OPTIMIZATION OF SUPERCRITICAL FLUID EXTRACTION OF BIOACTIVE COMPOUND FROM STROBILANTHES CRISPUS
MUHAMMAD AFIQ SYAMIM BIN RAHIMI 55201113563
FINAL YEAR PROJECT 2 PROPOSAL
Malaysian Institute of Chemical & Bio Engineering Technology University Kuala Lumpur
JANUARY 2016
Table of Contents
........................................................................... .................................................... ............................ .. 10 CHAPTER 1 ................................................. ........................................................................ ................................................. ........................ 10 INTRODUCTION ............................................... 1.1 Background Background of studies......................... studies................................................... .................................................... ............................ .. 11 1.2 Problems Problems Statement.................... Statement.............................................. .................................................... .................................... .......... 14 1.3 Significant Significant of Study ....................................................... ................................................................................. ............................ .. 16 1.4 Scope of Study.................................................. ........................................................................... ........................................ ............... 17 1.5 Objectives Objectives ................................................ ......................................................................... ................................................. ........................ 18
........................................................................... .................................................... ............................ .. 19 CHAPTER 2 ................................................. ..................................................................................... .............. 19 LITERATURE REVIEW . ....................................................................... 2.1 Bioactive Bioactive Compounds Compounds ........................................................... ............................................................................... .................... 19 2.2 Strobilanthes Strobilanthes Crispus ............................... ........................................................ ................................................. ........................ 22 2.2.1 Strobilanthes Strobilanthes Crispus Crispus characteristic characteristic ................................................... ................................................... 22 2.2.2 Uses of Strobilanthes Strobilanthes Crispus ........................................ ............................................................ .................... 23 2.2.3 Chemical and Physical Properties of Strobilanthes Crispus ............... 26 2.3 Diabetes Mellitus Disease................................................. ......................................................................... ........................ 27 2.4 Extraction Extraction Method ............................................. ....................................................................... ........................................ .............. 29 2.4.1 Conventional Conventional Extraction Extraction Method.................................................. ......................................................... ....... 29 2.4.2 Non-Convention Non-Conventional al Extraction Extraction Method ................................................. ................................................. 31
........................................................................... .................................................... ............................ .. 33 CHAPTER 3 ................................................. ........................................................................... ............................................. .................... 33 METHODOLOGY .................................................. 3.1 Outline of the methodology ......................... ......................... Error! Bookmark not defined. 3.2 Methodology Methodology ................................................ ......................................................................... ............................................. .................... 35 3.2.1 Sample Collection and Preparation Preparation .......................................... .................................................... .......... 35 3.2.2 Solvent preparation preparation ................................................ .......................................................................... ............................ .. 36 3.2.3 Sample Extraction Extraction .................................................. ............................................................................ ............................ .. 37 3.2.4 Sample Analysis Analysis ................................................. ........................................................................... ................................ ...... 38 3.2.5 Sample Data................................................... ............................................................................ .................................... ........... 39
ii
........................................................................... .................................................... ............................ .. 42 CHAPTER 4 ................................................. .......................................................................... ........................................ ............... 42 EXPECTED RESULT .................................................
CHAPTER 5 ........................................................................... ................................................. ........................ 43 CONCLUSION ..................................................
......................................................................... ............................................. .................... 44 RESEARCH PLAN ................................................
........................................................................... ................................................. ........................ 45 REFERENCES ..................................................
............................................................................ ................................................. ........................ 48 APPENDICES ................................................... Chemicals and Materials................................................. ........................................................................... ................................ ...... 49 Solvent Preparations Preparations.......................................... ................................................................... ............................................. .................... 50 Example of Calculation Calculation ......................................................................... ................................................................................... .......... 50
iii
List of Figures
Figure
Tittle
Page
1.1
The Prevalence of diabetes in adult by the age in Malaysia (IDF, 2015) 15
2.1
Strobilanthes Crispus plants in Malaysia (Lin, 2013)
23
2.2
Strobilanthes Crispus leaved sold in Malaysia (Lin, 2013)
24
2.3
The islets contain beta cell cells in human organ (NIH, 2014)
28
2.4
The ultrasonic-assisted extraction (University of British Columbia, 2012
30
2.5
The maceration method of plant extraction (Rahul, 2013)
30
2.6
The supercritical fluid extraction method (Suzan Mahdi & Altikriti, 2010) 31
2.7
The Pressurized liquid extraction (Department of Pharmaceuticals Sciences, 2015)
32
3.1
The outline of the methodology in this studies
34
3.2
The ideal gas law formula (Corbett, 2014)
36
List of Tables
Tables
Tittle
2.1
The Medical uses of Strobilanthes Crispus plants (GlobinMed, 2010)
2.2
The chemical and physical properties of the Strobilanthes Crispu
Page
25
(Ghasemzadeh, Jaafar, & Rahmat, 2015)
27
Project cost estimation for Final Year Project 1
48
2
Table of chemicals/materials chemicals/materi als and apparatus
49
3
Solvent Preparation for Ethanol
50
1
iv
ABSTRACT
Supercritical Fluid Extraction (SFE) has become one of the most popular green extraction techniques nowadays since it has demonstrated many advantages compared to traditional or conventional extraction process. Aspects such as improved selectivity, higher selection yields, better fractionation capabilities and lower environmental impacts have been crucial to the important growth of SFE. In this study, non-conventional Supercritical Fluid Extraction by using Tubular Batch Reactor was used to extract the bioactive compound from Strobilanthes Crispus besides using Supercritical Fluid Extractor.
Strobilanthes Crispus is an herbal medicine plant which is native to country from Madagascar to Indonesia. This plant are used in medicinal and to treat a variety of ailments in the various traditional systems of medicine. Phytochemical investigations have revealed that the plant contains polyphenols, flavonoids, catechins, alkaloids, caffeine, tannis, and compound known to possess health beneficial effects. Preclinical studies have shown that the plant possess antioxidant, anti-cancer, anti-diabetic, and anti-microbial activities.
Hence, the aim of this study was to identify the bioactive compounds in extracted Strobilanthes Crispus which could be used to treat diabetes mellitus disease. The bioactive compound which is quercetin will be analyzed to cure the diseases.
ABSTRAK
6
APPROVAL PAGE
We have supervised and examined this report and verify that it met the program and the University’s requirements for the Bachelor in Chemical Engineering Technology in Process.
Date:
Signature: ………………………………… Supervisor Name: Mdm. Nor Aini Bt Burok Official Stamp:
7
DECLARATION
I declare that this report was my original work and all references have been cited adequately as required by the University.
Date:
Signature: ………………………………… Full Name: MUHD AFIQ SYAMIM BIN RAHIMI ID Number: 55201113563
8
ACKNOWLEDGEMENT
Alhamdulillah, all praises to Allah SWT, the Almighty and may Allah’s peace and blessing be upon His Servant and Messenger Muhammad SAW and upon his family and Companions. Thanks to Allah whom with His willing giving me the opportunity to complete the final year project report.
I would like to express deepest gratitude to my advisor, Madam Nor Aini Binti Burok for her full support, expert guidance, understanding, and encouragement throughout my study and research. Without her incredible patience and timely wisdom and counsel, my thesis work would have been a frustrating and overwhelming pursuit. To my parents, no words that I could use to express my genuine appreciation for their endless support and invaluable expenditure of motivation. I would not have been able to complete this thesis without their continuous love and encouragement.
Last, but by no means least, my sincere appreciation goes to my sister Nur Farthiah Bt Mohd Adanan and all my friends who patiently helped in revising and correcting every single page of my thesis report. I also would like to thank The technicians of UniKL MICET, En.Shukri for guiding, sharing advice and giving strong cooperation to help me during handling equipment and apparatus in the lab. I hope that with all the knowledge and experiences that I gained from this project will be beneficial for me in the real working in the future.
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CHAPTER 1
INTRODUCTION
Overview
A bioactive compound is a substance present in natural source that having an impact or cause a response to the living organism, tissue or cell. Nowadays, it becomes a trend to used bioactive compound in geo-medicine, plant science, modern pharmacology, agrochemicals, cosmetics, food industry and nanobioscience. Based on the problem statement below, there were over a million cases of diabetes in Malaysia in 2015 and it getting increase year by year. Quercetin exhibits a wide range of biological functions and gives a lot of benefits to human health. Thus, the Strobilanthes Crispus leaves was used to extract quercetin that can be used to treat diabetes disease. The plants contained high amount of mineral content and vitamin C, B1, and B2. The extraction technique used in this study was non-conventional Supercritical Fluid Extraction by using Tubular Batch Reactor.
10
1.1 Background of studies
Bioactive compound are extra nutritional constituents that typically occur in small quantities in food and has an effect on living organism, tissue or cell. These compounds utilized as a part of extensive variety of conventional and present day applications. They are being intensively studied to evaluate their effects on health widely. Nowadays, it becomes a trend to used bioactive compound in geomedicine, plant science, modern pharmacology, agrochemicals, cosmetics, food industry and nano-bioscience. Bioactive compounds can have an influence on health and they are being studied in the prevention of cancer, diabetes, and other disease. Strobilanthes Crispus (pecah kaca pecah beling leaves) is a member of the
Acanthaceae family and is an herbal medicine plant which is native to countries throughout Madagascar region to Indonesia. Traditionally, it is known as “pecah kaca” in Malay, “daun picah beling” in Jakarta, and “kejibeling” in Java. (Chong, Koh, Kiong Ling, Chye, & Yew, 2014). The plants contained high amoun t of mineral content and vitamin C, B1, and B2. The plants is a well-known her in Malaysia with various pharmaceutical properties and contain several biologically active chemical constituents which are responsible for its pharmaceutical quality .The plant part will be used to extract the bioactive compound is the leaves. Phytochemical investigations have revealed that the plant contains polyphenols, flavonoids, catechins, alkaloids, caffeine, tannis, and compound known to possess health beneficial effects. Preclinical studies have shown that the plant possess antioxidant, anti-cancer, anti-diabetic, and anti-microbial activities and can gives an advantage in order to prevent the disease. (Nurraihana, Norfarizan Hanoon, & N.a, 2013) Extraction in chemistry is a separation process consisting in the separation of a substances from a matrix. There are two extraction methods can be used to extract bioactive compound which is conventional and non-conventional method. 11
The examples of conventional method are soxhlet extraction, macerations and hydrodistillation that had been used in order to retrieve bioactive compounds. Supercritical fluid extraction (SFE), and pressurized liquid extraction (PLE) are non-convetional methods that offers a more economical, environmental friendly and ozone-depleting emissions. Scalia et al., (1999) in their studies state that the conventional techniques to obtain plant extracts such as steam distillation and organic solvent extraction usually requires several hours or even days causing the extraction process require large amount of solvent. The solute or solvent separation may result in degradation of the thermo labile components and traces of the solvent may be present in the product, which it may reduce quality assessment of the extraction yield. Process optimization and antioxidant activity supercritical fluid extraction studies by (Yuefei, Le, Da, Shikang, Yuejin, & Ping, 2011) in their journal supercritical carbon dioxide extraction (SC-CO 2) of bioactive compounds from Ampelopsis grossedentata stems was to investigate extraction parameters such
as pressure, temperature, dynamic time and modifier. The best conditions obtained for SC-CO 2extraction of flavonoids was 250 bar, 40 °C, 50 min, and with a modifier of methanol/ethanol (1:3, v/v), and that for phenolics extraction was 250 bar, 40 °C, 50 min, and with a modifier of methanol/ethanol (1:1, v/v). This shows that methanol and ethanol are the best solvent to extract flavonoids and phenolics compounds. A comprehensive study on different methods of extraction was done by Vibha, Pallavi, & Devendra, 2012 by using Guajava Leaves for curing various health problems. This study was to introduce the different extraction processes with different solvent such as ethanol, methanol, ethyl acetate and water. The method used was Supercritical Fluid Extraction (SFE), Soxhlet Extraction, Steam Distillation and Ultrasound Extraction. The yield shows Soxhlet process gives highest global yield but separation of compounds is difficult in this method but in SFE gives better recovery of functional compounds.
12
Supercritical Fluid extraction (SFE) has been documented as an effective method for preparing bioactive products from plant materials. The combined liquidlike solvating capabilities and gas-like transport properties of supercritical fluids make them particularly suitable for the extraction of diffusion-controlled matrices such as plant tissues. The remarkably high selectivity can be achieve when the solvent strength of supercritical fluid can manipulated by changing pressure and temperature. Subsequently, based on previous studies, this study was conducted to extract bioactive compounds from
Strobilanthes Crispus by using non-
conventional extraction method which is supercritical fluid extraction (SFE) method. Ethanol was used to identify bioactive compounds content from Strobilanthes Crispus and acts as a supercritical fluid. Extraction parameter studied was solid loading, temperature, and extraction time to identify their effect on extracted Strobilanthes Crispus .
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1.2 Problems Statement
Cancer is a major public health problem worldwide with millions of new cancer patients diagnosed each year and many deaths resulting from this disease. Many scientist had been trying to develop the cure for cancer, unfortunately their attempt had failed. Chemotherapy remains the principal mode of treatment for various cancers and the patients need to spend a lot of their money for the treatment. Tamoxifen, non-steroidal, and anti-estrogen drug is used in the treatment of estrogen receptor that positive in breast cancer patients. This statement is supported by (Yaacob, et al., 2010) in his titled “Anticancer activity of a sub-fraction of dichloromethane extract of Strobilanthes crispus on human breast and prostate cancer cells in vitro” There is no treatment that can completely cure diabetes melli tus until today. Presently, insulin is used to treat diabetes mellitus type 1 and the pharmacological agents currently used for the treatment of type 2 include sulphnonylureas, biguanide, and acarbose. These agents however have restricted usage due to several undesirable side effect and failure to significantly the course of diabetic complications. (Bakar, 2005) According to the International Diabetes Federation (IDF) reports, there were 3.3 million cases of diabetes in Malaysia in 2015. The number of deaths in adults due to diabetes is 34,576. Based on the figure 1.1 below, the figure describes which are groups in population have the highest proportions of diabetes. The dotted lines is the distributions of diabetes prevalence by age of the world, the black line is the distribution for the region and the country region is plotted in the red line. Many middle and low income countries have more people under the age of 60 with diabetes compared to the world average.
14
Fig ure 1.1: The Prevalence of diabetes in adult by the age in Malaysia (IDF, 2015)
15
1.3 Significant of Study
The bioactive compound extracted from this study will be used for diabetes disease. The bioactive compound existed in the leaves was able to lower blood glucose level in strptozocin induced diabetic rats by extract of both fermented and unfermented leaves using hot water. It was found that the content of vitamins C, B1 and B2 with catechin, caffeine, tannin were the major contributors to the antioxidant activity. In order to enhance the defense system especially the incidence of degenerative diseases, the daily consumption of the tea could contribute towards it. The extraction technique use in this study was Supercritical Fluid Extraction (SFE). This techniques was widely used in industrial extraction operations because of regulatory and environmental pressures on hydrocarbon and ozone-depleting emission. Nowadays, the most currently available solvent free extraction system will utilize carbon dioxide (CO 2) which is generally considered as safe for solvent free extraction processes. In this study new technique were applied, SFE will be conducted by using tubular batch reactor. This reactor was design by using 15 mL bulkhead union (3/4” OD) with two steel caps (3/4” OD). In addition, this study will build up the people about the benefits of the leaves thus it can consume the leaves in their daily life since it can make as a tea. Traditionally, it is widely used to treat gastrointestinal and kidney diseases at a certain place around Malaysia.
1.4 Scope of Study
The Strobilanthes Crispus leaves was used in this study because it has been of much interest due to traditional claims of its anti-cancer properties and other diseases in this country. The leaves was collected from the area around Alor Gajah and will be followed with a few step until it is ready to use. In this study, supercritical fluids extraction method by using tubular batch reactor design was applied in order to extract the bioactive compound of Strobilanthes Crispus leaves. Ethanol was used as a supercritical fluid because it
is a very polar molecule due to its hydroxyl (OH) group with high electronegativity of oxygen allowing hydrogen bonding to take place with other molecules, thus it can attract polar and ionic molecules. Ethanol can dissolve both polar and nonpolar substances because it can attract non-polar molecules. The parameter studied in this project were types of solid loading, temperature and extraction time. Temperature use is 300 oC and 400 oC, solid loading 30% and 50%, and retention time 15min and 25min. The critical state temperature and pressure for ethanol are 243 oC and 63.18atm respectively. In order to identify the existence of bioactive compound in Strobilanthes Crispus leaves, Ultraviolet-Visible Spectrophotometer (UV-Vis) was used to
analyze the extractant. To determine the optimal yield bioactive compound in Strobilanthes Crispus , Response Surface Methodology (RSM) was used by using
statistical software which is Minitab 17.
17
1.5 Objectives
1. To extract the bioactive compound in Strobilanthes Crisus leaves. 2. To identify the bioactive compound found in Strobilanthes Crispus leaves. 3. To optimize the process condition of supercritical fluid extraction to obtain better yield using Response Surface Methodology (RSM).
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CHAPTER 2
LITERATURE REVIEW
2.1 Bioactive Compounds
Bioactive compounds by definition is a substance has an organic activity if it consumes direct effects on a living organism. These effects can both be advantageous or inconsistency on the substance and the bioavailability. It is usually linked with positive effects on an organism. (NPCR , 2016) Bioactive compounds have actions in order to promote good health in the body. Bioactive substances present as natural essential in food to provide health benefits beyond the basic nutritional value of the product. Many bioactive compounds have been discovered and have been widely in chemical structure, function, and are grouped accordingly. Phenolic compounds including flavonoids are present in all plants and have been studied extensively in tea, vegeta bles, fruits and others. Many phenolic compound have antioxidant properties, and it have been proved favorable effects of some disease such as thrombosis and tumorogenesis.
Bioactive compound in plants are compound produced by plants having pharmacological or toxicological effects in human and animals. Typically, bioactive compounds in plants are produced as secondary metabolites. Hence, the definition of bioactive compounds in plants in known as secondary plant metabolites. The secondary metabolites compounds in plants come out to be randomly synthesized. Most of the bioactive compound found have different characteristic and functions for example, flavonoids can protect against free radical generated during photosynthesis. It is slightly different with primary metabolites that course of compounds aimed at plant growth and development such as carbohydrates, amino acid, proteins, and lipids. It can be observe as the side track of biochemical products in plant cell and not needed for the plant daily functioning.
2.1.1 Flavonoid
Flavonoids or bio-flavonoids from the Latin word meaning yellow based on their color in nature. Chemically, it have general structure of a 15-carbon skeleton which consists of two phenyl rings A and B. According to the IUPAC nomenclature, it can be classified into flavonoids or bio-flavonoids, Iso-flavonoids, and Neoflavonoids. (McNaught, Wilkinson A., & Andrew, 1997) Flavonoids are abundant in plants, in which they perform several functions. They are essential pigments for producing the colors needed to attract pollinating insects. In higher order plants, flavonoids are also required for nitrogen fixation, UV filtration, cell cycle inhibition and can act as chemical messengers. Some flavonoids also inhibits certain spores to protect against certain plant disease. Flavonoids are plentiful in plants and are the most common type of polyphenolic compound found in human diet. The abundance of flavonoids coupled with their low toxicity relative to other plant compounds means they can be ingested in large quantities by animals including humans. The examples of foods that are rice in flavonoids include onions , blueberries, red wine, dark chocolate, and bananas. 20
Flavonoids, including quercetin are important in anti-inflammatories because they act as anti-oxidants which mean the literally fight the natural process of oxidation that takes place over time of the age. Quercetin can help stop damaging particles in the body that known as free radical which negatively impact on how the cells works.
2.1.2 Quercetin
Quercetin exhibits a wide range of biological function. Quercetin is a type of flavonoids anti-oxidants that’s found in plant foods including leafy greens, tomatoes, broccoli and others. Technically, it is considered as a plant pigment which is exactly found in deep colored, nutrient packed fruits and vegetables. They scavenge particles in the body known as free radicals which damage cell membranes, tamper with DNA, and even cause cell death. Anti-oxidants can neutralize the free radicals and it may reduce or even help to prevent some of the damage free radical causes. In test tubes, quercetin has strong anti-oxidant properties and the researches are not sure whether taking quercetin and many other anti-oxidants has the same effect on the body. (Ehrlich, 2015) Many of the benefits often hear attributed to anti-oxidants that refer to the effects associated with quercetin, some of them are supports normal respiratory. For example when respiratory system is irritated, redness, and swelling can result from the release of histamines and quercetin has been reported to have an antihistamine effect. From the previous research that have been done by Chirumbolo (2010), the lab test shown quercetin influences intracellular enzymes and may help inhibit histamine release. The study conducted by Perez Vizcaino and Duarte (2010) shown that the consumptions of flavonoids, specifically quercetin offer a dual benefits in promoting overall cardiovascular health and it encourages the blood flow very well. Next, researchers have witnessed that quercetin anti-oxidant action pretect against LDL
21
cholesterol oxidation and this may be useful because oxidation causes LDL cholesterol to stick to artery walls. Figure 2.1 below shows the structure of quercetin that is believe can give a lot of benefits to human body.
Fig ure 2.1: The structure of quercetin
2.2 Strobilanthes Crispus Strobilanthes Crispus is a member of the Acanthaceae family and is known
by the following names “Pecah Kaca” or “Pecah Beling” in Malaysia and “Kecibeling” or “Kejibeling” in Indonesia. It
is
a
Plant
that
originated
from
Madagascar and all the way to Indonesia and it grows rapidly in Malaysia. The leaves are used traditionally for anticancer treatment and diabetes mellitus disease. (Wikipedia, 2009)
2.2.1 Strobilanthes Crispus characteristic
The Strobilanthes Crispus is a woody spreading shrub that can easily reach more than a meter in height of the cultivation. The stem has a diameter of between 0.2-0.7cm with the external bark being purplish in color when young and brown 22
when matured. The leaves are elliptical in shape and have an attractive color which is glossy sheen. The surface of the leaves are also rough to the touch. The Strobilanthes Crispus leaves rarely produces flowers. It has rough in texture and
measures 5-8cm and 2-5cm wide. The buds are borne in leafy sheaths and the yellow flowers that emerge are said to be able reach up to 2cm in diameter. Figure 2.2 below shows the Strobilanthes Crispus plants were found in many places in Malaysia as it is easily to grow.
Fig ure 2.2: Strobilanthes Crispus plants in Malaysia (Lin, 2013)
2.2.2 Uses of Strobilanthes Crispus
2.2.2.1 Drinks In Malaysia, Strobilanthes Crispus plants were extracted in certain process in order to make it as healthy tea or herbal tea. The people believe especially Chinese race that this tea can relief cough, removal of kidney stones and urinary bladder problems. It is slightly bitter taste and has the sweet aroma. Figure 2.3 shows the example of Strobilanthes Crispus tea packet were sold in Malaysia
23
Fig ure 2.3: Strobilanthes Crispus leaved sold in Malaysia (Lin, 2013)
2.2.2.2 Traditionally Uses Strobilanthes Crispus plants been used traditionally to treat kidney stones.
The responsible for this action occurs because the phenolic compound in the leaf that was extracted. The phenolic compounds has inhibitory actions on calcium oxalate crystal growth. The most effective and the highest in phenolic compound content was found in the extraction by using methanol that acts as a solvent. This statement is supported by (Afrizal Itam, Ismail, & Abdul Majid, 2007) in his article titled “In vitro studies of calcium oxalate crystal growth inhibition of Strobilanthes Crispus extracts”. To further enhance its ability to erode renal stones, the aqueous
extract of the leaves of Strobilanthes Crispus also has it diuretic properties. In addition, Strobilanthes Crispus is used to treat constipation because it considered as a laxative. It is also used in the treatment of liver problems, hepatitis, and liver problems. The leaves part has always been used in a decoction process to produce the healthy water. Strobilanthes Crispus had been encouraged in the medication of poisonous bites including snakebites. The poultice of the fresh leaves can be directly applied 24
on to wounds caused by the bites of poisonous snakes or other animals. (Afrizal, 2008)
2.2.2.3 Medical Uses The use of Strobilanthes Crispus plants as medicine is not a new thing in Malaysia. This plants used by the local people to season food also yield medicinal compounds. As part of response to the threat of the disease in Malaysia, the use of the Strobilanthes Crispus leaves in cuisine is developed. Table 2.1 below shows the medical uses of Strobilanthes Crispus leaves.
Table 2.1: The Medical uses of Strobilanthes Crispus plants (GlobinMed, 2010) Requirement
Cytotoxic activity
Renal activity
Anti-oxidant activity
Uses
To induce the apoptosis in the liver cells through the mechanism action of the extraction process.
To treat kidney stones and its ability to erode renal stones.
To enhance the defense system especially towards the incidence of degenerative disease.
25
Anti-diabetic activity
Anti-obesity activity
Anti-viral activity
Both fermented and unfermented leaves of Strobilanthes Crispus leaves was able to lower blood glucose level.
It can induced the human body weight from the study of Strobilanthes Crispus extract on lipolysis.
It is found to be an efficiently in virucidal agent.
2.2.3 Chemical and Physical Properties of S trobi lanthes C ris pus
The different types of matter can be distinguished through two components which are physical and chemical properties. The physical properties of the Strobilanthes Crispus plants can be observed or measured without the changing
of composition while for chemical properties is the potential of the plants to undergo some chemical change or reaction by purity of its composition. Table 2.2 below shows the chemical and physical properties of the plants, it is very extensive to study the properties of the Strobilanthes Crispus plant in order to achieve the quality samples during the extraction process.
26
Table 2.2: The chemical and physical properties of the Strobilanthes Crispus (Ghasemzadeh, Jaafar, & Rahmat, 2015) Properties
Value
Low Temperature ( oC)
21oC-23oC
High Temperature ( oC)
32oC-33oC
Height above sea level (m)
3m-56m
Humidity (%)
76%-81%
Light intensity (umol/m 2/s)
54.6
Moisture
66.3%
Potassium (%)
51%
Calcium (%)
24%
Sodium (%)
13%
Iron (%)
1%
Phosphorus
1%
2.3 Diabetes Mellitus Disease
Diabetes mellitus is a group of metabolic diseases in which there are high blood sugar levels over a continuous period and commonly refer red to as diabetes. Frequent urination, increased thirst and increased hunger are the symptoms of high blood sugar. Diabetes can cause many complication to human health if left untreated. The long-term problems include stroke, cardiovascular disease, chronic kidney failure, and damage to the eyes. (Wikipedia, 2016) Diabetes is a disorder of metabolism that the body use to digest food for energy and it will breaks down to carbohydrates, sugars and starches found in many meals into glucose. Diabetes evolves when the body does not make enough insulin or is not capable to use insulin efficiently. Insulin is made in the pancreas and contains clusters of cells called islets. Beta cells within the islets make insulin and release it into the bloodstream. 27
Fig ure 2.4: The islets contain beta cell cells in human organ (NIH, 2014)
The assessment of the anti-hyperglycemic properties were investigated and the etnopharmacological value of the Strobilanthes Crispus plant such as the development of nutraceutical Strobilanthes Crispus herbal tea with condition fermented and unfermented. The hot water extract from the fermented and unfermented
reduced blood glucose and also improve lipid profile shown by the
result of the experiment which is tested on rats. The antihyperglycemic and antilipidemic properties are contributed by the presence of antioxidant and polyphenol content in the extracts. (Abu Bakar, Othman, & R Asmah, 2006) In addition, rats are used in lab research because of their physical similarities to human. Rats seem to analyze consequences in a similar way that humans and other primates do and this phenomenon is called metacognition. These similarities include physical, psychological, behavioral and social characteristics. (Kalish, 2010)
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2.4 Extraction Method
Extracting the bioactive compound from plant materials is the most appropriate
and
standard
method
used
in
different
sectors
such
as
pharmaceutical, food and chemical industries. Eventhough a lot of new methods have been established along with conventional method, there is no method that can be classified as standard for extracting bioactive compounds from plants. There are two extraction methods which are conventional and non-conventional. The methods depend on the critical input parameters, nature of plant matrix, chemistry of bioactive compounds, and scientific expertise.
2.4.1 Conventional Extraction Method
Conventional extraction method is synthesized in small quantities in nature that had been used several years ago which classified as the classical extraction technique to obtain bioactive compounds. It requires labor intensive and time consuming purification procedures which present as conjugate or mixture in extract.
2.4.1.1 Ultrasonic-Assisted Extraction (UAE)
In ultrasonic assisted extraction, the use of ultrasound is expediting the extraction which is a modified from maceration method. A vial is used to place the plant powder which later placed in an ultrasonic bath. Ultrasound is used to induce a mechanical stress on the cells through the production of cavitation in the sample. The solubilization of metabolites in the solvent is increased by the cellular breakdown and improves the extraction yields. The initial extraction of a small amount of material commonly use this method.
29
Fig ure 2.5: The ultrasonic-assisted extraction (University of British Columbia, 2012)
2.4.1.2 Maceration Processes The plant material cut into small pieces and moderately coarse powder. A closed vessels is used to place the substances and the selected solvent is added in a suitable ratio. Allowed to stand for seven days shaking occasionally and the liquid is strained off. The process is repeated for once or twice with fresh solvent. Finally, by using a mechanical press or a centrifuge, the last residue of extract is pressed out of the plant particles. Both initial and bulk extraction is suitable in this method. The main disadvantage of maceration is that the process can be quite time-consuming, taking from a few hours up to several weeks. (Singh, 2012)
Fig ure 2.6: The maceration method of plant extraction (Rahul, 2013)
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2.4.2 Non-Conventional Extraction Method
The non-conventional method was introduced since the conventional method is no longer suitable because of the long extraction time, costly, lot of solvent amount used, low selectivity and thermal decomposition. The production, detection, separation and characterization of bioactive compounds are able to be improved by using this method.
2.4.2.1 Supercritical-Fluid Extraction Supercritical fluids extraction is increasingly demanded in industry replacing organic solvents which is used in industrial extraction because of regulatory and environmental pressures on hydrocarbon and ozone-depleting emissions. Carbon dioxide (CO 2) which is considered as safe for solvent free extraction processes is utilized by most of the available solvent free extraction. By regulating its temperature and pressure, the liquid CO 2 is forced into supercritical state. Next, Supercritical CO 2 has solvent power and extracts predominantly lipophilic and volatile compounds. Gaseous CO 2 returns to CO 2 tank. The new extraction starts with circulating CO2 after a full round.
Fig ure 2.7: The supercritical fluid extraction method (Suzan Mahdi & Altikriti, 2010)
31
2.4.2.2 Pressurized Extraction The extraction cell is loaded with the powdered plant materials, which is placed in an oven. In order to fill the cell, the solvent is then pumped from a reservoir which is heated and pressurized at programmed levels for a set of period time. The cell is flushed with nitrogen gas, and the extract which is automatically filtered, is collected in a flask. To rinse the cell and solubilize the remaining components, a fresh solvents is used. To dry the material, a final purge with nitrogen gas is performed. This method offers a few advantages which are more economical and environmental friendly.
Fig ure 2.8: The Pressurized liquid extraction (Department of Pharmaceuticals Sciences, 2015)
32
CHAPTER 3
METHODOLOGY
Overview
This chapter discusses about the methodology that used in this study. The extraction process were done in this study was Supercritical Fluid Extraction (SFE) by using Tubular Batch Reactor. The preparation sample of the Strobilanthes Crispus, extraction process, and the sample analysis were discussed in this
chapter. The bioactive compound of the leaves were analyzed by using UltravioletVisible spectrophotometer (UV-Vis) while Response Surface Methodology (RSM) was used to get the optimal yield value of the leaves.
The outline of the methodology was plotted as shown in figure 3.1 before run the experiment to make sure the effectiveness of the study. Sam le collection of Strobilanthes Cris us leaves The leaves were cleaned and dried at 40 oC
The cleaned dried leaves were grounded and stored Preparation the volume of solvent (ethanol) by using Ideal-Gas Law formula PV=nRT
Ethanol Tem erature: 243 oC Pressure: 62.18atm
Supercritical Fluid Extraction (SFE) of bioactive compounds using Tubular Batch Reactor
Tested with different parameters
Type of solvents:
Ethanol
Temperature:
Extraction time:
300oC and o
400 C
15min and 25min
Solid Loading
30% and 50%
Analysis of bioactive compounds exist by using Ultraviolet-Visible Spectrophotometer (UV-Vis)
The experimental data obtained by using Response Surface Methodology (RSM)
Fig ure 3.1: The outline of the methodology in this studies 34
3.2 Methodology
The details of the experiment from this study were briefly explained in this section.
3.2.1 Sample Collection and Preparation
The Strobilanthes Crispus leaves can be found on riverbanks or abandoned fields. The leaves of the plants collected from the area around Alor Gajah, Melaka. The leaves were separated from the stalks and then completely washed with tap water. The leaves were rinsed with distilled water that had been supplied and dried in ventilated drying oven at 40 oC for 24 hours. The dried leaves were grounded in a dry mill blender to form a powder in order to increase the surface area of sample. The grinded fresh leaves were directly stored in air-tight bottles and dark place prior to extraction process. Figure 3.2 below shows the powder of the Strobilanthes Crispus leaves.
Fig ure 3.2: The powder form of the Strobilanthes Crispus leaves
35
3.2.2 Solvent preparation
In preparation of supercritical fluid, the volume of the solvent used in the reactor to extract Strobilanthes Crispus must be considered Ethanol was widely used in industrial and it is the second most important solvent after water. In this extraction process, ethanol was used as supercritical fluid solvent. By referring figure 3.3, the volume of the solvent used was calculated by using Ideal-Gas Law formula. In this case, the temperature and pressure of the solvent calculated must be above their supercritical state. Where the critical state temperature and pressure for ethanol are 243 oC and 62.18atm respectively. (Material Safety Data Sheet Ethanol, 2013)
Fig ure 3.3: The ideal gas law formula (Corbett, 2014)
36
3.2.3 Sample Extraction
These extraction methods are based on Supercritical Fluid Extraction (SFE) merthod. Since SFE equipment is not available at the laboratory, so Tubular Batch Reactor was designed to replace SFE equipment to extract anti-oxidants compound which is bioactive compound in Strobilanthes Crispus. Tubular batch reactor was designed by using 15mL bulkhead union (3/4” OD) with two steel caps (3/4” OD). This reactor was made of stainless steel 316 because it can adapt with high temperature and pressure. One end of the bulkhead union was closed with one steel cap. Then, Strobilanthes Crispus sample of 30% and 50% solid loading and the solvent was placed in the bulkhead union and closed with another steel cap at the other end of bulkhead union. Both end of bulkhead union must be closed tightly to avoid the reactor exploded while heating in the furnace, due to its high temperature and pressure.
Once the reactor filled with sample and solvent, the tubular batch reactor was placed in the furnace with different extraction time 15 and 25 minutes. The temperature of the furnace was set to 300 OC and 400 OC for each extraction time samples to convert the solvent to its supercritical fluid. The extracted sample was cooled in water bath immediately to avoid another reaction take placed in the reactor that can lead to the error of the data. After the reactor was cooled, the extractant was transfer to a universal bottle. The cap of the reactor does not open until the reactor cooled to avoid the sample burst out due to high pressure. Figure 3.4 and 3.5 below shows the tubular batch reactor in order to extract the bioactive compound from the leaves.
37
Fig ure 3.4: The 15ml bulkhead union of the tubular batch reactor
Fig ure 3.5: The steel cap of the tubular batch reactor
3.2.4 Sample Analysis
The
extracted
leaves
were
analyzed
by
using
Ultraviolet-Visible
Spectrophotometer (UV-Vis) to identify the bioactive compound in Strobilanthes Crispus. This equipment was left ON for 30 to 60 minutes before proceeding. At first, a cuvette is filled with quercetin and another cuvette is filled with absolute ethanol solvent. Before cuvette contain quercetin is placed in sample holder and cuvette contain ethanol is placed into reference holder compartment to run, the sides of the cuvette was wiped with soft tissues. This wavelength scan is run in order to determine lambdamax for the extracts. Then, both cuvettes filled with
38
blanks ethanol is scanned in order to get zero value of the instrument. To run the photometric scan, the cuvette contain chloroform placed in sample holder is took out and replace with the cuvette filled with standard solution which are 1.00ppm, 50.00ppm, 100.00ppm, 250.00ppm, and 500.00ppm respectively. The other blank is left in the reference holder for subsequent runs.
Next, each sample of 30 extracted was diluted with absolute ethanol with 0.1g sample in 10ml absolute ethanol. Photometric scan was conducted to get the absorbance results and the standard calibration graph was generated automatically by using the equipment. The purpose of photometric scan was to determine the concentration (single component) of the extract from the standard calibration graph.
3.2.5 Sample Data
Before the experimental procedure was run, basically Response Surface Methodology (RSM) was designated for the whole process. RSM is a collection of statistical and mathematical methods that are useful for the modelling and analyzing engineering problems. In this technique, the main objective is to optimize the response surface that is influenced by various parameters such as solid loading, temperature, and extraction time. Response surface also quantifies the relationship between the controllable input parameters and the obtained response surface. In this study, The Box Behnken Design (BBD) was employed for the experimental design chosen with a fraction factorial for three independent variables at three level or for finding out the relationship between the yield of extraction and the three variables. This design will be a first option because relatively few experimental combinations of the variables are acceptable to estimate complex response function. The various process parameters involved in total extraction yield of Strobilanthes Crispus solid loading 30% and 500%, temperature 300 oC and 400 oC,
39
extraction time 15 and 25 minutes with the level code as -1 (low) and +1 (high) respectively. Table 3.1 below shows the actual experimental parameters and the coded experimental BBD level used.
Table 3.1: The actual experimental and the coded level used. Level
Coded
Parameters
Factor
Low (-1)
High (+1)
Solid Loading (%)
A
30
50
Temperature (C)
B
300
400
Extraction Time (min)
C
15
25
RSM was applied to the experimental data using statistical software, Minitab17. The statistical terms and their definitions used in the software are well defined elsewhere. The linear and second order polynomials were fitted to the experimental data to obtain the regression equations. The sequential F-test, lack of fit test and other measures were used in selecting the best model. The total extraction yield of Strobilanthes Crispus was determined by using the equation below.
% = ( ) 100 Where Yextract is percentage of extraction yield, m extract is the crude extract mass (g) and m feed is the feed mass (g). The quadratic polynomial equation will be as the response surface with the equation below.
Yield = A0 + A1X1 + A2X2 + A3X3 + A4X1X2 +A5X2X3 + A6X1X3 + A7X12 + A8X22 + A9X32 Where, A0 is constant, A 1, A2, and A 3 is linear coefficients, A 4, A5, and A6 is cross product coefficients, A 7, A8, and A 9 is quadratic coefficients respectively. For this three-level or three-factorial Box-Behnken experimental design, a total of 30 40
experimental runs are needed. The RSM is carried out from the design of experiment (DOE). The parameters selected are inserted in Box-Behnken Design. Then, the number of replicates was changed to two in order to get a better spherical design and the value of the factors are inserted based on the table above. After that, the different value of each factors will appeared on the new sheet along with 30 standard runs. Figure below indicates the range of parameters that was produced by Box-Behnken design using Minitab17.
Fig ure 3.6: The range of parameters By Boh-Behnken design using Minitab17
41
CHAPTER 4
EXPECTED RESULT
42
CHAPTER 5
CONCLUSION
43
RESEARCH PLAN
44
REFERENCES
Abu Bakar, M., Othman, F., & R Asmah. (2006). Effects of Strobilanthes crispus Tea Aqueous Extracts on Glucose and Lipid Profile in Normal and Streptozotocin-Induced Hyperglycemic Rats. ResearchGate, 7-12. Afrizal. (2008). ANALYTICAL, BIOACTIVITY AND STABILITY STUDIES ON STROBILANTHES CRISPUS L. BREMEK AND SONCHUS ARVENSIS L. EXTRACTS. ANALYTICAL, BIOACTIVITY AND STABILITY STUDIES ON STROBILANTHES CRISPUS L. BREMEK AND SONCHUS ARVENSIS L. EXTRACTS AFRIZAL UNIVERSITI SAINS MALAYSIA , 34-35. Afrizal Itam, Ismail, Z., & Abdul Majid, A. (2007). In Vitro studies of calcium oxalate crystal growth inhibition of Strobilanthes Crispus extract. Malaysian Journal of Pharmaceutical Science , 96-97. Asmah Rahmat, & Mandana, B. (2009). IChemE. Supercritical carbon dioxide extraction of bioactive flavonoid from Strobilanthes crispus (Pecah Kaca) , 3. Bakar, M. F. (2005). Effects of Strobzlanthes Crispus crued and tea in streptozotocin included hyperglycemic rats. Universiti Putra Malaysia , 2425. Corbett, A. (2014). Chapter 12 Molecular Composition of gases . Retrieved from slideplayer.com: http://slideplayer.com/slide/266930/ Department of Pharmaceuticals Sciences . (2015). Retrieved from Universiti Basel: https://pharma.unibas.ch/home/
Ghasemzadeh, A., Jaafar, h., & Rahmat, A. (2015). Phtochemical constituents and biological activities of different extracts of Strobilanthes Crispus Bremek Leaves grown in different locations of Malaysia. Research Article, 3-4. GlobinMed . (2010). Retrieved from www.globinmed.com: http://www.globinmed.com/index.php?option=com_content&view=article&i d=83483:strobilanthes-crispus&catid=721:s IDF . (2015). Retrieved from International Diabeted Federation Wstern PAaific: http://www.idf.org/membership/wp/malaysia
Kalish, M. (2010). Similar Characteristics in Rats & Humans . Retrieved from eHow: http://www.ehow.com/info_8413774_similar-characteristics-ratshumans.html 45
Leo M, L Nollet, & Toldra, F. (2012). Handbook of Analysis of Active Compounds in Functional Food. eBook , 415-416. Leo, M., Nollet, L., & Toldra, F. (2012). Handbook of Analysis of Active Compounds in Functional Foods. eBook , 415-416. Lin, K. K. (2013). Strobilanthes crispus Tea - Teh Pecah Beling. Retrieved from http://www.sabahsnakegrassherb.com/: http://www.sabahsnakegrassherb.com/2013/03/strobilanthes-crispus-teateh-pecah.html Liza Md Salleh, Russly Abdul Rahman, Jinap Selamat, Azizah Hamid, & Zaidul, M. (n.d.). Optimization Of Extraction Condition for Supercritical Carbon Dioxide (SC-CO2) Extraction of Strobilanthes Crispus (PEcah Kaca) leaves by Response Surface Methodology. Food Processing and Technology , 3-6. Material Safety Data Sheet Ethanol. (2013, May). Retrieved from www.sciencelab.com: http://www.sciencelab.com/msds.php?msdsId=9923955
NIH. (2014, June). National Institute of Diabetes and Digestive and Kidney Disease. Retrieved from www.niddk.nih.gov: http://www.niddk.nih.gov/health-information/healthtopics/Diabetes/causes-diabetes/Pages/index.aspx NPCR . (2016, September). Retrieved from http://www.omicsonline.org/: http://www.omicsonline.org/scholarly/bioactive-compounds-journalsarticles-ppts-list.php
Rahul, B. (2013, January). Extraction of plant constituents. Retrieved from Slideshare: http://www.slideshare.net/rahulbs89/extraction-of-plantcontituents Singh, J. (2012). Maceration, Percolation and Infusion techniques of extraction of medicinal and aromatic plants. Central Institute of Medicinal and Aromatic Plants (CIMAP) , 7-8. Suzan Mahdi, & Altikriti, Y. (2010). Extraction of Natural Products. Retrieved from Biologiskt Aktiva Naturprodukter: http://www.fkog.uu.se/course/a/biolakt/biolakt-archive/BiolAkt%2020102/StudentpresentationerHT2010%20(kopia)/BiolAktHT2010_ExtraktionNat Prod_Yassir_Suzan/Extraction%20of%20natural%20products_files/Page4 70.htm University of British Columbia. (2012, March). Retrieved from wiki.ubc.ca: http://wiki.ubc.ca/Course:FNH200/2011w_Team20_Vanilla
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Wikipedia. (2009, February). Retrieved from https://en.wikipedia.org/wiki/Strobilanthes_crispa
Wikipedia. (2016, February). Diabetes Mellitus . Retrieved from wikipedia: https://en.wikipedia.org/wiki/Diabetes_mellitus Yaacob, N. S., Hamzah, N., Nik Mohamed Kamal, N., Zainal Abidin, S., Choon Sheen Lai, Viswwswaran Navaratnam, & Norazmi, M. (2010). Anticancer activity of a sub-fraction of dichloromethane extract of Strobilanthes Crispus on human breast and prostare cancer cells in vitro. BioMed Central , 10-42.
47
APPENDICES
Table 1: Project cost estimation for Final Year Project 1 Materials and Equipments
Quantity
Strobilanthes Crispus leaves
1kg
Price
RM10
Ethanol
1500mL
RM99
0.1% Formic acid
3500mL
Rm139
Acetonitrile
3500mL
RM139
15 mL ¾” OD Bulkhead Union
1
RM212.40
¾” OD Cap
2
RM68.60
TOTAL
RM668.00
48
Chemicals and Materials
Table 2: Table of chemicals/materials and apparatus Method
Chemicals / Materials
Apparatus
Scissors
Sample Collections and Preparations
Solvent Preparations
Strobilanthes Crispus
leaves
Tap Water
Glove
Grinder
Air tight Bottles
10 mL of measuring cylinder
Ethanol
Beaker
Union (Swagelok)
Ethanol
Sample Extraction
Strobilanthes Crispus
samples
Tap Water
Extracted Strobilanthes
0.1% formic acid
Acetonitrile
Distilled water
¾” OD Cap (Swagelok)
Furnace Thongs
Universal Bottles
High Performance Liquid
Crispus leaves Sample Analysis
15 mL ¾” OD Bulkhead
Chromatography (HPLC) Syringe
HPLC Waste Bottles
Gloves
49
Solvent Preparations
Ethanol Molecular Weight, Mr
= 58.08 g/mol
Critical Temperature, T critical
= 516.15 K
Critical Pressure, P critical
= 6.30 MPa (62.18 atm)
Density, ρ
= 789 g/L
Gas Constants, R
= 0.082057 L atm/mol.K
Volume of Reactor, V reactor
= 0.015 L
Table 3: Solvent Preparation for Ethanol Temperature, oC
Volume of Solvent, mL
300
1.61
700
1.61
Solid Loading, % 50 100 50 100
Mass of Sample, g 0.64 1.27 0.35 0.70
Example of Calculation
Ethanol Molecular Weight, Mr
= 58.08 g/mol
Critical Temperature, T critical
= 516.15 K
Critical Pressure, P critical
= 6.30 MPa (62.18 atm)
Density, ρ
= 789 g/L
Gas Constants, R
= 0.082057 L atm/mol.K
Volume of Reactor, V reactor
= 0.015 L
50
