INTRODUCTION
Background of the Study Neem – the legendary medicinal tree of India, has grown with the human
settlement settlement all over the country and has been an integral part of the Indian way of life for centuries. The history of the Neem tree is inextricably inextricably linked to the history of the Indian civilizationNeem !oundation, n.d". It is also known as #$ivine Tree%, #&ife giving tree%, #Nature's $rugstore%(ustNeem, )*++". t four or five years old, neem can produce flowers and fruit, but only after +* to +) years will it produce economically viable seed -uantities gro!orestyTree$atabase, n.d". very part of the Neem tree has health promoting benefits .They call Neem tree
as
the
#/illage
0harmacy%
because
of
its
medicinal
benefits
1elvester,+222".The leaf is also the part of the neem plant that plant that western medicine knows the most about. The ma3ority of scientific neem studies was done with neem leaf or neem leaf extracts $iscover Neem, )*+4". Neem bark has also been utilized for centuries by the Indian and sian cultures to treat numerous medical illnesses, prevent pregnancy and also as a potent pesticide (ernigan, )*+*". (ernigan added also that one of the ma3or uses of neem bark is as an insect repellent and it offers benefits benefits for humans because because there
is
no
need
to
use
harmful
pesticides
or
chemicals
on
crops............. crops. ......................... ......................... .......................... .......................... ....................... ........... 5os-uitoes are very common household insect . The life cycle of this insect insect is normally normally short short and breed in stagnan stagnantt water water accumul accumulate ated d in cans, cans, old tires, flower vases, pots, pails and other containers in the households 65 News, )*+4" . The female mos-uitoes feed on blood while male mos-uitoes feed
1
on plant 3uices and nectar beacause of having weaker mouth. lso, they are very common in houses during nightime. They not only disturb sleep but are also carriers of diseases. diseases. 7istorically, 7istorically, mos-uitoes mos-uitoes have been identified identified as the carrier of malaria and dengue and considered as the most deadliest and dangerous disease respectively brought about by a mos-uito bite 879, )*++" . They are also vectors of filariasis and encephalitis. &ately, the recent outbreaks of dengue fever which have killed countless of children and adults in many rural and urban places have been traced to mos-uitoes specifically specifically the female of Aedes aegypti . It is imperative that this insect be eradicated. In addition of this, it is preferred that this will be controlled: in a less costly manner and environmental friendly. ccording ccording to 1chumetterer 1chumetterer +2;+", Azadirachta Indica Neem" Neem" Tree Tree contains zadirach zadirachtin, tin, a limonoid, that has been reported to have adverse effect on endocrine system of a bean bettle, pilachna pilachna varivestis, and to cause sterility in the female insects. 1chluter 1chluter and 1chulz +2;4" +2;4" also reported this compound compound to cause degradation in larval epidermis preventing the larvae from molting. In addition, 1ridharan 1ridharan )**2" also said that zadirachtin zadirachtin is mainly responsible responsible for the insecticidal properties of the neem. Insects are perceptive to smell: they do not like the smell of neem oil. zadirachtin disrupts the growth and reproduction in most of the pest. It is one of the most potent growth regulators. It will repel or reduce the feeding of many species of pest insects as well as some nematodes. The The proble problems ms raise raised d earli earlier er serv served ed as reaso reasons ns why why the the resear research chers ers embarke embarked d to the study to find out the use of Azadirachta Indica Neem" bark extract and its effectivity as insecticide against mos-uito larvae.This is because neem tree are abundant in the 0hilippines and always available as a source of
2
on plant 3uices and nectar beacause of having weaker mouth. lso, they are very common in houses during nightime. They not only disturb sleep but are also carriers of diseases. diseases. 7istorically, 7istorically, mos-uitoes mos-uitoes have been identified identified as the carrier of malaria and dengue and considered as the most deadliest and dangerous disease respectively brought about by a mos-uito bite 879, )*++" . They are also vectors of filariasis and encephalitis. &ately, the recent outbreaks of dengue fever which have killed countless of children and adults in many rural and urban places have been traced to mos-uitoes specifically specifically the female of Aedes aegypti . It is imperative that this insect be eradicated. In addition of this, it is preferred that this will be controlled: in a less costly manner and environmental friendly. ccording ccording to 1chumetterer 1chumetterer +2;+", Azadirachta Indica Neem" Neem" Tree Tree contains zadirach zadirachtin, tin, a limonoid, that has been reported to have adverse effect on endocrine system of a bean bettle, pilachna pilachna varivestis, and to cause sterility in the female insects. 1chluter 1chluter and 1chulz +2;4" +2;4" also reported this compound compound to cause degradation in larval epidermis preventing the larvae from molting. In addition, 1ridharan 1ridharan )**2" also said that zadirachtin zadirachtin is mainly responsible responsible for the insecticidal properties of the neem. Insects are perceptive to smell: they do not like the smell of neem oil. zadirachtin disrupts the growth and reproduction in most of the pest. It is one of the most potent growth regulators. It will repel or reduce the feeding of many species of pest insects as well as some nematodes. The The proble problems ms raise raised d earli earlier er serv served ed as reaso reasons ns why why the the resear research chers ers embarke embarked d to the study to find out the use of Azadirachta Indica Neem" bark extract and its effectivity as insecticide against mos-uito larvae.This is because neem tree are abundant in the 0hilippines and always available as a source of
2
our raw material and the researchers decided to use the neem bark as the test plant since ma3ority of the scientific scientific studies studies in neem neem were done in neem leaf: leaf: and seeds is hard to find right now. STATEMENT OF THE PROBEM 6enerally, this study aimed to utilize the neem bark extract as insecticide
against mos-uito larvae. 1pecifically, it sought to answer the following -uestions< +. 8hat 8hat are the the chemic chemical al compo composit sition ion of Azadirachta Indica Neem" bark extract= ). re re neem neem bark bark extrac extractt derive derived d from from $eco $ecocti ction on 5ethod 5ethod and thano thanoll xtraction 5ethod effective as insecticide against mos-uito larvae= 4. Is ther there e a sign signif ific ican antt diff differ eren ence ce betw betwee een n Azadirachta Indica Neem" bark bark $ecoc $ecoctio tion n and and tha thano noll extra extracti ction on in terms terms of their their larvi larvicid cidal al activity= RESEARCH H!POTHESES 7*+< Azadirachta Indica (Neem) bark extracts derived from $ecoction 5ethod
and thanol xtraction 5ethod have no effect to mos-uito larvae. 7*)< There There is no signific significant ant difference difference between between Azadirachta Indica Neem" bark $ecoction and thanol extraction in terms of their larvicidal activity. SI"NIFICANCE OF THE STUD! This study was conducted in order for the researchers to find alternatives
on how to combat illness>carrying mos-uitoes by using inexpensive and natural insecticide from Azadirachta Indica Neem" bark extract. The other beneficiaries of the study are the following<
•
The government, to give them awareness to take necessary actions in addressing the problems such as conducting seminars in urban 3
and rural areas regarding regarding awareness awareness and and prevention of the boost
•
of health cases brought by mos-uitos. The educational educational institutions, institutions, with the help of teachers, teachers, to integrate integrate to their lessons the use of Azadirachta Indica as insecticide against
•
mos-uito larvae. The The commu communi nity ty,, espec especial ially ly to areas areas where where illne illness ss carrie carried d by mos-uitos is rampant, to help them lessen the number of cases and and thre threat at to thei theirr heal health th by teac teachi hing ng them them the the natu natura rall and and inexpensive way to fight illness>carrying mos-uitos with the use of Azadirachta Indica.
SCOPE AND IMITATION The research researchers ers focused focused on the use of Azadirachta Indica Neem" bark
extract extract as insectic insecticide ide against against mos-uito mos-uito larvae. larvae. The Azadirachta Indica Neem" bark was collected within the ?ity of @oronadal. The test insects were mos-uito larvae were chosen randomly since the researcher used ?omplete Aandomized $esign and considering their physical appearance after )B>B; hours. The study was conducted from (uly )*+4 to 1eptember 1eptember )*+4 which includes collection and preparation preparation of materials, gathering gathering of data, experimentation experimentation and observation observation of variables. Two methods were used for the extraction of Azadirachta Indica Neem" bark. xtraction with distilled water $ecoction xtraction 5ethod" and xtraction with 2CD ethanol solution thanol xtraction 5ethod". There were five treatments in each of the method of extraction, Treatment * T*", Treatment + T+", Treatment ) T)", Treatment 4 T4" and Treatment B 4
TB" consisting of three replicates in each treatment were prepared. T* is the negative control which contains +**D distilled water, T+ is the positive control which which contai contains ns +**D +**D li-ui li-uid d inse insecti ctici cide, de, T) cont contain ains s +*D +*D Azadirachta Indica Neem" bark extract and 2*D distilled water, T4 contains )*D Azadirachta Indica Neem" bark extract and ;*D distilled water, and TB contains 4*D Azadirachta Indica Neem" bark extract and E*D distilled water.
Def#n#t#on of Ter$% Ter$% Azadirachta Indica > the scientific name of neem tree. zadirachtin> zadirachtin> a complex tetranortri>terpenoid tetranortri>terpenoid limonoid from the neem tree that is
mainly responsible for the insecticidal properties of the neem. Fark –the outermost layer of the woody part of a neem tree that was used in the study as insecticide against mos-uito larvae. xtra xtract ct > is a subst substanc ance e made made by extracting extracting a a part of a raw material, material, often by using a solvent solvent such such as ethanol ethanol or or water . Insecticide > chemical substance used to kill insects used as the positive control in the study. ðal $osage C* &$C*" > is a measurement used in toxicology studies to determine the potential impact of toxic substances on different types of organisms. It is the median lethal dose of a substance, or the amount re-uired to kill C*D of a given test population. It used to determine the toxicity of the extracts used in the study. &imon &imonoi oids> ds> 0hytoc 0hytochem hemica icals, ls, abund abundant ant in citru citrus s fruit fruit and and othe otherr plants plants of the the families Autaceae and 5eliaceae. 5os-uito &arvae &arvae > is the larvae of various kinds of mos-uitoes that lay their eggs on the surface of the water. lso, the test insects used in the study to determine the effectiveness of the extract as insecticide.
5
Neem tree – is an evergreen tropical tree that belongs to the family 5eliacease 5ahogany". Itis a small to medium>sized treeup to +Cm 4* max." tall, with a round, large crown up to +*m )* max." in diameter: branches spreading: bole branchless for up to E.C m, up to 2* cm in diameter, sometimes fluted at base: bark moderately thick, with small, scattered tubercles, deeply fissured and flaking in old trees, dark grey outside and reddish inside, with colourless, sticky foetid sap. 5os-uito egg> is the first stage in life cycle of a mos-uito. ggs may be laid individually or connected together to form a #raft% type of structure 5os-uito> any of various two>winged insects of the family ?ulicidae, in which the female of most species is distinguished by a long proboscis for sucking blood. 5os-uito pupa> the comma>shaped stage in the mos-uito life cyle. This is the transition stage between the a-uatic stages of the mos-uito's life cycle and the terrestrial adult stage. 0robit nalysis > is commonly used in toxicology to determine the relative toxicity of chemicals to living organisms. It is used to determine the toxicity level of the zadirachta Indica Neem" bark extracts derived from $ecoction 5ethod and thanol xtraction 5ethod.
6
RE&IE' OF REATED ITERATURE
Azadirachta Indica
(Nee$ Tree)
Neem or 5argosa is a botanical cousin of mahogany. It belongs to the family 5eliaceae. The latinized name of Neem > Azadirachta indica > is derived from the 0ersian<zad G !ree, dirakht G Tree, i > 7ind G of Indian 9rigin which literally means< HThe !ree Tree of India'.Neem is an attractive broad>leaved, evergreen tree which can grow up to 4*m tall and ).Cm in girth. Its trunk usually straight is 4*>;* cm in diameter. Its spreading branches form a rounded crown of deep>green leaves and honey>scented flowers as much as )*m across Neem !oundation,n.d" Azadirachta Indica trees may start flowering and fruiting at the age of B>C years, but economic -uantities of seed are produced only after +*>+) years 9rwa et al., )**2"
Neem bark is taken from the neem tree which predominately grows in India and sia. Neem bark can be used 3ust off the tree or can be freeze dried or ground into a fine powder for use at a later date. Neem bark has been utilized for centuries by the Indian and sian cultures to treat numerous medical illnesses, prevent pregnancy and also as a potent pesticide (ernigan, )*+*" and (ernigan
7
also added that one of the ma3or uses of neem bark is as an insect repellent.
Che$#ca* Co$+o%#t#on of Azadirachta Indica
ccording to ?handy n.d.", the important chemical constituents of Azadirachta Indica are azardirachtin, +>maliantriol, salannin, nimbin, nimbidin and
others. The dry flowers on extraction with petroleum>ether yields waxy sub> stances like -uercetin, myricetin and b>sitosterol on extraction with alcohol they produce a pungent essential oil, green amorphores bitter toxic substance, nonacosane and a ses-uiter> pene and a fatty acid fraction. 7e also added that the different plant parts contain mostly terpenoids limonoids". The flowers and fruits are borne in axillary clusters and when ripe the smooth ellipsoidal drupes are greenish yellow and comprise a sweet pulp enclosing a seed. The seed consist of a shell and +>4 kernels which contain azadirachtin and its homologues 5ordue, )***". 5ordue added that both the bark and leaves also contain biologically active molecules but not high levels of azadirachtin which is found mainly in the seed kernels. The trunk bark contains nimbrin *.*BD", nimbinin *.**+D", nimbidin *.BD", nimbosterol *.*4D", essential oil *.*)D", tannins .*D", a bitter principle margosine and >desacetyl nimbinene ?handy, n.d. J Neem !oundation,n.d". The trunk bark contains nimbin, nimbinin, nimbosterol, tannins and a bitter principle margosine $abur, )*+4".The stem bark contains tannins +)>+D" and non>tannin ;>++D". The bark contains anti>inflammatory
8
polysaccharide consisting of glucose, arabinose and fructose at a molar ratio +<+<+ with molecular weight of ;,B**. The bark also yields an antitumor polysaccharide. Fesides polysaccharides, several diterpenoids, viz., nimbinone, nimbolicin, margocin, nimbidiol, nimbione, etc. have been isolated from stem bark and root bark Neem !oundation,n.d"
Neem has rightly been called 1arvaroghari. 5odern scientists have isolated more than +B* compounds from various parts of the Neem tree that have been evaluated for curative powers. ?laimed to be a H/illage dispensary' the following properties found in Neem, make it one of the best herbal medicines. 1ome of its chemical content are 1odium, 0otassium, 1alts, ?hloriphyle, ?alcium, 0hosphorus, Iron, Thiamine, Aiboflasium, Nicocin, vitamin ?, carotene and oxalic acid Aeadnd$igest.com, n.d.".
U%e% of Azadirachta Indica
Neem, identified by 879KLN0+2;2 as an environmentally powerful natural pesticide, is considered to be one of the most promising trees of the )+st century for its great potential in pest management, environment protection and medicine Nicoletti, )*+)". Azadirachta
indica
Neem",
an
herb extensively
used
in yurveda, Lnani and 7omoeopathic systems of medicine to treat many health related problems and ailments 7erbcyclopedia, )*++". Neem is also well known and used for its medicinal properties from the ancient period B*** F?": mainly
9
on the indications of yurveda medicine, being very popular, even revered in the Indian 1ubcontinent 6a3alakshimi 1. J bbasi 1.., )**B".
In practice, all parts are traditionally used for a variety of indications, but limiting to the ethnobotanical indications concerning the aim of this paper, we can recall the use of neem in indigenous medicine as a bitter tonic, antimalarial, antipyretic, anti>inflammatory, antihelmintic, and for antimicrobial and antiviral effects /arie, +22". It is also known to exert anticancer , antioxidant, wound> healing, and antimicrobial properties is also known to be one of these plants from which almost every part is used, from the Neem oil extracted from its seeds to the leaves and branches, to say the least neem benefits extend to various illnes of health 7erbcyclopedia, )*++". In the article of 0amantong )**;", she stated that the Neem tree, for the last B,C** years, healed hundreds of millions in India, frica and many other parts of sia. 9ne of the immediately perceivable impact of this antifungal, antibacterial and, perhaps, even antiviral king of the arboretums on the human body is its guaranteed ability to heal or cure many, if not all, skin diseases or epidermal problems ranging from dandruff, acne, psoriasis, ringworm, athlete's foot, warts, chicken pox, small pox and malaria. 8ong )*+)" also stated in her article entitled #Neem< 8hat should I know about it=# that Neem is said to help with a number of health problems, including asthma, constipation, cough, diabetes, gastric ulcers, indigestion, periodontal disease, and urinary tract infection. dditionally, neem is purported to reduceinflammation, improve liver health, alleviate pain, preserve eyesight, stimulate the immune system, and protect against heart disease. 10
In the article of Tandon n.d." entitled The Neem Tree, he wrote that neem leaves are used to treat chickenpox and warts by directly applying to the skin in a paste form or by bathing in water with neem leaves. In order to increase immunity of the body, neem leaves are also taken internally in the form of neem capsules or made into a tea. The tea is traditionally taken internally to reduce fever caused by malaria. This tea is extremely bitter. It is also used to soak feet for treating various foot fungi. It has also been reported to work against termites. In yurveda, neem leaves are used in curing neuromuscular pains. Neem leaves are also used in storage of grains.
$ifferent parts of Neem tree are being used extensively in manufacturing of soaps, skin creamsKlotions, shampoos, toothpastes, beauty aids and toiletries. In most cases Neem oilKextract is being used for making these cosmetics like soaps and tooth pastes etc (ohnson, )*+*". Neem twigs are used by millions of Indians as an antiseptic tooth brush. Its oil is used in the preparation of toothpaste and soap (ohnson, )*+* J 1hiva, )*+)".
#fe Cyc*e of Mo%,u#to
The length of the mos-uito life cycle varies between species and is dependent upon environmental conditions such as temperature and moisture 9rkin, )*+4". 7owever, the mos-uito goes through four separate and distinct stages of its life cycle and they are as follows< gg, &arva, pupa, and adult. ach
11
of these stages can be easily recognized by their special appearance lameda ?ounty 5os-uito batement $istrict, )*+4". Their life cycle which can vary in length depending on temperature and food resources. In the summer time it takes mos-uitoes 4>+* days to totally complete their life cycle from egg to adult Aenchie, )**E". The feeding habits of mos-uitoes are -uite uni-ue in that it is only the adult females that bite man and other animals. The male mos-uitoes feed only on plant 3uices. 1ome female mos-uitoes prefer to feed on only one type of animal or they can feed on a variety of animals. !emale mos-uitoes feed on man, domesticated animals, such as cattle, horses, goats, etc: all types of birds including chickens: all types of wild animals including deer, rabbits: and they also feed on snakes, lizards, frogs, and toads lameda ?ounty 5os-uito batement $istrict, )*+4".
In the life cycle of mos-uitoes an egg is the first stage. $epending on the mos-uito species, eggs may be laid individually or connected together to form a #raft% type of structure. The edes aegypti mos-uito lays her eggs individually, while the ?ulex pipiens mos-uito lays approximately )** eggs which she unites to form a raft 5os-uito5agnet.com, )*+4". It was also stated in the study of nderson and 7arrington n.d." entiled # Mosquito io!ogy for the "omeo#ner that a freshly laid egg is light in color and darkens within a few hours. 5os-uito eggs are oval and about +KB* th of an inch *.4Cmm" long. ggs are either deposited singly or as an egg raft depending on the type of mos-uito. standard egg raft is about +KB inch .4Cmm" long and contains +**>)** eggs. 1ome 12
species of mos-uitoes lay their eggs singly and deposit them directly on water or floating a-uatic vegetation. 9thers will lay their eggs on moist soil that is sub3ect to periodic flooding, or above the water line in natural and artificial containers. The number of single eggs laid per batch varies within and between mos-uito species and can range from * to )**. ggs will hatch into larvae within )B to B; hours 9rkin, )*+4".
The mos-uito eggs hatch into larvae or Mwigglers,M which live at the surface of the water and breathe through an air tube or siphon !reudenrich, )*+4". &arvae feed mostly on plant and animal debris in the water. This food is generally low in nutritional value. The nutrition provided by the blood>meal> enriched yolk is therefore important for development (&und-uist, 5. et.al n.d". The larvae filter organic material through their mouth parts and grow to about *.C to *.EC inches + to ) cm" long: as they grow, they shed their skin molt" several times. 5os-uito larvae can swim and dive down from the surface when disturbed !reudenrich, )*+4". ccording to Aey )*++", the larvae are enclosed in a hard, inflexible envelope that is absolutely essential for larval survival, so it can't 3ust be discarded. In order to grow, mos-uito larvae grow a new exoskeleton under the old one. This new exoskeleton is soft and flexible at first, thus allowing the larvae to grow. 8hen ready to molt, the larvae then shed the old exoskeleton and the new one hardens when exposed, to protect the larvae's internal organs. &arger larvae can be seen floating 3ust above the surface of infested waters.8ithin seven to +* days, larvae enter the pupal stage 9rkin, )*+4".
13
ccording to Aey )*++", the fourth developmental stage of mos-uito is when larvae molt into the comma>shaped stage called the pupa or tumblers". This is the transition stage between the a-uatic stages of the mos-uito's life cycle and the terrestrial adult stage. 0upa or pupae plural" live near the surface of the water, breathing through two horn>like tubes called siphons" on their back. nchanted&earning.com, )*+*". 5os-uitoes in this transitional stage are referred to as #tumblers,% describing how they propel through the water. In this stage, the mos-uito pupa rests without eating as it prepares to change into an adult mos-uito 5os-uito5agnet.com, )*+4". ccording to merican 5os-uito ?ontrol ssociation )*++", the pupal stage is a resting, non>feeding stage of development, but pupae are mobile, responding to light changes and moving tumble" with a flip of their tails towards the bottom or protective areas. In the study of !reudenrich )*+4", the stated that at the end of the pupal stage, the pupae encase themselves and transform into adult mos-uitoes.
&arvae and pupae usually cannot survive without water. If a water source evaporates before the larvae and pupae within it transform into adult mos-uitoes, those young often will die 9rkin, )*+4".
The newly emerged adult rests on the surface of the water for a short time to allow itself to dry and all its body parts to harden. The wings have to spread out and dry properly before it can fly merican 5os-uito ?ontrol ssociation, )*++". dult mos-uitoes are characterized by having long, slender, needle>like mouthparts proboscis", antennae, and legs. Their narrow wings are often
14
covered with minute scales nderson and 7arrington, n.d.". The adult male mos-utios emerge first from the pupae about ) days earlier than the females" and form a cloud over the standing water called a #nuptial cloud%. They wait for females to emerge and each female takes flight she enters the cloud and mates with a waiting male. The female only mates once in her life and holds the sperm for to fertilize all the eggs she will lay in her lifetime Thompson>Nicola Aegional $istrict, )*++". In the study of (ohnsen and Aenchie )**E", they cited that female and male mos-uitoes both re-uire carbohydrate sources nectar, plant exudates" throughout their life to maintain energy for ying, mating, and seeking hosts for bloodmeals. 9nly the female mos-uito takes a bloodmeal because she needs the extra protein to develop eggs. dult female mos-uitoes will then seek an animal on which to feed. !emales are capable of flying for miles 9rkin, )*+4". The process of taking a bloodmeal is how the mos-uito is able to vector viruses, protozoans, and helminthes worms" to humans and animals. 5ale mos-uitoes tend to only live a week or two while female mos-uitoes can live for up to a month and produce multiple batches of eggs. 1ome mos-uito species overwinter as blood fed females and can survive for multiple months (ohnsen J Aenchie, )**E".
Ha.#tat% of Mo%,u#to
ll mos-uitoes must have water in which to complete their life cycle. This water can range in -uality from melted snow water to sewage effluent and it can 15
be in any container imaginable. The type of water in which the mos-uito larvae is found can be an aid to the identification of which species it may be. lso, the adult mos-uitoes show a very distinct preference for the types of sources in which to lay their eggs. They lay their eggs in such places such as tree holes that periodically hold water, tide water pools in salt marshes, sewage effluent ponds, irrigated pastures, rain water ponds, etc. lameda ?ounty 5os-uito batement $istrict, )*+4".
-uatic
habitats
are
containers
in
which eggs
develop
into adult mos-uitoes. 5os-uitoes that transmit dengue lay eggs on the walls of water>filled containers in the house and patio. The eggs hatch when submerged in water and can survive for months. 5os-uitoes can lay dozens of eggs up to C times during their lifetime ?enters for $isease ?ontrol and 0revention, )*+)".
ccording to Autgers )**;", mos-uito habitats can be generally grouped into four types< Aunning 8ater, Transient 8ater, 0ermanent 8ater, or ?ontainer. 5os-uito species breed in running waters, such as streams. &arvae can be flushed out when stream volume increases, and to remain in the stream re-uires a
large
amount
Anophe!es species
of
energy. are
The
stream
tropical
genus
breeders.
In
$hagasia and
some
addition, Anophe!es
quadrimacu!atus% $u!ex territans% and &ranotaenia sapphirina have all been
found in streams, although they prefer other habitats. 1tream breeders will find vegetation along banks with which to anchor themselves or attempt to remain away from the main flow of the stream by seeking isolated eddies. Transient
16
water sources, such as flooded areas, snowpools, and ditches are used as breeding grounds for mos-uito species whose eggs can withstand desiccation, such as Aedes and 'sorophora. Their life cycles re-uire alternating periods of wet and dry. 9ther species, like an opportunistic $u!ex% might be able to pull off a single generation during an extended flooded period. Transient water generally show water -uality changes which results in various mos-uito species using the same pool over a period of time. 0ermanent waters also known as 1emi> permanent" are present for extended periods of time and support characteristic a-uatic vegetation. ?attail, rushes and sedges are typical freshwater swamp vegetation. 6enera associated with permanent water are Anophe!es% $u!ex% $u!iseta% $oqui!!ettidia% and &ranotaenia. ggs of these species are not
desiccant>resistant and must be laid directly on the water. Aedes adults will oviposit near the edge of the swamp, or within tussocks of vegetation, re-uiring later flooding to inundate the eggs for hatching. s with transient waters, there is a seasonal change in the vegetation, water -uality and mos-uito species present. ?ontainer water habitat can be found in both natural settings, such as water held by plants bromeliads" to artificial settings, such as water found in tires. The habitat of containers are based on the containers themselves. Treehole sites generally have tannin>enriched water which is characteristically clear, with rotting wood at the bottom. 5any treehole species now also use artificial sites, such as tires since they provide insulation against the weather and are more numerous. rtificial containers are a convenient mode of transporting a species of mos-uito outside of itOs natural range.
17
There is a great variety of man>made containers on backyards or patios that collect rain water or that are filled with water by people where dengue vectors thrive. $isposing of unused containers, placing useful containers under a roof or protected with tight covers, and fre-uently changing the water of animal drinking pans and flower pots will greatly reduce the risk of dengue infections. 8ater storage containers should be kept clean and sealed so mos-uitoes cannot use them as a-uatic habitats ?enters for $isease ?ontrol and 0revention, )*+)".
$u!ex and Anophe!es mos-uitoes
are
among
the
most
common
permanent water mos-uitoes. These mos-uitoes are most active when the average temperature is above E* degrees. Their eggs must stay in water in order to survive and usually will hatch within a couple of days, releasing larvae to begin the development process 5os-uito 8orld, n.d.".
5any permanent water mos-uitoes can also breed in containers that collect and hold water, such as wading pools, buckets or toys left outside5os-uito 8orld,n.d.".
Re*ated Stud#e%
The
neem
tree, Azadirachta
indica
has
well>known
insecticidal
8andscheer et al., )**B, &arvicidal action of ethanolic extracts from fruit endocarps of Me!ia azedarach and Azadirachta indica against the dengue 18
mos-uito Aedes aegypti " and insect growth regulatory I6A" constituents 1ukumar et al., +22+: Fatra et al., +22;: ?opping J 5enn, )***, Fotanical derivatives in mos-uito control< review", and has been used for centuries in India 1chmutterer, +22*, . 0roperties and potential of natural pesticides from the neem tree, zadirachta indica". /arious neem products have been studied extensively for their phytochemistry and exploitation in pest control programs 5ulla J 1u, +222, ctivity and biological effects of neem products against arthropods of medical and veterinary importance". Azadirachta Indica contains limonoids such as azadirachtin, salannin,
deacetylgedunin, gedunin, +E>hydroxyazadiradione, and deacetylnimbin showed strong antifeedant and growth inhibitor activity against larvae Nathan et al., )**C, fficacy of neem !imonoids on $napha!ocrocis medina!is(uen*e) (+epidoptera, 'yra!idae) the rice !eaffo!der ". Its main chemical composition is a
blend of 4 to B related compounds along with over )* lesser ones, which are e-ually as active. The general class of these compounds is triterpenes and within this category, the most effective are the limonoids, which are abundant in Neem oil 1anskrit $ocuments 9rg., nd, ". &imonoids are phytochemicals, abundant in citrus fruit and other plants of the families Autaceae and 5eliaceae. ?urrently limonoids are under investigation for a wide variety of therapeutic effects such as antiviral or /iricide, antifungal, antibacterial, antineoplastic and antimalarial. ?ertain limonoids are insecticides such as azadirachtin from the neem tree 9mnilexica, n.d., Meaning of +imonoid ". t least nine limonoids are effective in inhibiting insect growth, especially some of the most deadly varieties found in human health and agriculture 19
worldwide. 9f these limonoids, azadirachitin has been found to be the main ingredient for fighting insects and pests, being up to 2*D effective in most instances. It repels and disrupts the life cycle, however does not kill immediately, but is nonetheless one of the most effective growth and feeding deterrents ever examined 1anskrit $ocuments 9rg., nd, The Neem Tree". In the study of 5ordue )***" entitled # Azadirachtin from The Neem Tree Azadirachta indica, Its Action
Against Insects-% it was stated that
zadirachtin is the main component responsible for both antifeedant and toxic effects in insects. 9ther limonoid and sulphur>containing compound with repellent, antiseptic, contraceptive, antipyretic and antiparasitic properties are found elsewhere in the tree, e.g. leaves, flowers, bark, roots. In the 3ournal by 1cientific Technical and 5edical 9pen ccess (ournals )*+4" entitled Azadirachtin ound in the ark of Neem Tree ro#n in $hina , zadirachtin was
found in the bark of introduced neem tree Azadirachta indica A. /uss " grown in ?hina by T&? examination. The bioassays made with imported cabbage worm 0ieris rapae" and siatic corn borer 9strinia furnacalis" having typical poisoning reaction to azadirachtin indicated that the extracts from the crushed bark of neem tree contained a certain amount of azadirachtin. The content of azadirachtin in the bark of neem tree was tested and calculated to be *.C mg K g in dried bark by 70&?, those findings could be very helpful to directly use the substances from the neem tree as insecticide and the comprehensive utilization of the products of the neem tree.
20
1ridharan )**2" states in his study entitled # Neem Tree, Me!ia Azadirachta and Azadirachta Indica- that zadirachtin is mainly responsible for
the insecticidal properties of the neem. zadirachtin disrupts the growth and reproduction in most of the pest. It is one of the most potent growth regulators. It will repel or reduce the feeding of many species of pest insects as well as some nematodes. 1ridharan also added that azadirachtin is structurally similar to insect hormones called MecdysonesM, which controls metamorphosis in the insects as they pass from larva to pupa to adult. It affects the corpus cardiacum, an organ similar to the human pituitary that controls the secretion of hormones. 5etamorphosis re-uires a careful synchrony of many hormones and other physiological changes to be successful. zadirachtin because of its structural similarity to ecdysone will block the ecdysone's action in metamorphosis and the release of these vital hormones. Insects then will not molt. This of course breaks their life cycle. ccording to 1chumetterer+2;+, 0roperties and potential of natural pesticides from the neem tree, zadirachta indica", zadirachtin, a limonoid, has been reported to have adverse effect on endocrine system of a bean bettle, pilachna varivestis, and to cause sterility in the female insects. 1chluter and 1chulz +2;4" also reported in their study entitled # 0tructura! damages caused by neem in pi!achna 1eri1estis, a summary of histo!ogica! and u!trastructura! damage% this compound to cause degradation in larval epidermis preventing the
larvae from molting. In addition, 1ridharan )**2" also cited in his study entitled Neem Tree, Me!ia Azadirachta and Azadirachta Indica that zadirachtin is mainly
21
responsible for the insecticidal properties of the neem. Insects are perceptive to smell: they do not like the smell of neem oil. zadirachtin disrupts the growth and reproduction in most of the pest. It is one of the most potent growth regulators. It will repel or reduce the feeding of many species of pest insects as well as some nematodes.
METHODOO"!
This includes the research design, setting, the participants, sampling techni-ue, measures, research instrument, research procedure, and statistical treatment that will be used in the course of this study.
Re%earch De%#gn
This research used an experimental research design. This is an in-uiry on cause>and>effect relationships, and is conducted in a specialized setting, such as the laboratory, experimental unit or research center. The researcher controlled and manipulated the independent variable and randomly assigned the sub3ects to different conditions or situations Tan, )**". The researcher used the random sampling techni-ue in getting their test insects. Aandom sampling techni-ue is a techni-ue where each member of the population has an e-ual chance of being selected as sub3ect. The entire process
22
of sampling is done in a single step with each sub3ect selected independently of the other members of the population ?astillo, )**2".
The research design is divided into two variables< the dependent variable x" and the dependent variable y". The independent variable x" corresponds to Azadirachta Indica bark extract. The distilled water will be the negative control and the li-uid insecticide will be the positive control. The Azadirachta Indica neem" bark extract derived from decoction method and Azadirachta Indica neem" bark extract derived from ethanolic extraction method are the treatments that will be used: and three 4" treatments, one +" as the negative control, one +" as the positive control and will be conducted with three 4" replicates each. ach method of extraction will have the same concentration of distilled water extract. In treatment *, it is prepared with zero percent *D" extract of Azadirachta Indica bark and one hundred percent +**D " distilled water as the
negative control and its replicate respectively and for treatment one +" it is prepared with zero percent *D" extract of Azadirachta Indica bark and +**D li-uid insecticide and its replicate respectively: treatment two )" it is prepared of ten percent +*D" extract of Azadirachta Indica bark and ninety percent 2*D" distilled water and its replicate respectively: in treatment three 4" twenty percent )*D" extract of Azadirachta Indica bark and eighty percent ;*D" distilled water and its replicate respectively: in treatment four B" thirty percent 4*D" extract of Azadirachta Indica bark and seventy percent E*D" distilled water and 23
its replicate respectively.
The dependent variable y" represents the percentage
of mortality rate of mos-uito wriggler .
24
E"END FOR RESEARCH DESI"N
T*> *D of Azadirachta Indica Neem" bark extract and +**D distilled water as the negative control of the sample A+>first replication of T* A)> second replication of T* A4> third replication of T*
T+> *D of Azadirachta Indica Neem" bark extract and +**D li-uid insecticide as the positive control of the sample A+>first replication of T+ A)> second replication of T+ A4> third replication of T+
T)> the addition of +*D of Azadirachta Indica Neem" bark extract and 2*D distilled water to the sample A+>first replication of T) A)> second replication of T) A4> third replication of T)
25
T4> the addition of )*D of Azadirachta Indica Neem" bark extract and ;*D of distilled water to the sample. A+>first replication of T4 A)> second replication of T4 A4> third replication of T4
TB – the addition of 4*D of Azadirachta Indica Neem" bark extract and E*D of distilled water to the sample. A+>first replication of TB A)> second replication of TB A4> third replication of TB
MATERIAS AND E/UIPMENT
;*** grams of Azadirachta Indica bark ; liters of distilled water &i-uid insecticde 5os-uito wriggler +***m& glass beaker with stopper" ?oarse filter paper +,**m& 2CD thanol solution Aotary vaporator !unnel rlenmeyer !lask ?ornmill 6rinder 4* pcs C**m& calibrated measuring cups CP ;*mm 5agnifying glass
26
"ENERA PROCEDURE A0 Co**ect#on and Pre+arat#on of P*ant E1tract%
0lant extracts were obtained from the bark parts of the Neem plant Azadirachta Indica". ll these barks were collected from Farangay 1aravia, @oronadal ?ity.The barks were thoroughly washed under distilled water and air> dried for E days before grinding using a cornmill grinder and sent to Notre $ame 5arbel Lniversity>?hemistry &aboratory for phytochemical analysis and for decoction and ethanol extraction process.
Fark is separated from its wood
27
A0 2 Decoct#on E1tract#on Method
The air>dried Neem bark was Fark is thoroughly washed ground using a grinder. under distilled water before air> B**gm of the ground plant materials were divided into +**gm each and were put
in a +***m& glass beaker with stopper" containing B**m& of distilled water and boiled for 4*mins . The boiled extracts were allowed to cool, and then they were sieved using funnel and coarse filter paper.
A0 3 Ethano*#c E1tract#on Method
The plant extracts were carried out using ethanol by soaking B**gm of the ground materials in +, **m& of 2CD ethanol solution for )B hours, after which they were filtrated using funnel and coarse filter paper and were concentrated to dryness under pressure and controlled temperature B*>C* o?"
using rotary
evaporator.
B0 CUTURE OF TEST INSECTS
5os-uito larvae will be collected by catching mos-uitoes and placing
them
inside a mos-uito net with black containers so that the mos-uitoes can lay their eggs.
28
C0PH!TOCHEMICA ANA!SIS OF THE E4TRACTS
The 0hytochemical analysis of the extracts is based on the 0hytochemical 1creening by 6uevara )**C".
Te%t for A*ka*o#d%< Bm& of plant extract ethanol extraction method
were
evaporated to dryness using the evaporating dish placed on top of the beaker which was heated in a hot plate. Cm& of )5 7?& was being added to the residue and stirred before it was filtered in a testube. Then it was divided into three for confirmatory tests. The second one was added with $ragendorff's Aeagent, and the other one was added with 5ayer's Aeagent. The third one was added with 4m& and );D mmonia until alkaline and added with +*m& ?hloroform then divided into two test tubes. The first chloroform extract was evaporated and added with Cm& of )5 7?& and then was heated for ) minutes then filtered and separated into two portions and tested with $ragendorff and 5ayer's Aeagent. The other one was added with -ueous extract and )5 7?& then filtered and separated into two portions and tested with $ragendorff and 5ayer's Aeagent. 9bservation made for the formation of precipitation indicated positive results.
29
Te%t for Sa+on#n% (froth#ng te%t)5 )m& of plant extracts were diluted with
distilled water to )*ml each was added into test tubes and shaken vigorously. They were then allowed to stand on testube rack for + minute and observation made for the formation of stable froths which indicated positive results.
30
Te%t for F*a6ono#d%5 )m& of plant extract derived from
decoction and ethanol extraction method were evaporated to dryness using the evaporating dish placed on top of the beakers which were heated in a hot plate then cooled. 2m& of 0etroleum ether was added to each of the evaporated plant extract and sitrred.+*m& of ;*D ethanol solution was added to each of the evaporating dish then filtered. ach extract was divided into 4 test tubes for confirmatory test. The first test tube of each extract will be added with *.Cm& +)5 7?& and magnesium turnings. The content of the second extract with +) 5 7?l were put in separate beaker with water and place in a hot plate. The third testube served as the control. The occurrence of a red or orange colouration was indicative of the presence of flavonoids compounds.
31
Te%t for Tann#n%5 +*grams of plant material was evaporated to dryness using
evaporating dish. The residue was dissolved in )* m& of distilled water and C drops of Na?l solution then filtered. Then it was divided into 4 parts. The first one served as control. The second one was added with gelatin>salt reagent. The third one was added with three drops of +*D of !e?l4 were added to the filtrate. The appearance of blackish>blue or blackish>green colouration was indicative of tannins.
32
Te%t for
Anthra,u#none%5 ) evaporating dish were prepared for the test< one for the
extract obtained from $ecoction 5ethod and the other one for thanol xtraction 5ethod. )m& of each extract were placed into each evaporating dish and placed in a hot plate till dryness. The residue were added with +*m& of distilled water then filtrated. The filtrate were added with Cml benzene then divided into two tubes. 9ne served as control. The other one was added with Cm& ammonia solution. Aed colouration was indicative of anthra-uinones.
33
FO' CHART Azadirachta Indica
(Nee$) Bark E1tract#on Through Decoct#on Method
34
+**gm of the ground plant material were put in a +***m& glass beaker containing B**m& of distilled water.
Extraction of
$istilled water with ground plant material were boiled for 4* mins.
The FO' boiled CHART extracts were sieved using funnel and coarse filter paper. Azadirachta Indica (Nee$) Bark Through Ethano* E1tract#on Method
35
B**gm of the ground bark was soaked in i, **m& of 2CD ethanol solution for )B hours.
D0 TESTIN" FOR TO4ICIT! The extracts were concentrated to The soaked plant material were dryness using rotary evaporator. sieved using funnel and coarse Ten +*" larvae were added to each calibrated measuring cup containing filter paper.
varying concentrations of Azadirachta Indica Neem" bark extract. 5ortality rates were observed after )B hours. 5ortality rates were compared to determine the lethal effects and the level of toxicity. Thirty 4*" C**m& calibrated measuring cups will be prepared – +C cups was used for $ecoction 5ethod and +C cups was used for thanol xtraction 5ethod. The experiment were laid out using ?ompletely Aandomized $esign ?A$" with the following treatments< Treatment * T*", Treatment + T+", Treatment ) T)", 36
Treatment 4 T4", and Treatment B TB" consisting of three replicates in each treatment were prepared. T* contains
*D Azadirachta Indica Neem" bark
extract and +**D distilled water as negative control, T+ contains *D Azadirachta Indica Neem" bark extract and +**D li-uid insecticide as positive control, T)
contains +*D Azadirachta Indica Neem" bark extract and 2*D distilled water, T4 contains )*D Azadirachta Indica ( Neem" bark extract and ;*D distilled water, and TB contains 4*D Azadirachta Indica ( Neem" bark extract and E*D distilled water. dd +* mos-uito wrigglers in each of the glass of different concentration. 5ortality rates of mos-uito wrigglers in each glass was observed after )B hours with the use of Cx;*mm magnifying glass. 0robit nalysis and ðal $osage &$C*" was used to determine the toxicity level of the dosage. 0robit nalysis is commonly used in toxicology to determine the relative toxicity of chemicals to living organisms. This is done by testing the response of an organism under various concentrations of each of the chemicals in -uestion and then comparing the concentrations at which one encounters a response. s discussed above, the response is always binomial e.g. deathKno death" and the relationship between the response and the various concentrations is always sigmoid having the shape of letter 1" /incent, )**;". &$C* is a measurement used in toxicology studies to determine the potential impact of toxic substances on different types of organisms. It is the median lethal dose of a substance, or the amount re-uired to kill C*D of a given test population. It provides an ob3ective measure to compare and rank the toxicity of substances 7adley, )*+4".
37
FO' CHART OF THE TESTIN" FOR TO4ICIT!
+* larvae were added to each container containing varying concentrations of Azadirachta Indica Fark extract.
38
&arvae were exposed to extracts for )B hours.
5ortality rates were observed after )B hours.
E0 STATISTICA TREATMENT
In analyzing the data gathered for this study, the researcher will use 9ne> 8ay nalysis of /ariance N9/", Two>8ay nalysis of /ariance N9/" , T> test, 0robits nalysis and &$C*. To analyze variance between the effects of Azadirachta Indica Neem" Fark xtract obtained from $ecoction 5ethod and thanol xtraction 5ethod, 9ne>8ay nalysis of /ariance N9/" was used. This is a statistical procedure for testing mean differences among three or more groups by comparing variability between groups
to variability within groups Tan, )**". Two>8ay 39
nalysis of /ariance was also used. This is a means of comparing multiple levels of two independent variables. The two>way N9/ is grounded in the idea that there are two variables, referred to as factors, affecting the outcome of the dependent
variable.
To
N9/ assumes population samples are e-ual
in variance,
and
contain
be effective, normally
a
two>way
distributed, independent,
sample groups of
e-ual
size
Fusiness$ictionary.com".
The t>test was also used to determine the difference between the means of two methods of Azadirachta Indica Neem" bark extraction. This is a parametic statistical test that shows the difference between the means of two groups of values. The t distribution is commonly used with samples less than 4* units speras, )**C". 0robit nalysis and ðal $osage &$C*" will be used to determine the toxicity level of the dosage. 0robit nalysis is commonly used in toxicology to determine the relative toxicity of chemicals to living organisms. This is done by testing the response of an organism under various concentrations of each of the chemicals in -uestion and then comparing the concentrations at which one encounters a response. s discussed above, the response is always binomial e.g. deathKno death" and the relationship between the response and the various concentrations is always sigmoid having the shape of letter 1" /incent, )**;". &$C* is a measurement used in toxicology studies to determine the potential impact of toxic substances on different types of organisms. It is the median lethal
40
dose of a substance, or the amount re-uired to kill C*D of a given test population. It provides an ob3ective measure to compare and rank the toxicity of substances. 8hen comparing &$C* values, a lower value is regarded as more toxic, as it means a smaller amount of the toxin is re-uired to cause death 7adley, )*+4".
FO' CHART OF THE "ENERA PROCEDURE
?ollection of 0lant 5aterials and ?ulturation of Test Insects
0reparation of xtracts
0hytochemical 1creening 41
Test of Insects
9bservation and 6athering of $ata
RESUTS AND DISCUSSION OF DATA
0resented in this section are the results of the data gathered on the Azadirachta Indica N5" bark extract as insecticides against mos-uito
larvae.1pecifically, it contains the phytochemical screening analysis of the bark extract, the effectivity of neem bark extract derived from $ecoction 5ethod and thanol xtraction 5ethod as insecticide against mos-uito larvae, and the significant difference between Azadirachta Indica Neem" bark $ecoction and thanol extraction in terms of their larvicidal activity.The test involves the determination of the significant difference between the effects of Azadirachta 42
Indica Neem" bark extract obtained from $ecoction 5ethod and thanol
xtraction 5ethod by using N9/ nalysis of /ariance". T>test is used to show the difference between the means of two groups of values where the t distribution is commonly used with samples less than 4* units speras, )**C". 0robit nalysis was used to determine of Toxicity &evel of Azadirachta Indica Neem" bark extract and ðal $osage &$C*" will be used to determine its toxicity level.
Ta.*e 20 Phytoche$#ca* Screen#ng of P*ant E1tract Phytoche$#ca*
$ragendorff's Test &@&9I$1 5ayer's Test ?onfirmatory Test -ueous $ragendorff's 5ayer's ?hloroform $ragendorff's 5ayer's 109NIN1 !rothing !&/9N9I$1 Fenzopyrene &eucoanthocyanins TNNIN1 6elatin>1alt !erric ?hloride NT7AQLIN9N1 mmonia 1olution
Decoct#on
Ethano*
Method
E1tract#on
7 7
Method 8 8
7 7 7 7 7 8 8 7 7 8
8 8 8 8 8 8 8 8 8 8
43
egend%5 1light turbidity $efinite turbidity 7eavy precipitation Not performed
R" RR" RRR" >"
Table + shows the phytochemical screening results of the plant extract. It was shown in the table that the plant extract is positive in or contains alkaloids, saponins, flavonoids, tannins and anthra-uinones when it undergone different confirmatory tests which shown at the middle column of the table.
Ta.*e 30 Morta*#ty Rate% of Mo%,u#to ar6ae U+on E1+o%ure to Azadirachta Indica (Nee$)
Bark E1tract O.ta#ned Through Decoct#on Method0
12 10 8 6 4 2 0
Replicate 1 Replicate2 Replicate3
Table ) shows that among the three treatments Treatment ), Treatment 4, and Treatment B", Treatment B has the highest mortality rate of mos-uito upon exposure to the extract having +**D +* larvae" in Aeplicate + while Treatment ) has the lowest mortality rate having 4*D 4 larvae" in Aeplicate+.
Ta.*e 90 Morta*#ty Rate% of Mo%,u#to ar6ae U+on E1+o%ure to AzadirachtaIndica (Nee$)
Bark E1tract O.ta#ned Through Ethano*
E1tract#on Method0
44
12 10 8 Replicate 1
6
Replicate 2
4
Replicate 3
2 0 Treatment 0 (negative control)
Treatment 2
Treatment 4
Table 4 shows that among the three treatments Treatment ), Treatment 4, and Treatment B", Treatment 4 and Treatment B have the highest mortality rate of mos-uito upon exposure to the extract having +**D +* larvae" in all replicates while Treatment ) has the lowest mortality rate having )*D ) larvae" in Aeplicate ). Fased from the results on 5ortality rates of 5os-uito &arvae upon exposure to Azadirachta Indica Neem" Fark xtract from the table ) and 4, it was proven that both
Azadirachta Indica Neem" Fark xtract derived from
$ecoction 5ethod and thanol xtraction 5ethod are both effective as insecticide against mos-uito larvae.
Ta.*e :0 Ana*y%#% of &ar#ance Bet;een the Effect% of Azadirachta Indica (Nee$) Bark E1tract O.ta#ned fro$ Decoct#on Method and Ethano* E1tract#on Method0
Su$$ary
6roups ?olumn +
?ount
1um
verage
/ariance
+C
;+
C.B
+4.B 45
?olumn )
1ource of /ariation Fetween 6roups 8ithin 6roups Total
+C
+**
.E
+2.4;*2C
00
df
M0
'21a!ue
crit
+).*4444
+
+).*4444
*.E4B+
*.42;;+
B.+2C2E)
BC;.2444
);
+.42*B;
BE*.2E
)2
Table B shows that with the ! value of *.E4B+, the critical ! G B.+2C2E). 1ince the ! statistic is smaller than the critical value, we fail to re3ect the null hypothesis. Aemember from above, the null hypothesis was that all ) of these groupsO means were e-ual. 1o, we fail to re3ect that there is a significant difference between Azadirachta Indica Neem" bark $ecoction and thanol extraction in terms of their larvicidal activity. Therefore, it was concluded that there is no significant difference between Azadirachta Indica Neem" bark $ecoction and thanol extraction in terms of their larvicidal activity since they both have e-ual means.
Ta.*e <0 Te%t on the S#gn#f#cant D#fference Bet;een Azadirachta Indica (Nee$) Bark E1tract o.ta#ned fro$ Decoct#on Method and Ethano* E1tract#on Method0
$5 5 $ifference
N +C +C +C
5ean C.B* .E >+.)E
1t$ev 4. B.B* ).);)
1 5ean *.2C +.+B *.C;2
2CD ?I for mean difference< >).C4+, >*.**4" T>Test of mean difference G * vs not G *"< T>/alue G >).+C 0>/alue G *.*C* 46
Table C shows the difference between the means of two methods of Azadirachta Indica Neem" bark extraction. Lsing the ?ritical /alues for the t>
$istribution, first we must select the correct critical value from the table to compare with our calculated value. 8e do this by computing the degrees of freedom and the value is +.2E. 1ince our t>value t G ).+C" is larger than the tabled t>value t G +.2E" this means that there is a small chance that the population means are the same, and so it is reasonable to conclude that the means are different. 7ence, we re3ect the null hypothesis that there is no significant difference between Azadirachta Indica Neem" bark extract derived from $ecoction 5ethod and thanol xtraction 5ethod.
Ta.*e =0 Re%u*t% on e6e* of To1#c#ty U%#ng PROBIT ANA!SIS and ETHA DOSA"E (D<>) on Decoct#on Method of E1tract#on Accord#ng to FINNE!?S METHOD $ose 1timulus "
ctual 0ercent D"
0robit 0ercent
N
A
A"
$ifference
?hi>1-uare
4.+4 B .*CC ) E.4) C
*.4;
*.*B+B
>+.*CC)
*.+;42
*.4EC
*.*CC)
+
*.B
*.4+4
+*
B
+.4*+
*. C
*.*CC
+*
C
+.BEE+
*.;
*.E4)
+*
;
&$C*< +B.;BC; Table shows toxicity level of Azadirachta Indica Neem" bark extract derived from $ecoction 5ethod. It was shown that toxicity level of the extract is at dose + having a probit percent of *.4+4 and an &$C* of +B.;BC;. lower 47
value is regarded as more toxic, as it means a smaller amount of the toxin is re-uired to cause death. 7adley, )*+4".
Ta.*e @0 Re%u*t% on e6e* of PROBIT ANA!SIS and ETHA DOSA"E (D<>) RESUTS on Ethano* E1tract#on Method Accord#ng to FINNE!?S METHOD $ose 1timulus "
+ +.4*+
ctual 0ercent D"
0robit 0ercent
N
A
A"
$ifference
?hi>1-uare
*.4 +
+* +*
4 2.EC
* >*.)B2E
* *.**)
+
+*
2.EC
4 2.222 E +*
>*.)C
*.**)
*.4 *.2EC
+.BEE+ *.2EC &$C*< +*.;)2E
Table E shows toxicity level of Azadirachta Indica Neem" bark extract derived from $ecoction 5ethod. It was shown that toxicity level of the extract is at dose + having a probit percent of *.4 and an &$C* of +*.;)2E. lower value is regarded as more toxic, as it means a smaller amount of the toxin is re-uired to cause death. 7adley, )*+4".
48
SUMMAR! CONCUSIONS AND RECOMMENDATIONS
This section presents the summary of study and conclusions made from the study and the recommendations given by the researchers. Su$$ary of the Study
The main purpose of the study is to utilize Azadirachta Indica Neem" bark extract as insecticide against mos-uito larvae and to determine its effectivity. Two methods were used for the extraction of Azadirachta Indica Neem" bark. xtraction with distilled water $ecoction xtraction 5ethod" and xtraction with 2CD ethanol solution thanol xtraction 5ethod". 0hytochemical 1creening, which includes test for 1aponins, Tannins, lkaloids, !lavonoids and nthra-uinones, was performed to determine the chemical compounds present in Azadirachta Indica Neem" bark. 49
There were five treatments in each of the method of extraction, Treatment * T*", Treatment + T+", Treatment ) T)", Treatment 4 T4" and Treatment B TB" consisting of three replicates in each treatment were prepared. T* is the negative control which contains +**D distilled water, T+ is the positive control which contains +**D li-uid insecticide, T) contains +*D Azadirachta Indica Neem" bark extract and 2*D distilled water, T4 contains )*D Azadirachta Indica Neem" bark extract and ;*D distilled water, and TB contains 4*D Azadirachta Indica Neem" bark extract and E*D distilled water. Toxicity testing of the extract was determined through monitoring of the
mortality of ten larvae introduced to each calibrated measuring cup containing different concentration of the extract. 5onitoring and observation were made after )B hours. The data gathered from mortality of larvae in toxicity testing was treated by using Two>8ay N9/ nalysis of /ariance", 0robit's nalysis, &$C* ðal $osage C*" and T>Test. The significant difference between the effects of Azadirachta Indica Neem" Fark xtract obtained from $ecoction 5ethod and
thanol xtraction 5ethod was determined by using 9ne>8ay and Two>8ay N9/ nalysis of /ariance". T>test is used to show the difference between the means of two groups od values where the t distribution is commonly used with samples less than 4* units speras, )**C". 0robits nalysis was used to determine of Toxicity &evel of Azadirachta Indica Neem" bark extract and ðal $osage &$C*" was used to determine the toxicity level of the dosage.
50
The results from the tests indicated that based on the 0hytochemical 1creening Aesult, zadirachta Indica Neem" bark extract contains alkaloids, saponins, tannins, flavonoids and anthra-uinones. N9/ and T>test results show that both zadirachta Indica Neem" bark extracts derived from $ecoction 5ethod and thanol xtraction 5ethod are both effective and has no significant difference in terms of their larvicidal activity since they both have e-ual means. 0robit nalysis and &$C* shows that their toxicity level is at the lowest dosage for it only re-uires less amount of extract to cause death of the mos-uito larvae.
Conc*u%#on% fter a thorough analysis of the data gathered, the researchers concluded
that< +. zadirachta Indica Neem" bark extract contains insecticidal property against mos-uito larvae. ). zadirachta Indica Neem" bark extract derived from $ecoction 5ethod and thanol xtraction 5ethod are both effective as insecticide against mos-uito larvae. 4. Fased on the result from N9/ Test Aesult, there is no significant difference between Azadirachta Indica Neem" bark extract derived from $ecoction 5ethod and thanol xtraction 5ethod in terms of their larvicidal activity.
Reco$$endat#on%
The results show that Azadirachta indica (Neem) Fark xtract derived from $ecoction 5ethod and thanol xtraction 5ethod are both effective as 51
insecticide against mos-uito larvae by mixing B** grams of plant material to +, **ml of water or 2CD ethanol solution. The researchers recommend the following for further study and development of this study< +. To purify the extract of Azadirachta Indica Neem" bark to identify its component that is responsible for the mortality of mos-uito larvae with the use of 7igh>0erformance &i-uid ?hromatography 70&?" since a phytochemical screening was conducted. ). further study on the comparison of the different parts of Azadirachta Indica Neem" in terms of their larvicidal activity. 4. 5aking a soap or lotion out of Azadirachta Indica Neem" bark extract.
52
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