting apparatus of the honey bee (Apis mellifera L. var ligustica Spin.). Ph. D. Thesis, Hebrew University of Jerusalem, Israel, 1\0 p. 13. Tel-Zur D. and Lensky Y. (1995) Bioassay and apparatus for measuring the stinging response of an isolated worker honey bee (Apis mellifera L. var /igustica Spin.). Compo Biochem. Physiol. A, in press. 14. Lenky Y., Cassier P. and Tel-Zur (1995) The setaceous membrane of the honey bee (Apis mell!fera L.) worker's sting apparatus: structure and alarm pheromone distribution. J. Insect Physiol., 41, 7, 589-595. 15. Hepburn H. R. (1986) Honey bee and wax. Springer, Berlin, Germany. 16. Cassier P. and Lensky Y. (1995) Ultrastructure of the wax gland complex and secretion of beewax in the worker honey bee Apis mellifera L. Apidologie, 26, 17-26. 17. Kurstjens S. P., McClain E. and Hepburn H. R. (1990) The proteins of the beewax. Naturwissenchaften, 77. 34--35. 18. Winston M. (1991) The biology of the honey bee. Belknap Press, Harvard University Pres, Cambridge, MA, 281 p. 19. Cassier P. and Lensky Y. (1994) The Nassanov gland of the workers of the honey bee (Apis mellifera L.). Ultrastructure and behavioural function of the terpenoid and protein components. J. Insect Physiol., 40, 577-584. 20. Zupko K., Sklan D. and Lensky Y. (1993) Proteins of the honey bee (Apis mellifera L.) body surface and exocrine glands. J. Insect Physiol., 39, 41-46. 21. Renner M. and Baumann M. (1964) Uber Komplexe von subepidermalen Driisenzellen (Duftdriisen ?) der Bienenkonigin. Naturwissenchaften, 51, 68-69. 22. Sanford M. T. (1977) An ultrastructural study of the subepidermal glands of the queen honey bee (Apis mellifera L.). Ph. D. Thesis, University of Georgia, Athens, GA, 110 p. 23. Sanford M. T. and Dietz A. (1981) The fine structure of the subepidermal glands of the queen honey bee (Apis mellifera L.). 28th Congo Apimondia, Acapulco, 287-292. 24. Billen J. P., Dumortier K. T. M. and Velthuis H. H. W. (1986) Plasticity of honey bee castes: occurrence of tergal glands in workers. Naturwissenchaften, 73, 332-333. 25. De Hazan M., Lensky Y. and Cassier P. (1989). Effects of queen honeybee (Apis mellifera L.) ageing on her attractiveness to workers. Compo Biochem. Physiol., 93A, 777-783. 26. De Hazan M., Lensky Y. and Cassier P. (1991) The chemical composition of the pheromonal secretion from the tergal glands of the queen bee Apis mellifera L. 27. De Hazan (1986) The attractiveness, composition and structure of mandibular glands of the queen bee (Apis mellifera L.). M. Sc. Thesis, Hebrew University of Jerusalem, 34 p. 28. Hyams Y. (1988) Characterisation of pheromonal components in the tarsal and mandibular glands of the queen honeybee (Apis mellifera L.). M. Sc. Thesis, Hebrew University of Jerusalem, 42p. 29. Lensky Y., Cassier P., Rosa S. and Grandperrin D. (1991) Induction of balling in worker honeybees (Apis mellifera L.) by « stress» pheromone from Koschewnikow glands of queen bees: behavioural, structural and chemical study. Compo Biochem. Physiol.. Ser A. 100. 3, 585-594. 30. Espelie K. E., Butz V. M. and Dietz A. (1990) Dodecyl decanoate: a major component of the tergite glands of honey bee queens (Apis mellifera L.). J. Apicult. Res., 29,15-19. 31. Grandperrin D. and Cassier P. (1983) Anatomy and ultrastructure of the Koschewnikow's glands of the honey bee, Apis mellifera L. (Hymenoptera, Apidae). Int. 1. Insect Morphol. Embryol. 12,25-42. 32. Cassier P., Lensky Y., Rosa S., Grandperrin D., Teeshbee A. and Schlesinger R. (1990) Declenchement du comportement d'emballage des ouvrieres de l'Abeille domestique (Apis mell!fera L.) par la pheromone de « stress » des glandes de Koschewnikow de la Reine. Etude ethologique. structurale et chimique. Xth. Coli. Physiol. Insectes, Toulouse (France), 19-21 septembre. 33. Grandperrin D. (1981) La glande de Koschewnikow. Origine de la pheromone du dard chez I'ouvriere de l' Abeille (Apis mellifera L.). These 3eme cycle, Universite P. et M. Curie, Paris, 51 p. 34. Huber F. (1814) Nouvelles observations sur les Abeilles. Paschoud edit., Paris, Vol. I (362 p) et Vol. II (479 pl. 35. Maschwitz U. W. (1964) Alarm substances and alarm behaviour in social Hymenoptera. Nature, London, 204, 324--327. 36. Mauchamp B. and Grandperrin D. (1982) Chromatographie en phase gazeuse des composes volatils des glandes 11 pheromones des Abeilles : methode d'analyse directe. Apidologie, 13,29-37. 37. De Hazan M., Hyams Y., Lensky Y. and Cassier P. (l989b) Ultrastructure and ontogeny of the manibular glands of the queen honeybees, Apis mellifera L. (Hymenoptera, Apidae). Int. J. Insect Morphol. Embryol., 18, 311-320.

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38. Vallet A., Cassier P., Lensky Y. (1991) Ontogeny ofthe fine structure of the mandibular glands ofthe honeybee (Apis mellifera L.) workers and the pheromonal activity of the 2-heptanone. J. Insect Physiol., 37, II, 789--804. 39. Lensky Y., Cassier P., Notkin M., Delorme-Joulie C. and Levinsohn M. (\985) Pheromonal activity and fine structure of the mandibular glands of honeybee drones (Apis melli/era L.) (Insecta, Hymenoptera, Apidae). J. Insect Phusiol., 31, 4, 265- 276. 40. Butler C. G. (1966) Mandibular gland pheromone of worker honeybees. Nature, London, 212, 530. 41. Cassier P. and Lensky Y. (\991) Evolution du titre de I'hormone juvenile III. des ecdysterordes et d'une pheromone, la 2-heptanone, en relation avec Ie polyethisme des ouvrieres de I' Abeille domestique (Apis melli/era L. var. ligustica ) (Hymenoptera; Apidae). C. R. Acad. Sci. Paris, 312, serie III, 343-348.

19

ALARM PHEROMONES OF THE QUEEN AND WORKER HONEY BEES (APIS MELLIFERA L.) Yaacov Lenskyl and Pierre Cassicr2 ITriwaks Bee Research Center Hebrew University, Faculty of Agriculture 76100 Rehovot, Israel 2Universite Pierre et Marie Curie Laboratoire d Evolution des Etres organises 105 boulevard Raspail, 75006 Paris, France

Both queen and worker honey bees secrete specific alarm pheromones which trigger different behavioural patterns in each female caste.

1. THE ALARM PHEROMONE OF THE QUEEN The society of the honeybee is basically a monogynous one. Attempts to achieve cohabitation of multiple queens of identical or different ages in the same brood-nest, without being confined to a cage or a compartment, were unsuccessful due to mutual aggressive behaviour. Polygynous societies headed by multiple queens can be established when queens are separated by a mechanical barrier, such as a queen excluder (I, 2, 3, 4, 5). However, in the absence of a physical barrier in the brood nest, queen bees launch fatal battles until only one survives. Virgin queens are more aggressive than mated ones (6). Independently of the mutual aggressiveness of queens, worker bees also playa major role in the elimination of supernumerary queens in experimental or feral colonies. Multiple queens are tolerated in a bee colony only during the swarming period (7, 8,). Even following the severing of mandibular tips and stingers of the queens to prevent mutual aggression (9) and their successful introduction into a single brood chamber of a bee colony, all but one were balled by workers and eliminated 4 to 6 weeks later (8). It is generally believed that pheromonal secretions from queen mandibular glands affect the aggressive behaviour of workers towards queens (10, II). However, volatile compounds originating from the sting apparatus or its vicinity are likely to be involved in the mutual recognition of queens, as well as in evoking aggressive behaviour of workers towards queens ("balling"). 151

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Y. Lensky and P. Cassier

Worker Koschewnikow glands (WKG) secrete alarm pheromone (12, 13), but Koschewnikow glands of queens (QKG) may have a different function. Although Butler and Simpson (14) attributed to the secretion of QKG an olfactory non-specific attractive effect on workers, their function has not yet been established. The aggressive behaviour of workers towards queens is welI documented: balled queens frequently opened their sting chambers, protruding their stingers and sting sheath. Balling workers were attracted to the sting sheath and licked it (15). Also, a queen's abdomen is more attractive to workers than the head, presumably due to the secretion of tergal and Koschewnikow glands (16). It seems that some pheromonal secretions of queen bees may elicit: (a) aggressive behaviour between two queens, following mutual detection, which in general results in the death of one of them, and; (b) balling behaviour of worker bees towards either an introduced, or one of multiple queens in the nest, except during swarming season. The balling behaviour of workers has been explained by a "stress pheromone" hypothesis : a disturbed queen produces a "stress pheromone" that stimulates an attack on her (balling) and her own death (17, IS). However, the glandular origin and chemical composition of the "stress pheromone" have not yet been established. Studies were carried out to document the reaction of workers to extracts from QKG and WKG and to induce in workers balling behaviour of a "pseudoqueen" (PQ) treated with QKG. To avoid the effect of possible pheromonal secretions from queen mandibular, tarsal (19) and/or tergal glands (16), we used worker bees as (PQ), following their treatment with QKG extracts. To analyse the composition of QKG we used gas liquid chromatography coupled with mass spectrometry. The observations of the balling behaviour of the PQ by the sister workers were carried out in glass observation hives according to the procedure previously described (20).

1.1. Reaction of Workers to PQ Covered with either Ethanol Extracts

of QKG or WKG Extracts 1.1.1. PQ Covered with QKG Extract. Each introduced worker pseudoqueen was immediately surrounded by hive bees, who formed a dense ball around her, consisting of about 15-35 workers. The "ball" around the PQ persisted for 5 to 10 min, after which about two workers remained. Approximately 4 min later, they abandoned the PQ. Workers that participated in balling of the PQ displayed aggressive behaviour, such as grasping and biting of the wings and hind legs, as well as pulling. We also observed non-aggressive behaviour of workers participating in the ball, such as antennating and licking. Of 10 PQ, seven were balled; two were aggressively attacked following their introduction, but there was not any dense ball formation of workers; and one PQ succeeded in escaping from the hive and flew off after being attacked by workers. None of the nine PQ that were balled died. When the balling was terminated, the PQ could move around, but in most cases their wings were damaged. 1.1.2. PQ Covered with WKG Extract. Immediately after introduction, about 6--S workers approached the PQ, pushed her and arched their abdomen as during stinging, while others only antennated her and then abandoned her. The hive workers did not display their aggressive behaviour continuously: they would approach the PQ and then retreat. This specific behaviour lasted for about 1.0--1.5 min.

Alarm Pheromones of the Queen and Worker Honey Bees

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1.2. Reaction of Workers to Control-PQ 1.2.1. PQ Marked with White Paint Only. PQ marked with white paint only did not arouse any attention inside the hive. marked 1.2.2. PQ Marked with White Paint and Covered with 2 III of Ethanol. PQ with white paint and covered with 2 III of ethanol were inspected and antennated by about five workers for about 30 sec.

1.3. Gas Liquid Chromatography-Mass Spectrometry (GC-MS) Twenty-eight compounds were characterized according to their molecular weights, which ranged from 112 to 604. The compounds (C8HI6-C43H88) included: acids, alcohols, alkanes and alkenes. None of these compounds is present in workers' alarm pheromone, which is produced in WKG (13). We demonstrated by topically treating worker bees with QKG a specific balling behaviour toward "pseudo queen" workers similar to that displayed towards queens. Since the workers used as pseudoqueens were removed from and introduced into their own colonies, we eliminated the effect of a foreign colony odour, which could have been responsible for aggressive behaviour towards an introduced worker or queen. The aggressive reaction of hive workers toward a worker covered with WKG extract was similar to that of guardian bees towards an intruding foreign worker during which alarm pheromone is released. However, this reaction di~ not resemble the behavioral pattern of "balling" of a queen bee. We observed several violent and non-violent activities towards the pseudoqueens treated with QKG extracts, as described in the case of balled queens (18, 15). The onset of balling of a pseudoqueen covered with QKG extract would occur almost immediately following her introduction into the observation hive, whereas it took about 8.5 min for the behaviour to begin toward a foreign queen, as reported by Robinson (15). Moreover, the relatively short balling duration of pseudo queens (7 min), as compared to that of an intact queen (58 min, 16) may be due to the rapid evaporation rate of ethanol extracts from the body surface of a worker versus continued secretion from the gland and its release from the setaceous membrane in the queen bee. One may presume that QKG secretions play an important role in the mutual detection of two queens and the subsequent duels between them. When their sting chambers were sealed off and/or antennae were covered with wax, queens were unable to detect each other and did not fight (8). In addition, the attraction of workers to queens can be modified by sealing off their tergal glands and sting chamber with paraffin (16). The pheromonal secretion of the QKG which is released on the setaceous membrane did not elicit any behavioural pattern that is characteristic of worker alarm pheromone. It assumes the function of a "suicide" pheromone and thus seems to be instrumental in the maintenance of monogynous status of each bee colony: a supernumerary queen or a pseudoqueen covered with the QKG extract is balled and may be eliminated. The pheromonal secretion of the QKG released on the setaceous membrane did not elicit any behavioural pattern that is characteristic of worker alarm pheromone. It assumes the function of a 'suicide' pheromone and, thus, seems to be instrumental in maintaining the single-queen status of a colony: a supernumerary queen or a pseudoqueen covered with QKG extract is balled and may be eliminated.

154


The chemical composition of pheromonal components of the WKG and QKG is entirely different. We have not detected any of the alarm pheromone components of worker bees (21, 22, 23) in QKG extracts. We do not preclude the possibility that other queen bee pheromones, such as those originating from the mandibular (10, II) and tergal glands act synergistically with QKG secretions (unpublished observations).

2. THE ALARM PHEROMONE OF THE WORKERS Honey bee workers defend their colonies against many intruders that may attack their nests to rob nectar, honey, pollen, brood or adult bees. The defence systems are essential for colony survival (27,28). Guards at hive entrances intercept all bees entering the nest and inspect them with their antennae. They are able to distinguish intruders and robbing honey bees from different colonies using odour and other cues. The colony-specific odour that adheres to the body of a bee is partly under genetic control and partly dependent on the nature of food collected (26, 29). When disturbed by an intruder, guards display a characteristic behaviour, which has been classified into the following four stages: alerting, activating, attracting and culminating (30). During the alerting and culminating stages the guard releases alarm pheromones either by opening the sting chamber and protruding the sting shaft, or as a result of stinging. The alarm pheromones produced by glands associated with the sting apparatus tag the intruder and attract other workers to continue stinging the attacker. The stinging reaction can be enhanced by dark colours, rough textures and movement (1). After a worker has thrust her stinger into an intruder's body and detached herself from the sting apparatus, the sting continues to penetrate deeper while injecting venom and, at the same time, releasing alarm pheromones to alert and recruit other workers. The alarm pheromones of a worker are believed to consist of 2-heptanone secreted by the mandibular gland and other components secreted from glands associated with the sting apparatus. The intensity of colony defensive behaviour is affected by the age of the workers and colony strength, as well as by genetic and meteorological factors (31). The intensity of the defensive reaction can be evaluated by several tests that examine the behaviour of individual workers or groups of workers at hive entrances (31, 32). We have recently developed an apparatus and bioassay that applies only the volatile components of alarm pheromones to stimulate either individual workers in the laboratory or groups of workers inside observation hives or at hive entrances (19, 33, 34). Guards and foragers produce 2-heptanone (2-H) in their mandibular glands, which has two pheromonal functions: (a) Alarm releasing behaviour with a lower efficacy than that of the sting apparatus (35, 36, 37, 38) ; (b) Releasing stinging behaviour as effectively as iso-pentyl acetate (31, 39) mainly secreted by the Koschewnikow glands (13). However, 2-H is from 20 to 70 times less potent than the alarm pheromone derived from the stinger (36) 2-H also possesses some repellent properties affecting foraging bees (35, 40, 41). Indeed, when 2-H was applied to alfalfa flowers, a short term repellence was observed (42). We studied the role of2-H (34, 43) as an alarm pheromone.

Alarm Pheromones ofthe Queen and Worker Honey Bees

155

2.1. The Changes of 2-H Levels According to the Age and Function of Workers 2-H was detected (43) in the headspace of mandibular glands of emerging workers (0.1 1l1/headspace); its level increased progressively with age, as did the level of juvenile hormone III (44), and peaked in guards and foragers (7 1l1/headspace). According to previous reports, the secretion of 2-H begins only between two and three weeks after emergence (35, 45, 46, 47). This discrepancy may be explained as follows: in these reports the researchers had extracted 2-H with a solvent from crushed head capsules of workers, whereas we used the headspace (43) of volatile substances, which were released from dissected and homogenized mandibular glands prepared for GLC analysis without using any solvent. A positive correlation between the level of 2-H and the aggressiveness of Italian, Africanized bees and of their hybrids has been reported (47). Our analysis did not show any significant difference between the level of 2-H of workers obtained from 'docile' Italian colonies and those from hybrid (Apis mellifera ligustica x A. m. syriaca) 'aggressive' colonies. Moreover, the amount of 2-H produced by A m. adansonii in South Africa was similar to that of A. m. ligustica from Canada (46). It seems that the intensity of defensive behaviour of different bee races cannot be attributed to the amount of 2-H produced by mandibular glands of workers.

2.2. The Role of 2-H as an Alarm Pheromone and as a Forage-Marking Pheromone Although 2-H is generally considered to be an alarm pheromone, our results did not support this view. Even the synergistic effect of the compound on the alarm pheromones originating from the sting apparatus is negligible (34). On the other hand, guards and foragers are repelled by 2-H; for instance, it has a short-term repellent effect on foragers visiting flowers of wax-flower shrubs which have been treated with 2-H. Hence, it seems to act as a 'forage-marking pheromone'. Our results confirm previous and recent reports (48, 49,50,51,52).

2.3. The Sting Apparatus and Associated Glands The sting apparatus which serves efficiently as a mechanical and chemical defence organ, is contained within the sting chamber between the tergal and sternal plates of the seventh segment. Several glands and organs are associated with the sting apparatus of the worker, which produce blends of odours used for alarm and colony defence (28) The venom gland, venom sac and Dufour's gland have not been reported to produce pheromones (32). The Koschewnikow glands located on the upper part of the quadrate plates are involved in the secretion of an alarm pheromone (32, 53) that accumulates on the setaceous membrane (13, 37,54). Although this membrane is the source of an important blend of pheromones released when the sting is protruded, it has not yet been established that the membrane constitutes the glandular source of this chemical signal so characteristic of disturbed bees (32). Other sting glands can also participate in the production of sting alarm pheromones. More than 40 compounds have been identified in extracts of the worker sting apparatus and six major compounds are releasers of alarm behaviour (30, 32, 42, 55). How-

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ever, only Koschewnikow glands have been demonstrated to produce at least some of the alarm pheromone components (12, 13). The intensity of defence reaction (alarm, attraction and stinging) of guards stimulated with different parts of the sting apparatus decreases according to the following order: whole sting > setaceous membrane > sting sheaths > venom gland and venom sac> venom> Koschewnikow glands> Dufour's gland (43).

2.4. The Glands and Organs Involved in the Secretion and Distribution of Alarm Pheromones 2.4.1. Koschewnikow Glands. The chemical composition of pheromonal components of the WKG and QKG is entirely different. Mauchamp and Grandperrin (13) detected isoamyl acetate, isoamyl alcohol, hexyl acetate, nonanol, benzyl acetate, benzyl alcohol following the injection of the volatiles from WKG into a gas-liquid chromatograph without using solvents. 2.4.2. Sting Sheaths. The intensity of alarm behaviour elicited by the volatile components of the Koschewnikow glands was much lower than that elicited by sting sheaths. The volatile compounds released by both the sting sheaths and Koschewnikow glands trigger a recruiting activity and hence may have a synergistic effect. The chemical identity of the volatile components secreted by the sting sheaths and those that accumulate on the surface of the setaceous membrane is now being analysed by us. Stinging reaction of individual workers in the laboratory has been recorded only following their stimulation with setaceous membrane or with sting sheaths, but not with other glands or organs. Similar results were obtained with guards at hive entrances that were 'stimulated with sting glands and organs (33, 34). 2.4.3. Setaceous Membrane.

• Guards at hive entrances were exposed to the volatiles of setaceous membranes and their attraction, alarm and stinging reactions were recorded. The results are shown in descending order, as follows: • Attraction: setaceous membrane > sting sheaths > quadrate plates and Koschewnikow glands> venom glands and venom sac> Koschewnikow glands> Dufour's gland and pure venoms. • Alarm: sting sheaths> setaceous membrane> venom gland and venom sac. • Stinging: setaceous membrane > sting sheaths > quadrate plates and Koschewnikow glands> venom sacs. Of all the organs tested, the setaceous-membrane volatiles elicited the strongest defence reaction of the guards (20). The secretions present on the surface of the setaceous membrane contained isoamyl acetate, isoamyl alcohol, hexyl acetate, nonanol, benzyl acetate, benzyl alcohol (13). The setaceous membrane (tergum IX) is directly connected to the sting sheaths and is disposed all around the base of the sting. It is abundantly covered with cuticular infoldings and multiforked bristles, which considerably increase the surface of the evaporation area.The function of the setaceous membrane is to serve as a platform for the discharge of alarm pheromones that are secreted elsewhere and accumulate on its surface (56).


157

ACKNOWLEDGMENTS This review was prepared within the framework of the Agreement for Scientific Cooperation between the Universite Pierre et Marie Curie (Paris VI) and the Hebrew University of Jerusalem.

REFERENCES 1. Farrar e.L. (1953) Two queen colony management. Bee World 34, 189--194. 2. Loubel de I'Hoste E. (1959) La biruche. Laubet de l'Hoste, Toulouse. 3. Holzberstein 1.w. Jr (1955) Some whys and hows at two queen management. Glean. Bee Cult. 83, 344-347. 4. Wafa A. K. (1956) Two queen colonies for a plentiful yield of honey. safe wintering. means of propagation and swarming control. Bull. Fac. Agr. Cairo Univ. 98, 22. 5. Wallrebenstein W. (1955) Mein Beitrag zum Mehrmutterverfahren. 17th Int. Beekeep. Congr. 6. Darchen R. and Lensky Y. (1963) Etude preliminaire des facteurs favorisant la creation des societes polygynes d' Apis mellifica var. ligustica. Ann. Abeille 6,69-73. 7. Darchen R. and Lensky Y. (1962) Les societes polygynes de Reines (Apis melli/iea var. ligustica). e. R. Acad. Sci .. Paris, 255, 2300-2302. 8. Lensky Y.. Darchen R. and Levy R. (1970) L'aggressivite des reines entre e11es et des ouvrieres vis-a-vis de la creation des societes polygynes d'Apis mellifica L. Rev. Compo Animal. 4, 50-62. 9. Darchen R. (1960) L'ablation du dard des reines et des ouvrieres d'Apis mellifiea. e. R. Acad. Sci., Paris, 250, 934-936. 10. Gary N.E. (1962) Antagonistic reactions of workers honeybees to mandibular gland contents of the queen bee. Bee World 42, 14-17. 11. Yadava R.R.S. and Smith M. Y. (1971) Aggressive behavior of Apis melli/era L. workers towards introduced queens II. Role of mandibular gland content of the queen and releasing aggressive behaviour. Can. 1. Zool. 49, 1179-1183. 12. Grandperrin D. and Cassier P. (1983) Anatomy and ultrastructure of the Koschewnikow's gland of the honeybee, Apis melli/era L. (Hymenoptera: Apidae). Int. 1. Insect Morphol. & Embryol. 12,25-42. 13. Mauchamp B. and Grandperrin D. (1982) Chromatographie en phase ga7euse des composes volatils des glandes a pheromones des Abeilles: methode d'analyse directe. Apidologie 13, 29-37. 14. Butler e.G. and Simpson 1. (1965) Pheromones of the honeybee (Apis melli/era L.). An olfactory pheromone from the Koschewnikow gland of the queen. Ved. Pr. Wyz. Us. Vcel. Dole 4, 33-36. 15. Robinson G.E. (1984) Worker and queen honeybee behaviour during foreign queen introduction. Insectes Soc. 31, 254-263. 16. De-Hazan M., Lensky Y. and Cassier P. (191\9) Effects of quecn honeybee (Apis melli/era L.) ageing on her attractiveness towards workers. Compo Biochem. Physiol. 93A, 777-783. 17. Yadava R.R.S. and Smith M.Y.(1971b) Aggressive behaviour of Apis melli/era L. workers towards introduced queens. 111. Relationship between the attractiveness of the queen and worker aggression. Can. J. Zool. 49, 1359--1362. 18. Yadava R.R.S. and Smith M.Y. (J 971 c) Aggressive behavior of Apis melli/era L. workers towards introduced queens I. Behavioural mechanisms involved in the release of worker aggression. Behaviour 39, 211-236. 19. Cassier P., Finkel A. and Lcnsky Y (199 I) The chemical composition of tarsal gland secretions of honeybee, Apis melli/e.·a (L.) queens, workers and drones. (personal communication). 20. Lensky Y, Cassier P., Rosa S. and Grandperrin D. (1991) Induction of balling in worker honeybees (Apis melli/era L.) by stress pheromone from Koschewnikow glands of queen bees: behavioural, structural and chemical study. Compo Biochem. Physiol. 100A, 3. 585-594. 21. Boch R., Shearer D.A. and Stone B.C. (1962) Identification of isoamyl acetate as an active component in the sting pheromone of the honeybee. Nature 195, 1018-1020. 22. Pickett J.A., Williams I.H. and Martin A.P. (1982) (2)-II-Eicoson-I-ol, an important new pheromonal component from the sting of the honeybee, Apis melli/era L. (Hymenoptera, Apidae). J. Chern. Ecol. 8, 163-175. 23. Free J.B., Ferguson A.W. and Simpkins J.R. (1988) Honeybee responses to chemical components from the worker sting apparatus and mandibular glands in field tests. 1. Apic. Res. 28, 7-2 I.

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24. De-Hazan M. (1986) The attractiveness, composition and structure of mandibular glands of the queen honeybee (Apis melli/era L.). M.Sc. Thesis. Hebrew University of Jerusalem. 25. Finkel A. (1983) Biological effects and chemical characterization of the foot-print substance in the honeybee (Apis melli/era L.). M.Sc. Thesis. Hebrew University of Jerusalem. 26. Hyams Y. (1988) Characterization of pheromonal components of the tarsal and mandibular glands of the queen honeybee (Apis melli/era L.). M.Sc. Thesis. Hebrew University of Jerusalem. 27. Lensky Y, Cassier P. and Tel-Zur (1995) The setaceous membrane of the honey bee (Apis mellifera L.) workers sting apparatus and alarm pheromone distribution. J. Insect Physiol. 21, 589-595. 28. Winston M.L. (1987) The biology of the honey bee. Harvard University Press, Cambridge, 281 pp. 29. Crane E. (1990) Bees and beekeeping: science, practice and world resources. Heinemann Newnes, Oxford, UK,614p. 30. Collins A.M. and Blum M.S. (1983) Alarm responses caused by newly identified compounds derived from the honeybee sting. J. Chern. Ecol., 9, I, 57~5. 31. De Hazan M., Hyams J., Lensky Y. and Cassier P. (1989) Ultrastructure and ontogeny of the mandibular glands of the queen honey bee, Apis mellifera L. (Hymenoptera, Apidae). Int. J. Morph. Embryol. 18, ~, 311-320. 32. Blum M.S. (1992) Honey bee pheromones. In Graham, I. M.(ed.) The hive and the honey bee. Dadant & Sons; Hamilton, pp 373-400. 33. Cassier P., Tel Zur, D. and Lensky Y. (1994) The sting sheaths of honey bee workers (Apis melli{era L.) : structure and alarm pheromone secretion. J. Insect Physiol. 40, 233. 34. Tel-Zur D. (1993) Alarm pheromones of the sting apparatus of the honey bee (Apis mellifera L. var. ligusfica) PhD Thesis, Hebrew University of Jerusalem. Israel, II Op. 35. Boch R. and Shearer D.A. (1967) 2-heptanone and 10 hydroxy frans-dec-2-enoic acid in the mandibular glands of worker honeybees of different ages. Zeitsch. Vergl. Physiol. 54, I-II 36. Boch R., Shearer D.A. and Petrasovits A. (1970) Efficacies of two alarm substances of the honey bee. J. Insect Physiol. 16, 17-24. 37. Maschwitz U.W (1964) Gefarhrenalarmstoffe und Gefaharenalarmierung bei sozialen Hymenopteren. Z. Vergl. Physiol. 47, 59~55. 38. Shearer D.A. and Boch R. (1965) 2-heptanone in the mandibular gland secretion of the honey-bee. Nature 206,530. 39. Free lB., Simpson l (1968) The alerting pheromones of the honey bees. Z. Vergl. Physio. 61, 361-365. 40. Melksham K.J., Jacobsen N. and Rhodes J. (1988) Compounds which affect the behaviour of the honeybee, Apis melli{era L.: a review. Bee World 69,104-124. 41. Simpson J. (1966) Repellency of the mandibular gland scent of worker honey bees. Nature 209, 531-532. 42. Rieth J.P., Wilson WI. and Levin M.D. (1986) Repelling honeybees from insecticide-treated flowers with 2-heptanone. l Apicult. Res. 25, 78-84. 43. Vallet A., Cassier P. and Lensky Y. (1991) Ontogeny of the fine structure of the mandibular glands of the honeybee (Apis mellifera L.) workers and the pheromonal activity of 2-heptanone. l Insect Physiol. 37, 789-804. 44. Cassier P. and Lensky Y. (1991) Evolution du titre de I'hormone juvenile III, des ecdysterojdes et d'une pheromone, la 2-heptanone, en relation avec Ie polyethisme des ouvrieres de I'abeille domestique, Apis mellifera L. (Hymenoptera, Apididae). e. R. Acad. Sci. , Paris, III , 312, 343--348. 45. Crewe R.M. (1976) Aggressiveness of honey bees and their pheromone production. South Afr. J. Sc. 72, 7, 209-212. 46. Crewe R.M. and Hastings H. (1976) Production of pheromones by workers of Apis mellifera adansonii. J. Apicult. Res. 15, 149-154. 47. Kalmus H., Ribbands e.R. (1952) The origin of the odours by which honeybees distinguish their companions. Proc. Roy. Soc. B 140, 5Q.-59. 48. Butler e.G. (1966) Mandibular gland pheromone of worker honeybees. Nature 212, 530. 49. Free J.B., Pickett J.A., Ferguson A.W., Simpkins J.R. and Smith M. (1985) Repelling foraging honeybees with alarm pheromones. J. Agr. Sc. 105,255--260. 50. Robertson G.E. (1968) A morphological and functional study of the venom apparatus in representatives of some major groups of Hymenoptera. Austral. J. Zool. 16, 133--166. 51. Snodgrass R.E. (1956) Anatomy of the honey bee. Cornell University Press, Comstock Publishing Associates, Ithaca, NY, 334 pp. 52. Giufra M. and Nunez J.A. (1992) Honeybees mark with scent and reject recently visited flowers. Oecologia 89, 113-117. 53. Cassier P. and Lensky Y. (1992) Structure et role social de quelques glandes exocrines a secretion pheromonale chez I'abeille domestique, Apis mellifera L. (Hymenoptera, Apididae). Annee BioI. 31, 61-95.


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54. Ghent R.L. and Gary N.E. (1962) A chemical alarm releaser in honey bee stings (Apis mellifera L.). Psyche, 69, I, 1--6. 55. Collins A.M. and Blum M. S. (1982) Bioassay of compounds derived from the honeybee sting. J. Chern. Ecol. 8, 463-470. 56. Lensky Y. and Cassier P. (1995) The alarm pheromones of queen and worker honeybees. Bee World 76, 3, 119-129.

20

PROTEIN TRAFFIC BETWEEN BODY

COMPARTMENTS OF THE FEMALE HONEY

BEE (APIS MELLIFERA L.)

Yoseph Rakover 1 and Yaacov Lensky 2 IOtorhinolaryngology Department Central Emek Hospital Afula, Israel 2Triwaks Bee Research Center Hebrew University of Jerusalem, Faculty of Agriculture Rehovot, Israel

ABSTRACT Several "anatomical compartments" of the female honey bee body have bee studied: I. : Exocrine compartments (the venom gland and the head glands). 2. : Internal organs (ovaries and fat body). 3. : The haemolymph that bathes the above mentioned compartments. To explore the protein traffic from the haemolymph to the exocrine or external body compartments immunological and electrophoretic methods were used. The studies did not show any immunological identity between the proteins of larval food, venom and haemolymph but most of the haemolymph antigens were common with those of ovaries and fat body. A tentative model of the protein traffic between honey bee body compartments is proposed: a. Some compartments are enveloped by a cellular layer (venom and head glands) which prevents the macromolecular traffic between these compartments and the haemolymph. b. Some other compartments are lined by a cellular layer (fat body and ovaries), allowing macromolecular traffic between organs through the haemolymph. The traffic of macromolecules between the body compartments of the female honey bee may serve as a biological model and help in understanding the process in medical and pharmacological disciplines.

1. INTRODUCTION Protein traffic between body compartments is one of the most important process in animals to maintain the biological balance or the homeostatic status and to trigger defense 161

162

Y. Rakover and Y. Lensky

Fat body

Postcerebral gland

Ovary Hypopharyngeal gland

Oocyte

Thoracic gland

Mid-gut

: Sting Venom Venom gland sac

Figure I. Longitudinal, schematic section of a worker honey bee, showing the exocrine glands and the internal organs (modified after Ribbands, 1953)(2).

Figure 2. The collection of haemolymph from the dorsal vessel of a worker with a fine glass capillary.

Protein Traffic between Body Compartments of the Female Honey Bee

163

mechanisms. So, a comprehensive study of this process is important particularly in physiological, pathophysiological and medical disciplines. In mammals, numerous investigations have been done about protein traffic between body compartments, for example traffic between the cerebrospinal fluid and the blood (1). Whereas in mammals the blood flows in a close system, in insects the "blood" (haemolymph) flows in an open system, baths tissues and organs which suggests a different model to study the protein traffic. The female honey bee, a new biological model with a clear anatomical compartmentalization, allows to study the protein traffic (Fig. 1). So, the protein components of three body compartments of the female honey bee are compared by immunological and electrophoretic methods: 1. The exocrine glands: the venom gland, the venom sac and the sting apparatus that secrete and drain venom; the hypopharyngeal and mandibular glands that drain their secretions through ducts and groves via the mouthparts to the exterior as a larval food. 2. The internal organs: ovaries and fat bodies. The vitellogenins are synthesized in the fat body, released to the haemolymph, selectively taken up by the oocyte through several cellular and acellular envelopes (tunica propria, external sheaths), then deposited as vitellin platelets. The oocyte membranes are secreted by the follicle cells (3). 3. The haemolymph that bathes the above mentioned compartments.

We examined the existence of separate body compartments with specific proteins and the possible uptake of haemolymph proteins by the body compartments. This biological model may help in understanding more about the protein traffic or its blocking between body compartments.

2. MATERIALS AND METHODS 2.1 Preparation of Samples for Analyses Italian honey bee (Apis mellifera L. vaL ligustica) adult workers were obtained from the apiary of the Triwaks Bee Research Center, Rehovot. Haemolymph was collected from the dorsal vessel of workers with a fine glass capillary (Fig 2.). Mandibular and hypopharyngeal glands were dissected from head capsules of nurse workers. Ovaries and fat bodies were removed from laying workers. Guardians were captured at hive entrances. The sting apparatus, venom gland and venom sac were removed fro IT their bodies. The venom sac membrane was punctured at several sites and the venom oozed out. Royal jelly was collected from queen cells after the removal of 3 to 4 day-old larvae.

2.2 Preparation of Antisera Antisera were prepared in rabbits against haemolymph and venom of adult workers, royal jelly, and ovaries oflaying queens (4, 5).

2.3 Immunological Analyses We used double diffusion and immunoelectrophoresis methods for the immunological analysis (5).

164


2.4 Electrophoretic Analyses Polyacrylamide gradient slab gel electrophoresis with SDS (SDS-PAGE) and isoelectric focusing were used for the electrophoretic analysis (4).

3. RESULTS 3.1 Are Haemolymph Proteins Incorporated into the Secretory Products of Exocrine Glands? To establish whether haemolymph proteins participate in the composition of proteinaceous secretions of the exocrine glands we analyzed them using electrophoretic and immunological methods. SDS-PAGE. The electrophoretic separations of the peptide components of the royal jelly, venom and haemolymph on SDS-PAGE slabs are recorded in Fig. 3 (samples Nos. 5, 4, 3, respectively). Royal jelly was separated into 33 bands, venom into 10 bands and haemolymph into 36 bands. Although several minor bands in the three samples shared similar Rjs , no major haemolymph proteins were present either in royal jelly or in venom.

Isoelectric Focusing. Samples of haemolymph, royal jelly and venom were separated on PAG-plates (Fig. 4 samples Nos 5, 4, 3, respectively). Haemolymph proteins were separated into 29 bands within pI range of 4.0-7.2; royal jelly into 26 bands within pI range of 4.7-9.2 and venom into 3 bands within pI above 11.0. pI values of venom fractions corresponded neither to haemolymph nor to royal jelly. Several fractions of haemolymph and royal jelly have similar pI's. The results of electrophoretic analyses indicate that most of the protein constituents of the three samples were not shared in common. A small number of bands had similar R, or pI values. Which of these bands represented identical proteins is considered in the next section, using immunological methods and specific antisera. Immunoelectrophoresis. Samples of royal jelly, venom and haemolymph were analyzed by immunoelectrophoresis using antisera against royal jelly, venom and haemolymph (Fig. 5). Royal jelly (well 2) formed 16 precipitation lines following absorption with antiserum against royal jelly (A) and one diffuse line with antiserum against haemolymph (B), which seems to show a reaction of non-identity, as demonstrated below by double-diffusion analysis. Venom (well 3) formed 6 precipitation lines with antiserum against venom (C) and 2 lines (Nos 7 and 8) with antiserum against haemolymph (b). The position of these two lines neither corresponded to venom nor to haemolymph precipitation lines. This may indicate a reaction of partial- or non-identity. Haemolymph (well 1) formed 24 precipitation lines with antiserum against haemolymph (B). Haemolymph proteins were not precipitated by antiserum neither against royal jelly (A) nor against venom (C). Double Diffusion. Samples of haemolymph, royal jelly and venom were absorbed with antiserum against royal jelly, haemolymph and venom. With the antiserum against royal jelly (central well A) five precipitation lines were formed with royal jelly (well No.2) and one line of non-identity, forming a spur precipitated with haemolymph (well


165

- - -- --- -- - -- - -- •-- • ..• -•- - ~

-

-

-

-

--

--

-

-

-

-

-

-

-

- -

Figure 3. SDS-PAGE (5- 15% gradient) protein separation of ovaries (I), fat body (2), haemolymph (3), venom (4), royal jelly (5), ovalbumin (6), Rnase (7) and phosphorylase b (8).

No. I) (Fig. 6). With the antiserum against haemolymph (central well B) 15 precipitation lines were formed with haemolymph (wells No. I). This antiserum precipitated neither the antigens of royal jelly (wells No.2), nor the antigens of venom (wells No.3) (Fig. 7).Then, with the antiserum against venom (central well C) three precipitation lines were formed with venom (well No.3), but none with haemolymph (well No.1) (Fig. 8). The immunoelectrophoresis and double diffusion results demonstrated that no identical antigens were shared by royal jelly, venom and haemolymph. These data confirm and

166


Band 1 No.

--

(-)

pH

11.0

9.2

--

8.5 8.0

-

7.5 7.3

"

7.0 6.8 6.5 6.0 5.5 5.2 5.0 4.5 4.0

55

(+)

0

2

3

5cm

Figure 4. Isoelectric focusing of fat body (I), ovaries (2), venom (3), royal jelly (4) and haemolymph (5). PAGplates, pH range 3.5-9.5.

extend the results of the electrophoretic analyses. It is suggested that the head glands, the venom glands and the haemolymph are separate compartments with regard to macromolecular traffic.

3.2 Are Proteins Shared in Common by Haemolymph, Fat Body and Ovaries? To determine the presence of haemolymph proteins in the fat body and ovaries of laying workers, electrophoretic and immunological methods were used.

SDS-PAGE (Fig. 3, Samples 1, 2 and 3 Respectively). Ovaries were separated into 22 bands, fat body into 21 bands and haemolymph into 36 bands. Both ovaries and fat body shared, each, 17 bands of similar Rf s in common with haemolymph. Thirteen of these bands were common to all three samples. Ovaries and fat body shared in common 20 bands. Isoelectric Focusing (Fig. 4, Samples 2, 1 and 5, Respectively). Ovarian proteins were separated into 18 bands within pI range of 4.5-7.8; fat body into 19 bands within pI range of 4.5-7.2 and haemolymph into 29 bands within pI range 4.0-7.2.


:L'" .

,.

•

}

.~ ~

--

1

167

c

Figure 5. Immunoelectrophoretic analysis of haemolymph (l), royal jelly (2), and venom (3) . Absorbed with antisera against royal jelly (A), haemolymph (8) and venom (C).

Figure 6. Double-diffusion analysis of haemolymph (I) and royal jelly (2) prepared in the surrounding wells. Absorbed with antisera against royal jelly (Al in the center well.

Figure 7. Double-diffusion analysis of haemolymph (I), royal jelly (2) and venom (3) prepared in the surrounding wells. Absorbed with antisera against haemolmph (B) in the center well.

Figure 8. Double-diffusion analysis of haemolymph (I 1 and venom (3) prepared in the surrounding wells. Absorbed with antisera against venom (e) in the center well.


169

In general, the results confirmed those of SDS-PAGE, with small differences in the number of common bands between the three samples. The results of electrophoretic separations show that most (about 14) of the proteins of ovaries, fat body and haemolymph are shared in common. The identity of the proteins of the three samples was further analyzed by immunological methods using specific antisera. Immunoelectrophoresis. The identity of haemolymph, fat body and ovaries proteins was examined by Immunoelectrophoresis using antisera against haemolmph (B) and ovaries (A) (Fig. 9). Haemolymph formed 26 precipitation lines with antiserum against haemolymph and 12 lines with antiserum against ovaries. Ovaries formed 16 lines with antiserum against haemolymph and 7 lines with antiserum against ovaries. Fat body formed 14 lines with antiserum against haemolymph and 7 lines with antiserum against ovaries. It also emerges from the results that: haemolymph and fat body shared 14 lines precipitated by anti haemolymph serum and 5 lines in common when precipitated with antiserum against ovaries. Haemolymph and ovaries formed 5 common lines with anti haemolymph serum and 6 common lines with antiserum against ovaries. Double-Diffusion. Samples of ovaries, fat body and haemolymph were absorbed with antisera against ovaries or haemolymph. With the antiserum against ovaries (central well A) most of the lines formed by ovaries (well 2) and haemolymph (well I) merged, except for line No.5 which formed a spur. The major line (Nos 2 and 3) was not formed by the fat body (well 3) (Fig. 10). With the antiserum against haemolymph (central well B) 16 lines were formed by haemolymph (well I) which had in common with ovaries (well 2) 7 lines and 6 lines with fat body (Fig. 11). The results of immunoelectrophoretic and double diffusion analyses confirm the identity of several protein components of haemolymph, fat body and ovaries, which was indicated by the electrophoretic analysis. The data suggest that contrary to the above described separate compartments of exocrine glands, the ovaries and fat body cannot be considered as such, because of their association with haemolymph proteins.

DISCUSSION Since neither antiserum against venom precipitated.haemolymph proteins nor antihaemolymph serum formed precipitates with venom, no bands with similar Rf or pI values could be detected, it is concluded that the venom gland and the venom sac are separate compartments. The venom contains pharmacologically active components (6), which are lethal whenever a honey bee is stung. When a drop of venom is removed with a glass capillary from the sting of a donor bee and immediately injected into its body cavity, the bee dies instantly (Rakover, unpublished observation). It seems that the venom gland and sac walls serve as a macromolecular barrier at least with regards to the traffic of venom macromolecules. The fact that no identical antigens were shared by royal jelly, venom and haemol-ymph indicates that the head and venom glands are separate protein compartments from one another and from haemolymph proteins. It seems therefore that the glandular excretory proteins are not taken up from the haemolymph , except for their precursors, but that they are synthesized in situ. Contrary to the exocrine gland' proteins, the immunochemical and the electrophoretic separations of haemolymph, fat body and ovaries revealed that most of their proteins were identical. As a tentative model of protein traffic, the body compartments of the honey bee seem composed of two main parts:

170


Figure 9. Immunoelectrophoretic analysis of haemolymph (I), ovaries (2), and fat body (3). Absorbed with antiserum against ovaries (A) and haemolymph (8).

a. Compartments enveloped by a cellular layer which acts as a protein barrier and prevents macromolecular traffic between the venom glands or the head glands and the haemolymph. b. Compartments lined with a cellular layer which allows a macromolecular traffic between one organ (the fat body or the ovaries) and the haemolymph. Separate fluid compartments have been described in the honey bee pupal molting space ( 7) and in the queen bee spermatheca (8).

Protein Traffic between Body Compartments ofthe Female Honey Bee

171

Figure 10. Double-diffusion analysis ofhaemolymph (I) , ovaries (2) and fat body (3) charged in the surrounding wells. Absorbed with antiserum against ovaries (A) in the center well.

Figure 11. Double-diffusion analysis of haemolymph (l), ovaries (2) and fat body (3) charged in the surrounding wells. Absorbed with antiserum against haemolymph (B) in the center well.


172

The barrier to protein traffic in some insect tissues is related with the presence of separate and scalariform junctions (9). In mammals, the protein traffic between compartments is also composed of two main parts: a. A barrier and prevention of macromolecule traffic by tight junction. For example the blood brain barrier (10) or the pancreatic duct epithelium (11, 12). b. Compartment which are lined with a cellular layer, permitting macromolecular traffic between one organ and another such as between liver epithelial cells (13) or in the vestibular labyrinth (14). The macromolecular\ traffic can be done via gap junction or by receptor mediated protein transport (15).

REFERENCES I. Van-Deures B., Moller M., Amtorp O. (1978) Uptake of horseradish peroxidase from C.S.F into the choroid plexus of the rat with special reference to transepithelial transport. Cell. Tiss. Res. 233 , 215--234 2. Ribbands R. (1953) The Behaviour and Social Life of Honey Bees. pp. 55-63; Bee Res. Assoc., London. 3. Engelmann F. (1980) Insect vitellogenin: identification, biosynthesis and role in oogenesis. [n : Advances in [nsecs Physiology. Ed : Trehence JE., Berridge MJ. and Wigglesworth VB. Academic Press, London, 14, pp.49-108. 4. Lensky Y, Skolnik H. (1980) [mmunochemical and electrophoretic identification of the vitellogenin proteins ofthe queen bee (Apis mellifera L.). Compo Biochem. Physiol. 66B, 185-193. 5. Lensky Y., Rakover Y (1983) Separate protein body compartments of the worker honeybee (Apis mellifera L.). Compo Biochem. Physiol. 75B: 607-615. 6. O'Connor R., Peck ML. (1978) Venom of Apidae. In: Handbook of Experimental Pharmacology. Ed: Bettini S. Springer, Berlin. 48, pp. 613-615. 7. Lensky Y., Rakover Y (1972) Resorption of moulting fluid during the ecdysis of the honeybee. Compo Biochern. Physiol. 41B, 521-531. 8. Lensky Y, Alumot E. (1969) Proteins in the spermathecae and haemolymph of the queen Bee. Compo Biochern. Physiol. 30, 569-575. 9. Noirot-Timothee c., Noirot C. (1980) Separate and scalariformjunctions in arthropods. [nt. Rev. Cytol. 63, 97-140. 10. Lane NJ. (1991) Morphology of glial blood-brain barriers. Ann. N.Y Acad. Sci. 633, 348-362. II. Arendt T. (1991) Penetration of lanthanum through the main pancreatic duct epithelium in cats following exposure to infected human bile. Dig. Dis. Sci. 36, 75-81. 12. Satir P., Gilula NB. (1973) The fine structure of membranes and intercellular communication in insects. In : Ann. Rev. Entomol. Ed: Smith RF., MittlerTE.VoI18, p. 143-166. 13. Mesnil M., Asamoto M., Piccoli C., Yamasaki H. (1994) Possible molecular mechanism ofloss ofhomologous and heterologous gap junctional intercellular communication in rat liver epithelial cell lines. Cell Adhes. Commun. 2, 377-384. 14. Kikuchi T., Adams JC., Paul DL.. Kimura RS. (1994) Gap junction systems in the rat vestibular labyrinth: immunohistochemical and ultrastructural analysis. Acta Otolaryngol. (Stockh.) 114, 520-528. 15. Gitlin JD., Gitlin D. (1974) Protein binding by specific receptors on human placenta, murine placenta and suckling murine intestine in relation to protein transport across these tissues. J. Clinic. Invest. 54, 1155--1166.

21

EFFECTS OF FEEDING, AGE OF THE LARVAE, AND QUEENLESSNESS ON THE PRODUCTION OF ROYAL JELLY Nuray Sahinler 1 and Osman Kaftanoglu 2 lMustafa Kemal University Faculty of Agriculture Hatay-Turkey 2Cukurova University Faculty of Agriculture 01330 Adana-Turkey

ABSTRACT The effects of feeding, the age of the larvae and queenlessness on the acceptance rates and royal jelly production were studied. The average acceptance rates were 65.0±0.82 % in queenright cell builders and 87.1±1.08 % in queenless cell builders. Feeding colonies with pollen substitute increased the acceptance rates significantly (P0.05). The age of the larvae was also important on the acceptance of the cells. The acceptance rates of I or 2 days old larvae were higher than that of3 days old larvae in both queenless and queenright colonies. In queenright cell builders the average royal jelly yields were 153.7±4.27 mg per cell when they were fed with sugar syrup and 185.3±5.68 mg when pollen substitute was given besides sucrose syrup. In the queenless cell builders the average yields were 189.3±9.11 mg in the sugar syrup fed and 225.6±14.52 mg in the pollen substitute fed colonies. In general royal jelly yield was much higher in queenless cell builders than that of qucenright. Feeding colonies with pollen substitutes in addition to sucrose syrup increased the royal jelly yield by 36 % in queenright colonies and 40 % in queenless colonies. The best result were obtained by grafting one day old larvae in queenless cell builders that were fed with pollen substitute and sucrose syrup.

1. INTRODUCTION Royal jelly is the whitish cream like secretion from the food glands of the young worker honey bees. It is used to feed the queen bee and young larvae in the colony. It 173

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N. Sahinler and O. Kaftanoglu

shortens the developmental period of the larvae and causes the differentiation of the worker larvae into queen. It prolongs the life of the queen, stimulates cell divisions and promotes tissue regeneration. There is no genetical difference between the worker bees and queen bees raised from the same colony. The only difference comes from nutrition of the larvae during the development. The queen larvae are fed with royal jelly throughout the larval stage, while the older worker larvae are fed with the mixture of nectar and pollen. As a result queen bees develop within 16 days, they are bigger, their ovaries and the spermathecae are fully developed. They can live up to 2-3 years and can lay 1500-2000 eggs per day. On the other hand worker bees develop within 21 days, live 35-40 days, their ovaries are not developed and their spermathecae are not functional. Royal jelly has become a very popular bee product for the last 5--6 years. There is a big demand for fresh and good quality royal jelly in the country. It has been used for the healthy growth of children and for many maladies as folk medicine by elderly people, by impotent couples, by patients suffering from cancer and other diseases in order to increase appetite, stimulate the immune system and keep the body stronger. Most of royal jelly is imported from China and other countries. There are over 3.5 million colonies and 40.000 beekeepers in Turkey. We have a great potential in producing good quality bee products such as honey, royal jelly, pollen, propolis, bee venom etc., having rich flora, suitable climate and genetic richness of bees in the country. However most of the beekeepers produce only honey and do not know how to produce royal jelly. Therefore we have started a program to train beekeepers to produce quality bee products. Since the value of royal jelly is much higher than honey and other bee products, it is an excellent source of income for the beekeepers. We decided to work on the factors affecting the royal jelly production in order to initiate and spread the royal jelly production in the country. We have studied the effects of feeding and the age of the larvae on the grafting rates and royal jelly yield in queenless and queenright colonies. We are currently studying the effects of different genotypes and season on the production of royal jelly in subtropical climate.

2. MATERIALS AND METHODS This study was conducted at the Cukurova Region in Turkey, during july-September of 1995. Total of8 cell builders were used during the experiment. One half of the colonies were queenless and the other half were queenright. They were also divided into 2 groups and they were either fed with syrup or with syrup and pollen substitute. Pollen substitute was prepared by mixing 4 parts of soybean flour, I part dried skim milk and making a cake with sugar syrup. About 250 grams of pollen substitute was placed on top of the frames and they were replaced with the fresh one every week. Queenless starter colonies were prepared by dequeening the colonies and rearranging the frames in the brood chamber as; honey, sealed brood, open brood, open space for larvae transfer, open brood, sealed brood, honey and feeder. The supers and extra frames were removed from the hives and the bees were shaken to the brood chambers in order to have strong one-story free flying starter colonies. All the queenless cell builders were inspected regularly and the natural queen cells were removed. In order to strengthen the colonies adult worker bees and/or frames of sealed brood were added to the queenless cell

Production of Royal Jelly

175

builders or new queenless colonies were prepared every 15 days. Grafting was repeated every 2 days, and royal jelly was harvested with 48 hour intervals. Queenright starter colonies were prepared by placing a queen excluder above the brood chamber confining the queen and rearranging the frames as in the queenless cell builder. Empty frames were exchanged with the brood frames between the brood chambers and supers as the brood emerged. Queen cell cups were made according to Laidlaw (I). Pure beeswax was melted in a double-jacketed tray. The wax was dipped first into a soap-solution, and the excess water was shaken off. The stick was then dipped into the melted wax 3 or 4 times to a depth of about 8-10 mm. After the last dip, the formed cups were fixed to a grafting bar and submerged in clean and cold water, where the cell cups were separated from the mold. By this way 15 cell cups were prepared on a grafting bar. Grafting was done in a tent or a room near the apiary. One drop of diluted royal jelly was placed to the bottom of the queen cell cups when the new cell cups were used (2). The larvae were lifted gently with some royal jelly from the cells and transferred to the bottom of the cell cups by using a grafting needle. A clean wet towel was placed over the cell cups in order to prevent the larvae from drying. One frame with three bars of cells was put into the space in the cell builders as soon as the grafting was finished. One day old larvae were grafted to the top bar, 2 days old larvae to the middle bar and 3 days old larvae to the lowest bar. All the frames were removed from the cell builders 2 days after grafting. The accepted cells were counted and the acceptance rates were determined. Later all the larvae in queen cells were removed by using a pair of fine forceps. Royal jelly was harvested by using a special plastic royal jelly collecting spoon and they were placed in a separate vial, weighed on an electronic balance and the average royal jelly yield was determined.

3. RESULTS AND DISCUSSION 3.1. Acceptance Rates 3.1.1. Queenright Cell Builders. The acceptance rates In queenright cell builders were summarized in Table 1. The acceptance rates in the queenright colonies were rather low. The average acceptance rates were 65.1±1.23 % in the syrup fed and 64.9±1.I0 % in the pollen substitutes fed colonies. Addition of pollen substitute did not increase the acceptance rates in queen-

Table 1. Acceptancc ratcs (%) in quecnright colonies

Fed with syrup Age of larvae

N

I Day old 2 Days old 3 Days old Total/Average

150 150 150 450

x± S; 68.7 66.7 60.0 65.1

± ± ± ±

1.96 1.68 1.81 1.23

Fed with syrup and pollen substitute Max.- Min.

N

67~80

150 150 150 450

67~73

53--67 53~80

'Different letters indicate significant differences among the means (P
x±S, 68.0 66.7 60.0 64.9

± ± ± ±

1.61 1.68 1.48 1.10

Max.- Min. 60--73 60--73 53--67 53~73

Overall Average

x±S,

68.4 66.7 60.0 65.0

± 1.24 a* ± 1.16 a ± 1.14 b ± 0.82

176


Table 2. Acceptance rates (%) in queenless colonies Fed with syrup Age of larvae

N

I Day old 2 Days old 3 Days old Total/Average

150 150 150 450

x±S"

Fed with syrup and pollen substitute Max.-min.

88.7 ± 1.96 85.4 ± 2.45 77.9 ± 1.82 84.0 ± 1.43**a

80--100 73--100 73--S7 73--100

N 150 150 150 450

x±Sx 94.6 93.9 82.1 90.2

± ± ± ±

1.64 1.53 1.82 1.41 **b

Max.-min. 87-100 87-100 73-87 73--100

Overall average

x±S,

91.7 ± 89.7± 80.0 ± 87.1 ±

1.42a* 1.42 a 1.34 b 1.08

'Different letters indicate significant differences among the means (P
right colonies (P>O.OS). These colonies probably collected enough fresh pollen for the development of the larvae. The acceptance rates oflarvae grafted at 1,2, 3 days were found to be 68.4±1.24 % 66.7±1.l6% and 60.0±1.l4 % respectively in queenright colonies. There was no significant difference between the acceptance rates of I or 2 days old larvae (P>O.OS); however, the acceptance rates of 3 days old larvae were lower than that of young ( I or 2 days old) larvae (P
3.1.2. Queenless Cell Builders. In general the acceptance rates in queenless colonies were better than the queenright colonies (P
3.2. Royal Jelly Production 3.2.1. Queenright Cell Builders. The royal jelly production in queenright colonies was summarized in Table 3. The average royal jelly yield per cell in queenright cell builders that were fed with sugar syrup and pollen substitute were found to be IS3.7 ± 4.27 mg. and 185.3 ± 5.68 mg.

Production of Royal Jelly

177

Table 3. Royal jelly yield per cell (mg) in queenright colonies Royal jelly yield (mg) Age of the larvae I Day old 2 Days old 3 Days old Total/average

Syrup

Syrup + pollen substitute

x±S,

x± S,

% Increase

200.4 ± 11.21 a 187.7±8.91 a 167.8 ± 6.88 b 185.3 ± 5.68 b

36 17 4 20

147.2 159.9 160.9 153.7

± 7.53 ± 7.91 ± 6.85 ± 4.27

c* b b a

Average

x±S; 173.8 ± 8.96 170.9 ± 7.04 164.4 ± 4.79 169.5 ± 4.08

*Different letters indicate significant differences among the means (P<0.05).

respectively. There was a 20 % increase in royal jelly production in the pollen substitute fed colonies compared to syrup fed colonies. Feeding colonies with sugar syrup was not enough for commercial royal jelly production. Addition of pollen substitute increased the royal jelly yield significantly (P<0.05). Proteins in pollen substitute increased the activities of the mandibular and hypopharyngeal glands of the young worker bees to produce more royal jelly. The average royal jelly yield increased from 147.2 mg to 200.4 mg when I day old larvae were grafted and the cell builders were fed with pollen substitutes in addition to sugar syrup. The age of the larvae also affected the royal jelly yield significantly (P<0.05). There was an average of 36 % increase in royal jelly yield when 24 hours old larvae were grafted. Similarly, royal jelly yield increased by 17 % when 2 days old larvae were grafted and 4 % when 3 days old larvae were grafted provided that they were fed with pollen substitute.

3.2.2. Queenless Cell Builders. The royal jelly production in queenless colonies was summarized in Table 4. The average royal jelly yield was 189.3 ± 9.11 mg in sucrose syrup fed colonies and 225.6 ± 14.52 mg in pollen substitute fed colonies. As seen in queenright colonies addition of pollen substitute significantly (P<0.05) increased the royal jelly yield in queenless cell builders too. The addition of pollen substitute increased royal jelly yield by 19 % in queenless colonies. This difference was even higher (46 %) when 1 day old larvae were grafted. More royal jelly was harvested when I day old larvae were grafted than that of 2 or 3 days old. In general royal jelly yield depends on the genotype of the bees, condition of the cell builders, availability of the food, season and duration of the cells in the cell builders etc. The effects of genotype on the production of royal jelly was investigated in China and an Table 4. Royal jelly yield per cell (mg) in queenless colonies Royal jelly yield (mg) Age of the larvae 1 Day old 2 Days old 3 Days old Total/average

Syrup

Syrup + pollen substitute

x±Si

x±S;

194.9 ± 16.15 b 206.2 ± 20.12 b 166.8 ± 6.77 c 189.3±9.11* a

284.8 200.0 194.9 225.6

± 27.33 a ± 19.93 b

± 16.56 b ± 14.52** b

Average % Increase 46 0 17 19

x±S; 239.8 203.1 180.8 180.8

*Different letters indicate significant differences among the means (P<0.05), **(P
± 18.57 ± 13.80 ± 9.29 ± 9.29

178


average of 0.375 ±0.03 gr. royal jelly was harvested from Zau A line, 0.232 ±0.03 gr. from Carpathian bees and 0.347 ±0.06 gr. from US Italian bees during 15/5-311811992. The average royal jelly yield changed from 137.9 mg to 200.4 mg in different years when 24 hours old larvae were grafted and royal jelly was harvested at 48 hours intervals(3). The royal jelly content increased to 327.5 mg in 1989,469.5 mg in 1980, and 346 mg in 1991 when it was harvested at 72 hours intervals. In our study royal jelly yield was less than the results obtained from the royal jelly producing lines in China. However, there are several races and ecotypes of honeybees in Turkey and they are different from each other both morphologically and physiologically(S-7). There are also great variations among the ecotypes of Anatolian honeybees in terms of colony development, honey yield, wintering ability, gentleness and disease resistance(8). The effects of different races and ecotypes on the production of royal jelly is also under investigation. As a result the acceptance rates and royal jelly production were much higher in queenless colonies than queenright colonies. Feeding cell builders with pollen substitute in addition to sugar syrup increased the royal jelly yield significantly. The best results were obtained by grafting one day old larvae in queenless cell builders that were fed with pollen substitute and sugar syrup.

REFERENCES I. Laidlaw, H.H., (1979). Contemporary Queen Rearing. Dadant and Sons Hamilton,IIlinois. 2. Giil, M.A., Kafianoglu, D. (1990) Effects of grafting techniques on the quality of queen bees (Apis mel/ifera L.) raised under <;:ukurova Region conditions. <;:ukurova Univ. J. of Science and Engineering 4(2):41-53 (In Turkish) 3. Shibi, c., Fuhai , L., Shengming, H., Puxiu, L., Study on Relationship Between the Yield of Royal Jelly and the age of grafted Larvae. Bee honey. Royal jelly. Environment. China 1993. P: 67-81. 4. Shibi, c., Shengming , H., Fuhai , L., Puxiu , L., Studies on the Relationship Between the Bee Races and the Yield of Royal Jelly. Bee honey. Royal jelly. Environment.China. 1993. P:4{}-53. 5. Ruttner, F. (1988). Biogeography and taxonomy of honey bees. Springer, Verlag, Berlin. 293 pp 6. Dogaroglu, M.(l981). Tiirkiye'de yeti~tirilen onemli ari irk ve tiplerinin Cukurova Bolgesi kosullarinda performanslarinin karsilastirilmasi. Ph.D. Thesis, C.U. Ziraat Fakultesi, Adana, Turkey. Unpublished. 7. Giiler, A., Kafianoglu, D., Bek, Y., Yeninar, H. (1996 ) Discrimination of some Anatolian honeybee (Apis melli/era L.) races and ecotypes by using morphological characteristics. Submitted for publication. 8. Giiler, A., Kaftanoglu, D. (I 996)The performance of Anatolian honeybee (Apis melli/era L.) races and ecotypes in migratory beekeeping. Submitted for publication.

22

THE USE OF ROYAL JELLY DURING TREATMENT OF CHILDHOOD MALIGNANCIES Osman Kaftanoglu' and Atilla Tanyeli 2 'Faculty of Agriculture 2Faculty of Medicine University of Cukurova 01330 Adana, Turkey

ABSTRACT Eight children who have malign diseases such as acute leukemia, lymphoma and hepatoblastoma were included in this preliminary study. All the patients had I gram of royal jelly (RJ) before breakfast once a day for one month. During this period complete blood counts, general condition and weights of the patients were recorded. The values of the same patients before and after the use of RJ were examined as controls. The patients did not have GM-CSF and G-CSF while they were having RJ. The average hematocrit values did not differ before and after the use of RJ. The average white blood cells increased from 2,857±388 to 3,732±366; neutrophils from 1,489±367 to 2,647±620; and lymphocytes from 1,137±180 to 1,665±255 after using RJ. The general conditions (having an appetite and feeling better) increased and weight gain were observed after RJ administrations. The patients who had RJ gained an average of 1.737±0.725 kg weight after one month.

1. INTRODUCTION The occurrence of different types of childhood malignancies such as leukemia, lymphoma and solid tumors are increasing every year due to genetic factors, viral infection, environmental pollution and other factors. Patients have to go to the doctors and gct chemotherapy and radiotherapy for the proper treatment. There are mainly 3 types of blood cells in human body, namely the red blood cells, white blood cells and thrombocytes. The majority of the blood cells are red blood cells, and their major function is to transport oxygen from the lungs to the tissues. The percentage of the red blood cells is called the hematocrit and the normal hematocrit is 45 per cent in healthy children. 179

180

O. Kaftanoglu and A. TanyeH

The second type of blood cells is a white blood cell or leukocyte. It is called white because it is not colored by hemoglobin. White blood cells have several different functions but the most important of these is to protect the body against foreign organisms. Most of the white blood cells such as the neutrophils, the eosinophiles and the basophiles are formed in the bone marrow along with the red blood cells. The neutrophils are the most important white blood cells for protecting the body against acute invasion by bacteria. These cells are about 12 microns in diameter and can pass rapidly through the pores of the capillaries, enter the tissue spaces and attack almost any agent that may be causing tissue damage. Neutrophils have the ability to ingest or phagocytize particles that are foreign to the tissues including bacteria and tissue particles. They contain digestive enzymes that are capable of digesting most ingested bodies and proteins of bacterial bodies. A neutrophil can phagocytize and digest 5 to 25 bacteria before it becomes exhausted and dies. Lymphocytes are produced from the lymph nodes and other lymph tissues instead of bone marrow and released into the blood. They have different functions. A large proportion of the lymphocytes are specially sensitized cells which are the products of the immunity system of the body. These cells attack specific foreign invaders of the body such as parasites and some cancer cells, etc. Leukopenia is the decreased number of white blood cells in the circulation. This usually results from damage to the bone marrow by toxic reaction to drugs, or by ionizing radiation from x-ray or nuclear bomb exposures. When the number of white blood cells falls extremely low the body becomes unprotected against bacterial invasion. If the person is not treated with antibiotics or other bacteria-resisting drugs he usually will die of fulminating infection within several days. The acute leukemia is the results of accumulation of early myeloid or lymphoid precursors in bone marrow, blood and other tissues. Acute leukemia presents with features of bone marrow failure (anemia, infections due to leukopenia and hemorrhage due to thrombocytopenia) and mayor may not include features of organ infiltration by leukemic cells (blasts). Infections are often bacterial in early stages and fungal infections are particularly common in patients with prolonged periods of neutropenia. The disease is divided into two main subgroups, acute myeloblastic leukemia (AML) and acute lymphoblastic leukemia (ALL). The acute lymphoid leukemia (ALL-Ll) is the most common malignancy among children. The patients' parents usually try different folk medicines, herbs or plants and expect help from them besides medicinal treatment such as chemotherapy and radio-therapy. Most of the patients or their parents insist on using royal jelly in order to feel better, increase appetite, gain strength against bacterial and viral infections. Therefore we decided to have a preliminary study on the effects of RJ on childhood malignancies.

2. MATERIALS AND METHODS Eight children between the age of 4--7 years and have malign diseases such as acute leukemia (ALL), lymphoma (NHL) and hepatoblastoma were included in the study. They had 1 gr. of RJ once a day before breakfast for one month. The average weight and the general condition of the patients were observed and the hematocrit counts, average white blood cells, neutrophils lymphocytes and thrombocytes were determined before and after the use of RJ. The patients did not have granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF) while they were having RJ.

Royal Jelly and Treatment of Childhood Malignancies

181

Table 1. The age, sex, disease of the patients and the effects ofR] on weight and appetite Weight

Appetite

Patients

Age

Sex

Disease

Before RJ

After RJ

2 3 4 5 6 7 8 Mean

5.5 7.0 4.0 5.0 4.0 6.0 7.0 6.0 5.6

F M M M F M M F

ALL-LI NHL Hepatoblastoma ALL-LJ ALL-LJ NHL NHL ALL-L3

18.2 19.6 17.0 13.7 13.0 19.0 22.0 20.0 17.81±1.1

18.7 21.0 18.0 14.9 14.3 23.0 27.5 19.0 19.55:!: 1.5

Before RJ

After RJ

-,+ + + + + + + +

-,+ -,+ -,+ +

Royal jelly was produced in queenless cell builders which were fed with 50 % sugar syrup and pollen substitute throughout the season. One day old larvae were grafted every 3rd day and RJ was harvested 48 hours after grafting. The larvae were lifted gently with some RJ by using a grafting needle and transferred to the queen cell cups. A clean and wet towel was put on the cells in order to prevent larvae from drying. As soon as the grafting was done the bars with the cell cups were put on a frame and placed into the cell builders. Two days after grafting they were taken from the cell builders. The larvae were removed from the queen cell cups by using a pair of fine forceps and RJ was collected with a special plastic spoon. It was either used fresh or stored in deep-freeze at _18°C.

3. RESULTS The age, sex and the disease of the patients and the effects of RJ on appetite and general conditions were summarized in Table 1. As seen from the table, RJ increased the appetite in all the patients. Seven of them gained and only one lost weight during treatment. They gained an average of 1.737±0.725 kg. weight after one month. An the parents of the patients' also indicated that RJ increased the general condition of the children and they become more resistant to the viral and bacterial infections. Consequently they have continued giving RJ to the children. The average hematocrit values and trombocytes are summarized in Table 2. The average hematocrit values did not differ before and after the use of RJ. However, the average trombocytes increased by 23.4 % from 188,000 to 232,000 cells per cubic millimeters after using royal jelly. The average white blood cells, neutrophils, and lymphocytes before and after RJ applications were summarized in Table 3. The average white blood cells increased from 2,857±388 to 3,732±366; neutrophils from 1,489±367 to 2,647±620; and lymphocytes from 1,13 7± 180 to 1,665±255 after using

Table 2. The average hematocrit values and trombocytes of the patients Trombocytes

Hematocrit

X±S,

Before RJ

After RJ

% Increase

Before RJ

After RJ

% Increase

34.9 ± 1.48

35.2::1: 1.07

0.8

188,000

232,000

23.4

182

O. Kaftanoglu and A. Tanyeli

Table 3. The average white blood cells, neutrophils, and lymphocytes

x ± S, White blood cells Neutrophils Leukocytes

Before RJ

After RJ

% Increase

2,857 ± 388 1,489 ± 367 1,137 ± 180

3,732 ± 366 2,647± 620 1,665 ± 255

30.6 77.8 46.4

RJ. In other words the white blood cells, neutrophils and leukocytes increased 30.6 %, 77.8 % and 46.4 % respectively.

4. DISCUSSIONS Royal jelly is a very rich and nutritious bee product. There are many carbohydrates, vitamins, proteins, lipids, sterols, essential amino acids, minerals and trace elements such as zinc, selenium, etc. which may increase the appetite and regulate metabolism. It also stimulates cell divisions and propagation resulting the growth of the organisms. Almost all the patients' appetite increased and they gained weight after RJ application Royal jelly also has a similar effects on silkworm (Bambyx mari) and other animal species. Dietary addition of royal jelly increased and quickened the development of silkworms Bambyx mari (I). Afifi et al (2) indicated that royal jelly increased the growth of guinea-pigs. The guinea-pigs were injected subcutaneously each day with RJ solution at doses of 100, 200 or 300 mg/kg body weight and the average gain in body weight of the animals was 136.2, 144.7, and 150.5 gr. respectively; whereas weight gain was only 119.5 gr. in water injected control group animals. The authors indicated that differences between the control and treatment groups were highly significant. Royal jelly and its component 10-Hydroxy deconoic acid have also strong antibacterial effects(3) . The bactericidal effects of 10-Hydroxy deconoic acid was also confirmed by other researchers (4). Fujiwara et al (5) found a potent antibacterial protein in RJ and they named it as royalisin. It was stated that in bioassays royalisin had a potent antibacterial activity against Gram-positive bacteria at low concentrations, but not against Gram-negative bacteria. The antibacterial and antiviral properties of RJ may have prevented the patients from getting bacterial and viral infections. All the patients did not have any cold or other infections during the observations. Royal jelly has anti-tumor effects(6) and it is effective against slowly growing tumors (e.g. solid tumors) but not against rapidly growing ones (e.g. leukemia L 1210 or P 388). It also stimulates the immunoglobulin production by the lymphocytes and increases the IgM and IgG in patients with breast cancer(7) . Because of all these positive properties RJ has been widely used by the patients who are suffering from childhood malignancies, any type of cancer and other disorders. It is a rich, supplementary bee product that increases the appetite, keeps the organism stronger, stimulates the production of white blood cells and prevents the patients from getting bacterial, fungal or viral infections during chemotherapy and radio-therapy. It stores the liver and kidney functions and lessens the adverse effects of the chemicals on the organs during chemical treatments. However it should be kept in mind that RJ by itself is not a drug or medicine to cure any type of malignancies therefore the patients should not only rely on RJ. It may also cause allergic reactions especially on the asthmatic patients. They should

Royal Jelly and Treatment of Childhood Malignancies

183

have allergy test before taking RJ. As a result of this preliminary study we plan to have more patients to investigate the RJ effect for patients who have cancer and conduct double blind experiments to eliminate the placebo effect.

REFERENCES I.

2.

3. 4.

5.

6. 7.

Saikatsu, S., Ikeno, K., Hanada, Y, Ikeno, T. (1989) Physiologically active substances in oral secretions produced by the honey bee- effects of royal jelly on the silkworm. Ohu Univ. Dental J. 61(3):1-4 (In Japanese) Afifi, E. A., Khattab, M.M., EI-Berry, A.A., Abdel-Gawaad, A.A. (1989) Effect of royal jelly on guinea-pig growth. Proceedings of the Fourth International Conference on Apiculture in Tropical Climates, Cairo, Egypt, ~IO Nov. 1988. Blum. M.S., Novak, A.F.. Taber. S. (1959). 10-hydroxy-2-decenoic acid. an antibiotic found in royal jelly. Science 130(3373):452-453 Serra Bonvehi, J., Escola Jorda, R. (1991) Studie iiber die mikrobiologischc qualitat und bakteriostatische aktivitat des weiselfuttersaftes (Gelee Royale):Beeinflussung durch organische sauren. Deutsche Lebcnsmittel-Rundschau 87(8):25~259 Fujiwara, S .. Imai, 1.. Fujiwara. M .• Yaeshima. T.. Kawashima. T.. Kobayashi. K. (1990) A potent antibacterial protein in royal jelly. Purification and determination of the primary structure of royalisin. J. BioI. Chern. 265( 19): 11333-11337 Tamura. T., Fujii, A., Kuboyama, N. (1987) Anti-tumor effects of royal jelly. Nippon Yakurigaku Zasshi 89(2)73-80 (In Japanese) Yamada, K., Ikede. I., Maeda. M .• Shirahata. S .• Murakami. H. (1990) Effect of immunoglobulin production stimulating factors in foodstuffs on immunoglobulin production of human lymphocytes. Agricultural and Biological Chemistry 54(4): I 087-1089.

23

THE ROLE OF HYMENOPTEROUS VENOMS IN NATURE Eli Zlotkin Department of Cell and Animal Biology Institute of Life Sciences The Hebrew University of Jerusalem Jerusalem 91904, Israel

VENOMS Venom is defined as a mixture of substances which are produced in specialized glandular tissues in the body of the venomous animal and introduced by the aid of a stinging-piercing apparatus into the body of its prey or opponent in order to paralyze and/or kill it. The academic interests in the topic of venoms (Toxinology) is substantially based on considerations concerning (a) public health, dealing with clinical pathological problems of human envenomation and (b) pharmacology-neuropharmacology, which views venoms as a potential source of useful substances for medicine, industry and biological research [1]. Our approach to venoms is directed by eco-zoological and ecochemical considerations which view a venom mechanism as an unique specialization and adaptation of the venomous animal in order to solve a certain vital problem. In the present article hymenopterous venoms are compared to those of other organisms on the basis of the role that they fulfill in nature. With this background and from the ecological ecochemical point of view the vast majority of the venomous animals (such as snakes, many spiders, venomous snails, and various coelenterates) are slow, and even static, predators which feed on freshly killed prey comprised of mobile and relatively vigorous animals. Such a drastic difference in the locomotory capacity of the venomous predator when compared to its potential prey demands special adaptations for prey encounter. These include, firstly, behavioural adaptations in the form of ambush hunting tactics and, secondly, the development of the venomous apparatus for the quick immobilization of the prey at the earliest moment after encounter and with relatively low doses. The latter is expressed in the nanomolar or lower range of concentrations of the active substances in the body of the affected animal. The goal of fast action at low doses is achieved with the aid of two devices. The first is a physical device, the stinging instrument, enabling the direct introduction of the venom 185

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into the circulation or in close proximity to the critical excitable nervous and neuro-muscular target tissues which control and operate the animals locomotory system. The second is a chemical device in the form of the neuroactive constituents of venom, the so-called neurotoxins. In this presentation a neurotoxin is defined as a substance which affects the function of the excitable tissues due to a specific recognition and binding affinity to given sites in these tissues [2]. The various neurotoxins so far obtained from various animal venoms are commonly classified according to their effects and sites of action in nervous systems. They are thus defined as ion channel toxins which modify ion conductance, presynaptic toxins which affect neurotransmitter release, and postsynaptic toxins which interfere with the binding and the resulting expression of neurotransmitters [1 ,3]. A very common characteristic of the various biologically active venom constituents, including neurotoxins, is their protein-polypeptide nature. This chemical characteristic has a double significance. Firstly, polypeptides, through their high diversity of covalent structures and the resulting spatial arrangements, may reveal a highly diverse array of functional specificities such as site-directed and selective neurotoxicity. Secondly, polypeptide is the most readily available structure for adaptive modifications through the genetic machinery. To summarize, the venomous animal appears as a slow obligatory predator which feeds on fast animals. The hymenopterous insects indeed possess venoms which satisfy the above definition but they are neither slow nor predatory.

VENOMOUS HYMENOPTERA Fig. I presents the systematic definition and allocation of the hymenopterous insects. The Hymenoptera comprise a major order of holometabolous insects. More than 100,000 species of this order are know [4] the majority of which can be easily identified by the characteristic "wasp waist" and the two pairs of well developed transparent wings. As shown, (Fig. I), the sting possessing hymonopterans (Apocryta) are subdivided into the terebrants (parasitic wasps) and aculates which are further divided into solitary and social hymenoptera. In the different groups of Hymenoptera the venomous apparatus is a modified ovipositor and exhibits a fundamental similarity of structure [5~8, (Fig.2)]. The sting is hollow and composed of a dorsally located stylet and two barbed lancets which form the ventrolateral boundaries of the ventral venom canal. The two lancets possess grooves along their entire length which fit to two guide-rails of the stylet. The sting is protruded and set in motion by a complex arrangement of levers consisting of three pairs of plates and their specific musculature. During the action of stinging the lancets slide along their track-and-groove connection to the stylet and drive thc sting shaft deeper into the wound. The liquid injected through the sting originates basically from two main secretory entities: the branched venom gland and the "dufour" gland - the so-called "acid" and "alkaline" glands respectively in bees and social wasps. The venom gland is drained into the venom sac connected by a duct to the stylet bulb successively leading to the ventral venom canal. With this background several peculiarities related to the different division of Hymenoptera may be mentioned. In Aculeata, the ovipositor is converted into a sting and the egg passage opens to the exterior at its base. In Terebrantia the ovipositor retains its egg-laying function [5,9]. In honeybees the venom sac is devoid of any musculature. Venom flow is attributed to the pumping function of the back-and-forth movement of the lancets coupled to a successive valve action of lobes at the lancet bases [7]. The sting loss of honeybee

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188

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workers when encountering vertebrates is attributed to specific and purposeful mechanisms absent in other groups of social Hymenoptera. The venom apparatus of the terebrant parasitic wasps is characterized by a multiplicity of glands, the exact function of which has not been clarified so far [11]. In ants (Formicidae) several subfamilies have deviated morphologically from the above general pattern. The subfamilies Formicinae and Dolichorinae have lost the major portion of the sting shaft as well as associated sclerites. In Dolichorinae the function of the poison gland reservoir was replaced by anal glands which synthesize a variety of low molecular weight organic compounds and are devoid of protein components [12-13].

HYMENOPTEROUS VENOM IN NATURE In both the aculeate and the terebrant groups of solitary wasps the venom system is primarily employed for securing food for their offspring. This basic function, however, is performed with different patterns of prey encounter and stinging behaviour. The relatively large aculeate forms hunt prey which roughly equals their own size; the terebrants encounter a prey which is in the range of about 3 orders of magnitude larger than their own size such as fifth instar larvae of the wax moth encountered by the Bracon parasitic wasps [14]. For details on the biology of, and prey predator interactions of, solitary wasps the reader is directed to the references [8,9] and [38]. The Sphecidae are the most intensively studied among the aculeate solitary wasps [16,15]. In most cases the prey, composed of insects and spiders, is more or less deeply paralysed by the injection of the venom. Once the wasp has grasped the prey with its legs it immediately tries to insert the sting. Wasps which prey on Hymenoptera or adult holometabolous insects typically apply only one sting, through either the thin intersegmental membranes on the base of the legs or the central integument between head and prothorax. Sphecids preying on Orthoptera or larval insects particularly caterpillars, typically sting several times. The insertion of the sting is guided by tactile stimuli [17] sensed by mechanoreceptors located on the stings sheaths. In the group of terebrant parasitic solitary wasps the venom system of Braconidae has received the greatest attention [11]. Parasitism by Braconidae may be either internal or external. The former occurs in free-living hosts (such as beetles, caterpillars, aphids) and the latter predominates in hosts that live in confined quarters (such as digging caterpillars and wood-boring coleoptera). In contrast to the external parasitoids the internal ones seldom, even temporarily, paralyse their free-living hosts. The external parasitoids may be subdivided into those which induce a temporary paralysis and others which induce a permanent one. The latter were studied in detail through the venomous system of Braconidae (Bracon hebe tor, Bracon brevicornis and Bracon gelechiae [11,14]. Bracon wasps require for their development lepidopterous larvae such as Ephestia, Plodia or Galleria. The female wasp paralyses its host by injecting venom through its stinging apparatus and feeds upon the haemolymph released from the wound made in the host integument. The male wasp does not feed on the host's haemolymph. Oviposition is independent of the stinging and feeding behaviour, the eggs being deposited on, or very near, the paralysed larvae. After leaving the egg, the wasp larvae cling to the host, and they, too, feed upon the haemolymph by puncturing the integument [14]. The venoms of social Hymenoptera, mainly those of honeybees and social wasps, have attracted attention due to their medical significnce, either harmful [18,19] or beneficial [20--22]. It is commonly accepted that the venoms of social Hymenoptera are primar-

Role of Hymenopterous Venoms in Nature

189

ily employed for defensive purposes [11,23,24,12]. This concept is supported by the following considerations:

1. According to their feeding habits the Apidae are herbivorous (nourished by nectar and pollen) wasps and ants are omnivorous and do not consume living prey. 2. Bees and social wasps are exclusively diurnal and are warningly coloured, possessing the yellow and black stripes typical for other aposematic animals. 3. The nests of social Hymenoptera are static, immobile stores of food (including their progeny) which has to be defended. The venom is supposed to serve as the most effective device for defence [12]. 4. The stinging behaviour of social Hymenoptera is often performed in a communal, organized manner associated with volatile low molecular alarm, recruitment pheromones [25-27] such as isoamyl acetate in honeybees [28,29] or aliphatic ketones in Vespa orientalis [30]. Such an organized and mobilized defence was shown to occur in honeybees against mammals [18] as well as insects (Vespa mandarinia). Adapted patterns of a mobilized active defensive behaviour were also demonstrated to exist in social wasps [26,33]. While stinging, the wasps, like bees, grip the victim tightly with their legs and mandibles. Wasps and hornets try to sting the enemy several times in succession. Unlike bees, there is usually no sting autonomy in Vespidae. 5. The latter in bees was suggested to serve as a specific adaptation developed by selection which is directed against the largest enemies of the honeybee, the honey- and brood- mammals and birds. This is expressed in a preformed breaking point, strong barbs in the lancets, and the inclusion of the last ganglion in the ripped-out sting enabling further ejection of venom. Several modes of attack behaviour also imply a defensive specialization against mammals [10,26,27]. 6. A convincing argument in favour of the defensive role of hymenopterous venoms is supplied by their composition and pharmacology. The most characteristic property of a defensive venom was suggested to be its pain-producing (algogenic) capacity. In this aspect, hymenopterous venoms possess an efficient array of pain-producing agents [33,24,12]. Since this presentation deals with the ecological ecochemical implications of hymenopterous venoms rather than the, more conventional, pharmacological clinical aspects the interested reader can find complementary information in references [38-47).

VESPIDAE-THE OPTIONAL HUNTERS Social wasps, however, also employ their stings for offensive purposes related to food-collecting. They are optional, occasional food hunters. Insects serve as the main target of this activity. Caterpillars, mantids, bees, bugs, grasshoppers, beetles, butterflies and moths were observed to be taken into the nests of wasps and hornets as food [34-37,19]. Systematic, organized invasions of Vespa mandarinia on to Apis mellifera bees in Japan were observed and described [31], ending with the seizure of pupae and brook as food. Some of the Vespa species in Japan are highly predatory on Polistes wasps and attack their nets, mainly after emergence of the young males and queens. V. mandarinia attacks Vespula and Vespa species usually in the same manner as it attacks bees [37,35]. The chemistry of vespid venoms, generally, favours the concept of a defensive role. This is expressed in the presence of pain-producing biogenic amines (such as serotonine,

190

E. Zlotkin

histamine, dopamine, etc.) and wasp kinins [40]. Internal release of histamine in the body of the envenomated animal is induced by mast cell degranulation performed by a series of short, strongly basic, peptides such as the MCD-peptide in bees [49], "Mastoparan" in wasp venoms [50,56,57,58,60], basic kinins [51]] and finally by lytic peptides such as the amphipathic (detergent-like) melittin of bees [52,53] and similar lytic peptides from venoms of wasps [52] and ants [54]. The effects of these agents are strengthened by phospholipase A2 which interacts synergically with lytic factors, and is known to occur in hymenopterous venoms [52,55]. Non proteinaceous algogenic factors have been shown to occur in formicid ant venoms in the form of formic and piperidine alkaloids [24]. With this background and the above-mentioned information on the predaceous activity of social wasps, certain data concerning the neurotoxicity of wasp venoms is of interest. It was shown [59] that the venoms of Vespa mandarinia, Vespa xanthoptera and Vespa analis insularis, when assayed in a crustacean neuromuscular preparation, suppressed both the excitatory and inhibitory postsynaptic potential (EPSPs and IPSPs). A component of the venom separated by Sephadex G-50 chromatography caused the depression of EPSPs while IPSPs were unaltered. Since no appreciable changes were found in the potential, conductance and sensitivity of the postsynaptic membrane, the above blockage of the excitatory transmission may be attributed to a presynaptic effect [59]. The venom of Vespa mandarinia has served as the source for the isolation of a neurotoxin which blocked (1000{, M) the action potential in a lobster nerve through the reduction of sodium current. This tetrodotoxin-like action was induced by a polypeptide of molecular weight about 20 kD with a pI of 9.1 and devoid of enzymatic or haemolytic activity [60]. As shown in Fig. 3, the purification of the neurotoxin was performed by two steps of gel filtration and cation exchange column chromatography. As shown in Fig. 4, the suppressory presynaptic origin of the toxicity was suggested by the ability of the toxin to block the excitatory junction potential in lobsters skeletal muscle without affecting the muscles resting potential [60]. An induction of presynaptic block of synaptic transmission in the insect CNS was recently [61] demonstrated by a wasp glycosylated peptide, vespulakin in 1 and its synthetic analogues. All analogues have different effects on synaptic transmission in the cockroach sixth abdominal ganglion namely: at first, a direct and reversible block of excitatory cholinergic nicotinic transmission with a concurrent activation of the inhibitory GABA-ergic system and, secondly, a delayed irreversible block of the transmission [61]. To summarize, the venoms of the omnivorous Vespidae possess neurotoxic polypeptides which seem to fulfill a role in prey paralysis, similar to the "classical" venom systems (see above).

SOLITARY ACULEATES-THE PROVIDERS In the solitary aculeate wasps the venoms of Sphecidae have received much attention, mainly through the study of the venom of Philanthus triangulum. Field observations and prey records for many species of sphecid wasps suggest that their venoms affect a large number of insect species from different orders, as well as spiders. In spite of the fact that P. triangulum preys exclusively upon honeybees it may paralyse, in practice, all other insects so far tested by forced stinging, and these include 37 genera from 15 families from seven orders, as well as spiders [8,16]. Philanthus venom induces a flaccid paralysis which is substantially due to a blockage of neuromuscular transmission and does not af-


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Figure 3. Purification of a neuTtoxin from hornet venom. Upper: Column chromatography on Sephadex G-50 of Vespa mandarinia venom supernatant. 69 ml of hornet venom supernatant was applied to a column (i.d. 4.1 X 98 cm) of Sephadex G-50 equilibrated with 50 mM acetate buffer. pH 5.6. Each fraction collected was at 15mLltube, and the flow rate was 189 mLih. Toxic activity appeared in peak B (fractions 43-60). The fractions were pooled and concentrated for further purification. (a) is an eluting position of chymotrypsinogen Aon the same column. lower: CM-Sephadex column chromatography of the peak B obtained after the Sephadex G-50 chromatography. The concentrated sample (ca. 10 ml) was applied 10 a column (i.d. 2 X 33 cm) of M-Sephadex equilibrated with 50 mM acetate buffer, pH 5.6. Elution was performed with a rigid line~r gradient of NaCI from 0 to 0.8 M. Fractions of 4 ml were collected at a flow rate of 30 mLih. The toxic activity was found on peak H, and the active fractions (fractions 50-55) were combined and then concentrated for further studies. The inset is polyacrylam ide disc gel electrophoresis of MDT X in J3-alanine-glycine buffer at pH 4.3(a) and in NaDodS0 4 with reduced conditions (b).

fect the visceral musculature [66,9]. In addition, the venom of Philanthus is able to elucidate Post-synaptic as well as central [67,68] neurotoxic effects. The post synaptic effects of Philanthus venom have attracted attention. A toxic fraction [62,63 ] was examined on the methathoracic extensor tibiae locust muscles accompanied by an iontophoretic application of L-glutamate, the excitatory neurotransmitter of insect motor nerves [64]. The study of Clark et a\. [69] has revealed that the toxic fraction

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blocks the locust muscle glumate receptors following their activation. The fraction was shown to suppress both the iontophoretic glutamate potential (Fig. 5) and the excitatory junction potentials in a glutamate receptor activation dependent manner. The rate of recovery from the blocking effects was reduced by activating the muscle either by iontophoretic application of the neurotransmitter or by electrical stimulation of the motor nerve. Complementary assays by single channels by patch clamp analyses have supported the conclusion that the Philanthus fraction blocks open channels gated by both junctional and extrajunctional glutamate receptors on locust muscle. Figure S. Effect of Philanthus toxic fraction on L-glumate potential evoked by glutamate iontophoresis at excitatory neuromuscular junctions of locust extensor tibiae muscle . i. saline containing 0.1 V im I of Philanthus toxic fraction was introduced (i) during repetitive applications of 1.5 nC iontophoric glutamate doses. Note the reduction in response amplitude. After 1.5 min exposure to Philanthus toxic fraction toxinfree saline was introduced (,(.) ; ii , following 10 min wash with toxin-free saline. the response was restored to control amplitude. Saline containing 0.1 V /ml was reintroduced, but iontophoretic application of glutamate was discontinued for I min and then resumed. The amplitude of the first response at the end of the I min period was nearly identical to the (ii) 205 control amplitude (cf.I). Note the decline in amplitude of response to subsequent glutamate doses. Measurements were made at the resting potential of the muscle fibre , -60 m V . Calibration bars: potential (vertical), 4 mY; times (horizontal), 20s.


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The chemical identification of the factor responsible for the above open channel blocking was achieved by Eldefrawi et al. [70] by two steps of chromatography on a reversed phase column by HPLC system (Fig. 6). The resulting toxin (PTX) was shown to serve as a potent antagonist of transmission at quisqualate-sensitive glutamate synapses of locust leg muscle . This philanthotoxin (PTX-433) has been purified, chemically characterized, and subsequently synthesized along with two closely related analogues. It has a butyrylltyrosyllspermine sequence and a molecular weight of 433 (Fig. 7). Its two ana-

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Figure 7. The chemical structures of the natural philanthotoxin, PTX-433, and two isomers PTX-334 and PTX343. Taken from [70].

logues, PTX-343 and PTX-334 (the numerals denoting the number of methylenes between the amino groups of the spermine moiety), are also active on the glutamate synapse of the locust leg muscle; PTX-334 was more potent and PTX-343 was less potent than the natural toxin. Such chemicals are useful for studying, labeling, and purifying glutamate receptors and may become models for an additional class of therapeutic drugs and possibly insecticides.

THE INSECT SELECTIVE VENOM OF A PARASITIC BRACONID WASP Parasitism by hymenopteran species is a complex phenomenon, affecting the behavior, physiology and development of its host insect. The adult female parasitoid has the capability of regulating the host for the advantage of her offspring in order to provide a suitable source of nutrition and dwelling. Ectoparasitoids often rely on the venomous material to regulate their hosts. Venomous substances of ectoparasitoid often suppress the hosts host cellular defense reaction [810, inhibit its growth [82] or arrest its larval-larval molting [83]. The venom of Braconidae served as the major object of study [11]. Braconid and other terebrant parasitic wasps appear to be incapable of stinging vertebrate animals. Their selective toxicity to insects was demonstrated by the following series of evidence: (1) Host preference and host sensitivity assays indicated a unique selectivity of braconid venoms to lepidopterous insects [71]. The unusu!!l selectivity of Bracon (=Microbracon=Habrobracon) hebetor venom to insects was demonstrated by its ability to distinguish not only between related groups of insects but also between species belonging to the same order [71].


195

(2) A second, however indirect, indication of specificity follows from the high toxicity of the authentic venom to the susceptible species. Beard [14] reported that one part of Bracon hebetor venom in 2 x 10 8 parts of Galleria mellon ella haemolymph was sufficient to cause permanent paralysis. Bracon brevicornis venom in a dose of 26.8 x 10~10 ml also caused 100% paralysis in Galleria, Angasta and Plodia larvae [72]. (3) An additional indication of specificity is supplied by the unique pharmacology of the Bracon venom. It was shown [73-76] that the flaccid paralysis induced by this venom is a consequence of (a) a presynaptic blockage of the excitatory glutaminergic transmission in the insect skeletal neuromuscular junction; (b) the normal persistence of the inhibitory (GAB Anergic) neuromuscular transmission, which is not affected by the venom, and contributes to the former effect an additional, so-called, autopharmacological intoxication. The heart and visceral musculature are not affected [14,11], thus enabling the continuous prolonged survival of the paralysed prey. (4) The final, and most convincing, proof of the insect selectivity of the braconid venoms is supplied by a series of neurophysiological studies with non-insect preparations. Rathmayer and Walther [77] report that they could not find any activity of Bracon hebetor venom on neuromuscular transmission of a spider, crab and crayfish. The uniqueness of the pharmacological selectivity of Bracon venom is further emphasized in light of the fact that it has been almost established that L-glutamate is the excitatory neurotransmitter in crustacean as well as in insect neuromuscular junctions. No effect of the Bracon venom has been found on the cholinergic neuromuscular transmission of vertebrates such as in frog nerve muscle [78] and rat diaphragm [76] preparations. The presynaptic blocking action of the Bracon venom is exemplified by the neuromuscular examination of the effect of two active fractions A and B isolated by column chromatography from the crude venom (Fig. 8). The elementary effect comprises the suppression of the evoked junction potentials which is accompanied by the reduction in the frequency of the miniature excitatory post synaptic potentials (m.e.p.s.p) without affecting their amplitude and the muscle resting potential. These data suggest a presynaptic suppression in the release of the excitatory neurotransmitter. The purification and characterization of insecticide toxins from venom glands of the parasitic wasp Bracon hebetor was recently reported by Quistad et al [80]. The potency of venom from Bracon hebetor against lepidopterous larvae has been known for over 40 years, but previous attempts to purify and characterize individual protein toxins have been largely unsuccessful. Three protein toxins were purified from venom of this small parasitic wasp and the amino acid sequences of 22-31 consecutive residues at the amino-terminus were determined. These relatively large toxins (apparent molecular mass 73 kDa) were labile under many isolation techniques, but anion-exchange chromatography allowed purification with retention of biological activity. Two purified toxins were quite insecticidal (LD50 < 0.3 microgram/g) when injected into six species of lepidopterous larvae. On a molar basis, one toxin (Brh-l) has the highest known biocidal activity against Heliothis virescens larvae(LD50 = 2 pmol/g).

MELITTIN AS PAIN PRODUCER The clinical and therapeutic aspects of the honey bee venom are extensively studied [38,46]. However, when dealt from the ecological point of view, the Apinae are strictly herbivorous animals and the venom serves exclusively for defensive purposes [44]. The

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latter aspect is strongly supported by the algogenic (pain producing) capacity of bee venom. The composition of bee venom is presented in Table I. Pain production by bee and wasp venom was attributed substantially to biogenic amines and the histamine releasing peptides [44 and above]. However, the study of Prince et al. [86] reveals that the pain producing factor of bee venom is Melittin and offers an interesting explanation of its algogenic mode of action. On the average a single bee sting contains 0.5 /-II of volume and 50 /-Ig of dry matter. Subcutaneous injections of 2 /-II volume of the separate bee venom components, in physiological saline at their respective doses as in a sting, have shown that only melittin yields the sharp pain associated with a honey bee sting. As shown in Fig. 9, melittin is an amphipathic peptide of 26 amino acids composed mainly of hydrophobic amino acid residues with a positively charged polar C-terminal segment (Fig. 9). The amphipathicity of melittin is supplied not only by its primary structure but also by its spatial arrangement. In the latter the hydrophobic residues are oriented to one side through which melittin interacts with the membranal phosopholipids [85] .


197

Table 1. The major components of honeybee (Apis mellifera) venom Class of molecule

Component

Proteins Peptides

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Sugars Phospholipids a-amino acids Volatile compounds (pheromones)

% of venom

Hyaluronidase Phospholipase A, Melittin Secapin MCD peptide Tertiapin Apamin Procamine Small peptides (less than 5 a.a) Histamine Dopamine Noradrenaline a-aminobutyric acid glucoseFructose

10-12

Molecular weight

41000 20000 12000 (as tetramer) 3000 2500 2500 2000 600 <600 150 150 150 150 180 700 150 200

50 0.5--2.0 1-2 0.1 1-3 1-2 13-15 0.5--2.0 0.2-1.0 0.1...{J.5 0.5 2 5 I

4--8

Taken from [46J.

When interacting with membranes in the absence of a membrane potential melittin appears in its monomeric form. However, the presence of a membrane potential causes the conductance of the melittin-incorporated bilayer to increase. At a fixed voltage the conductance increases with a fourth power of the melittin concentrations suggesting that four melittin monomers associate to form a tetrameric anion selective pore. The speculation of Prince et al [84] attributes the pain producing capacity of mel itt in to its voltage dependent ionophoric properties. The mammalian skin is supplied nociceptors which are afferent sensory nerve fibers which are excited by various mechanical, physical and chemical stimuli. The nociceptor maintains a resting potential (+70 mV out),

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198

E.Zlotkin

so when melittin molecules encounter nociceptors they form their tetrameric pores, collapse the resting potential and thus trigger a wave of depolarization down the nerve. The transient depolarization results in the melittin tetramer dissociation to the inactive monomers. When the nerve cell membrane repolarizes the melittin reaggregates into its ionophoric tetramer which in turn depolarizes the membrane again. The above oscilatory mode of repolarization and depolarization induces a chronic stimulation of the nerve cell resulting in pain. If this model is correct the relatively short period of pain following a bee sting may be caused by the diffu~ion and escape of the individual melittin molecules.

REFERENCES 1. Shier, WT. and Mebs, D. (Eds.) Handbook of Toxinology, Marcel Dekker, N.Y., 1990. 2. Zlotkin. E. In: Comprehensive Insect Physiology, BiochemistlY and Pharmacology (Kerkut. E.A. and Gilbert, L.I., Eds.) Vol. 10, Pergamon, Oxford. 1985, pp. 499-546. 3. Hucho, Fand Ovchinnikov, Y.A. (Eds.) Toxins as Tools in Neurochemistry, de Gruyter, Berlin, 1983. 4. Berland, L.B. Superordre de Hymenopteroides. In: Traite de Zoologie, Vol. X(2). Masson, Paris, 1951. 5. Imms, A.D. A General Textbook of Entomology, Methuen, London, 1951. 6. Snodgrass, R.S. Principles olInsect Morphology. McGraw-Hill, New York and London, 1935. 7. O'Connor, R. and Peck, M.L. Venoms of apidae. In: Arthropod Venoms, Edited by S. Bettini. Springer, Berlin and New York, 1978, pp. 613--{559. 8. Rathmayer, W Venoms of Sphecidae, Pompilidae, Multillidae and Bethylide. In: Anthropod Venoms, Edited by S. Bettini, Springer, Berlin and New York, 1978, pp. 661--690. 9. Piek, T. and Simon-Thomas, R.T. (1969) Parlysing venoms of solitary wasps. Camp. Biochem. Physiol. 30, 13-31. 10. Maschwitz, V.Wl. and Kloft, W (1971) Morphology and function of venom apparatus of insects: bees, wasps, ants and caterpillars. In: Venomous Animals and the Venoms, Vol. 3, Venomous Invertebrates. Edited by W Bucherl and E. Buckley. Academic Press, New York, pp. I-56. II. Beard, R.L. (1978) Venoms of Braconidae. In: Arthropod Venoms, Edited by S. Bettini, Springer, Berlin and New York, pp. 778-800. 12. Schmidt, 1.0. (1982) Biochemistry of insect venoms. Ann. Rev. Ent. 27, 339-368. 13. Blum, M.S. and Hermann, H.R. (1978) Venoms and venom apparatuses of the Formicidae. In: Arthropod Venoms, Edited by S. Bettini, Springer, Berlin and New York, pp. 801-899. 14. Beard, R.L. (1952) The toxicology of Habrobracon venom: a study of natural insecticid. Conn. Agric. Exp. Stn. Bull. 562. 15. Rathmayer, W (1962) Das Paralysierungsproblem beim Bienewolf Philanthus triangulum F. (Hym. Sphec.) Z. Vergi. Physiol, 45, 413-462. 16. Rathmayer, W (1962) Paralysis caused by the digger wasp Philanthus. Nature (Land.), 196, 1148--1151. 17. Steiner, A.L. (1962) Etude du comportement predateur d'un hymenoptere sphegien: Uris nigra Vd.L. (= Notogonia pomplijormis pz), Ann. Sci. Nat. Zool. (Ser. 12) 4. 1-126. 18. Murray, 1.A. (1964) A case of multiple bee stings. Cent. Afr. J. Med., 10, 249--251. 19. Edery, H., Ishay, 1. Lass, I. and Gitter, S. (1972) Pharmacological activity of oriental hornet (Vespa orientalis) venom. Toxicon 10, 13-23. 20. Beck, B. Bee Venom Therapy, Appleton-Century, New York, 1935. 21. Banks, B.E.C., Rumjanek, FD., Sinclair, N.M. and Vernon, C.A. (1976) Possible therapeutic use ofa peptide from bee venom. Bull. Inst. Pasteur 74,137-144. 22. Schultz, .R., Loos, M., Bub, F and Arnold, P.I. (1980) Differentiation of hemolytically active-fluid-phase and cell-bound human Clq by an ant venom-derived polysacharide. J. Immunol. 124, 1251-1257. 23. Bucherl, W. Venomous chilopods or centipedes. In: Venomous Animals and their Venoms. Vol. 3 Venomous Invertehrates, Edited by W Bucherl and E. Buckley, Academic Press, New York, 1971, pp. 169--195. . 24. Blum, M.S. Chemical Defenses of Arthropods, Academic Press, New York and London, 1981. 25. Mashwitz, V. (1964) Gefahrenalarmstoffe und Gefahrenalrmierung bei sozialen Hymenopteren. Z. Vergl. Physiol. 47, 596--{)55. 26. Maschwitz, U. (1966) Alarm substances and alarm behavior insocial insects. Vitam. Horm. 24, 267-290. 27. Blum, M.S. and Brand, 1.M. (1972) Social insect pheromones: their chemistry and function. Am. Zool. 12, 553-576.


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28. Boch, R. and Shearer, D.A. (1966) Iso-pentyl acetate in stings of honeybees of different ages. J. Apic. Res. 5,65-70. 29. Morse, R.A., Shearer, D.A., Boch, R. and Benton, A.W. (1967) Observations on alarm substances in the genus. Apis. J. Apic. Res. 6,113-118. 30. Saslavsky, H., Ishay, J. and Ikan, R. (1973) Alarm substances as toxicants in the oriental hornet colony, Life Sci. 12, 135-144. 31. Matsura, M. and Sakagami, Sh. F. (1973) A bionomic sketch of the giant hornet Vespa mandarinia, aserious pest of Japaneses epiculture. 1. Fac. Sci. Hakkaido Univ. (Ser. VI) 19. 125-162. 32. Gaul, A.T. (1953) Additions to vespine biology XI defence flight. Bull. Brooklyn Ent. Soc., 48, 35-37. 33. Edery, H., Ishay, J., Gitter, S. and Joshua, H. Venoms ofvespidae. In: Arthropod Venoms, Edited by S. Bettini, Springer, Berlin and New York, 1978, pp. 691-771. 34. Rivnay, E. and Bytinski-Salz, H. (1949) The oriental hornet (Vespa orientalis L.) its biology in Israel. Bull. Agric. Res. Sta. Rechovot, 51. 36-42. 35. Kuhlhom, F. (1961) Uber das Verhalten sozialer Falten wespen (Hymenoptera: Vespidae) beim Stalleinflug, innerhalb von Viehstallen und eim Fliegenfang Z. Angew. Zool. 48, 405-422. 36. Ishay, J. Bytinski-Salz, H. and Shulov, A. (1967) Contributions to the bionomics of the oriental hornet Vespa orientalis. Isr. J. Ent. 2, 45-\06. 37. Sakagmi, Sh. and Fukushima, K. (1957) Some biological oservations on a hornet Vespa tropica var pulchra, with special referene to its dependence on Polites wasps. Treubia 24, 73-82. 38. Piek, T. (Ed.) Venoms of Hymenoptera. Academic Press, New York, 1986. 39. Akre, R.D. and Reed, H.C. Biology and distribution of social Hymenoptera. In: Handbook of Natural Toxins, Volume 2 (Anthony T. Tu Ed.), Marcel Dekker, New York, 1984, pp. 3--48. 40. Shipolini, R.A. Biochemistry of bee venom. In: Handbook of Natural Toxins, Volume 2 (Anthony, T. Tu, Ed.) Marel Dekker, New York, 1984, pp. 49--86. 41. Levin, I.w. Vibrational studies of model membrane - melittin interactions. In: Handbook ofNatural Toxins, volume 2 (Anthony, T. Tu, Ed.) Marcel Dekker, New York, 1984, pp. 87-108. 42. Nakajima, T. Biochemistry ofvespid venoms. In: Handbook of Natural Toxins, volume 2 (Anthony, T. Tu, Ed.) Marcel Dekker, New York, 1984, pp. \09--1 34. 43. Piek, T. Pharmacology of hymenoptera venoms. In: In: Handbook of Natural Toxins, volume 2 (Anthony, T. Tu, Ed.) Marcel Dekker, New York, 1984, pp. 135-186. 44. Zlotkin, E. Toxins derived from arthropod venoms specifically affecting isnects. In: Comprehensive Insect Physiology Biochemistry and Pharmacology, volume 10 (G.A. Kerkut and L.I. Gilbert, Eds.) Pergamon Press, Oxford, 1985, pp. 499--541. 45. Piek, T. Insect venoms and toxins. In: Comprehensive Insect Physiology Biochemistry and Pharmacology, volume \0 (G.A. Kerkut and L.l. Gilbert, Eds.) Pergamon Press, Oxford, 1985, pp. 595-634. 46. Hider, R.C. (1988) Honey bee venom: a rich source of pharmacologically active peptides. Endevour, 12, 60--65. 47. Schmidt, J.O. (1995) toxinology of venoms from the honeybee genus Apis. Toxicon, 33, 917-927. 48. Nakajima, T. Pharmacological Biochemistry ofvespid venoms. In: Venoms of the Hymenoptera (Tom Piek, Ed.) Academic Press, London, 1986, pp. 309--324. 49. Breithaupt, H. and Habermann, E. (1968) Mastzelldegranulierendes Peptid (MDCD-Peptid) aus Bienengift: Isolierung biochimische und pharmako10gische Eigenschaffen. Naunyn-Schmiedebergs Arch. Pharmakol. Exp. Pathol., 261, 252-270. 50. Hirai, Y., Kuwada, M., Yasuhara, T., Yoshida, H. and Nakajima, T. (1979) A new mast cell degranulating peptidehomologous to mastoparan in the venom of the Japanese hornet (Vespa Xanthoptera). Chem. Pharm. Bull., 27,1945-1946. 51. Yasuhara, T. Hoshida, H. and Nakajima, T. (1977) Chemical investigation of hornet (Vespa xanthoptera Cameron) venom. The structure of a new bradykinin analogue "vespakinin". Chem. Pharm. Bull., 25, 936-941. 52. Haermann, E. and Jentsch, J. (1967) Sequenzanalyse des Melittins aus den tryptischen und peptischen spaltstucken. Hoppe-Seylers Z. Physiol. Chem" 348, 37-50. 53. Hegner, D., Schummer, U and Schnepel, G.H. (1973) The interaction ofa lytic peptide, melittin with spinlabeled membranes. Biochim. Biophys. Acta, 291, 15-22. 54. Bemheimer, A.W., Avigad, L.S. and Schmidt, J.O. (1980) A hemolytic polypeptide from the venom of the red harvester ant Pogomyrmex arbatus. Toxicon, 18,271-278. 55. Hoffman, D.R. (1994) Alergens in hymenoptora venoms. XXVI: The complete amino acid sequences of two vespid venom phospholipases.lnt. Arch Allergy Immonol .. 104, 184--190. 56. Nakahata, W., Ishimoto, H., Mizuno, K., Ohizumi, Y. and Nakanishi, H. (1994) Dual effects of Mastoparan on intracellular free Ca+2 concentrations in human astrocytoma cells. Br. J. Pharmacol. tt2, 299--303.

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57. Radermecker, M., Louis, R., Leclecq, M., Weber, T., Corhay, J.L. and Bury, T. (1994) Cytokine modulation of basophil histamine release in wasp venom allergy. Allergy, 49, 641~44. 58. Dohtsu, K., Okumura, K., Higiwara, K., Palma, M.S. and Nakajima, T. (1993) Isolation and sequence analysis of pep tides from the venom of Protonectarina sylveirae (Hymenoptera, Vespide). Nat. Toxins, I, 271-276. 59. Kawai, N. and Hori, S. Effect of hornet venom on crustacean neuromuscular junctions. In: Animal. Plant and Microbial Toxins. Vol. 2 (A. Ohsaka, K. Hayashi and Y. Sawai, Eds.), Plenum, New York, 1976, pp. 309-317. 60. Abe, T., Kawai, N. and Niwa, A. (1982) Purification and properties of a presynaptically acting neurotoxin, Mandaratoxin, from hornet ( Vespa mandarinia). Biochemistry, 21, 1693-1697. 61. Piek, T., Hue, B., Le Corronc, H., Mantel, P., Gobbo, M. and Rocchi, R. (1993) Presynaptic block of transmission in the insect CNS by mono and di galactosyl analogues ofvespulakinin I, a wasp (Paravespula maculifrons) venom neurotoxin. Compo Biochem. Physiol. C, lOS, 189-196. 62. Piek, T., May, T.E. and Spanjer, W Paralysis of insect skeletal muscle by the venom of the digger wasp Philanthus triangulum F. In: Insect Neurobiology and pesticide action, Soc. Chern. Indust., London, 1980, pp. 219-226. 63. Piek, T. and Spanjer, W. Effects and chemical characterization of some paralysing venoms of solitary wasps. In: Pesticide and venom neurotoxicity (D.L. Shankland, R.M. Hollingworth and T. Smyth, Eds.), Plenum, New York, 1978, pp. 211-226. 64. Usherwood, P.N.R. and Machili, P. (1968) Phrmacological properties of excitatory neuromuscular synapses in the locust, J. Exp. BioI., 49, 341-361. 65. May, T.E. and Piek, T. (1979) Neuromuscular block in locust skeletal muscle caused by a venom preparation made from the digger wasp Philanthus triangulum F. from Egypt. J. Insect Physiol., 25, 68~91. 66. Rathmayer, W (1966) The effect of the poison of spider and digger wasps on the prey. Mem. Inst. Butantan Simp. Int., 33, 651~57. 67. Piek, T., Mantel, P. and Jas, H. (l980a) Ion-channel block in insect muscle fibre membrane by the venom of the digger wasp Philanthus triangulum F. J. Insect Physiol., 26, 345-349. 68. Piek, T., May, T.E. and Spanjer, W. (I 980b) Paralysis oflocomotion in insects by the venom of the digger was Philanthus triangulum. In: Insect Neurobiology and Pesticide Action (Neurotox 79). Society of Chemical Industry, London, pp. 219-226. 69. Clark, R.B., Donaldson, P.L. Oration, K.A.F., Lambert, J.J., Piek, T., Ramsey, W, Spanjer, Wand Usherwood, P.N.R. (1982) Block oflocust muscle glutamate receptors by 8-Philanthotoxin occurs after receptor activation. Brain Res., 241, 105-114. 70. Eldefrawi, A.T., Eldefrawi, M.E., Katsuhiro, K., Mansur, N.A., Nakanishi, K., Oltz, E. and Usherwood, P.N.R. (1988) Sstructure and synthesis of a potent glutamate receptor antagonist in wasp venom. Proc. Nat!. Acad. Sci. USA, 85, 4910-4913. 71. Drenth, D. (1974b) Susceptibility of different species of insects to an extract of the venom gland of the wasp Microbracon hebetor (Say). Toxicon, 12, 189-192. 72. Tamashiro, M. (1971) A biological study of venoms of two species of Bracon. Tech. Bull. Hawaii Agric. Exp. Stn., 70. 73. Piek, T. and Engels, E. (1969) Action of the venom of the wasp Microbracon hebetor Say on larvae and adults of the moth Philosamia cynthia Hubn. Compo Biochem. Physiol,. 28, 60~ 18. 74. Piek, T., Mantel, P. and Engels, E. (1971) Neuromuscular block in insects caused by the venom of the digger wasp Philanthus triangulum L. Compo Gen. Pharmacol., 2, 317-331. 75. Walther, C. and Rathmayer, W (1974) The effect of Habrobracon venom on excitatory neuromuscular transmission in insects. J. Comp. Physio., 89, 23-38. 76. Piek, T., Veenendaal, R.L. and Mantel, P. (I 982b) The pharmacology of Microbracon venom. Compo Biochern. Physiol,. 72C, 303-309. 77. Rathmayer, W. and Walther, C. Mode of action and specificity of Habrobracon venom. In: Animal. Plant and Microbial Toxins (A. Ohsaka, K. Hayashi and Y. Sawai, Eds.) Vol. 2, Plenum Press, New York, 1976, pp.290-307. 78. Deitmer, J.W (1973) Die Wirkung des Giftes der Schlupfwespe Habrobracon hebetor (Say) auf die neuromuskulare Ubertragung am Sartoriusmuskel des Frosches. Diplomarbeit, Uniersitat Bonn. 79. Spanier, W., Grosu, L. and Piek, T. (1977) two different paralyzing preparations obtained from a homogenate of the wasp Microbracon hebetor (Say) Toxicon, 15,413-421. 80. Quistad, O.B., Nguyen, Q., Bernasconi, P. and Leisy, D.J. (1994) Purification and characterization of insecticidal toxins from venom glands of the parasitic wasp, Bracon hebetor. Insect Biochem. Mol. Bioi, 24, 955-961.


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81. Hayakawa, Y. (1994) Cellular immunosupressive protein in the plasm ofa parsitized insect larvae. J. BioI. Chern .. 269, 1453Cr14540. 82. Soldevila, A.!, and Jones, D. (1994) Characterization of a novel protein associated with the parazitation of lepidopeteran hosts by an endoparasitic wasp. Insect Biochem. Mol. Bioi., 24, 29-38. 83. Coudron, T.A. (1996) Endocrinologically active venom proteins of ectoparasitic wasps. Toxicon, 34, p. 302. 84. Prince, R.C., Gunson, D.E. and Scarpa, A. (1985) Sting like a bee! The ionophoric properties of mel ittin. Trends in Biochemical Sciences, 10,99. 85. Banks, B.E.C. and Shipolini, R.A. Chemistry and pharmacology of honeybee venom. In: Venoms of Hymenoptera (Tom Piek, Ed.) Academic Press, London, 1986, pp. 330-403. 86. Tu, A.T. (Ed.) Handbook of Natural Toxins Vol. 2, Marcel Dekker, Inc., New York, 1984.

24

EFFECT OF AP AMIN AND MELITTIN ON ION CHANNELS AND INTRACELLULAR CALCIUM OF HEART CELLS G. Bkaily, M. Simaan, D. Jaalouk, and P. Pothier MRCC Group in immuno-cardiovascular interactions Department of Anatomy and Cell Biology Faculty of Medicine Universite de Sherbrooke Sherbrooke, Quebec Canada, Jl H 5N4

INTRODUCTION Several toxins have been reported to be highly specific blockers of a single type.of ionic channel. A good example of this is tetrodotoxin (TTX), a highly specific fast Na+channel blocker l . Like TTX, scorpion toxins have become important tools for the study of Na+ channels 2 However, different scorpion venoms have different types of action on this channeI 2-4. Saxotoxin (STX) was also reported to be a specific fast Na+ channel blocker l,56. Some natural toxins do not inhibit the fast Na+ channel but rather activate or open this type of channel. These include gonioporatoxin (GPT)7, batrachotoxin (BXT)8 and grayanotoxin (GTX)9. Several other types of toxins were found to be specific for different types of K+ channels. Examples are charybdotoxin (ChTX), apamin, dendrotoxin, noxiustoxin and gaboon viper venom 10.11. Our laboratory, as well as others, reported that some toxins may specifically affect the L-type Ca 2 + channels 12 as well as the early embryonic fast transient (ft) slow Na+ channels lJ - 15 in heart muscle. Another toxin, co-conotoxin (co-CgTx), was reported to block the L-type and N-type Ca 2 + channels but with only transient inhibitory effects on T-type Ca 2+ channels in neurons and not in heart muscle l6 . Maitotoxin (MTX) was found to activate a new class of voltage-independent Ca 2+ channel or an entirely modified form of voltage-gated Ca 2+ channel in heart cells l7 .

SPECIFIC BLOCKADE OF L-TYPE Ca2+ CHANNELS BY APAMIN Recently, apamin was reported to specifically block the L-type lea in old embryonic chick heare 2 ,18,19. This toxin, at a very low concentration (lO-loM), decreased the over-

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shoot and the duration of the action potentials recorded from isolated 19-day-old embryonic chick hearts (Fig.lD) and blocked completely the slow Ca 2+ action potential of old embryonic single heart cell reaggregates (Figures I A-C). This bee venom toxin decreased the slow Ca 2+ action potentials l2 and the L-type Ca 2+ current l9 in a dose-dependent manner (Fig. IE). Very low concentrations of this toxin (l0- 12 M) decreased L-type lea amplitude by 10% while a 50% blockade of this current was achieved at to- 10M apamin (Fig. I E). At this low concentration (I O- loM), apamin also decreased the L-type lea by 50% in 20-weekold single human foetal heart cells l9 . Apamin decreased the L-type and the tail current within 5 min . This toxin had no effect on the TTX-sensitive IN a and the T -type l ea of heart muscleI2.1 5.19.

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205

APAMIN BLOCKS THE SPONTANEOUS INCREASE OF [Cah Using Fura-2 Ca2+ measurement techniques, spontaneous increase of intracellular Ca2+ and contraction of heart cells were found to be blocked by the highly potent Ca2+ channel antagonist PN200-11O (Bkaily et aI., unpublished results). Using Fura-2 Ca2+ measurement technique20 , apamin (l0-7M) did not affect the resting total intracellular free Ca2+ concentration ([Cal) of quiescent ventricular heart cells from human and chick origin. However, in spontaneously contracting ventricular heart cells, apamin was found to decrease in a dose-dependent manner the amplitude of the spontaneous increase of [Ca], and contractility of heart cells without affecting the frequency of spontaneous beating. Using Fluo-3 confocal microscopy in order to monitor both spontaneous increase of cytosolic ([Ca]J and intranuclear ([Ca]n) free Ca2+ 20.21 during spontaneous contraction, 1O-9M apamin was seen to decrease the amplitude of cytosolic and nuclear spontaneous Ca2+wave without affecting the frequency of the increase of [Cal wave and the spontaneous contraction of heart cells. Figure 2 illustrates an example of the effect of apamin on two attached isolated ventricular heart cells (Figs. 2A and B) of 10-day-old chick embryo. As can be seen, superfusion with lO-9M of apamin decreased within 3 min. the amplitude ofthe spontaneous Ca2+wave through the cytosol and the nucleus by 50% and slightly increased the basal level of the spontaneous increase of [Cal in both attached cells (Figs. 2A and B). Increasing the concentration of apamin up to 1O-7M largely decreased the spontaneous amplitude of total [Cal (Figs. 2A and B) and further increased the basal level of this amplitude without affecting the frequency of the spontaneous increase of[Cal (Figs 2A' and B'). Superfusion (in presence ofapamin) with the Ca2+ chelator, EGTA, completely blocked the remaining small spontaneous increase of [Cal and decreased basal [Cal to near control levels (Figs. 2A' and B'). Figure 3 demonstrates an individual measurement of a spontaneous increase of [Ca]c and [Ca]n' As seen in this figure, the resting level of cytosolic [Ca]c is lower than that of the nucleus20 •21 • Spontaneous entry of Ca 2+ into the cytosol is rapidly buffered by the nucleus and the peak amplitude of the spontaneous increase during the Ca2+ wave is higher in the nucleus than that in the cytosol (Fig. 3A). The amplitude of the Ca2+ wave in both the cytosol and the nucleus follow the same pattern of increase and decay (Fig. 3A). Superfusion with 1O-7M apamin largely blocked the amplitude of the spontaneous increase of both [Ca]c and [Ca]n and increased the basal level of [Ca]n without affecting the basal level of [Cal (Fig. 3A and B).

MODULATION OF SLOW Na+ CHANNEL FUNCTION BY APAMIN AND MELITTIN In order to determine the state of membrane differentiation of cells, comparison should be made of the properties of intact hearts at different stages of development in situ. The electrical properties of the heart undergo sequential changes during developmenez-24 . Myocardial cells in young chick hearts (2-3 days in vivo) possess slowly rising (10-30 Vis) action potentials (APs) preceded by pacemaker potentials. The upstroke is generated by Na+ influx through slow Na+ channels which are insensitive to TTX and Mn2+ (Figs. 4A and 4B). Kinetically fast Na+ channels which are sensitive to TTX make their initial appearance at about day 5, and increase in density until about day 18. The maximal rate of rise of the AP (+Vrna) increases progressively from day 3 to day 18, where the adult V max of approximately 150 Vis is attained. From day 5 to day 7, fast Na+ channels coexist with

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Figure 3. Time-lapse rapid scan of a spontaneously contracting chick myocyte loaded with 13.6 ~M Flu03-AM. The cell was scanned continuously every 320 msec at a 512 x 512 resolution and O.IS ~m pixel size. In this instance, the Ca" wave had a 3 sec duration. Graphs on the left represe nt the spontaneous Ca'+ waves through the cytosolic and intranuclear spaces. In the absence of apamin, the increase in free Ca 2+ fluorescence intensity levels is particularly higher in the nuclear region which remains elevated even after cytosolic Ca'+ has returned to basal levels. Cytosolic and nuclear Ca 2+ waves follow the same profile of a fast increase followed by a smaller component. 8) In prese nce of apamin, the amplitude of the Ca" waves of both cytosolic and nucl ear regions largely decreased and is associated with an increase in basal nuclear Ca'+ level. Images are shown as pseudocolored representations according to an intensity scalc from 0-255 illustrated on Figure I. Time span: 3.3 sec. The scalebar on the lower right of each time frame represents 5 microns. Cell number is cp27S-I. (Bkaily et aI. , unpublished result s).

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5 min CONTROL Figure 4. Characteristics of the acti on potentials (APs) and the inward currents in single 3-day-old embryonic chick hearts. (A-B) Action potentials and inward currents (B) recorded from single cells by switching between current clamp and voltage clamp modes. (A,B) The APs and the inward slow current of single 3-day-old embryonic chick ventricular cells in culture were insensitive to 10-5M TTX and 2 mM Mn 2" . (C,D) Blockade of APs inward currents by melittin (C) and apamin (D) in single heart cells from 3-day-old embryonic chick. The frequency of stimulation was 0.02 Hz. HP ~ holding potential and VS ~ voltage step. Current traces in panels (C) and (D) were taken from two different single cells. (C) A high concentration of melittin (I 0-4M) completely separated I" from I" (st) in a typical 3-day-old embryonic chick heart single cell that showed overlapping of slow currents. Digital subtraction of I" (s) (in the presence of 10-4 M melittin) from the I" (sts) component (in the presence of 10-sM melittin) shows that I" (st) is blocked by 10-4M melittin. Al so. digital subtraction of I" (sts) (in the presence of 10-sM melittin) from the total control inward current shows the presence of a slow inward current that inactivates rapidly (/" (ft)). (Modified from refs. 12 and 15).

a large complement of slow Na+ channels. TTX reduces Vmax to the value observed in 2day hearts, i.e. 10--20 VIs, but the APs persist. After day 8, the APs are completely abolished by TTX and depolarization to less than -50mV now abolishes excitability. This indicates that the AP-generating channels consist predominantly of fast Na+ channels, most of the slow Na+ channels having been lost (functionally) so that the remaining numbers are insufficient to support regenerative excitation. Single heart cells from 3-day-old embryonic chick heart cells exhibit three types of slow Na+ inward currents l 5 and can be easily separated by melittin (Fig. 4C). The first type, a fast transient (ft) slow Na+ inward current (/si(ft)' is activated from a holding potential (HP) of -80 mV and shows fast activation and inactivation (Figs. 4B-D). The maximal mean ft l ea activated from a HP of -80 mV with a voltage step (VS) to -20 mV averaged 93.5 ± 14.5 /lAI /lF I5 • This value is lower than that reported for the TTX-sensitive fast lNa in heart muscle. The low current density of the TTX-insensitive ft slow I Na may explain in part the low Vmax of these single cells. The ft slow I Na is responsible for the rising phase of the slow action potential in 3-day-old embryonic chick hearts (Figs. 4A and 4B). This type


209

of ft slow I};a exists during the development of the foetal human heart and disappears completely at a foetal age of 20 weeks. In both 3-day-old chick embryonic heart cells and in 10-19-week-old foetal human heart cells, the decay phase of the ft slow INa fits well with the sum of two exponentials 15 as in adult animal cardiac celIs 25 .26 . The kinetics of the ft slow INa in young embryonic heart cells is similar to that Qfthe TTX-sensitive INa 26 or the TTX-insensitive 27 and TTX-resistane S,29 Na+ currents lO • The TTX- and Mn 2+-insensitive ft inward slow Na+ channel in young embryonic heart seems to share a few characteristics with the L-type Ca2+ channel: (I) it is completely insensitive to the fast Na+ channel blocker, TTX; (2) it is highly sensitive to one calcium blocker, apamin (Figs. I and 4); and (3) it is highly permeable to the divalent cation Ba2 + . However, these two types of channels do not seem to share many other properties such as: (1) threshold and reversal potential 11 ,14; (2) insensitivity of the ft slow INa to Mn"+, Ne+, Cd"+, C0 2+, La l + and [Ca]o; (3) low sensitivity of the ft slow inward Na+ current to verapamil, D-600, (-) D-888 and nifedipine; (4) high sensitivity of the ft slow INa to melittin (Fig. 4C); (5) high permeability of the ft slow INa to Na+ and Lt; (6) stability of the current in whole-cell voltage clamp conditions; the ft slow inward Na+ current is much more stable that of I ca 11 ,14; (7) the time-course of the ft slow INa activation is different from that reported for the T-type Ca 2+ channels in heart muscle 31 .l 2 . This TTX- and Mn 2+-insensitive ft slow inward Na+ channel resembles the TTX-resistant fast Na+ channef 9 only in that it is permeable to Na+ and Lt and impermeable to Ca 2+. The coexistence of this channel with the fast Na+ channel in adult heart cells could contribute to an increase in intracellular Na+, which may force the Na+-Ca 2+ exchanger to pump Na+ ions out, thus creating a calcium influx through this exchanger. Such a phenomenon would allow a Ca2 + overload in heart cells. A close look at ventricular heart cells of the newborn normal hamster shows only the presence of the fast Na+ current. However, ventricular cells of the newborn cardiomyopathic hamster (one to two days old) show mainly the presence of the ft slow Na+ current. In fetal human ventricular cells, as in heart cells from one-day-old cardiomyopathic hamster, there is a TTX- and Mn 2 +-insensitive, ft slow inward Na+ current. This ft slow Na+ current in both fetal human and newborn cardiomyopathic hamster ventricular cells is sensitive to apamin and melittin. This slow Na+ channel, continues to be functional during the development of the cardiomyopathic hamster. The ft slow Na+ channel was also detected in older cardiomyopathic hamster ventricular heart cells, However, a number of single cells show a decrease in this type of current with increasing age of the cardiomyopathic hamster. This decrease in the number of ventricular heart cells with functional ft slow Na+ channels may correlate with the development of intracellular Ca 2 + overload and focal myocardial necrosis in this pathological model. Apamin and melittin could be used to study the contribution of the slow Na + to the pathology implicating this type of channel. Molecular cloning of TTX-resistant rat heart Na+ channel isoform, as well as the primary structure, functional expression, and molecular cloning of human heart TTX-insensitive slow Na+ channel, was recently reported. Cardiac cell line (MCM I), which originates from a transgenic mouse, shows a TTX-insensitive Na+ current that seems to be similar in origin to the TTX-insensitive ft slow sodium current in fetal heart cells and hamster cardiomyopathic heart cells.

DISCUSSION Earlier studies showed that apamin blocked the slow Ca 2 + and slow Na+ action potential by respectively inhibiting the L-type Ca 2+ channel and fast transient slow Na+ chan-

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nel in heart cells. Using Fura-2 micro fluorometry and laser confocal microscopy, it was demonstrated that apamin does effectively block Ca2+ influx through the L-type Ca2+ channels. Recent results in the literature reveal that the heart L-type Ca 2+ channel possesses an apamin binding site 33 . These results strongly suggest that the main effect of apamin in heart function is due to its high potency blockade of the L-type Ca 2+ channel. Thus, apamin could be used as highly potent L-type Ca2+ channel antagonist with more specificity to this type of Ca 2+channel than other well known Ca 2+ antagonists. Like melittin, apamin was also found to block the ft slow Na+ channel in heart. This type of channel seems to be functional in hereditary cardiomyopathy as well as in Duchenne muscular dystrophy. Also, this type of channel appears to be functional in certain nerve cells. Recent cloning of the apamin binding protein in brain synaptosomes suggested a non significant homology with any known ion channels or receptors. which may suggest that the gene (Kcal 1.8)34 is likely to encode a protein associated not only with the small conductance Ca2 +-activated potassium channel (which does not exist in heart cells) but also with the slow Na+ channel.

ACKNOWLEDGMENTS Supported by MRCC grant MAll722 to Dr. G. Bkaily. Dr. Bkaily is a Merck FrosstFRSQ Professor. The authors thank Ms Mireille Dussault for her secretarial assistance.

REFERENCES 1. Mille O. (1975) The receptor for tetrodotoxin and saxitoxin. A structural hypothesis. Biophys. J. 15, 615-{i19. 2. Wheller KP., Watt DO., Lazdunski M. (1983) Classification ofNa+ channel receptors specific for various scorpion toxins. Pflugers Archv - Eur. 1. Physiol. 397,164-165. 3. Carbone E., Prestipino G., Franciolini F., Dent MA. Possani LO. (1984) Selective modification of the squid axon Na+ currents by Centruroides noxius toxin 11-10. J. Physiol. 79, 179-184. 4. Yatani A., Kunze DL. and Brown AM. (1988) Effects of dihydropyridine calcium channel modulators on cardiac sodium channels. Am. 1. Physiol. 254, 140-147. 5. Kao CY. and Nishiyama A. (1965) Actions of saxitoxin on peripheral neuromuscular systems. 1. Physiol. 180, 50-{)6. 6. Strichartz G. (1984) Structural determinants of the affinity of saxitoxin for neuronal sodium channels. Electraphysiological studies on frog peripheral nerve. J. Gen. Physial. 84, 281-305. 7. Nishio M., Muramatsu I., Kigashi S., Fujiwara M. (1988) Effects of goniopora toxin on the action potential and membrane currents of guinea-pig single ventricular cells. Naunyn-Schmiedebergs Arch. Pharmacol. 337,440-446. 8. Garbcr SS. (1988) Symmetry and asymmetry of permeation through toxin-modified Na+ channels. Biophys. J. 54, 767-776. 9. Seyama 1., Yamada K., Kato R .. Masutani L Hamada M. (1988) Grayanotoxin opens Na+ channels from inside the squid axonal membrane. Biophys. J. 53, 271-274. 10. Castle NA., Haylett OG., Jenkinson OH. (1989) Toxins in the characterization of potassium channels. Trends in Neurosci. 12, 59--65. II. Carbone E. and Swandulla D. (1989) Neuronal calcium channels: kinetics, blockade and modulation. Prog. Biophys. Malec. BioI. 54, 31-58. 12. Bkaily G., Sperelakis N., Renaud J.-F., Payet MD. (1985) Apamin, a highly specific Ca'+ blocking agent in heart muscle. Am. J. Physiol. 248, H961-965. 13. Bkaily G., Jacques D., Yamamoto T., Sculptoreanu A., Payet MD., Sperelakis N. (1988) Three types of slow inward currents as distinguished by melittin in 3-day-old embryonic heart. Can. 1. Physiol. Pharmacal. 66, 1017-1022.


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14. Bkaily G., Peyrow M. Yamamoto T., Sculptoreanu A., Jacques D. and Sperelakis N. (1988) Macroscopic Ca'+-Na+ and K+ currents in single heart and rabbit aortic cells. Mol. Cell. Biochem. 80.59-72. 15. Bkaily G., Jacques D., Sculptoreanu A., Yamamoto T., Carrier, D., Vigneault D. and Sperelakis N. (1991) Apamin, a highly potent blocker of the TTX- and Mn'+- insensitive fast transient Na+ current in young embryonic heart. J. Mol. Cell Cardiol. 23, 25-39. 16. McCleskey EW., Fox AP., Feldman DH., Cruz IJ., Olivera BM., Tsien RW., Yoshikami D.(l987) Omegaconotoxin: direct and persistent blockade of specific types of calcium channels in neurons but not muscle. Proc. Nat. Acad. Sciences of USA. 84, 4327-4331. 17. Kobayashi M., Ochi R., Ohizumi Y. (1987) Maitotoxin-activated single calcium channels in guinea-pig cardiac cells. Br. J. Pharmacol. 92, 665-671. 18. Bkaily G. Single heart cells as models for studying cardiac toxicology in: In Vitro Methods in Toxicology (Jolles G and Cordier A. Editors) Academic Press London 1992 pp.289-334. 19. Bkaily G., Sculptoreanu A., Jacques D., Economos D. and Menard D. (1992) Apamin, a highly potent fetal L-type Ca'+ current blocker in single heart cells. Am. J. Physiol. 262, H463-471. 20. Bkaily G., Gros-Louis N., Naik R., Jaalouk D. and Pothier P. (1996) Implication of the nucleus in excitation contraction coupling of heart cells. Mol. Cell. Biochem. 154, 113-121. 21. Bkaily G., Pothier P., D'Orleans-Juste P., Simaan M., Belzile E, Jaalouk D. and Neugebauer W (1996) The use of confocal microscopy in the investigation of cell structure and function in heart, vascular endothelium and smooth muscle cells. Mol. Cell. Biochem. In press. 22. Sperelakis N. Change in membrane electrical properties during development of the heart in: In vitro methods in toxicology. (Zipes DP., Bailey Je. and Elharrar V. Editors) Martinus Nijhoff Publisher The Hague 1980 pp.221-262. 23. Kojima M. and Sperelakis N. (1983) Calcium antagonistic drugs differ in ability to block the slow Na+ channels of young embryonic chick hearts. Eur. J. Pharmacol. 94, 9-18. 24. Bernard e. Establishment of ionic permeabilities of the myocardial membrane during embryonic development of the rat in: Development and physiological correlates of cardiac muscle. (Lieberman M. and Sano T. Editors) Raven Press N.Y. 1988 pp. I 69-1 84. 25. Brown AM., Lee KS., Powell T. (1981) Sodium current in single rat heart muscle cells. J. Physiol. 318, 479-500. 26. Follmer CH. Ten Erck RE., Yeh JZ. (1987) Sodium current kinetics in cat atrial myocytes. J. Physiol. (Lond) 384,169-197. 27. Anderson PAY. (1987) Properties and pharmacology of a TTX-insensitive Na+ current in neurones of the jellyfish cyancea capillata. J. Exp. BioI. 133,231-248. 28. Bossu J-L., Fletz A. (1984) Patch-clamp study of the tetrodotoxin-resistant sodium current in group C sensory neurones. Neurosci Let, 5 I, 241-246. 29. Ikeda S. Schofield GG. (1987) Tetrodotoxin-resistant sodium current ofrat nodose neurones: Monovalent cation selectivity and divalent cation block. J. Physio!. (Lond) 389, 255-270. 30. Sperelakis N., Shigenobu K. and McLean M.J. Membrane cation channels: changes in developing hearts, in cell culture, and in organ culture in: Developmental and physiological correlates of cardiac muscle. (Lieberman M. and Sano T. Editors) Raven Press N.Y. 1976 pp.209-234. 31. Bean BP. (1985) Two kinds of calcium channels in canine atrial cells. Differences in kinetics, selectivity, and pharmacology. J. Physio!. (Lond). 86, 1-30. 32. Fox AP., Nowycky Me., Tsien RW. (1987) Kinetic and pharmacological properties distinguishing three types of calcium current in chick sensory neurones. J. Physio!. (Lond) 394, 149-172. 33. Schetz JA. and Anderson PAY. (1995) Pharmacology of the high-affinity apamin receptor in rabbit heart. Cardiovasc. Res. 30, 755-762. 34. Sokol PT., Hu W, Yi L., Toral J., Chandra M. and Ziai MR. (1994) Cloning of an apamin binding protein of vascular smooth muscle. J. Protein Chern. 13, 117-128.

25

BEE VENOM IN TREATMENT OF CHRONIC DISEASES Th. Cherbuliez 1209 Post Road Scarsdale, New York 10583

Apitherapy, the medicinal use of the products of the Hive of the Honey Bee has appeared in the middle East and in China approximately at the same time, some two to three thousand years ago. It currently includes the use of honey, Bees wax, propolis, royal jelly, larvae, in addition to bee venom. The latter, Bee Venom Therapy (BVT), only, is the subject of this presentation. It should be understood that BVT applies as part of an overall approach which includes nutrition, vitamins supplements, minerals and exercise. These will not be reviewed here. If BVT has been known and used in parts of the world for more than twelve centuries, its current use is limited to certain countries, such as China, Korea in the Far East, Romania, with an extensive clinical program, Bulgaria and Russia. In the United States the use of venom is officially limited to di-sensitization.

CLINICAL RESEARCH In the United States, Dr Christopher Kim in New Jersey, has a National Institute of Health approved protocol for treatment with bee venom for chronic pain. He has treated more than 2,000 patients with pain refractory to conventional therapies, and who carried indications like arthritis, tendinitis, fibromyositis, neuritis, neuralgia, painful scars. Dr John Santilli in Connecticut has a project, funded by the National MS Society for the investigation of Bee Venom therapy for MS. Dr Ph. Singer, in Michigan has a project treating eight MS patients. Dr Klinghart in New Mexico routinely includes BVT in his clinic's treatment protocols. Typically researchers use venom in ampules. This venom has been extracted from bees, having them sting on a material that allows the retrieval of the venom. The venom then is dried, cleaned, weighted and dissolved in known amounts of liquid. This technique permits the application of the double bind approach to research, as one has created solutions that produce about the same subjective experience as bee venom and therefore can be used as placebo. However the question of whether there is a real equivalence between a given amount of venom from an ampule, where all the doses are 213

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identical to each other on the one hand and the same amount delivered by live bees has not been answered. Two factors are at play here. First during the process the handling of bee venom it is dried and then re-solved. The live bees injects a venom that has not been manipulated and has kept all of its constituents, including some possibly very volatile ones. Further when one gets, say, ten bees, on gets ten slightly different "hits" as opposed to ten identical ones. Indeed, we know that the venom's composition varies slightly with the bee's age and with the bee's genetics, possibly also with the bee's feeding source. These variables can affect bees from a same colony taken at the same time.

Publications The literature on Bee Venom is very varied and quite rich. The American Apitherapy Society (AAS) has a database containing some 630 titles of publications in english from the last 30 years of which 450 were published in the last 5 years. This database is updated yearly and is available to the membership Apimondia publishes regularly a journal, Apiacta, that includes articles on apitherapy. It is projected that an issue of this journal reserved for apitherapy will be published annually. The AAS, counting about 600 members has as its mission the collection and dissemination of information about the medicinal use of the products of the hive. It publishes four times a year a Journal, runs workshops on Apitherapy. The Chair of the Standing Committee on Apitherapy of Apimondia is currently held ad interim by an AAS member.

Clinics There are few clinics in the world devoted strictly to Apitherapy. On a trip organized by the AAS in 1993, our group visited one of them in Beijing, China .. Linked to the Beijing Technological Institute, and operating since 1990 this Clinic accepts patients with various therapies, but shifts them to BVT upon taking charge of the treatment. The range of diagnoses treated in the Clinic is difficult to assess as the concepts used are expressed in traditional chinese medicine terms. We did see arthritics, and one patient who gave the history and the symptomatology of MS. The latter diagnosis does not exist in China. At the time of our visit we heard that that there had been two anaphylactic reactions; both were treated with epinephrine, with a favorable outcome. The diagnoses of these 2 patients could not be retrieved. No statistical studies were available. However the Director of the Clinic stated that the treatments were mostly successful. In Bucharest, Romania, I visited the Center for Apitherapy and research. The staff includes 14 medical specialists, working part time and 2 full time researchers. The latter have several tasks: to develop new combinations and formulas of bee products, mostly following a request from one of the specialists, to control the quality of their preparations.

COMPOSITION OF BEE VENOM Bee Venom includes some forty components, of which a dozen have been extensively examined. 1 They include 11 peptides, 5 Enzymes, 3 physiologically active Amines, Carbohydrates, Lipids and amino-acids. Of the peptides, the most represented are Melittin, Apamin, Mast Cell Degranulating Peptide and Adolapin. All together, they have systemic actions: anti-inflammatory, anti-fungal, anti-bacterial, anti-pyretic, stimulating ACTH,

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stimulating vascular permeability. The Enzymes, act on the cardio-vascular system and locally at the point of administration of the venom. In the most summarized way one can say that Bee Venom acts on the immune system, redirecting some of its faulty mechanism. Bee venom acts on all afflictions influenced by cortisone, however without any of the side effects of the drug.

PRINCIPLES OF ACTION OF BEE VENOM There are three frames of reference in our theoretical view of the action of the venom. I) General, as the venom acts on the hypophyso-cortical axis to stimulate the adrenal cortex. This is more a construct than an established fact, as human experiments did not support this view. However clinic has shown that conditions sensitive to cortisone do respond, frequently to BVT. 2) Locating a sting on an acupuncture point has demonstrated the combined effect of the local stimulation and the acupuncture action. 3) In joints and muscle diseases, the local effect of the venom is the most powerfully demonstrated.

INDICATIONS FOR BEE VENOM Chronic conditions can be defined as stable conditions, maintained over a significant period of time. The intervention of bee venom challenges the elements of maintenance and therefore shifts the status from chronic to acute. The acute condition gives the organism a new assisted opportunity to find a more physiologic equilibrium, i. e. an opportunity to heal itself. The action on the immune system is confirmed by the cluster of illnesses amenable to BVT: Arthritis, of all types, lupus, endarteritis. Throughout history, the chronic conditions the most frequently treated have been and still are all forms of arthritis and conjunctive tissues pathology. In the last ten years an important new indication has been considered, Multiple Sclerosis. It is estimated that about 4,000 people suffering from MS in the United State have been or are treated with BVT. In Brazil the main indication for BVT is asthma. The Chinese have an impressive list of afflictions responding to BVT, covering a number of neurological conditions, joints and connective tissues, benign tumors of the skin, functional conditions such as masculine sexual impotence and neurosis. Some viral conditions such as shingles can respond favorably. A promising avenue lies in the treatment of painful scars, and of treatment of scars in condition of diminished functioning and of pain. Circumstances have shown that premenstrual syndrome react often favorably to venom. So do many cases of chronic fatigue syndrome (CFS). For the latter it is not known whether the long lasting type of CFS also responds. Which ones of the different types of depression respond well to BVT is to be determined. A number of successful treatment of carpal tunnel syndrome have been treated byBVT

COUNTER-INDICATIONS TO BVT As a general rule conditions that lead to instability of the person:

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• • • • • • •

Uncompensated allergies Cardio-vascular conditions Open tuberculosis Syphilis? Gonorrhea unstable, insulin dependant diabetes use of B-blockers

ADMINISTRATION OF BEE VENOM There are many ways of administering the venom. The most frequently used is with live bees, for reason of economy and practicality. This way of delivery will be implied throughout this presentation. I have a The amount delivered with this technique varies between 0.1 and 0.3 milligrams of venom. The amount delivered can be approximately controlled by the time one leaves the sting of the bee in the skin. Practical as this way is, it does not allow an exact knowledge of how much venom a person receives, and for this reason, researchers favor administering the venom from ampules. Bee venom can be obtained in known concentration to be injected intradermally. The conventionally accepted weight of a "dose" is 0.1 milligram. Venom can also be given through unbroken skin as a liquid, which is forcefully rubbed into the skin (a chinese technique) and through electrophoresis or as a salve, often mixed with other active agents such as camphor. Finally it can be applied as inhalations.

ACTUAL TREATMENT As a physician licensed in the state of New York, I am very careful to remind people-they already know this!-that BVT is not an approved treatment modality. Hence it falls under the rubric of research an no payment is due or accepted. I do not recommend this therapy, I study it and teach it application Most of my BVT practice consists of initiating people to BVT. That is getting them started, and launched. This is the frame I will use in the representation that follows. People accepting bee venom therapy have already, by this very choice indicated that they espouse a certain philosophy of life. They [at least in the United State] do not belong to the main stream. They often are desperate. They have suffered for years, spent great amount of time and money, sometimes with little results. But, more important, they are people agreeing to becoming responsible for their own therapy, accepting the fear [which is significant] of the stings and the pain [to which one gets somewhat accustomed] that accompanies nearly every sting. They also accept to change significantly their way of therapy. BVT takes place in a relationship. This is due in part only for technical reasons, many stings are administered in places the person stung cannot reach easily, but more importantly, the element of pain is always present and requires emotional support. This aspect of the therapy is so important that I strongly recommend that anyone starting BVT does it with someone else. The first session begins with acquiring a certain sense about the person seeking BVT. What their condition is, their general state of health, any relevant particularities, their support system, their relation to pain, their level of determination, and their resources: how will they procure the bees, whom or what will they have to guide and advise them in the course of their therapy. Then comes the teaching them about safety, fol-


217

lowing the schema of the next paragraph and the application of the protocol fitting their condition. It is essential that following the first stinging session, the person stung has a 48 hour available and knowledgeable contact to refer to with any question or problem

SAFETY Any treatment with venom starts (or should start) with a test of allergy. One convenient way to do so is to have a bee sting on the wrist, the sting being removed a fraction of a second after the bee stung. In the following minutes the person stung is observed. It is essential that the climate of the stinging session be comfortable and relaxed. I usually take this time to explain the different reactions to the venom, to show a Epinephrine Kit give principle of its use and if necessary write a prescription for such a kit. In addition, all people involved in the therapy should be tested. Bee Venom has an unusual therapeutic/toxic ratio. The amount of bee Venom administered in one session varies from a fraction of one dose to 20 dependant on the indication. Treatments infrequently exceed 20 stings at one time. A healthy person can usually tolerate 100 stings. (I am not here counting with the toxic components, that is the fact that sometimes with relatively small amounts of venom", one can see intravascular hemolysis and kidney failure*). This gives a ratio of 1 to 5. or better in most cases. When thinking of safety, one must however include the element of allergy in general and anaphylaxis in particular. It is generally accepted in the United States that 3 % of the population is allergic to Bee Venom. I believe that this figure includes a number of non-honey bee venom reactions, as people often call "bee" any insect that flies and stings. The US statistics inform us that about 40 people die each year in the States of anaphylactic reaction to stinging insects. Mostly these accidents happen out doors. We do not know how many of these events are due to honey bees. Charles Mraz, the first one to treat an MS patient in the United States with bee venom, indicatedt that in the 65 years of his experience with treating arthritics with bee venom he has never encountered an anaphylactic reaction. This either speaks for a very low proportion of strongly allergic people, and/or perhaps, for the proposition that arthritics do have a modified immune system, that does not create anaphylaxis.

REACTIONS TO BEE VENOM Reactions can be classified in four categories, according to location and to latency from the time of the sting to the onset of clinical manifestations.

Local-Immediate Reactions For this purpose, "local" means that the reactions include the site of the sting, no matter how widely spread they may be. They follow by seconds the administration of venom. Well known, they include Pain, Swelling, Redness, Warmth. They are harmless unless the swelling is located at such • This is the case of a man stung hy 175 yellow jackets. not honey bees but not of a woman having receives 65 honey bee stings t personal communication

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a place as to create mechanical difficulties. Intentional stinging includes attention to this factor.

Local-Delayed Reactions They occur hours later. Itching can be quite unpleasant, but responds usually to cold or to Preparation H (an Anti-hemorrhoidal salve). Changing the technique of stinging by early removal of the sting, say after one minute, often prevents itching. Occasionally one sees a major swelling, mostly in the arms or legs, which can last for days. They are easily confused with cellulitis, and are dues, when they last more then a few hours, to trouble in the drainage of the area. A mechanical solution like elevation of the affected limb or compression bandage is often all what is needed. Rarely one see a reaction revive in an old site stung long ago, with stings applied elsewhere.

General-Immediate Reactions "General" in this sense refers to reactions distant from and not necessarily including the sting site. This is the one that gives us concern. Onset is within minutes, usually, less than five. The first complaint can be summarized by the statement "I don't feel right". It can then take different forms such as nausea, dizziness, urge to defecate or urinate, urticaria, general oedema, general weakness. It can be followed by itching of the palms and the soles, which show nothing. Symptoms like itchy eyes, scratchy throat can follow. If the process continues, breathing troubles come next, (and this is the moment to use the epinephrine kit). The process, left unchecked, can lead to cardio-vascular collapse and death. This picture may be interrupted at any time, and lead to progressive return to normal. Relaxation and reassurance are important to observe as they tend to abort the process. Without being an absolute rule the following has frequently been observed: the longer the latency between the sting and the beginning of a generalized reaction the more benign and slow the clinical process.

General Delayed Reactions Frequently observed in ongoing treatment with BV. It typically takes place in the second week. The most frequent picture is of flue-like manifestations, with gastro-intestinal predominance, fever and general malaise. Often it is followed by a clear abatement of symptoms and has therefore received the not quite earned name of "healing crisis".

PROTOCOLS There are no generally accepted protocols in BVT for the treatments of these conditions, but, the majority of BVT practitioners adhere to certain principles. I will represent some of them in the following descriptions of approaches to illnesses. For this purpose I will use the application of bee venom with live bees. As indicated previously treatment starts with general knowledge of the person, his/her condition, and safety considerations. For the first session, my protocol for all conditions accepts one test sting plus 2 complete stings, that is two stings where the sting is left several minutes in place. For people wishing it, one can cool the place of the sting.


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This cuts considerably the initial, most acute pain. The most practical object to do this with is a small all-metallic can, kept for this purpose in the freezer. For subsequent stinging session, the protocol allows for three more stings than were received on the previous time. So that if the person received, in addition to the test two stings the first time, they may get 5 the second time. If they did get 5 they may have 8. These numbers are only ceilings not to exceed, they never represent obligation. Symptomatic improvement and tolerance to the therapy is the guide to the number of stings and their duration.

SPECIFIC PROTOCOLS Arthritis. The selection of the places to sting is done near the affected joints, seeking places sensitive to pressure, often called "trigger points". Most frequently, after painful or disabling manifestations have abated, a lower "maintenance dose" is adequate. Multiple Sclerosis. This is to be considered a systemic illness, even though, at least sometimes, the symptom picture is localized. Hence the general notion that an MS patient will have to be stung "everywhere". The selection of the points to sting will follow anatomical, or acupunctural principles. This means that one will sting along the nerve paths, with much stinging at the emergence of the nerves, along the spine as well as following acupuncture ways. One of the most frequently presented symptom of MS is fatigue, and this is one that generally responds quite well to the venom, as people speak of increased energy. The timing of the stinging, morning or evening is also determined by individual responses. Some people sleep better and are more rested when they sting in the evening, others need their stings in the morning so as to have their strength to go to work. Some .people will report that they stings until they feel relaxed!

ADVERSE REACTIONS AAS has collected 67 adverse reactions to Bee venom. Here, adverse refers to any reaction that worries the person who has it. Although none of them were judged to be potentially lethal, several received epinephrine, and 9 were admitted either to emergency rooms, or to inpatient status in hospital. The major lesson taught by this series is the importance of information. The majority of the hospitalization might have been avoided if the persons had been informed of the course of reactions and their handling. The issue of what people should do when they have experienced an adverse reaction has been insufficiently studied. The principle is however that they should make certain that they are not at risk. The procedure to achieve this goal is not well determined. It is usually left to immunologists, who often do not appreciate the value of BVT and are convinced that the patient takes unnecessary risks. It is to be noted however that the recommended dose of maintenance bee venom following de-sensitization is 100 microgram), that is one dose! So, here may be the indication that there are ways of reconciling the two opposites, official and alternative medicines.

CONCLUSION There is, in the United States a general increase in the development of alternative medicine as well as acceptance by the authorities of the poeple's right to choose their treatment even if their choice is un-approved.

Th. Cherbuliez

220

AAS has developed a protocol allowing the rating of severity of symptoms in arthritis and in MS. This protocol lends itself for following up people as they progress with their treatment with bees, and of following a control group left unstung. The need for communications amongst practitioners of all countries is more pressing and more promising than ever.

REFERENCES 1. Kim CM-H Bee venom and bee acupuncture therapy 1992 pp515 2. Bousquet-J. Huchard-G Michel-F-B Toxic reaction induced by hymenoptera venom Ann-AIlergy 1984 May 52(5). p 371--4 3. Bousquet-J Immunotherapy with Hymenoptera venoms. Position paper of the working Group on Immunotherapy of the European Academy of AIlergy and Clinical Immunology in Allergy 1987 Aug 42 (6)

pp40H3

26

APITHERAPY IN ORTHOPAEDIC DISEASES Franco Feraboli Orthopaedic and Traumatologic Department Ospedale Civile di Cremona-Italy

For about the last seven years I have been using bee products (honey, pollen, propolis and venom) for the treatment of orthopaedic and traumatological diseases.

HONEY I began employing honey as topical dressing in 8 cases of serious limb compound fractures, with muscle and skin loss. I used sterile gauzes spread with acacia and lime honey and applied on the areas concerned. Honey was taken directly from unsterylized pots. Before application some samples were taken in order to submit them to a bacteriological test, and all proved negative. In the first and more difficult case of tibial compound fracture I applied the gauzes spread with lime honey immediately after the surgical operation on the soft tissues injury. The dressing was changed every day: each time it was soaked with serum secreted from the injured tissues. There was no purulent secretion, and the healing process carried on regularly. In order to stimulate further the cicatrization, after 10 honey dressings I applied refined beet sugar, and then tiny small skin auto grafts (taken from the patient's thigh) obtaining complete re-epithelisation three months after the trauma. Honey acid's pH, between 3.5 and 5.5, and its hygroscopic properties cause injury essudation (1). This could be the reason for the excellent results obtained and for the lack of infections for such serious injuries.

POLLEN I often suggest pollen as the main element in a vegetarian diet which I use in order to reduce the overloading in arthritic patients, expecially those suffering in the lower limb joints. I have analyzed pollen composition, and obtained these results: water 12.28 %, lipids 5.59 %, protides 17.04 %, ashes 2.05 %, extracts unazotized 63,04 %. 221

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F. Feraboli

From these results you could say that pollen composition is like that of an hamburger without the fats (2). The only real problem is due to the special taste of pollen, essential for diet success, but unpalatable to some. In fact a satisfying taste allows the patient to transfer to pollen the psychological problems arising from an eccessive and unbalanced nutrition. Where this occured the results were excellent: haematological tests performed six months and one year after the beginning of the diet always showed completely normal values also in those patients who had hepatic, anemic and dislipidemical problems.

PROPOLIS It has been used in 9 cases of extremely serious bone infections and 14 cases of soft tissue infections. All the patients had previously had, without any success, a treatment of surgical cleaning and antibiotic therapy. The bacteria isolated were in 6 cases Staphylococcus Aureus, in 2 cases Pseudomonas Aeruginosa and in I case Escherichia Coli. For their treatment I used only solid rough propolis and dissolved propolis. The solid propolis was used when the lesion was wide or with undermined edges. The liquid preparation was employed in case of deep septic lesion with thin fistolous channels. Through these I have injected from 5 to 10 ml of diluted propolis each day. Every 3-4 days I executed a careful flush with hydrogen-peroxide, in order to take out from the septic focus the remains of pro polis, which tend to collect in the abcess cavity. The results have always been excellent. After a first phase of 2-3 days, in which the purulent secretion increased, there was a progressive reduction of the secretion, a diminution of hyperpyrexia when present, and a slow but progressive reduction of haematochemical values of inflammation (VES and PCR). I have very much appreciated the cicatrising properties of propolis (3), expecially the quick reduction of the abcess cavity volume and the consequent reduction of the places where bacteria could hide.

BEE VENOM My great interest for the bee world began studying the so-called "antirheumatic properties" of bee venom, part of folk medicine. Therefore I read many publications on this topic (4). They all attribute the anti-inflammatory and antirheumatic qualities of the venom to the stimulation of the axis hipophysis-adrenal glands, with consequent cortisol release (5). Then I experimentally examined cortisol release in ten volunteer patients, who submitted to 4 consecutive days of apitherapy. They were stung by 5 bees each on average. I carried out four daily blood samples (at 9 a.m., 2 p.m., 6 p.m. and 10 p.m.) in order to measure the cortisol level. During the first day no venom treatment was carried out in order to measure the basal cortisol level. My results for the following 3 days of treatment did not demonstrate any significant increase of the cortisol. Only 3 very emotional patients showed a slight cortisol increase.

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Table 1. Record of cases Disease Knee arthrosi s Post traumatic ankle arthrosis Back pain Epicondylitis Shoulder pain Peripheral neuropathies Metatarsalgia and Hallux valgus Gout arthropathies Psoriathic arthropathies Undifferentiated arthritis Rheumatoid arthritis Achille tendinitis

Number of cases

26 4 34 15 31 6 23 8 2 25 12 3

Improved

No improvement

17 3 27

9

8

7

20 6 19

11

7

4

8

2 19 7 2

6 5

Although this test could undermine the mainly accepted theory for the cause of venom effectiveness I started to employ it for the treatment of some kinds of diseases as can be seen in the Table 1. The healing rate was 68 %. The patients treated for arthritis and arthritic pathologies benefitted from a periodic treatment (from 6 months to 2 years in length) consisting of 5---6 therapeutic sessions that have always relieved the painful symptomatology. In these cases the follow up goes from a minimum of 3 months to a maximum of 6 years (average 3.4 years). It is worthing bearing in mind that 8 of the 17 patients with knee arthritis, which improved with apitherapy, were then submitted to surgery to replace the joint, damaged by the inflammatory arthritic process, with a prosthesis. I did not consider 3 cases I treated 7 years ago for hip arthritis. In this kind of pathology bee venom is completely ineffective, as several colleagues who also use this therapy told me. I employed in almost all the cases the sting of live bees applied on the trigger points, with an average of 6 stings for each session (min. 1, max. 130). This figure depends on not the kind of pathology but on the time employed for solving the painful symptomatology. Recently I have utilized in some patients the lyophilized bee venom from Simic's Apitronic Service. It seemed extremely useful and practical: using the syringe I can choose the depth of the injection, allowing new therapeutic possibilities (6). My first results concerning the effectiveness of this product are extremely interesting. I have observed side effects only in 3 patients: one difficulty in breathing spontaneously solved itself, and two cases of cutaneous rashes were treated by antihistamine tablets. Generally (94 % of the cases) the biggest problem caused by apitherapy was itching. I had good results in controlling this symptom by honey, spread on the treated area and left there for 5-20 minutes. I was very impressed by the quick and effective treatment of metatarsalgia in the valgus great toe. The pain in this area is caused by the irritation of the great toe dorsal digital nerve, crushed between the first metatarsal head and the shoe. The venom injected near the first metatarsal head reaches quickly the subcutaneous digital nerve. This action constitutes the effectiveness of the venom: in fact in my opinion it has as its main target nervous tissue.

F. Feraboli

224

In order to verify the venom action on nervous tissues I tested some laboratory animals such as mice, fish and invertebrates. The venom injection in the lumbar area of the mouse (2 mg of venom in a mouse of 20g weight) causes the temporary paralysis of the posterior limbs, while some venom drops in a gold fish acquarium (2 mg of venom /1 It of water /25 g fish weight) causes total motor incoordination to the point of paralysis. Finally venom administered to caterpillars induces immediate paralysis. These simple tests are confirmed by Menez's experimental data (7), demonstrating that hymenoptera toxins work at the synaptic and neuromuscolar level, stopping some ionic channels, expecially those ofCa 2+ and K+ (8). The specific action, therefore, on the nervous system with the stopping or slowing of the pain is clearly evident in the treatment of the hallux valgus, and in the local anesthetic effect following the first quick painful phase after the bee sting, referred to by almost all the patients. But this theory by itself cannot explain totally the good results obtained. On examining the cases treated I noticed that most of the successes were obtained in those cases where there were clearly the classical signs of inflammation: rubor, tumor, calor, dolor and functio laesa. It seems nonsensical to use an inflammatory substance like bee venom, to treat an inflammation. Nevertheless in these cases the apitherapy was very effective and the healing was very quick. Ifwe want to give credit to the homeopathic philosophy we can confirm that the indications for using bee venom are acute inflammations, when there are these symptoms: redness, heat, swelling, pricking and burning pain, worsened by heat and improved by cold (9,10). Acute arthritis is the archetypal rheumatological disease where the use of bee venom is indicated. In this disease there is in fact redness and swollen joints with stabbing, burning and very bad pain. Moreover the joints are stiff and the pain worsened by every small movement. These symptoms are also present in the valgus great toe and in the gout arthropathies, where apitherapy is strongly indicated. I don't believe that the bee venom has special antiinflammatory properties; rather I think that it has an important analgesic property. The first and immediate effect of the apitherapy, as the patients told me, was always the reduction or the vanishing of the pain, although in some cases the inflammation did not disappear. The latter progressively vanished during the treatment or after a few days from the end of the therapy. Interpreting this data in a psychoneuroimmunological perspective (the only one able to bring together the many sides of apitherapy, the cultural, the psychological, the chemical for example) we could assume that the mitigation of the pain influences the central nervous system, modulating favourably the peripheral inflammation. Finally, I think that the bee products are an important support to the medical practice and especially bee venom. It is, in fact, with precise indications and after a careful diagnosis, an effective alternative to the often indiscriminate use of cortisone.

REFERENCES 1. Nahmias F. La miel cura y sana. Editorial De Vecchio Barccllona, 1987.

Apitherapy in Orthopaedic Diseases 2. 3. 4. 5. 6. 7. 8. 9. 10.

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Iannuzzi J. (I 993) Pollen: food for honey bee - and man? Am. Bee J. 133, 557-563 Kaal J. Natural medicine from honey bees (Apitherapy). Kaal's Printing House, Amsterdam, 1991. Malone F. Bees don't get arthritis. Academy Books Rutland. 1979. A.A.S. 1977-1985 Apitherapy Society proceedings. American Apitherapy Society, Inc. Simics M. Bee Venom: exploring the healing power. Apitronic Publishing, Calgary, 1994. Menez A. (1994) La struttura delle tossine degli animali velenosi. Le Scienze. 305, 56--62. Habermehl G.G. Venomous Animals and their Toxins. Springer, Berlin 1981. Hodiamont G. Remedes et venins du regne animal en homeopathie. Chez l' Auteur, Bruxelles, 1984. Desmichelle G., Manson J., Droudard J.M. Homeopathie en Rhumatologie. Maloine Paris 1991 pp. 34--36. 130--131.

27

THE MONITORING OF POSSIBLE BIOLOGICAL AND CHEMICAL CONTAMINANTS IN BEE PRODUCTS Boris A. Yakobson The Kimron Veterinary Institute Bet Dagan, P.O.B. 12,50250, Israel

Recent significant increases in trade together with elevated awareness in health issues, especially the interrelationship between environmental pollution and food, has resulted in the development of international standards for contaminants in meat, milk and other agricultural products. Consumption of honey and other bee products is especially vulnerable to even a suspicion of hazardous contamination, as they are regarded as "health" products and are not essential for human nutrition. There are three main purposes for monitoring honey and bee products: consumer health protection, international competition and better producfquality. In this review "contaminant" refers to any chemical substance which should not occur in natural "clean" honey, beeswax, propolis, and other bee products. The main classes of contaminants are classified as: inadvertent, microbial, drug and acaricides, and poisonous plants. An example of inadvertent contaminants may be pesticides such as monocrotophos on maize fields which can be found even when the major source of nectar is an uncontaminated source. The most important microbial contaminant of honey is Clostridium botulinum, a ubiquitous bacterium (4). Therapeutic drugs and acaricides are frequently used to control and eliminate bee diseases. On rare occasions bees collect nectar from plants which contain plant toxins, resulting in potentially toxic honey (e.g. Rhododendron and romedotoxin or Nerium oleander) (12). Depending on the source of contamination, an alternative classification of contaminants may be considered: environmental, disease and pest control related, storage, handling and processing related (3). It is increasingly accepted to make a careful monitoring of possible contaminants in honey, wax, pollen, royal jelly, propolis and bee venom. Compared to the available literature concerning the analysis of pesticide residues in grains, fruits, vegetables, animal products, soils and water, very little has been published addressing residue analysis in bees, honey and beeswax (1,5,10,21) . . When pesticide residue laboratories are confronted with the analysis of bees and hive products for a particular chemical, the normal response is to adapt a method developed for some other matrix. These adaptations, when successful, are rarely published. 227

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B. A. Yakobson

Thus each laboratory, over time, has developed its own particular set of techniques for analysis of bees and hive products with no standardization between laboratories (8). The range of chemical residues found in bees and hive products is not limited to agricultural chemicals. Bees are notorious for collecting materials contaminated with chemicals and bringing them back to the hive. These include environmental pollutants, radioactive chemicals, heavy metals, inorganic compounds and industrial wastes. Honey bees have been used to monitor local pollution. Regarding the import/export regulations it should be noted that no accepted international detailed specific code for product quality yet exists. A general requirement for honey to be free of any foreign organic and inorganic matter, as well as free from mould, insects, brood and grains of sand has been applied. It is also required that honey is to originate from disease-free apiaries. The analysis of chemical residues is a rapidly evolving field with new and improved instrumentation being frequently introduced. Recently the major advance has been to increase ability to separate compounds through the use of high resolution chromatography. These techniques have contributed to the continuous lowering of detection limits for residues. Gas chromatography-mass spectrometry (GC-MS) is currently the method of choice for a large number of compounds. On the horizon is the greater usage of the argon plasma detector which can quantify a wide number of elements simultaneously. Falsification of honey is an old problem. It will not be considered here, except to mention that the laboratory methods to detect it are well documented (6). Regarding possible microbial honey contamination, C. botulinum should be mentioned. C. botulinium is an ubiquitous bactearium and widespread as dust. Its spores are present in and on a wide variety of agricultural products such as fruits and vegetables. Botulinum toxins are among the most highly toxic substances known. Diet changes after 6 months of age affect the flora of infant intestines so that Clostridium botulinum cannot colonize. On July 5, 1978, the world's largest honey-producing group, the Sioux Honey Association, warned that there may be a risk of infant botulism if honey is fed to infants under one year of age. Contaminated honey has been proven to cause infant botulism very rarely, but has not been shown conclusively to be a significant risk factor. Since honey is not necessary for the nutrition of infants, it is recommended not to be given to infants up to one year of age (4,7,11,16,18). Pesticides remain the major contaminant of concern for honey originating from cultivated plants (oranges, cotton, etc.). Recently, group-specific detection methods for residues have been developed, and the following groups are monitored: organochlorides, organophosphates, carbamates. Varroasis causes a major problem for beekeeping. A wide range ofacaricides is used worldwide to control this pest (9,15,17). In Israel fluvalinate is mainly used and therefore a screening program to examine the level of this acaricide is conducted at a national level (23). Fluvalinate in honey has not been detected, however an early accumulation in wax and propolis has been shown (levels range between 0.2 and 2.4 ppm). The established tolerance for fluvalinate by EPA (Environmental Protection Agency of the USA) is 0.05 ppm for honey. Since beeswax is used in cosmetic products and fluvalinate is a known allergen, a tight control and monitoring program is strongly recommended. A report by Liakos, B. described contamination of Greek honey and wax with malathion and coumaphos (13). Rare, but documented cases of malicious application of home insecticides and pet parasitic ides for bee extermination have occurred. In such cases high residue levels were detected in wax and honey. Various insecticides were found in the wax from wild bee nests, when inappropriate chemical substances were used to remove bees.

Monitoring of Contaminants in Bee Products

229

Antibiotics such as tetracycline, oxytetracycline, chloramphenicol, sulfathiazole, fumagillin may also be found in bee products (19,20). Their origin stems from inappropriate use of these drugs for therapy of bacterial and protozoal bee diseases, when the therapy takes place within honey collection time or 30 days prior to it. A residue monitoring program was initiated in Israel during the years 1995/96. Contamination of honey with streptomycin, tetracycline, sulfonamides, fluvalinate and amitnaz was investigated. No residues of any of these compounds were found. Residues of up to 5 ppm of fluvalilvate were found in the recycled wax. Chemical substances such as ethylene dibromide, phosphine and methyl bromide used in wax moth control, can find their way into bee products. Our experience shows that use of CO 2 in portable PVC built fumigation chambers eliminates the need for anti-wax moth chemical treatment (22). A number of contaminants can enter into bee products during storage, handling and processing. A recent case of increased residue levels of zinc, was described in the Australasian Beekeeper Journal. The zinc residues apparently came from drums, pipes or tanks (2). Wood protectants used in hive manufacturing can also be a source of contamination. Use of non-feed plastics for storage and processing may lead to honey contamination too. A polluted environment, containing such radionuclides as Sr 132 and/or such heavy metals as As, Pb, Cd, Hg, etc., was demonstrated on several occasions to be a source of bee product contamination. For example, 2 peaks of increased level of radioactivity appeared after the Chernobyl disaster in honey and pollen (14). The first peak was a result of direct radioactive exposure. Several months later a secondary effect, through radiopolluted soil and vegetation appeared. Long term monitoring of polluted areas is clearly required in such cases. Regarding pollen, one should pay attention to botanic composition, poisonous plants, visible mould and fungi, mycotoxins, allergens and physical cleanliness. Propolis accumulates environmental pollutants, drugs and in hive waste products to a high degree. Therefore propolis for human consumption should be harvested only from designated hives, which did not undergo any chemical treatment. Royal jelly and bee venom are products with a high protein content. Therefore, special measures such as aseptic collection, rapid refrigeration and strict hygiene of collectors should be taken. The final product must meet bacteriological safety requirements determined as: total bacterial count less than 200 CF/g. Escherichia coli and Staphylococcus aureus should not be detectable in I g, while Vibrio cholera and Salmonella spp. should not be detectable in 100 g. Also no residues of antibacterial drugs should be detectable from royal jelly products. In summary, our experience and data from the literature suggests the following measures to be taken to reduce bee product contamination: I. Education of beekeepers regarding proper bee hive location. 2. Judicious use of pesticides, acaricides, and antibiotics (amount and timing). 3. Systematic monitoring at all level - producer, packer and national organizations.

REFERENCES I. Anon., (1989) Codex Alimentarius commission procedual manual FAO/WHO, FAO, Rome, 44-45. 2. Anon., (1992) Zinc residues found on exported honey. The Australasian Beeneefer II, 215. 3. Anon., (1993) Pesticide residues in Food Report FAO, Rome. 141-149.

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B. A. Yakobson

4. Beetlestone E, (1994) Botulism spores and honey. Am. Bee 1. 134(7) 471-472. 5. Con cellon Martinez A. (1995) Public health and residues of Veterinary medicines in foods Alimentaria, (265) 87-100. 6. Crane, E. (1975) Honey, W. Heinemann Ltd., London 314-325. 7. Fenicia L., Ferrini A.M., Aureli P., and Pocecco M. (1993) A case of infant botulism associated with honey feeding in Italy. Eur. 1. Epidemiol. 9(6) 671-673. 8. Flamini, C., Robin, S. (1985) Methode de dosage des residus d'amitraze dans Ie miel. Bulletin d'Inforrnation des Laboratories des Services Veterinaries 18; 47-54. 9. Infantidis, M.D. et al. (1988) Effectiveness of aqueous solution of malathion against Varroa mite applied in field experiments. Prceedings of a meeting of the EC Experts 203-208. 10. Kastner B., Groschuer P. (1994) Examining bee honey with regard to quality and residues. Dtsh. Lelensm. Ruudsch. 90(5) 146--150. II. Kothare S.Y., Kassuer E.C., (1995) Infant botulism: a rare cause of coloric ileus. Pediatr-Radio. 25 (I) 24-26. 12. Ktochmal, C. (1994) Poison honeys. Am. Bee. 1.134(8); 549-550. 13. Liakos B. (1983) Studies on toxic residues of malathion in honey. Mellenike Kteniatrike 2611 0 308-313. 14. Molrahn, D., Assmann-Werthmuller, U. (1993) Caesium radioactivity in several selected species of honey. Sci. Total Enrion. 130-131. 15. Nansen H. and Petersen 1.H., (1988) Residues in honey and wax after treatment of bee colonies with bompropyl ate. Tidsskr. Planteavl., 1-6. 16. Sakaguchi G., et al (1990) Distint characters of Clostridium botulinum type A strains and their associated with infant botulism in lapan. Int. 1. Food Microbiol. 11(314) 231-241. 17. Slaveezki, Y., Gal H., and Lensky Y. (1991) The effect offluvalinate applicaiton in bee colonies on population levels of Varroa jacobsoni and honey bees (Apis mellifera L.) and on residues in honey and wax. Bee science 1:189--195. 18. Spika 1.S. et al (1989) Risk factors for infant botulism in the United States Archives of Disase in Childhood 64(6) 871-872. 19. Takeba K., Fujinuma K., Nakazawa H. (1995) Honeybee diseases and drug residues, Foods, Food Ingred. 1. lpn (165) 63-72. 20. Usleber E., et al (1995) Detection of Streptomycin in honey by enzyme immunoassay using solid phase extraction and immunoaffinity chromatography. 46(4) 94-96. 21. Weitzman, R.1. (1994) Veterinary drug residues. Commission of the European Communities 1014. 22. Yakobson B.A. et al. (1990) Application of carbon dioxide in a portable fumigation chamber to control bee wax moth. Proceedings ofthe International Symposium on Recent Research on Bee. Bee Pathology, Gent, Belgium 192-193. 23. Yakobson B.A., and C. Efrat (1988) Recent developments in bee disease control in Israel. Proceedings of International Bee Research Association, 4th International confearence of Agriculture in Tropical Climates, Cairo, Egypt.

28

REA VY METALS IN PROPOLIS Practical and Simple Procedures to Reduce the Lead Level in the Brazilian Propolis

Nivia Macedo Freire Alcici CONAP---Cooperative Nacional dos Produtores de Apitoxina Rua dos Pampas 644--Belo Horizonte--30.410-580 Minas Gerais, Brazil

1. INTRODUCTION In order to improve the quality of our propolis, many kinds of analysis carried out and, after a critical study of the results, the lead content in our propolis called our attention.

2.LEAD RESEARCH IN BRAZILIAN PRO POLIS 2.1. Objective To discover the origin of the lead in Brazilian propolis and determine what has to be done to reduce or eliminate the lead in our product.

2.2. Justification Brazil has an expressive production of propolis. Through CONAP the country exports propolis to some different countries, specially Japan. The Japanese market has given our propolis the status of one of the best in the world. This title is not unfounded. The great variety of our plants, the climate, the soil, the pollution free areas and the species of our bees (apis melifera-africanized bees) are decisive factors for the quality of our propolis. CONAP is a national cooperative of beekeepers and our work aims at the beekeepers' awareness as to how important their work is to keep the quality of our products. In our struggle for better products, a lot of very hard work has been done on research as this is the best way to guarantee quality and give our clients confidence in our products. 231

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N. Macedo Freire Alcici

3. PROPOLIS Propolis is a kind of balsam collected by the bees from the buds and leaves of different types of trees and plants. The bees mix this balsam with substances derived from pollen and different types of active enzymes. The enzymes are secreted by glands situated in the head and thorax of the insect' . Propolis is soft and sticky at warm temperatures and can be moulded to fill holes and gaps or spread over surfaces. At cool temperatures and as it ages propolis becomes brittle and hard2• Propolis is the most effective means of applying plant-derived therapeutic substances to the human body and was already used by the Romans and Egyptians. It has always played an important role in traditional folk medicine, and there is clear evidence of growing interest of modem medicine in propolis '. Great part of the studies about propolis compounds are based on the extraction of alcohol-soluble fractions of the resin including wax (insoluble in alcohol). As for the relative proportions of these fractions, the propolis compounds in different regions seem to be very similar as shown in Table 1, which compiles results of propolis analysis done by different authors in the first half of the century. In Table 2 some biological activities of some compounds of pro polis can be seen.

4. LEAD The body of knowledge concerning the individual metals is extremely irregular. Notably, lead, mercury and cadmium have been studied most intensively. But no metal has been more intensively studied from a toxicological point of view than lead. And no metal has presented a broader range of problems, both in regard to the multiplicity of routes of entry and in regard to the spectrum of organs and systems affected in man and in domestic and wild animals as well. Among the heavy metals, lead is one the most used in industries. Its diversified use is mainly attributed to its malleability and resistance to corrosion. Despite the introduction of industrial hygienic measures, lead is still responsible for the high incidence of intoxication among workers. Recent studies have shown that even a person who is not occupationally exposed to lead can absorb about 28 to 38 ug of this metal a day: the ingestion of contaminated food contributes with 20 to 30 ug; breathing atmospheric air with 6 ug and drinking contami-

Table 1. Range ofthe relative proportions among different fractions extracted from propolis Fraction

Proportion

Resin Wax Balsam* Volatile oils Soluble in alcohol Insoluble in alcohol

50-80 15-40 03-11 0-10 05-13 traces to 13

'Corresponds to the fraction that can be extracted with 70 Gl alcohol.

233

Heavy Metals in Pro polis

Table 2. Propolis compounds and some relative biological activities2 Propolis compounds (popular names)

Biological activities

Galangin Pinocembrin

capacity to destroy or halt the multiplication of numerous types of bacteria (B.subtilis, Proteus bulgaris, B. alvei and E. coli B)

Pterostilbene Pinocembrin Acid p-cumaric methyl ester Acid caffeic ester

antimycotic

Ferulic acid

against gram negative and positive organisms

Pectolinarigenin Quercetin Kaempferide

espasmolitic

Luteonin

protection against gastric ulcers

Acaretin

capillary strengthening

nated water with 2 ug 10. Nowadays it is estimated that more than 4,000 tons of lead are consumed yearly in the world. The highest level of exposure occurs principally among people working in lead smelters. The various processes involved in refining lead result in the generation of metal fumes and deposition of lead oxide dust in the workers occupational environment. Some other sources of exposure:

• Mining, foundry and lead refinement: Lead can be found in a great variety of ore like Galena (PbS), Cerusita (PbC0 3) and Anglesita (PbS0 4 ). Galena is the main lead ore, being its deposits generally associated to other metals, specially Zinc (Zn). • Production of antidetonators: In order to produce antidetonators, lead organic compounds are used, specially tetraethila lead which is added to gas as an anti detonator agent. • Manufacture of paint, varnish, enamel and colouring: Some salts and oxides of lead are largely used in this sector in different ways. The most used is the red oxide (PbP). The others are ZCuO Pb0 2 (Copper plumbanate) and lead cromate (PbCr0 4 ). For example, PbCr0 4 is used in the paint employed in traffic signs . • Manufacture of electric cables, tubes and metal plates: The use of lead to manufacture tubes is decreasing and it is being replaced by plastic. Tubes made oflead are used for water supply, sewage systems and pipelines to hospitals and laboratories. Metal plates have a large use in civil construction, sound insulation, manufacture of anti-radiation screens, etc. Besides, lead compounds can be employed to produce press types, glass and ceramics, plastic, projectiles, insecticides, packages. Other manufacture operations, too numerous to describe here, result in varying degrees of lead exposure. In the general population, the major hazard is for young children who chew and swallow objects contaminated with lead-containing paint, flaking paint on walls and woodwork.

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For all practical purposes, there are two forms of lead: the first is organic lead in which the various salts and oxides are considered to act identically once absorbed into the systemic circulation. The second form is alkyl lead, notable tetraethil lead and tetramethil lead. The metabolism of the organic form of lead has been studied intensively, not only in animal models but also in man. It is assumed that lead ions dissociate to some degree and are absorbed and distributed in the body in the same manner regardless of environmental origin. The major routes of lead absorption are the gastrointestinal tract and the respiratory system. Small amounts of lead may also be absorbed from the intake or abraded skin when applied in high concentration 6.

5. METHODOLOGY Method: spectrophotometry of atomic absorption 7--l!. Sample preparation and analysis • • • • •

Weigh 3.0 g of sample. Ash at 600°C for 4 hours. Leave it to cool. Add to the ashes 5.0 ml of concentrated HCI and about 15.0 ml of distilled Hp. Heat in electric plate until complete dissolution of the residues is reached. Filter in paper filter for a 25.0 ml balloon washing the container distilled water. Complete the volume. • Dose the lead in spectrophotometer of atomic absorption with flames.

6. OUR EXPERIENCE Some routine analysis of our crude propolis showed an incidence rate of lead which was considered high and so called our attention. In Brazil there is no official legislation concerning propolis specifically. But we needed a standard to evaluate our products. The Brazilian association that regulates and controls the rates of incidental additives in foods-ABIA-has determined that the maximum level of lead allowed in manufactured products of animal origin should be 2 ppm. In the United States, for example, the maximum quantity of lead allowed in fish is 2.6 mg/Kg and in France 2.5 mg/Kg9. As our propolis is used worldwide as raw material for the manufacture of food supplements, medicines, soft drinks, we have adopted 3.0 ppm as the maximum reference value lead for our crude propolis. And then research started to be carried out aiming at reducing and eventually eliminating the lead from our products. What is the origin of the lead in the Brazilian propolis? What has to be done in order to reduce or eliminate lead in our products? These are our questions and the number of variables involved in the process of evaluating results, in this case, is very large. So the following aspects were chosen as our guidelines: • • • • •

Location of the hive; The kind of paint used on the cover; The use or not metallic spacers; The use or not of metallic screens to collect propolis; The use or not of metallic queen excluder;

Heavy Metals in Pro polis

235

• The use or not of nails and clamps; • The kind of material which the tools to collect propolis are made of; • The kind of material used to store propolis after the collection. First of all, some samples from different areas were chosen. (It is important to explain that CONAP is a national cooperative which congregates more than 200 beekeepers in 17 states of Brazil. As you know, Brazil is a very big country and has an enormous variety of vegetation, including different kinds of forests.) The samples were carefully chosen so as to include representatives of the different characteristics of propolis in different areas.

7. ANALYSIS RESULTS 7.1. It could be observed that the propolis from apiaries located in areas near big cities presented higher level of lead (2.6 to 4.5 ppm) comparing to the propolis from areas far from pollution (1.6 to 2.02 ppm). 7.2. Propolis collected in pollen collector was also analysed (therefore propolis with no contact with the hive) and in some cases we found lead levels different from what was expected, once it was hoped to find less than 2.0 ppm. in this kind of propolis because it had had no contact with possible contaminant agents (2.71 to 3.1 ppm) 7.3. From the same amount of propolis (from the same beekeepers) pieces of propolis covered with paint were separated and analysis was carried out comparing these with pieces of propolis free of paint. The results (in ppm) were amazing and are shown below, exemplifying 6 samples from different areas: Sample

Propolis free of paint

Propolis covered with paint

S. P-l S. P-2 S. MG-6 S. MG-7 S. MG-8 S. SP-12

2.09 3.38 2.12 3.79 1.87 2.00

30.43 20.42 47.98 52.08 19.03 45.01

7.4. Due to the results found in item 3 above, it was decided that it would be essential to analyse propolis from hives covered with paint comparing to that from hives not covered with paint. All the propolis tested came from the same apiary and it was cleared of all evident impurities before the analysis. The hives not covered with paint were covered with a mixture of kerosene and either beeswax or paraffin, in the proportion 6:4. Propolis from hives not covered with paint 1.07 to 2.21 ppm

Propolis from hives covered with paint 3.71 to 4.12 ppm

7.5. The analysis carried out with propolis collected in metallic screens to collect propolis presented lead level from 2.46 to 3.71, and the propolis covering nails, clamps and wires presented results that varied from 2.62 to 3.94 ppm. (It was not possible to carry out a great quantity of analysis in this case as this kind of propolis is not easy too obtain from the beekeepers)

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N. Macedo Freire Alcic!

7.6. Many times during propolis collection it gets wet with honey. When that was the case, as well as when it was propolis was collected too fresh, the beekeepers used to lay it on newspaper so that the humidity could be absorbed. Besides that, it was common to receive propolis coming from the countryside wrapped in newspaper. This product always presented lead level between 4.86 and 6.48. 7.7. Frequently, propolis was received by mail from countryside beekeepers packed untreated colour garbage plastic bags (blue, green, orange, grey or black) and this propolis systematically presented lead level between 3.10 and 3.61. 7.8. As for metallic spacers, it was observed that hardly any Brazilian beekeeper uses them.

8. OUR CONCLUSIONS 8.1. The location of the hives is very important. The hives should be located in pollution free areas, far from big cities, factories and mining areas. Along the process of gasoline combustion, 70% of the lead added is expelled into the atmosphere in the form of inorganic lead particles, usually bromine chloride of lead. The dust of factories and mining areas contributes to pollute the air in big cities and around them and certainly contaminates the plants which bees visit to collect propolis. The high level of pollution can explain the lead level in the propolis collected in pollen collectors. This propolis does not have any contact with the paint covering the hive, with screens or tools. The lead comes from nature, certainly from the pollutant dust on plants. 8.2. Paint is a decisive factor for the increase of the lead level in propolis. Paint or organic coverings are a very complex composition of organic substances. Mimio (PbP 4) is a red powder used in paint and colour paper. Lead carbonate (PbOH2)2'2PbC03 (alvaiade) was much used in paint in the past, but is very toxic so its use has been forbidden for this purpose. Nowadays it is used only to dilute the other colours. Lead chromate (PbCr04 ) is a beautiful yellow colouring used in painting. This compound of lead constitutes the pigment of many kinds of paint and is sometimes used to colour toys for children. The kind of paint (oil paint or water paint ??) was also evaluated: when the beekeepers cover their hives with paint (usually water paint as it is cheaper), the rain can dissolve it and contaminate the propolis. In this case it is not possible to see the paint mixed with the propolis as the dissolved pigments are slowly incorporated into the propolis and the bees do not stop propolinizing on top of it. When oil paint is used, the action of nature against the hive is not so drastic, but when the beekeepers collect the product, usually they scrape some propolis covered with paint or with splinters of wood covered with paint. The contact of propolis with newspapers and colour bags contaminates the product too since different compounds of lead are used in colour pigments. Propolis is a very active substance and it is known that many acids are present in its composition, like ferulic acid. The easy solubility of lead by acids, even weak ones, and particularly by organic acids present in various substances of natural origin, also has here a significant role as it allows the attack of undesirable components of a container or package containing lead compounds. 8.3. Although the number of analysis carried out in relation to the use of metal in screens, tools and wires may not be conclusive, it certainly is elucidating and has stimulated further research on the types of metal they are made of. In general, silver metals other than stainless steel undergo galvanoplastic processes. These are chemical processes that consist in the deposit of thin layers of metal, one on top of the other. It can be done with Zinc (Zn) or Tin (Es) and has a large use in the protection

Heavy Metals in Propolis

237

of iron objects (like nails) or wires (like the ones used in the frames and screens of hives). Many times lead or zinc alloys are used in the electrodes of electrodeposition. It is important to remember that not rarely zinc deposits are associated to deposits of lead ore. Many times metal purification is not perfect and contamination is frequent. In wires, thin plates, tools and screens, 0.5 % of lead is found when they are submitted to tin galvanoplasty, and 2.0 % when submitted to zinc galvanoplasty. In many types of solders used in the manufacture of tools, 30 to 75% oflead is found. A fusion alloy containing 85% of lead is used in the manufacture of press types and in the manipulation of composition machines Therefore, if these metals, Os susceptible to contamination, are replaced by stainless steel, considered as the ideal metal for the manufacture of utensils in food industries since it is more inert and more resistant to corrosion, the rate of contamination by lead is likely to decrease.

9. OUR SIMPLE PROCEDURES TO REDUCE THE LEAD LEVEL AND THE OBTAINED RESULTS First of all, the beekeepers of the cooperative were summoned to a meeting at eONAP, and in that opportunity they were shown the research results and explained the reasons why they were being asked to do some changes in the propolis collection method. At the same time instructions for the new procedures were published in our journal and sent to all our beekeepers in the national territory. 9.1. The hives have to be placed in pollution free areas, far from railways, roads, factories and possibly contaminated lakes, rivers and streams. 9.2. Preferably do not use paint to cover the hives. If it is used, it should be oil paint. We recommend a mixture of kerosene oil and beeswax or paraffin to cover the hive, in the proportion 6:4. This mixture can reduce the level of lead in up to 58.9 %. 9.3. Change the metallic screens to collect propolis for plastic screens, preferable without colour. 9.3.1. If possible, change the material of which the queen excluder is made (for stainless steel, for example). 9.3.2. Replace the wire that holds the wax to the frame for nylon or stainless steel. 9.4. Avoid using nails and clamps in the frames or in the box. Use dovetail work. 9.5. Do not use tools (spatula or chisel) which have "pits"(points of corrosion) or solder. Preferably, use wood or stainless steel tools. 9.6. Never put the collected propolis in contact with newspapers. If propolis is impregnated with honey, put it near or on top of the hive and the bees will lick all the honey. If propolis is fresh and has a high percentage of humidity, put it to dry at room temperature protected from light. 9.7. After collecting propolis, put it in sterile transparent and non-toxic plastic bags. (Do not use colour garbage bags).

10. FINAL CONSIDERATIONS Although it was difficult for some beekeepers to change their queen excluders, or not to use nails and clamps in the hives, the great majority followed these procedures

238


strictly. Many of them no longer paint their hives and are applying the new techniques to new apiaries, and all of them have been more careful about the quality and use of tools. None of them use newspapers and colour bags to store the product any more. Nowadays, most of the crude propolis that comes from different parts of the country presents lead level under 3 ppm. Then it can proudly be stated that the fact that we have been able to transmit to the cooperative's members little bits of knowledge and to show them the importance of quality in their work (keeping in mind that in Brazil many beekeepers are simple uneducated peasants) was responsible for our success in obtaining a better quality product. Besides the tests with our crude products, tests have also been carried out with our propolis tincture and it has been certified that our struggle has been worthwhile since the results found have indicated lead levels between 0.07 ppm and 0.84 ppm. It is important to point out that, in addition to these simple procedures for the handling of the product by the beekeepers, the manufacturing process at the cooperative is also decisive for the quality of the final product. Regardless of size, grade or origin, all the propolis that arrives at CONAP is cleaned by hand, grain by grain, Os that all remaining impurities such as nails, clamps, paint, wood splinters, etc. can be spotted and removed. None of this would have been possible without the close interaction and mutual trust between our technical body and our suppliers, simple but enthusiastic countrymen.

REFERENCES I. Kaal,1. Natural Medicine from Honey Bees .Vert van Aoitherapy. Amsterdam Drukkag Kaal, 1987. 2. Dadant, C.c., Beeking Equipment in : The Hive and the Honey Bee (Graham B. Editor), 1992. 3. Ghisalberti, E.L. Propolis: a review. Nedlands, University of Western Australia, Department of Organic Chemistry 4. Wells, EB. Hive product uses--propolis. Part II Michigan, Kalamazoo: 521-3, 542. 1976 5. Wade, C. Propolis nature's energizer. 24 p. 6. Casarett and Doull's. The Basic Science of Poison. Toxicology, p. 415-416, 2nd Ed., 1980. 7. AOAC--Official Methods of Analysis of the Association of Official Analytical Chemistry. 8. Determination of heavy metals in foods by means of spectrophotometry of atomic absorption. Atomic Absorption Newsletter Vol 14, No.3. May-June, 1975 9. Fabre, R. e Truhaut, R., Toxicologia. Atlantida Editora, S. A .R.L., Coimbra p. 769-789, 1977 10. Laowerys, R.R. Industrial Chemical Exposure -Guidelines for Biological Monitoring, Davis, Biochemical Publications, \983, pp 27-37

29

ACARICIDE RESIDUES IN BEESWAX AND HONEY S. Bogdanov, V. Ki1chenmann, and A. Imdorf Federal Dairy Research Institute Bee Department 3097 Liebefeld, Switzerland

1. INTRODUCTION The acaricides Folbex VA (active ingredient, a.i. bromopropylate) , Perizin (coumaphos), Apistan (fluvalinate) and Bayvarol (flumethrine) are used in Switzerland for varroa control in autumn, after the bee season. These acaricides are in world-wide use and there are many reports, dealing with their residues in honey and beeswax (1-8). The varroacides are non-polar and contaminate mostly the beeswax. Most investigators have not examined their long term effects on the quality of honey and beeswax. As varroacides are used for a long term varroa control, it is important to study their level in honey and beeswax after repeated use. In the present study we report on the contamination level of these acaricides in brood- and honey combs, sugar feed, honey and in new recycled beeswax. We try to answer the following questions: 1. What is the acaricide level in honey and in beeswax after one normal treatment and after repeated or permanent varroacide treatments? 2. What is the behaviour of the acaricides when old combs are recycled into new beeswax? 3. What is the acaricide level in recycled beeswax in Switzerland?

2. MATERIALS AND METHODS 2.1. Materials All reagents were of analytical purity grade. C-J8 SPE (Solid Phase Extraction) Baker 7020 06 disposable columns, mounted on a Baker-l 0 SPE extraction manifold with vacuum. 239

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S. Bogdanov et al.

Honey, Sugar Feed and Comb Samples. All samples were taken after specific acaricide treatments (see below) Sugar feed and combs: From each comb, containing also sugar-feed. about 10 cm2 were scratched with a spoon down to the foundations. The sugar-feed and the comb were separated by sieving. The sugar-feed was analysed as honey. Commercial Swiss beeswax: samples of all the major Swiss beeswax producers (9). Wax of beekeepers with alternative Varroa treatment (10): the wax was taken from beekeepers, who have not used synthetic varroacides for at least 3 years.

2.2. Methods Extraction ofHoney Acaricides. 109 of honey were dissolved in 15 ml of ethanolwater 2:3. The Baker C-18 columns were activated with one volume of ethanol, followed by one volume of water. The honey solution was then passed under constant vacuum. The column was dried for 0.5 hour and is then eluted with 2 ml of ethyl acetate and 2 ml of hexane. The solvent is evaporated under vacuum and the residue is dissolved in 2 ml of isooctane, ready for analysis. Extraction of Wax Acaricides. The extraction and analysis of bromopropylate, coumaphos and fluvalinate in wax was essentially as described (11): Extract 1 g of sample with 10 ml hexane, eliminate high molecular compounds by repeated freezing and centrifugation, purify on florisil columns, elute with petroletherhexane (l: 1); evaporate to dryness and redissolve in 2 ml of isooctane, ready for analysis. Determination by Gas Chromatography. Inject I III honey- or wax extract by an autosampler on-column in 30 m DB I(wax analysis) or 30 m DB5 (honey analysis), both J+W, 0.25 mm id, 0.25 11m film thickness. The GC analysis was done either with a Carlo Erba MEGA Series or with a Hewlett Packard 5890 chromatograph, both equipped with an ECD detector. Quantification was done by the external standard method. The recoveries were between 80 and 100 %, the detection limits were 0.003 mg/kg in honey and 0.4 mg/kg in wax for all acaricides Flumethrin analysis by HPLC of selected wax comb samples was done by Bayer, Leverkusen, Germany. Acaricide Treatments. All treatments were carried out in autumn, after the sugar feeding of the bee colonies. Generally beekeepers in Switzerland renew their combs every spring with 2 -3 foundations per hive. Fo/bex VA: One treatment was with 4 strips per year in "Swiss bee hives". One strip contains 0.4 g bromopropylate. Samples were taken from apiaries with I to 5 treatments. Each year there is one Folbex treatment per apiary. Apistan: The treatment was carried out in Dadant hives with 3 Apistan strips during 4 weeks. One Apistan strip contains 900 mg of fluvalinate. We did a special experiment with 2 bee colonies: the control colony was treated once during 4 weeks and then the strips were taken away. In the other hive the strips remained for 13 months (permanent treatment).

241

Acaricide Residues in Beeswax and Honey

Table 1. Data of the four acaricides used in this study Acaricide

In use since

Active ingredient (ai)

mg ai per treatment

Time of treatment

Folbex VA Perizin Apistan* Bayvarol*

1982 1987 1991 1991

bromopropylate coumaphos fluvalinate flumethrine

1600 32 1600* 14.4*

autumn autumn, winter August, September August, September

·Only a small pan of the active ingredient is released during the treatment.

Perizin: The treatments were carried out in "Swiss bee hives". One treatment was with 50 ml containing 32 mg coumphos. Samples were taken from hives, with I to 5 treatments. Each year there are 1-2 treatments per apiary. Bayvarol: Treatments were carried out with 4 strips per hive during 4 weeks. One strip contains 3.6 mg flumethrin. The samples were taken from apiaries with two treatments.

3. RESULTS AND DISCUSSION 3.1. Brood Comb Wax 3.1.1. Folbex VA. Fig. I shows the accumulation of Folbex residues in the brood comb wax in dependence of the number of Folbex treatments. The values in the diagram are the combined residues of bromopropylate (BP) and its metabolite dibromobenzophenone (BBP). A part of bromo propyl ate (about 20 %) disintegrates into dibromobenzophenone during the burning of the Folbex strips for the treatment. The concentration ratio in

5

years of treatment (4 strips per year) Figure 1. Bromopropylate residues in brood comb wax after repeated Folbex VA treatments. (2 foundations per year.)

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S. Bogdanov et al.

70

mg / kg

60 50

40

r--

-

30

20 10

o

- II Oct

Nov _

...

Dec

Jan

..J Feb

Mar

4 weeks treatment

•

r-- -

-

f---

r-- -

-

I-

~

Apr

May

Jun

-

Jul

--

Aug

L--

Sep

Permanent treatment 13 months

Figure 2. Fluvalinate residues in comb wax after a normal and a permanent Apistan treatment. (Beginning of treatment: 7/9/88.)

wax between bromopropylate and its metabolite was about 5: I and remained constant for all wax samples. There is a significant correlation (p=0.025) between the number of Folbex treatments and the amount of the residues found. The residues were 2 to 5 times lower if 3-4 foundation per colony and year were replaced (2). 3.1 .2. Apistan. In Fig. 2 the wax residues offluvalinate after one treatment (control) and after a permanent Apistan treatment are shown. The residues values in the comb of the control varied between 0.2 and 7.3 mg/kg with an average of l.9 mg/kg. When the same Apistan strips are kept in the colony all the time, the fluvalinate residues increase with the duration of the strip exposition to reach a constant level of about 40-60 mg/kg at the end of the experiment. If the strips are left in the colony for one more year the residues stay at the same level (results not shown). Propolis samples taken from the colony with the permanent Apistan treatment had the same fluvalinate level as the comb wax. 3.1 .3. Perizin. The coumaphos residues do not show an increasing tendency with increasing number of Perizin treatments (table 2 ). An explanation of this behaviour might be the sampling problem. We take the comb wax randomly as pieces form each comb of the hive . If the distribution of the acaricide is not even, these samples might not re The Perizin solution, used for the treatment, is poured between the combs and possibly it can not be evenly distributed among the brood combs. The residues after Folbex and the Apistan treatments seem to be more evenly distributed. 3.1.4 Bayvarol. We have the data of only one study. After two normal treatments the residues in the brood combs were: average 0.051, minimum 0.026 (= analytical detection limit) and maximum 0.176. The residues are lower by a factor of 40 than after the comparable product Apistan (see 3.l.2)


243

Table 2. Coumaphos residues in brood comb wax after repeated Perizin treatments Number of treatments Mean Minimum-maximum n

3.8 0.4-11.9

10

2

5

7.4 1.6--14.2 4

5.8 2.2-13.8 4

Further details: see Methods

3.2. Distribution of Acaricides between Wax Combs, Sugar Feed and Honey The contamination level in the wax and in the honey depends theoretically on two factors: a. lipophilicity of the acaricide b. the acaricide released during the treatment The lipophilic character of each acaricide can be determined by creating the ratio between its concentration in the brood comb, respectively the honey comb and the feed, respectively the honey (see table 3). The greater the ratio, the more lipophylic the substance. The lipophilic character of the acaricide decreases in the following order: fluvalinate > bromopropylate > coumaphos The more lipophilic the substance the more the wax is contaminated and the less the sugar feed and the honey. Table 3 shows the residues in brood combs, honey combs, sugar feed and honey after acaricide treatments. The acaricide levels, found in these products decrease in the following order: brood comb wax> honey comb wax » sugar feed;:: honey. Wax. The residues in the brood comb wax are larger than those in the honey comb wax with all acaricides used. The contamination level of both brood comb and honey comb wax after one normal treatment decreases in the following order:

Table 3. Acaricide residue levels in brood comb wax, honey comb wax, sugar feed and honey Product bromopropylate

fluvalinate

coumaphos

Number on treatments

*

3 Brood comb wax Honey comb wax Sugar feed Honey Honey tolerance limit Ratio brood comb wax I feed Ratio honey comb wax I honey

47.8 2.4 0.03 0.01 0.1 1600 240

116.7 0.07 0.02

2.9 ;<;0.1 ;<;0.003 ;<;0.003 0.01

1670

24.8 14.0 ;<;0.003 ;<;0.003 8300 4670

The values in mg/kg originate from different experiments with the acaricides.

'The hive was treated during II months with Apistan. (For details see Methods and text).

2 3.8

0.7 0.013 0.015 0.05 290 54

7.4 0.4 0.010 0.004 740 100

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S. Bogdanov et al.

bromopropylate > fluvalinate

~

coumaphos » flumethrin

Feed and Honey. The concentration of the acaricide in the feed and in honey is a result of the equilibrium of the active ingredients between the sugar and the wax compartiments. The more hydrophilic the product and the more active ingredient is released, the greater the feed and the honey contamination. The acaricide residues in the feed and in the honey is on the average 2,500, respectively 1700 times smaller than in the brood combs, respectively in the honey combs .. The contamination of honey and feed after one acaricide treatment decreases in the following order:

bromopropylate

~

coumaphos> fluvalinate

Although much more bromopropylate is released into the colony after one treatment than coumaphos (a factor of 50), the honey residues after treatments with either acaricides are similar, because coumaphos is much less lipophilic. These results show, that the acaricide level in honey did not reach values above the tolerance limits in honey (see table 3). The danger for honey contamination, and also of exceeding the tolerance limit is greatest after the treatment with Perizin, because this acaricide has the weakest lipophilic character. We did not measure Bayvarol residues in honey. But according to the experience of Bayer, the honey flumethrine levels after Bayvarol treatments are below the detection limit of 0.003 mg/kg.

3.3. Recycling of Beeswax Brood comb wax was spiked with a certain amount of active ingredient and was then melted under different laboratory conditions (see table 4). The recovery of new wax under all conditions was 25 %. The results are summarised in table 4. The acaricide concentration in the new recycled wax was on the average 1.7 times highe~ than in the old combs. Boiling for a longer period of time and at higher temperatures (autoclave) had no effect on the acaricide concentration. The acaricide enrichment into new beeswax might be explained by the better solubility of the substances in the wax matrix than in the comb debris.

3.4. Acaricide Level in New Beeswax All beeswax, used for new foundations is produced by recycling old combs. In table 5 the residue level in commercial beeswax and in wax of beekeepers using an alternative Varroa control since at least 3 years after the last synthetic acaricide treatment are summarised. The residues in wax from beekeepers using alternative varroa control are much lower than those in the commercial beeswax. They are on the average 11 times lower for bromopropylate and fluvalinate and at least 3 times lower for coumaphos.

4. CONCLUSIONS 1. After acaricide treatments the comb wax is contaminated, while in honey there are very low residues. After repeated or longer treatments the residues in the


245

Table 4. Recycling of acaricide contaminated old comb wax into new beeswax Bromopropylate mglkg

Coumaphos mg/kg

Fluvalinate mg/kg

Flumethrine mglkg

19.6 36.0 34.6 34.8 34.0 1.8

14.8 28.9 27.8 27.5 27.9 1.9

17.0 26.9 26.5 27.1 24.3 1.6

20.3 34.8 33.4 34.4 31.2 1.6

Combs before recycling I hour boiling 3 hours boiling I hour autoclave (140 0 C) 2 hours autoclave (140 0 C) Enrichment factor wax/comb Results in mg/kg are averages of a duplicate.

comb wax rise, while those of honey remain relatively low. With the exception of Perizin, for most acaricides there is no danger of exceeding the honey tolerance limits. 2. In a recycling study of acaricide contaminated combs the acaricide concentration in the new recycled wax was on the average 1.7 times higher than in the old combs. Boiling for a longer period and at higher temperatures had no effect on the acaricide concentration. 3. The commercial samples during 1994 contained all three acaricides (without flumethrine) in the range between 0.2 and 4.8 mg/kg. The residues in beeswax from beekeepers, using alternative varroa control since at least 3 years are much lower than those in the commercial beeswax. 4. While the residues found in wax are not toxic to bees, the permanent presence of subtoxic amounts might lead to Varroa resistance.

SUMMARY In Switzerland the acaricides Folbex VA (bromopropylate), Perizin (coumaphos), Apistan (fluvalinate ) and Bayvarol (flumethrine) are used for varroa control after the bee season in autumn We studied the contamination level of these acaricides in brood combs, sugar feed, honey and in new beeswax. All samples were analysed by gas chromatography with ECD detection. After one normal acaricide treatment in autumn the brood comb wax was contaminated by all acaricide with residues ranging from 0.03 to 48 mg/kg. The degree of contamination decreases in the order:

Table 5. Acaricide levels in mg/kg of Swiss commercial (comm.) beeswax and wax from beekeepers using alternative varroa control since at least 3 years (altern.) Bromopropylate Comm. Average Minimax % positive n samples

2.1 0.7-4.2 100 7

Alter. 0.2 0.1-0.4 20 5

Fluvalinate

Coumaphos Comm. l.l 0.5-1.2 100 7

Alter. 0.4 0.4 0 5

Comm.

Alter.

2.3 1.5-3.4 100 7

0.5 0.4--1.3 20 5

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S. Bogdanov et at.

bromo propyl ate > fluvalinate "" coumaphos » flumethrin The residue level in the honey comb wax was on the average 10 times lower than in the honey combs. These residues increase with increasing number or longer duration of treatment. The residues in the sugar feed were much smaller than in the corresponding brood comb wax and varied from 0.004 to 0.04 mg/kg. The residues in honey were all beyond the tolerance levels and varied from 0.003 to 0.015 mg/kg. The greatest danger for honey contamination comes after the treatment with Perizin. where after one treatment one third of the tolerance level was reached. In a model study we examined the behaviour of the acaricides during the recycling of old comb wax into new beeswax. Old comb wax was spiked with a certain amount of active ingredient and was then melted under different conditions. The acaricide concentration in the new recycled wax was in average 1.7 times higher than in the old combs under all conditions (longer boiling times and higher temperatures). During 1994 the commercial samples in Switzerland contained all three acaricides, flumethrine excepted, in the range between 0.2 and 4.8 mg/kg. The residues in beeswax from beekeepers using alternative varroa control for at least 3 years are much lower than those in the commercial beeswax. Because the acaricides mentioned contaminate beeswax, and to a smaller extent also honey, we developed in Switzerland alternative methods for Varroa control, using oxalic-, lactic- and formic acid. With the organic acids no problematic residues in wax and honey are expected in the long run.

REFERENCES 1. Bogdanov, S.; Imdorf, A.; Ki1chenmann, Verena; Gerig, L. (1990) Ruckstande von Fluvalinat in Bienenwachs, Futter und Honig. Schweiz. Bienenztg., II3. 130-134 2. Bogdanov, S.; Imdorf, A.; Kilchenmann, Verena; Gerig, L. (1990) Riickstande von Folbex in Wachs, Futter und Honig. Schweiz. Bienenztg., 113, 250-254. 3. Klein, E., Weber, w., Hurler, E. and Mayer, L. (1986) Gaschromatographische Bestimmung von Bromopropyl at und 4.4-Dibrombenzophenon und verschiedenenen Akariziden in Honig und Wabenwachs. Detusche Lebensm Rundschau. 82, 185-188 4. Thrasyvoulou, A.T. and Pappas, N. (1988) Contamination of honey and wax with malathion and coumaphos used against the varroa mite. J.Apic.Res. 27, 55--61 5. Lodesani, M., Pellacani, A., Bergomi, Carpana, E .. Rabitti and T. Lasagni, (1992) Residue determination for some products used against varroa infestation in bees. Apidologie, 23. 257-272 6. Hansen, H. and Petersen, H. (1988) Residues in honey and wax after treatment of bee colonies with bromopropylate. Danisch Res. Service Plant Soil Science, Report No. 1921 7. Wallner, K., Nebeneffekte bei Bekiimpfung der Varroamilbe. (1995) Die Riickstandssitutation in einigen Bienenprodukten. Bienenvater, II 6. 172-177 8. Bogdanov and Kilchenmann, V. (1995) Acaricide residues in beeswax: long-term studies in Switzerland, Apidologie. 26, 320-321 9. Bogdanov and Ki1chenmann, V. (1993), Akarizide in Schweizer Mittelwanden - erste Ergebnisse. Schweiz. Bienenzeitung, II6, 559-580 10. Imdorf, A., Charriere, J.D., Maquelin, c., Ki1chenmann, V. and Bachofen, V. Alternative Varroa Control. American Bee J. 136, 189--193 II. Zimmermann, S., Gierschner, K.H. and Vorwohl, G. Bestimmung von Brompropylat, 4,4 - Dibrombenzophenon, Coumaphos und Fluvalinat in Bienenwachs. Deutsche Lebensmittel Rundschau, 89, 341-343

30

JUDGING THE QUALITY OF HONEY BY SENSORY ANALYSIS Michel Gonnet Institut national de la Recherche agronomique 84914 Avignon, France

SUMMARY The sensory analysis constitutes an essential basis for the determination of food quality. It is an indispensable and complementary part of the traditional laboratory physico-chemical analysis. The results are scored for judgment. The sensory analysis contributes to an improved definition and characterization of honey graders may refine their unbiased evaluation of the tested product. The suggested procedures can be used at honey shows, or as an attribution of special honey qualities, in addition to chemical analysis. Basic knowledge of honey properties and qualities, or of their absence, may contribute to the improvement of honey preparation, aiming at its preservation at a high quality. The originality of this novel technique of honey tasting should provide consumers with a better knowledge of honey.

INTRODUCTION The sensory or organoleptic analysis is based on evaluation and scoring ofthe qualities of consumed foods by visual, olfactory, gustatory and tactile perceptions. Actually, this procedure has been standardized to enable a semi-quantitative and objective scoring. (1,2) Honey is a natural product originating from flowers' nectar. Since the ecological and floral conditions change in each season of the year, honey is not a uniform, constant and stable product. Moreover, the definition of the quality of a food and in particular of honey, is a subjective one and several criteria can be used according to the nutritive, dietetic, hygienic and organoleptic qualities, as well as the therapeutic and symbolic values of the honey. Chemical analysis gives information on the freshness floral origins, the chemical composition of honey, its nutritional and dietetic values. Nevertheless, the chemical analyses are insufficient to provide complete and objective information about this food. The well defined methodology includes honey judging score sheets. Furthermore, to be efficient, the tested samples 247

248

M. Gonnet

must be selected from honeys belonging to the same categories and to be compared with a well defined and a characteristic honey reference. (3)

1. METHODOLOGY The methodology is based on the following procedure. Thirty to forty grams of honey to be tested are poured into a wine glass with a foot and a stem (capacity: 200 ml) that allows the direct observation of the following criteria: colour, transparency, cleanliness, viscosity, crystallization and smell intensity of monofloral or multifloral origins, animal or vegetal nature. The tests must be carried out in a quiet sound proof, correctly illuminated room (200--400 lux), without odours, where a pleasant average temperature (20°C) and a good relative humidity (60%) are maintained. The beekeepers or the professional tasters perform their tests in rooms with pastel coloured walls. They dispose of drinking water and of inert plastic spoons to taste small amount of 10 to J5 honey samples to be tested. To avoid supersaturation and sensorial fatigue they have during the breaks to eat a juicy and slightly acid apple. Also, the tasters must have information concerning the floral origin, the technology used in the beeyards to remove honey for extraction and other procedures used during and after honey extraction, packaging, storage, etc. The honey test consist mainly of three phases. During the first one the taster evaluates the visual qualities of honey: uniformity of appearance, colour, cleanliness, clearness, fluidity, consistency of solid or crystallized honeys, etc. During the second phase he smells the honey to detect the Table 1. Honey judging score sheet Ordinal system for multiple samples Date: Jury N°:

Place: Taster's name: 1. Superior +

2. Superior 3. Satisfying 4. Limited Disqualifications: XX = Quite strong

X= minor

XXX = Very strong

(Encircle the number of crosses corresponding to

th~

intensity level)

Disqualifications

Sample N°

Evaluation

Visual

Olfactive

Gustative

Tactile

XXX

XXX

XXX

XXX

XXX

XXX

XXX

XXX

XXX

XXX

XXX

XXX

XXX

XXX

XXX

XXX

Qualities Deficiencies

Score

249

Judging the Quality of Honey by Sensory Analysis

primary and secondary odours: intensity, animal-like or vegetal-like characters, fruity or resinous, etc. During the third phase honey is tasted, dissolved with the saliva and warmed up in the mouth to body temperature; it is the gustatory or savours test which complement the previous ones. The gustatory buds allowed us to distinguish between 4 savours: salty, sweet, acid and bitter. Also, during this phase the taster can determine some of the tactile qualities of the honey (consistency, unctuosity, granulation) and of its more subjective parameters: roughness, aggressivity, astringence. (4)

2. THE HONEY JUDGING SCORE SHEETS All the information acquired during each sensory test can be transducted on didactic sheets, using hierarchic classification, established by trained tasters. Then, at the end of the honey show or competition, a final and reliable score must be attributed both by each taster and by the jury (3 to 5 persons). Two scoring systems can be used. The first one, called the ordinal system (Table 1), allows to quickly reduce the number of samples to be tested. The trained tasters can freely evaluate, from 1 to 8, the visual, gustatory, olfactory and tactile disqualification of honey samples. Thus the samples that obtained a score below the median ordinal score are eliminated from the competition. (1) The second scoring system is a cardinal one (Table 2). The final score (max.: 20) results from the arithmetical addition of the partial score attributed to the visual (0-5), olfacTable 2. Honey judging score sheet for one samples (cardinal system) Origin of honey

Taster's Name

Date:

Sample N°

Quality

Jury N°

I

Qualities

Deficiencies

Tests

Sensations

Visual

Appearance: neatness and uniformity

+

=

-

Colour

+

=

-

=

-

Cleanliness

~

II

Olfactive

Odour

+

=

-

III

Gustative

Aroma

+

=

-

Primary and secondary flavours

+

=

-

Consistence unctuosity granulation

+

=

-

IV

Tactile

General evaluation of qualities and deficiencies

Score 0-5

0-4

l'-'

0-3

Final score

*

(+) (=)

H

Encircle the selected sign No deficiency Slight deficiency Strong deficiencies

0-20

250

M. GODDet

Table 3. Honey Competition Recapitulative scoring sheet of the jury according to ordinal

classification Jury N°

Honey Sample Scores by Jury N°

Samples

N°

I

2

3

4

5

Final Score

Classification according to the lowest score in the row

Medals: gold, silver, bronze

tory (0-4), gustatory (0-8) and tactile (0-3) evaluations. At the end the taster is requested to summarize the remarks 0 the characteristics of the assessed honey sample. (5) A recapitulative scoring sheet of the jury (Table 3) indicates the relative classification obtained and the medals that will be attributed to the best samples. (5)

3. TRAINING OF TASTERS AND THE USE OF THE SENSORY ANALYSIS TO PROMOTE HONEY QUALITY We recommend that beekeepers should be trained in the methodology of sensory analysis to be able to assess correctly the kind of honey they are producing and to improve their production methods. (6) The jury should consist of no less than three members, and there should be preferably 4-5 tasters. A jury composed of experts is selected as well as a jury of layman is randomly selected from a given population. (4) Honey is an excellent natural food. Its flavour and taste vary according to the abundance of flowering plant species and the region. Until now, the chemical analysis of honey does not include these parameters. However they can be detected and defined by the above described sensory analysis. (7)

Judging the Quality of Honey by Sensory Analysis

251

REFERENCES I. Gonnet M. and Vache G. (1979) Technique de degustation des miels et recherche d'un systeme de notation et de classification objectif pour apprecier la qualite par l'analyse sensorielle. XXVII Congo Intern. d' Apiculture Apimondia, Athenes, lOp. 2. Gonnet M.and Vache M. (1986) Le gout du mie1, UNAF, Paris, 110 p. see also English translation: The taste of honey, Apimondia, Bucarest. 159 p. 3. Gonnet M. (1987) La definition sensorielle d'un miel: complement indispensable it l'analyse traditionelle. Abeilles de France 719, 410-412. 4. Gonnet M. (1988) La degustation, tout un art. Rev. Franc. Apicult. 472,129-\32; 473,181-185. 5. Gonnet M. (l993a) Methodologies d'evaluation sensorielle des miel et de hydromels. Presentation des fiches de notations nouvelles. Abeilles de France 781, 171-176. 6. Gonnet M. (1993c) Methodologies promotionelles pour les miels. Abeilles de France 786, 417-421. 7. Gonnet M. (1991) Le miel: approche d'une appreciation sensorielle visant it une meilleure definition de la qualite du principal produit de I'abeille. Abeilles de France 757, 81-85.

31

METHODS FOR THE CHARACTERIZATION OF THE BOTANICAL AND GEOGRAPHICAL ORIGIN OF SOME BEE PRODUCTS AND FOR THEIR QUALITY CONTROL Giancarlo Ricciardelli D' Albore Agricultural Entomology Institute University ofPerugia-Italy Borgo XX Giugno, 74, 06121 Perugia, Italy

1. INTRODUCTION Melissopalynology (the microscopic analysis of honey) together with organoleptic analysis has been used for the past one hundred years for the characterization of the botanical and geographical origin as well as the quality control of some bee products (9). The majority of the research in this field has been on honey and pollen; recently royal jelly, propolis and bees wax have also been studied (11 and 10). The analytical methods, however, cannot be considered definitive; in fact during the last twenty years many research groups have been working on the standardization of the methods; today some are still active and we can expect that further modifications and improvements will be made. This paper is the result of over thirty years of personal experience. Contacts with foreign researchers and many stages in foreign countries have contributed to my knowledge of this topic. The first aim of this work is to describe syntetically the methods and the evaluation standards; the second is to criticize each method in order to estimate its value and eventually its limit; particularly for melissopalynology, it would be interesting to single out the most important elements of the environment and of the hive that affect the amount of pollen in the honey which may sometimes produce problems for the analyst.

2. MATERIALS AND METHODS Many samples of bee products were analyzed for the national and international studies; many researchers collaborated in this field; since 1985 wc havc also provided the Ital253

254

G. Ricciardelli D' Albore

ian beekeepers, the honey industries and the merchants with an analysis service; so the analysed samples have been very numerous.

2.1. Microscopical Analysis of Honey The methods for the qualitative and quantitative analysis of pollen in honey is well known and exhaustively described by Louveaux et al. 1978. The methods allow the botanical (unifloral or multifloral honeys) and geographical origin of the honey to be determined. Both methods (fresh or acetolyzed pollen) are efficacious, but the fresh method is faster. The microscopical analysis of royal jelly is carried out as follows: I g of sample is dissolved in distilled water and then a 5% potassium solution is added. The sample is centrifuged, the sediment is sucked up and transfered to a microscopic slide as is done in honey analysis. In the sample fresh and acetolyzed pollen is found. For propolis 250 mg are dissolved in a solvent (for ex. ethyl alcohol plus acetone plus chloroform in equal parts); after boiling and centrifuging, ethyl alcohol is added to the sediment; after a second centrifugation the sediment is acetolyzed (2) and finally transfered to a microscope slide as above in the reported methods. For the pollen two samples of pollen are weighed (each five grams); the loads are divided by their colour and weight again; the loads are then dissolved separately with water; the sample is sucked up with a Pasteur pipette and transfered to a microscope slide as above (3) and fresh pollen is analyzed. In the beeswax analysis the problem is to eliminate all the wax and the impurities, saving the pollen; so five grams of wax are dissolved in a solvent (Chloroform); after centrifugation, ethyl alcohol is added to the sediment, then further treatments and the slide preparation are the same as for propolis. In this case the acetolyzed sample is easier to analyze and all impurities are eliminated.

2.2. Organoleptical Analysis For honey the most used methods are the French and the German ones; the former is too subjective, while the second is more objective and is hence preferred. For both methods our senses are used to give an opinion on the following parameters: colour, purity, odour, flavour and touch; each evaluation is based on the presence or absence of imperfections; finally one judges the quality. The German method (DIB) is very meticulous particularly the appearance, because it is not important that a honey is of high quality, if it is presented in faulty food-containers (impurities or rust under the cover, twisted label. etc.) (5). Briefly for the other bee products the organoleptic analyses are as follows: • royal jelly: colour, consistency, odour, flavour, impurities (above all little wax pieces); • propolis: colour, odour, flavour, impurities (mostly little pieces of hive wood), presence of various sticky materials (sometime tar); • pollen: colour intensity, odour, flavour, impurities, dryness . • wax: colour, odour, impurities.

3. DISCUSSION For melissopalynology it is necessary to have a very good actuopalynologist, who is able to determine all the pollen grains in a honey and to interpretate its pollen spectrum.

Botanical and Geographical Origin of Some Bee Products

255

3.1. Honey The pollen quantity in the sediment of a honey is due to some important factors: 3.1.1. Primary Contamination. The gatherers soil themselves with pollen, partially falling into the nectar; this pollen will be transfered with the nectar and will be lodged in the small cells of the combs. This contamination is extremely variable; it depends on the foraging distance, so the Zander valve may pick up the pollen grains, which are in the ingluvies. It also depends on the pollen size and on the exine roughness (big and spinulouse pollen grains are picked up more easily). Moreover it depends on the presence of other polyleptical pollinators on the flower (for ex. the bumblebees leave pollen in the nectar, coming from other species visited earlier; this contamination has been considered only recently). Finally it depends on the flower morphology and the plant type; particularly a flower with little pollen, or aborted pollen or lacking pollen, extrafloral nectaries, a high quantity of small pollen or a low quantity of big pollen; besides typical flower morphologies, which inhibit the primary contamination; monoecious or dioceious species, typical behaviour of the honeybee (for ex. nectar sucking without contact with the anthers) etc. (14). 3.1.2. Secondary Contamination. In the hive the continuous movement of the workers, the trophallaxis, etc. increase pollen contamination of the nectar; also pollen of polIiniferous species are part of this contamination; finally, it comes in the air current, which makes the combs rich in anemophilous pollen (14). 3.1.3. Tertiary Contamination. Essentially it depends on the bee-keepings techniques such as the method for removing honey from the combs (for ex. percolated, centrifuged or pressed honey), the kind of hive and other beekeepings practices. Finally for the above mentioned factors the pollen may be hypo or hyperrepresented in the honey; sometimes it may be represented or "not pertinent", i.e. not coming from a nectariferous flower only (12, 7 and 14). All these factors, the frauds and the filtered honeys make the judgement of an analyst difficult (botanical origin). Generally for determination of the geographical origin, there are no problems, saving the mixtures of different origin, which need expert experience. Yet for a correct judge the analyst needs the organoleptic and some physico-chemical analyses too. In the sediment of the honeydew honeys prevalently we will find many anemophilous pollens, spores and others parts of the "fungi imperfecti". The organoleptic honey analysis, helpful for the microscopical analysis, allows the quality of a honey to be evaluated and how it looks to the consumers. As mentioned above the German method accurately analyzes the container, the impurities, the colour, odour, flavour and the crystallization of a honey; when a honey crystallizes it turns to make account the cream honey. When an analyst evaluates the quality of a honey practically he procedes as follows: first, the microscopic analysis (botanical and geographical origin, presence of impurities such as hairs or wool thread, eventually the presence of yeasts and so on); the second is the organoleptic analysis; then generally the freshness of honey is evaluated with some chemical analyses (HMF and ID); rarely for very difficult honeys or suspicious frouds further chemical analyses are considered (addition of sugar, artificial honey and so on). Because of the many factors which influence the quantity and quality of pollen, the analyst

256


Figure 1. Pollen spectrum of an Australian honey: a) Banksia; b) Eucalyptus; c) Echium .

.f

< •

.;

,~

~

~

(,

0

·0

.,

....

'eJ

,--I

t;'!

b r

C

Figure 2. Pollen spectrum of an Ethiopian honey: a) Lannea; b) Samanea.


257

has accurately to correct and compensate their percentages. The organoleptic analysis in these cases may be very helpful. When a honey comes in a big industry, the main analyses are as follows : container condition, honey homogeneity, impurities, organoleptic analysis, colour, moisture and hydroxymehilfurfural (mg/Kg) .

3.2. Pollen The microscopic analysis allows the botanical and geographical origin of a mixture to be perfectly. For the organoleptic analysis procedes as follows : the colour is observed; it must be intense , that is the product must be stored in a dark and cool room; one smells the pollen (it must smell of dry hay; when it smells slightly acid it is not usefull). Impurities are looked for and a load is pressed between two fingers (if to hard it could be heated in an oven and this is an imperfection, because the sugars may be caramelled; if to soft the moisture is to high, so the product may spoil). The pollen moth (Plodia interpunctella Hb.- Lepidoptera, Heterogynidae) may be eliminated conserving the product at 1°C; sometimes it may be necessary to disinfest the pollen as done for food-stuffs (CCI4) . For the microscopic analysis there are no limits, saving the mixtures (geographical origin).

3.3. Royal Jelly The microscopic analysis is very difficult, because there are many broken or bad pollen grains in the sediment (larvas digestion and regurgitation) (13); yet it is possible to

Figure 3. Pollen spectrum ofa tropical African honey: a) Vernonia; b) Triumfeua; c) Gramineae.

Figure 4. Pollen spectrum of a Senegalese honey: a) Elaeis; b) Combretaceae.

Figure 5. Pollen spectrum of a Tropical African honey: a) Terminalia; b) Palmae.


Figure 6. Pollen spectrum of a Spanish honey: a) Brassica ; b) Genista; c) Rubus; d) Stachys.

Figure 7. Pollen spectrum of a Venezuelan honey: a) Hyptis; b) Eupatorium; c) Avicennia; Mimosa pudica.

259

Figure 8. Pollen spectrum of a Venezuelan honey : a) Grewia ; b) Protium; c) Xantoxylum; d) Schinus.

Figure 9. Pollen spectrum ofa Zambian honey: a) Brachys/egia; b) Coffea ; c) Scrophulariaceae.


261

determine the geographical origin and the presence of impurities (wax pieces). For the organoleptic analysis one checks the colour, odour, flavour, impurities; unfortunately it is difficult to evaluate it freshness; so chemical analyses are needed.

3.4. Pro polis For the microscopic analysis the geographical origin can be determined, but not the botanical. For the organoleptical analysis the colour is observed (frequently yellow and brown); black propolis may indicate that the gatherers collected other material; the odour (propolis odour is typical), the impurities (hive wood pieces, from the traditional propolis collecting such as propolis scraping off); today beekeepers recover the propolis with a suitable plastic net.

3.5. Bee Wax From the microscopic analysis it is possible to determine the geographical origin; in the organoleptic the colour and the odour only are important; further chemical analyses allow the ascertainment if other synthetic waxes have been added (paraffin and so on). The factors to be considered are: the melting point, viscosity, saponification value, ester value and others.

3.6. The Quality Trade-Mark Recently the European Community (CEE) has issued a new regulation (CEE 1992). Practically this regulation, which should be extended to all foreign countries, privileges the qualities and the guaranteed origin of the products, such as from the time this is realized by other food-stuffs (for ex. wine, ham, etc). The Community allows that a bee product has European trade-mark of quality and guaranteed origin for the consumers, on the condition that the product is free of imperfections and adulteration; at the same time it is necessary to determine the origin of this product (country, region, mountains or valley and so on). So the geographical origin has the same importance as the quality; therefore, there is the possibility of emphasizing the value of the product of a country. For the determination of the geographical origin, it is necessary to analyze representative samples of a territory for three consecutive years because the final pollen spectra have to be steady and meaningful; it is also necessary to specify the marker pollens, typical and unique for them honeys of one country. This new analytical technique must be trusted in order to achieve a more remunerable market for our bee products, prevalently of the multifloral honeys, sometime partially less advertised in comparison with the unifloral honeys (Ricciardelli D' Albore 1994).

4. CONCLUSION The following conclusions can be made: • now we have all the analytical techniques for the determination of the botanical and geographical origin of a bee product (particularly honey); • the microscopic analysis has some limits, but fortunately the organoleptical analysis is very useful in these cases;

262


• the organoleptic analysis by mean of our senses allows to determine the quality and the freshness of a bee product to be determined; • the physical-chemical analysies may be helpfull in some difficult cases; prevalently, in order to define if a product is fresh and not adulterated, the most important analyses are the hydroxymethilfurfural (mg/Kg), the Diastase index and the moisture percentages; rarely H.P.L.C. chromatography and electrical conductivity; • the analyses should always be carried out to compensate one another; • the European Communitys' regulations on the quality and the origin of a bee product should be very important for the trade-mark of the products; • based on our experiences, our Institute is certainly available to help in this field, such as we have recently done in Venezuela, Brazil, Italy, North and Central Affica. • next year we will start with a big project for the quality control of honeys in Argentina too; • a perfect honey can be produced only if the beekeepers use modern techniques and if at the same time, before the commercialisation, the honey 'is accurately controlled.

REFERENCES I. CEE (1992) Regolamento n.2081192 relativo alla protezione delle indicazioni geogratiche e delle denominazioni d'origine dei prodotti agricoli e alimentari. Gazz.Uff. CEE 24/07/92 - 208: 1-8. 2. Erdtman G. Pollen morphology and plant taxonomy. Angiospermes I Almqvist and Wicksell. 1952 pp.6-IO. 3. Louveaux J. (1958-1959) Researches sur Ie recolte du pollen par les abeilles. Ann. Abeille 3, 115-188; 4 , 197-221; 1,5-111. 4. Louveaux J., Maurizio A., Vorwohl G. (1978) Methods of mel is so palynology. Bee World 59(4), 139-157. 5. Maurizio A., Evenius 1., Vorwohl G. Der Honig. Eugen Ulmer Vig. Stuttgart. 1975 pp. 169-185. 6. Ricciardelli D'Albore G. (1979) L'origine geographique de la propolis: Apidologie 10 (3),241-267. 7. Ricciardelli D' Albore G.(1992) Considerazioni e problcmi inerenti alle analisi del miele in !talia con particolare riferimento alla melissopalinologia. L'Ape n. arnica 5, 5-7. 8. Ricciardelli 0' Albore G. (I994) Caratterizzazione organolettica e geografica dei mieli della Coop. Agric. "II Bosco" di Pontremoli (MS). L'Ape n. arnica XVI (4),31-33. 9. Ricciardelli D'Albore G. Textbook of mel is so palynology. Apimondia, 1996a (in print). 10. Ricciardelli D'Albore G. (1996b) L'origine geografica della cera d'api. L'ape n. Arnica, (in print). II. Ricciardelli 0' Albore G., Battaglini Bernardini M. (1978) Origine geographique de la Gelee royale. Apidologie 9(1), 1-17. 12. Ricciardelli 0' Albore G., Persano Oddo L. Flora apistica italiana. Ed. 1st. Spero per la Zool. agr. Firenze 1978. 13. Ricciardelli D' Albore G., Tonini D'Ambrosio M. (1977) Considerazioni sui pollini della gelatina reale: Ann. 1st. Spero Zool. Agr. V,2-31. 14. Vorwohl G. (1995) Moeglichkeiten und Grenzen der mikroskopischen Honiguntersuchung. Bienenvater 6, 276-283.

INDEX

Abdominal scent (Nassanov) glands, 137, 140, 141 Abdominal tergites exocrine glands, 137, 139--140 Acacetin, 21 Acacia honey, 77, 78 Acaretin,233 Acaricides, 239-246 N-Acetyl-beta-glucosamidase, 95 Acidity: see pH Acid p-cumaric methyl ester, 233 Acid phos'phatase, 95 Actinomyces pyogenes, 33 Aculeate wasps, 190--194 Acupuncture point, application of sting-venom to, 215 Africanized bees, 155 Aggression, 2-heptanone and, 155 Alarm pheromones, 140, 145, 147, 151-156 queen, 151-154 workers, 154--156 Alkaline phosphatase, 95 Alkaloids in honey, 80 Allomones, 137 Almond (Prunus duleis) pollen, 94--95 American propolis, antimicrobial activity, 108 alpha-Amino butyric acid, 197 Analytical methods: see Quality control Andromeda toxins in honey, 79--80 Anti-atherosclerotic activity of pollen and, 95-96 Anti-browning agent, honey as, 89--91 Anti-gonadotrophic hormone, queen substance as, 147 Anti-inflammatory activity of venom chronic diseases, 213--220 orthopedic diseases, 222-224 Antimicrobial activity honey, 19,27-36 non-peroxide activity, 28-29, 32, 39--47 potential uses, 30--36 properties, 27-30 sources of honey, 78 of tropical bees, 8 pollen, 94--95 propolis, 8, 107-112 compounds and relative biological activities, 233 orthopedic diseases, 222

Anti-neoplastic activity pollen, 96 propolis, 112-119 royal jelly, 179--183 Antioxidant activity honey from bees fed with medicinal plant extracts, 49--54 pollen, 96-99 Antiseptic dressing: see Wound healing Apamin, 197,214 chemical activity and pharmacology, 22 and ion channels, 203-209, 210 Apis eerana, 1,2, 3, 197 clarifying agent production by, 90 propolis,8 Apis dorsata, I, 2, 5 Apisjlorea, I, 2 Apis laboriosa, 90 Apis melli/era, I, 2, 3, 4 clarifying agent production by, 90 as introduction to new world, 12 propolis, 8 venom components, 197 Apis melli/era anatoliaca, 176 Apis melli/era ligustica, 161-172 Apis mellifera ligustica x Apis melli/era syriaca, 155 Apistan (fluvalinate), 239, 240. 242, 243, 244, 245 Arachidonic acid, 96 Arbustus unedo, 79 Arbutin, 79 Argentine propolis, anti-Helicobacter pylori activity, 109, III Amart (tarsal) glands, 137, 138--139, 143 Artepillin C (3-prenyl-4-hydroxy cinnamie acid), 108, 109,110,115-119 Arthroderma, 108 Arthroderma benhamiae, 108 Aspergillus, pollen mold, 95 Asthma, 21 Aureobasidium pullulans, 95 Australian honey pollen spectrum, 256 Autoimmune disorders, venom therapy in, 21, 22, 213--220

263

264 Avicennia, 259 Azalea toxins in honey, 79---,'l0 Bacillus, 95 Bacillus cereus, 108 Bacillus pyocyaneus, 95 Bacillus subtilis, 95 Bacteria antimicrobial activity: see Antimicrobial activity oral flora, honey and, 65-71 Bacteroides, 108 Balling behavior, 147, 151, 152 Banksia, 256 Bayvarol (flumethrine). 239, 241, 242, 243, 244, 245 Bee brood: see Brood Bee origin of antibacterial substances of honey, 40, 45 Bee physiology: see Physiology of bees Bee pollen: see Pollen Bee species earliest historical records, I, 2, 3, 4, 5 sources of antibacterial honey, 78 Benign prostatic hyperplasia, 96 Benzoic acid, 142, 146 Benzyl acetate, 139, 156 Benzyl alcohol, 29, 139, 156 Beverages, propolis, 125-127 Bifidobacterium, 108 Biogenic amines, 189-190,197 Biological response modifiers, 113~114, 115 Bitter honey, 79 Blood vessels, 21, 233 Bluegum (Eucalyptus) honey, tooth enamel effects, 66-71 Bombyx mori (silkworm), 182 Botanical origin: see Plant origin Botulism, 31, 227, 228 Brachystegia, 261 Braconidae, 188, 194-195 Brassica, 259 Brassica campestris pollen, 94 Brazilian propolis anti-Helicobacter pylori activity, 109, III antimicrobial activity, 108, 109 against Helicobacter pylori, 109, III against MRSA, 1l1~1l2 cytotoxic effects of Artepillin C, 115-119 lead in, 231~238 Bromophylate (Folbex VA), 239, 240, 241~242, 243, 244,245 Bronchomel antioxidant properties, 49-54 Brood historical records of use, 10 social insects collecting and producing, 2 Buccal apparatus exocrine glands, 137 Burns: see Wound healing

Caffeic acid, 21 Caffeic acid ester, 21, 233

Index Calcium channels, venom and, 203~204, 205 Cancer: see Anti-neoplastic activity Candida albicans, 68, 70 Cape honey bee, 65 Capillaries, 21,233 Caprylate esterase-lipase, 95 Cardiac cell ion channels, venom and, 203~21 0 Cariogenesis, honey and, 65-71, 73~75 Carotenoids. honey, 79 Carrot honey, 77, 78 Cassie senna, 80 Catalase, 29, 53 Chamomile honey, 79 Chemical analysis for quality control, 255 Chemical composition and medical applications, 15-25 honey, 19-20,29 pollen, 16-19,94 propolis, 20--21 royal jelly, 23-24 venom,21~22, 197 wax, 24 Chemical contaminants in bee products heavy metals in propolis, 231~238 monitoring, 227~229 Chestnut honey, 78 Childhood malignancies, royal jelly in treatment of, 179-183 Chilean propolis, 122~1124 Chinese honey antioxidant properties, 49-54 Chinese propolis anti-Helicobacter pylori activity, 109, III Cholera, 33 Chronic diseases, venom therapy in, 21, 22, 213-220 action of venom, 215 administration of, 216 adverse reactions, 219 composition of venom, 214 contraindications, 215-216 indications for, 215 mode of action, 215 protocols, 218--219 reactions to, 217~218 research, 213~214 safety, 217 treatment, 216-217 Chrysin, III Cistus ladanifer pollen, 97 Clarif'ying agent, honey as, 89-91 Clerodane diterpenoid, 112~119 Clinical applications orthopedic diseases, 221 ~224 pollen, 19 propolis anti-tumor activity, 112~1l9 royal jelly in childhood malignancy, 179-183 venom therapy in chronic diseases, 213~220 Clostridium botulinum, 32, 227, 228 Coffea, 261 Combretaceae, 258

Index Coridothymus capitatus (thyme) honey, 77, 78, 80 COlynebacterium equi, 108 Cosmetics, propolis in, 121-124 Cotton honey, 78 Cyclic hydroxamic acid, 96 Cyclododecane, 143, 146 Cyc1o-oxygenase, 96 Cytotoxic effect, propolis, 112-113 Dairy animal mastitis, 32-33 Dandelion honey antibacterial activity, 44--47 2-Decanal, 143, 146 Dental enamel, honey and, 65-71, 73-75 Dermatomycoses honey and, 34-36 propolis and, 108 Dermomel antioxidant properties, 49-54 Diastase, 44, 47, 53 Dicarboxylic acid, 23 Diethyl-I,2-benzene dicarboxylate, 143, 146 2,4-Dihydroxy-2H-I A-benzoxazin-3( 4H)-one, 96 I ,4-Dihydroxybenzene, 29 3,5-Dimethoxy-4-hydroxybenzoic (syringic) acid, 29 2,2-Dimethyl-6-carboxy ethenyl-2H-I benzopyran, 108,109 1,2-Dodecadiene, 143, 146 2- Dodecanol, 143, 146 Dopamine, 189, 197 Dressings: see Wound healing Dufour's glands, 155 Echinacea angustifolia, 80 Echium, 256 Economic importance, overview of, 1-12 bee brood, 10 bee species, I, 2, 3, 4, 5 changes in through time, 11-12 honey, 2, 4, 5-8 pollen, 9-10 propolis, 8--9 royal jelly, II venom, 10-11 wax, 8 Eicosapentenoic acid, 96 Elaeis, 258 Enterobacter, 108 Enterobacter aerogenes, 108 Epidermophyton, 35-36 Erica australis pollen, 97 Erica honey, 78 Escherichia coli, 28, 66, 222 bee pollen and, 95 honey and, 31, 34 Essential oils, 79 Ethiopian honey pollen spectrum, 256 Eubacterium, 108 Eucalyptus, 256 Eucalyptus corbicular pollen, 94-95

265

Eucalyptus globulus pollen, 97 Eucalyptus honey, 44--47, 77, 78 Eucalyptus pollen, 97 European Community quality trade-mark, 261, 262 Exocrine glands of honey bees alarm pheromones, 151-156 protein traffic between compartments, 161-172 structure and secretory products, 137-148 Koschewnikow glands, 145, 147 mandibular glands, 147-148 Nassanov gland, 141 Renner's gland, 141-145, 146 sting sheath glands, 139-140 tarsal glands, 138--139 wax gland complex, 140 Fat body, 166-169, 170,171 Fats: see Lipids Fatty acids pollen, 96 royal jelly, 23 wax, 24 Flavonoids honey, 19,20,39, 79 pollen, 95, 96-97. 99 propolis, 8, 20, 21. 108--112 Floral origin: see Plant origin Fluoride in honey. 70 Fluvalinate (Apistan), 239, 240. 242, 243, 244, 245 Folbex VA (bromophylate). 239, 240, 241-242, 243, 244,245 Food processing, honey in, 83-87, 89-91 Foot-print secretions. 138 Forage marking hormone, 155 Free radical scavingers: see Antioxidant activity Fungal skin infections honey as treatment for, 34-36 propolis compounds active against, 108 Fungi, pollen molds, 94-95 Galangin, III, 233 Gamma irradiation of honey, 32, 33 Gangrene, 32 Gastric ulcers: see Ulcers, peptic Gastroenteritis, honey as treatment for, 34 Genal glands, 137 Genista, 259 Geographical origin, characterization of. 263-262 Geranium honey, 79 German propolis antimicrobial activity, 108 Germination of plant seeds, propolis and, 129--134 Gluconic acids, 23 Glucose oxidase, 53 beta-Glucosidase, 95 Glycosides, honey, 79 Gout, 21 Gramineae, 257 Grewia, 261

266

Hawthorn (Crataegus oxycantha), 80 Heart cell ion channels, venom and, 203-210 Heat inactivation of antibacterial substances of honey, 45 Heavy metals in propolis, 231-238 Helicobacter pylori, 19 honey and, 33-34 propolis and, 108, 109, III Hemolymph, protein traffic, 161-172 2-Heptanone, 147, 154-156 Hesperidin, 79 Hexadecanoic acid, 143, 146 9-Hexadecenoic acid, 142-143, 146 Hexyl acetate, 139, 156 Histamine, 22, 189, 197 Historical overview, 94; see also Economic importance, overview of Honey as antimicrobial agent non-peroxide antibacterial activity, 28-29, 32, 39-47 in orthopedic diseases, 221 properties and uses, 27-36 chemical composition, 19-20 food processing applications, 83-87, 89-91 historical records of use, 2, 4, 5-8 from medicinal herbs, 49-54, 77~0 quality control acaricide residues, 239-246 organoleptic analysis, 247-250, 254 pollen analysis, 254, 255, 256, 257 social insects collecting and producing, 2 and wound healing antimicrobial properties, 28-29, 32, 39-47 histological assessment, 57-62 Honey ants, 2 Honeydew honey, 44-47, 79 Honey wasps, 2 Hyaluronidase, 197 Hydoxyfatty acids, royal jelly, 23 Hydrogen peroxide, honey and antimicrobial activity, 28 factors in degradation of, 29 and wound healing, 62 Hydroxamic acid, 96 2-Hydroxybenzoic acid, 29 10-Hydroxy-trans-2-decenoic acid, 147 2-Hydroxy-3-phenylpropionic acid, 29 Hyptis,259 Immune system propolis and, 112-119 royal jelly and, 173, 174 venom therapy, 21, 22 Inhibine number, 29 Inhibines, 39-47 Invertase, 44,47, 53 Ion channels, mechanism of venom action, 190, 203-210

Index

Irradiation of honey, 32, 33 Isoamyl acetate, 139, 156 Isoamyl alcohol, 139, 156 Isopentyl acetate, 147, 154 Juvenile hormone, 147, 155 Kaempferide, 233 Kairomones,137 Kinins, 189 Klebsiella pneumoniae, 33 Korean propolis, 122 Koschewnikow glands, 137, 140, 143, 145, 147, 151-153,156 Labial glands, 137 Lactobacillus, 108 Lannea, 256 Laryngomel antioxidant properties, 49-54 Lavender honey, 44-47, 77, 78 Lead levels in propolis, 231-238 Leptospermum scoparium: see Manuka honey Leucine aminopeptidase, 95 Leukemias and lymphomas, pediatic, 173-178 Lime honey, 77, 78, 79 Linolenic acid, 96 Lipids pollen, 94 royal jelly, 23 wax, 24 Lipids levels in blood, pollen and, 95-96 Lipoxygenase, 96 Luteonin, 233 Macrophage function, propolis and, 113-114, 115 Majorana syriaca (majoram) honey, 79, 80 Mandibular glands, 137, 143, 147-148, 154 Manuka (Leptospermum scoparium) honey, 29 antibacterial substances, 39 gastroenteritis treatment, 34 non-peroxide activity, 32 skin infections, fungal, 34-35 Mast cell degranulating peptide, 22, 214 Mastitis, dairy animals, 32-33 Maternal nutrition, pollen and, 94 Mead,91 Medical applications: see Chemical composition and medical applications; Clinical applications Medicinal plants as bee food, antioxidant properties of honey, 4954 honeys from, 77-80 Melipona interrupta grandis, 7 Melipona subnitida, 78 Melittin, 21, 22, 214 and ion channels, 205-209, 210 role in nature, 195-198 Metastasis, propolis and, 113-114, 115

Index

Methicillin-resistant Staphylococcus aureus (MRSA), 31,108 5-Methyl-cyclohexanal, 143, 146 Methyl-3,5-dimethoxy-4-hydroxybenzoate (methyl syringate), 143, 146 3-Methyl-2,6-dioxo-4-hexenoic acid, 143, 146 Methyl ester of coumaric acid, 233 Micrococcus lysodeikticus, 108 Microscopic analysis, 254--262 Microsporum, 35-36,108 Microsporum gypseum, 108 Mimosa honey, 78 Mimosa pudica, 259 Monitoring of contaminants, 227-229 Moulds, pollen, 94--95 Multiple sclerosis: see Chronic diseases Mycobacterium phlei, 108 Nassanov (abdominal scent) glands, 137, 140,141 Neoplasia: see Anti-neoplastic activity Nerium oleander, 227 Neurotoxic venoms, 190--194 Neurotoxins: see Venom New Clerodane Diterpenoid, 112-119 New Zealand honeys, 29-36, 39: see also Manuka honey Nocardia asteroides, 33 Nonanol, 139, 156 Noradrenaline, 197 Nutrient composition pollen, 16, 17, 18,94 royal jelly, 23 9, 12-0ctadecadienoic acid, 143, 146 9-(Z)-Octadecenoic acid, 143, 146 Oleander, 227 Onion honey, 78 Oral flora, honey and, 65-71 Orange honey antibacterial activity, 44--47 Organoleptic analysis, 247-250, 254 Osmotic effect of honey, 27-28, 39 Palmae, 258 Pectolinarigenin, 233 Pediatric malignancies, royal jelly in treatment of, 179-183 Penicillium corylophilum, 95 Penicillium cruslosum, 95 Peptic ulcers: see Ulcers, peptic Perizin (coumaphos), 239, 241,242,243,244,245 Pesticides, 227-229, 239-246 Pesudomonas aeruginosa, 222 pH

honey, 28, 40, 44 royal jelly, 23 and tooth enamel erosion, 65 Phenolics pollen, 96 propolis,20

267 Pheromones, 137, 151-156; see also Exocrine glands of honey bees Philanthus triangulum, 190--194 Phosphoamidase,95 Phospholipase Al2], 22, 197 Physiology of bees alarm pheromones, 151-156 exocrine gland structure and secretory products, 137-148 protein traffic between compartments, 161-172 royal jelly production, factors affecting, 173-178 Phytochemical factors: see Plant origin Pinocembrin, 21,29, 111,233 Pinostrobin, 21 Plant germination, propolis and, 129-134 Plant origin honey, 28--28 antioxidant properties of honey produced from bees fed with medicinal plant extract, 49-54 and bacterial inhibition, 44--45 and flavonoid profile, 39-40 historical record, 5, 7 medicinal herbs, 77-80 methods of analysis for quality control, 253-262 pollen and antioxidant activity, 97 and nutrient composition, 94 propolis, 129 Plant toxins, 79-80, 227, 229 Plebiasp bees, 78 Pollen anti-atherosclerotic activity, 95-96 antibiotic activity, 94--95 anti-neoplastic activity, 96 antioxidant activity, 96--99 composition, properties, and applications, 16--19, 93-99 historical records of use, 9-10 nutritive value, 93-94 organoleptic analysis, 254 in orthopedic diseases, 221-222 social insects collecting and producing, 2 Pollen analysis, 254, 255,256 Polyphenol oxidase, 90 3-Prenyl-4-dihydro cinnamoloxy cinnamic acid, 108, 109,111-112 3-Prenyl-4-hydroxy cinnamic acid (Artepillin C), 108, 109,110,115-119 Procamine, 197 Propolis beverage products, 125-127 chemical composition, 20--21 cosmetic products, 121-124 heavy metals in, 231-238 historical records of use, 8-9 Japanese research status, 107-119 antimicrobial properties, 107-112 immune activation and anti-tumor activity, 112-119

268 Propolis (cont.) microscopic analysis, 261 organoleptic analysis, 254 in orthopedic diseases, 222 and seedling germination, 129-134 social insects collecting and producing, 2 wound dressings, hypoallergenic formula, 101105 Protein traffic, 161-172 Proteus mirabilis, 31 Proteus vulgaris, 95 Protium, 261 Prunus dulcis (almond) pollen, 94--95 Pseudomonas, 108 Pseudomonas aeruginosa, 28, 31 Pterostilbene, 233 Quality control, 86 botanical and geographical origin, methods for characterization, 253-262 lead in propolis, 231-238 monitoring contaminants, 227-229 pesticides, 239-246 sensory analysis, 247-250 Queen alarm pheromones, 151-154 exocrine glands: see Exocrine glands of honey bees and royal jelly production, 173-178 Queen substance-like compound, 147 Quercetin, 21, 233 Radionuclide contaminants, 229 Ragwort honey, 80 Ranunculus sardous pollen, 97 Rape honey antibacterial activity, 44-47 Raphanus raphanistrum pollen, 97 Red clover honey, 78 Regulation of product quality, contaminant monitoring, 227-229 Renner's (tergal) glands, 137, 140, 141-145, 146 Rheumatoid arthritis: see Chronic diseases, venom therapy in Rhizopus nigricans, 95 Rhododendron honey, 44-47, 227 Rhododendron toxins in honey, 79-80 Romedotoxin, 227 Rosemary honey, 78 Royal jelly antigens, 165, 166 chemical composition, 23-24 factors affecting production of, 173--178 historical records of use, II microscopic analysis, 257, 261 organoleptic analysis, 254 social insects collecting and producing, 2 treatment of pediatric malignancies, 179-183 Rubus, 259

Index Saccharase, 53 Safety: see Quality control Sage honey, 78, 80 Salivary flow, and cariogenesis of honey, 73-75 Salix atrocinerea pollen, 97 Salmonella typhi, 66 Salmonella typhimurium, 28, 31, 33 Salviafruticosa,80 Salvia ojJicinalis, 78, 80 Samanea, 256 Sarcina lutea, 43--46 Savory honey, 78 Schinus, 261 Scleroderma, 21 Scrophulariaceae,261 S. dublin pollen. 95 Secapin, 197 Seed germination. propolis and. 129-134 Senecio jacobaea toxins, 80 Senegalese honey pollen spectrum, 258 Sensory analysis, 247-250, 254 Serotonin, 189 Serratia marcescens, 31 Serum lipids. pollen and, 95--96 Setaceous membrane glands, 137. 139-140, 156 S. gallinarum pollen, 95 Shigella, 66 Shigellosis, 33 Silkworm (Bombyx mori), 182 Silver sulfadiazine, 19, 58, 59, 62 Skin: see Wound healing Skin infections, fungal honey and, 34--36 propolis and, 108 Social insects role of venom. 188--189 substances produced and collected by, 2 Sodium channels, venom and, 205--209, 210 South African honey, 65--71 Spanish honey pollen spectrum, 259 S. pullorum pollen, 95 Stachys, 259 Standards: see Quality control Staphylococcus aureus. 31, 32, 33, 65, 66, 68. 222 honey antibacterial activity studies, 39-47 methicillin-resistant: see Methicillin-resistant Staphylococcus aureus Sterilization of honey with gamma irradiation, 32, 33 Sting apparatus, 154, 155--156 Sting appratus exocrine glands, 137 Stinging behavior, 140, 154 Stingless bees honeys, 7-8 propolis,8 substances produced and collected by, 2 Sting sheath glands, 137, 139

Index Sting sheath phermones, 156 Storage effects on antibacterial substances of honey, 45 Streptococcus, oral flora, 65-66, 68, 70, 71 Streptococcus agalactiae, 33 Streptococcus anginosus, 66, 68, 70, 71 Streptococcus dysgalactiae, 33 Streptococcus gordon ii, 68 Streptococcus pyogenes, 28, 31 Streptococcus salivarius, 68 Streptococcus sanguis, 68 Streptococcus sobrinus, 68 StreptoLOcCUS uberis, 33 Stress pheromone, 152 Striped African honey bee, 65 Sugar versus honey as food product, 84--85 Sulfur-containing topical drugs, 19, 58, 59, 62 Sulfur dioxide, honey anti-browning properties versus, 91 Sunflower honey, 78, 79 Switzerland, pesticides used in, 239--246 Syngergistic phermones, 147, 148 Tannins, honey as anti-browning agent, 11 Tansy ragwort toxins in honey, 80 Taraxacum pollen, 97 Tarsal glands (glands of Amart), 137, 138-139, 143 Taste testing, 247-250 Tergal (Renner's) glands, 137, 140, 141-145, 146 Terminalia,258 Terpenes honey,29 propolis,21 Tertiapin, 197 Tetradecanoic acid, 143, 146 Tetratetracontane, 143. 146 Thermoactinomyces intermedius, 108 Thistle honey, 78 Thyme (Coridothymus capitatus) honey, 77, 78, 80 Tilia honey, 77, 78, 79 Tinea infections, 34--35 Tooth decay, honey and, 65-71, 73-75 Toxins, 79--80,227-229 Trichophyton, 35-36 3,4,5-Trimethoxybenzoic acid, 29 Triumfetta, 257 Tropical African honey pollen spectrum, 257, 258 Tropical bee honey, 7-8, 12 Tumor suppression: see Anti-neoplastic activity Turkish propolis, 129--134 Ulcers, peptic, 233 honey and, 19--20, 33-34 propolis and, 21 Ulex europaeus pollen, 97

269 Varroacides, 239 Venezuelan honey pollen spectrum, 259, 260 Venom gland, 155 antigens, 165, 166 historical records of use, 10-11 mode of action, ion channels, 203-210 role in nature, 185-198 Aculeate wasps, 190-194 Braconidae, 188, 194--195 mellitin,195-198 social hymenoptera, 188-189 venomous hymenoptera, 186-188 Vespidae, 189--190 social insects collecting and producing, 2 Venom sac, 155 Venom therapy in chronic diseases, 213-220 in orthopedic diseases, 222-224 Vernonia, 257 Vespidac, 189--190 Vespulakinin-l, 189 Vibrio cholerae, 33 Vibrio parahemolyticus, 33 Viper's bugloss honey, 39, 40 Vitamins pollen, 16, 17. 18,94 royal jelly, 23 Wax acaricide residues, 239--246 chemical composition, 24 historical records of use, 8 microscopic analysis, 261 organoleptic analysis, 254 with propolis, 8, 21 social insects collecting and producing, 2 Wax gland complex, 137, 140 White broom (Retama raetam), 80 Workers alarm pheromones, 154--156 exocrine glands: see Exocrine glands of honey bees protein traffic studies, 161-172 Wound healing honey and, 19 antiseptic dressing, antibacterial activity, 30-32 histological assessment, 57-62 propolis and, 101-105 Xanthine oxidase, 51,52-53 Xantoxylum,261 Yeasts, 95 Yersinia enterocolitica, 33 Zambian honey pollen spectrum, 261

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