ABSTRACT
Phytochemical investigation of the leaves of Peucedanum winkleri. H. Wolff, revealed the presence of carbohydrates, free reducing sugar, cardiac glycoside, saponins, steroids, flavanoids, alkaloids, tannins and triterpenes. The extracts were screened for their antimicrobial activity against Staphylococcus aureus, Methicillin resistant Staphylococcus aureus, Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Corynebacterium ulcerans, Klebsiella pneumoniae, Streptococcus pyogenes, Candida albicans, Candida tropicalis, Candida krusei and Proteus mirabilis. The result indicated that the extracts inhibited the growth of one or more test pathogens. The ethyl acetate extract showed a broad spectrum of antimicrobial activity.4-(4-Chlorophenyl)-3-cyclohexyl-1-methyl-6-(2-naphthoyl)-1,2,3,4-tetrahydro-1,2,4,5-Tetrazine was isolated from the ethyl acetate extract of the plant leaves by means of Vacuum liquid chromatography (VLC), Thin layer chromatography (TLC) and Preparative thin layer chromatography (PTLC) using a solvent mixture of 1:1 v/v petroleum-ether and ethyl acetate. The stucture was established mainly by MS, IR, 1H and 13C NMR including DEPT spectroscopy. Also the isolated compound was able to inhibit the growths of some of the microorganisms used for the studies. The result of the study justified the use of the plant against several infectious diseases.
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TABLE OF CONTENTS
1.0 INTRODUCTION ………………………………………………………………………………..1
1.0.1 Background of the study ……………………………………………………………………..1
1.1 Alkaloids ……………………………………………………………………………………………………. 6
1.1.1 Phenolic compounds…………………………………………………………………………………….6
1.1.2 Steroids …………………………………………………………………………………………………….7
1.1.3 Glycosides …………………………………………………………………………………………………7
1.1.4 Terpenoids ………………………………………………………………………………………….7
1.1.5 Tannins …………………………………………………………………………………………………… 8
1.1.6 Flavonoids …………………………………………………………………………………………………9
1.1.7 Saponins …………………………………………………………………………………………………….9
1.2 Historially important natural products ……………………………………………………….10
1.2.1 Morphine …………………………………………………………………………………………………11
1.2.2 Quinine …………………………………………………………………………………………………..11
1.2.3 Penicilin …………………………………………………………………………………………………..11
1.2.4 Vinblastine and Vincristine ………………………………………………………………………..12
1.2.5 Pacilitaxel ………………………………………………………………………………………………..12
1.3 Importance of natural products to drug discovery ………………………………………12
1.4 Aims and objectives of the research ……………………………………………………………14
1.4.1 Aims ……………………………………………………………………………………………………… 14
1.4.2 Objectives ……………………………………………………………………………………………… 14
1.4.3 Justification of the research ……………………………………………………………………….14
1.4.4 Scope and limitation of the research ………………………………………………………….. 15
2.0 LITERATURE REVIEW ………………………………………………………………………….16
2.1 The Apiacaea (alt. Umbelliferae) ………………………………………………………………..16
2.2 Some important members of the Apiacea, their medicinal
effects and compounds isolated from them ……………………………………………….20
2.3 Some important members of the Peucedanum genus, their medicinal
effects and compounds isolated from them ……………………………………………….27
2.4 Pencedanum Winkleri, H. Wolff species ……………………………………………………….41
2.5 Bacterial agents ………………………………………………………………………………………….42
2.6 Phytochemical screening …………………………………………………………………………….45
2.7 Isolation of constituents ……………………………………………………………………………..46
2.8 Identification and characterization of isolated constituent …………………………..46
2.9 Biological screening…………………………………………………………………………………… 47
2.9.1 Antimicrobial screening………………………………………………………………………………47
3.0 MATERIALS AND METHODS …………………………………………………………………49
3.1 Materials used for extraction and phytochemical test …………………………………..49
3.1.1 Reagents used for extraction ……………………………………………………………………… 49
3.1.2 Reagents used for phytochemical test …………………………………………………………..49
3.2 Materials used for antimicrobial test …………………………………………………………..51
3.2.1 Microorganisms used for antimicrobial test ………………………………………………….52
3.3 Materials for vacuum liquid chromatography (VLC) ………………………………….53
3.3.1 Materials for thin layer chromatography (TLC) …………………………………………….53
3.3.2 Instrumentation and general techniques ……………………………………………………….53
3.4 Sample collection, identification and preparation ………………………………………..54
3.5 Extraction of plant material ……………………………………………………………………….54
3.6 Phytochemical screening of plant material ………………………………………………….55
3.6.1 Test for carbohydrates ……………………………………………………………………………….55
3.6.2 Test for cardiac glycosides …………………………………………………………………………55
3.6.3 Test for Anthroquinones derivatives …………………………………………………………….56
3.6.4 Test for saponins ………………………………………………………………………………………..57
3.6.5 Test for steroids and triterpenes ……………………………………………………………………57
3.6.6 Test for flavanoids ………………………………………………………………………………………57
3.6.7 Test for tannins …………………………………………………………………………………………..57
3.6.8 Test for Alkaloids ……………………………………………………………………………………….58
3.7 Antimicrobial screening of crude methanol extract ……………………………………….58
3.7.1 Minimun inhibition concentration (MIC) of crude methanol extract ………………….59
3.7.2 Minimun bacteriocidal/fungicidal concentration (MBC/MFC) of
crude methanol extract …………………………………………………………………………………60
3.8 Vacuum liquid chromatography (VLC) ………………………………………………………..61
3.9 Thin layer chromatography (TLC) ……………………………………………………………….61
3.10 Spectroscopic measurements ……………………………………………………………………….62
4.0 RESULTS/DATA PRESENTATION AND ANALYSIS ……………………………….63
5.0 DISCUSSION ………………………………………………………………………………………………85
6.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS ……………………….93
REFERENCES ………………………………………………………………………………………………..95
CHAPTER ONE
.0 INTRODUCTION
1.0.1 BACKGROUND OF THE STUDY
Medicinal plants have always been associated with cultural behaviours and traditional knowledge. The renaissance of interest in plant products has been stimulated by the use of plant extracts in chronic conditions for which conventional drugs is perceived to offer very little specificity in its target (Rhiouani et al., 1999). This has resulted in the use of large number of medicinal plants with curative properties to treat various diseases (Verpoorte, 1998). Nearly 80% of the world‘s population relies on traditional medicines for primary health care, most of which involve the use of plant extracts (Akindele and Adeyemi, 2007).
The blind dependence on synthetics is over and people are returning to the naturals with hope of safety and security. Also, the development of adverse effects and high microbial resistance to the chemically synthesized drugs, has forced men into ethnopharmacognosy. More so, in our local situation, degree of ignorance and illiteracy had forced many to abandon or neglect pharmaceutically formulated drugs in favour of locally prepared herbal remedies coupled with the fact that pharmaceutical products are increasingly being faked. Thus, the herbal products today symbolise safety in contrast to the synthetics that are regarded as unsafe to human and environment (Joy et al., 2001). Herbs are staging a comeback, herbal ‗renaissance‘ is happening all over the globe and people returning to the naturals with hope of safety and security. By and large, the public is gradually drifting towards acceptance and usage of herbal preparations.
In Africa, traditional healers and remedies made from plants play important role in the health of millions of people (Adotey et al., 2012). The users of these remedies, found literally thousands of phytochemicals from plants as safe and broadly effective alternatives with less adverse effects. The Pharmacognosy Society of Nigeria supports the acceptance of herbal remedies or treatment of ethnomedicinal practice along with conventional orthodox health care system (Ndukwe, 2004). This is largely due to the fact that plants used for traditional medicine contain a wide range of substances that can be used to treat chronic as well as infectious diseases (Duraipandiyan et al., 2006).
Biological organisms particularly plants produce two distinctly different types of chemical products. The first type, primary metabolites, which consists of compounds such as sugars and proteins that are common to most organisms and are essential for functional metabolism. Secondary metabolites, on the other hand, are chemicals unique to a single species or related group of organisms. Not until the 1990s that scientists fully realize that these secondary metabolites are more than mere leftovers from an organisms metabolic processes. These chemicals can function as communications tools, defense mechanisms or sensory devices. The biological activity of these chemicals is beneficial to the organism that produces them, but it is often harmful to other species, including humans (Swerdlow, 2000). This toxicity can adversely affect the functions of the entire human body or only a specific biological process, such as the growth of cancer cells (Yoder, 2005). Also, many beneficial biological activity such as anticancer, antimicrobial, antioxidant, antidiarrheal, analgesic and wound healing activity of plants have been reported. In this way, certain foreign, naturally produced chemicals can act as powerful drugs when administered at the proper concentration (Yoder, 2005).
Natural products are important in health care. They can be used as starting materials for semisynthetic drugs. The main examples are plant steroids, which led to the manufacture of oral contraceptives and other steroidal hormones. Today, almost every pharmacological class of drugs contains a natural product or natural product analog (Eba, 2005). Similarly, it represents an excellent resource for the identification of new lead structures (Newman and Cragg, 2007).
It is estimated that 25% of all prescriptions dispensed in the USA contained a plant extract or active ingredients derived from plants. It is also estimated that 74% of the 119 currently most important drugs contain active ingredients from plants used in traditional medicine for health care (Farnsworth et al., 1985), these traditional medicines are primarily plant-based (De-Pascual-Teresa et al., 1996). Another study of the most prescribed drugs in the USA indicated that a majority contained either a natural product or a natural product was used in the synthesis or design of the drug (Wakelin, 1986). Similarly, about 121 drugs prescribed in USA today come from natural sources, 90 of which come either directly or indirectly from plant sources (Benowitz, 1996). Forty-seven percent of the anticancer drugs in the market come from natural products or natural product mimics (Newman and Cragg, 2007).
Tropical and subtropical Africa contain between 40,000 – 45,000 species of plants with a potential for development and out of which 5,000 species are used medicinally (Van Wyk, 2008). Still there is a paradox that in spite of this huge potential and diversity, the African continent has only contributed 83 out of the 1100 classic drugs globally (Van Wyk, 2008).
It is a fact that traditional systems of medicine have become a topic of global importance. Although modern medicine may be available in many developed countries, people are still turning to alternative or complementary therapies including medicinal herbs. Yet, few plant species that provide medicinal herbs have been scientifically evaluated for their possible medical applications (Adotey et al., 2012). Herbs had been priced for their medicinal and therapeutic effects, flavouring and aromatic qualities for centuries (Joy et al., 2001). Similarly, the herbal drugs contain many chemical compounds naturally. In many cases, traditional healers claim the good benefit of certain natural or herbal products. But, it is only a few herbs, their extracts and active ingredients and also, the preperation containing them that their safety and efficacy data are available (Adotey et al., 2012). No dought, plants extracts either as pure compounds or as standardized extracts, provide unlimited opportunities for new drug discoveries because of the unmatched availability of chemical diversity (Cosa et al., 2006). However, it is a common practice among chemists that the content of any unlabelled bottle in the laboratory should be discarded. In this way, a truelly practicing chemist should dissociate himself or herself from uncharacterized drug no matter how effective the drug may be (Ndukwe, 2004). As such, It is therefore essential to separate out those compounds which are responsible for therapeutic effect and characterise them. They are called active constituents or principles.
Phytochemical screening is very important in identifying new sources of therapeutically and industrially important compounds such as alkaloids, flavonoids, phenolic compounds, saponins, steroids, tannins, terpenoids etc (Akindele and Adeyemi, 2007). Also, Isolation is a part of natural product chemistry, through which it is possible to separate different components and biologically active ones which can be incorporated as
ingredients in the modern system of medicine. Modern medicine has largely confined itself to the isolation or synthesis of single active ingredient for the treatment of spercific disease (Shoge, 2010). Chromatographic techniques are widely used for the separation, isolation and purification of chemical constituents from natural drugs (Devi et al., 2012).
Clinical trials is an essencial aspect of drug discovery. It is necessary to establish and demonstrate the effectiveness of bioactive compounds (Natural drugs) to verify their effeciency. Clinical trials directed towards understanding the pharmacokinetics, bioavailability, efficacy, safety and drug interactions of newly developed bioactive compounds and their formulations (extracts) require a careful evaluation. Clinical trials are carefully planned to safeguard the health of the participants as well as answer specific research questions by evaluating for both immediate and long-term side effects and their outcomes which are measured before the drug is widely applied to patients (Sasidharan et al., 2011). Going by the slogan that every drug is a poison, the poisonous nature of every drug necessitates that its prescription must be followed. Even at that, reports exist where manunfacturing companies withdraw their products from circulation because of adverse reports recieved from dispensers of such drugs (Ndukwe, 2004).
Many plants are chemically very variable depending on the locality where they are found with some of the constituents occurring only at certain seasons of the year (Adelani, 2007). As such, plants synthesize a bewildering variety of phytochemicals but most are derivatives of a few biochemical motifs. Some of them include : Alkaloids, Phenolics.
1.1 Alkaloids
Alkaloids are the largest single class of secondary metabolites known. They are those basic substances which contain one or more nitrogen atoms as part of a ring system that shows marked physiological effect on man and animals (Harbone, 1973). One of the most common biological properties of alkaloids is their toxicity against cells of foreign organisms.. These activities have been widely studied for their potential use in the elimination and reduction of human cancer cell lines (Norobi et al., 1994). Large numbers of drugs are derived from plants containing alkaloids. Many of the earliest isolated compounds with biological activities were named alkaloids due to their ease of isolation. Many alkaloid have marked effects on the central nervous system and this has led to the development of powerful pain killer medications (Kam and Liew, 2002). Caffeine is an alkaloid that provides a mild lift whereas alkaloid in datura causes severe intoxication and even death. Other medicinal uses of alkaloids includes: as an analgesic, antimalarial, antihypertensive, remedy for gout and remedy for cough (Wikipedia).
1.1.1 Phenolic compounds
Phenolics are polyhydroxyl benzene derivatives. They include anthocyanins that give grapes their purple colour. Phytoestrogens from soy and the tannins that give tea its astringency. Natural phenolic compounds play an important role in cancer prevention and treatment. Their various bioactivities are responsible for their chemopreventive propertties (Huang et al., 2010).
1.1.2 Steroids
Steroids are important drugs used as hypotensives, cardiac depressants, sedatives and anti-dysentric agents (Tijjani et al., 2011). Okwu (2001) reported the relationship of steroidal compounds with various anabolic hormones including sex hormones. Steroids have equally been reported to have antimicrobial activity (Quinlan et al., 2000) and comfirmed to have antiviral properties (Neumann et al, 2004).
1.1.3 Glycosides
This is a general word that embraces all the many and varied combinations of sugars and aglycones or genins. The usual link between the sugar and aglycone is an oxygen linkage between the reducing group of a sugar and alcoholic or phenolic hydroxyl group of the aglycone (Isah, 2007). The aglycone is a molecule that is bioactive in its free form but inert until the glycoside bond is broken by water or enzymes. An example is the cyanoglycosides in cherry pits that release toxins only when bitten by a herbivore (Shoge, 2010).
1.1.4 Terpenoids
They are built up from terpene building blocks. Each terpene consists of two paired isoprene units. Based on the number of isoprene units, the names monoterpenes, sesquiterpenes, diterpenes and triterpenes are derived. Monoterpenes are responsible for fragrance of rose.
Many triterpenes and their aglycone have been reported to have varied uses as antiulcerogenic, anti-inflammatory fibrinolytic, antipyretic, analgesic and anti-edmatous in action (Ndukwe et al., 2005).
1.1.5 Tannins
This is an old term denoting substances present in plant extracts that were able to combine with protein of animal hides, prevent their putrefication, and convert them into leather. This definition excluded simpler phenolic substances present with tannins such as gallic acid , catechin and chlorogenic acid etc. i.e, They have tanning property. It has also been claim to have some protective values against toxins when taken orally. They are equally claimed to have anti-viral and anti-tumor properties (Harbone and Boxter, 1993). Tannins is an important plant metabolite because of its remarkable uses therapeutically to strengthen a weakened heart (Isah, 2007). Similarly, tannins are reported to have various physiological effects like anti-irritant, antisecretolytic, antiphlogistic, antimicrobial and antiparasitic effects. Phytotherapeutically tannin-containing plants are used to treat nonspecific diarrhoea, inflammations of mouth and throat and slightly injured skins (Westendarp, 2006; Trease and Evans, 2000).
Two groups of tannins are usually recognized. They are hydrolysable and non hydrolysable tannins. Hydrolysable tannins are of pharmacological importance because of their antiviral and antitoxic properties. Non hydrolysable tannins have been used in medicine to aid the healing of wounds and burns because of their antiseptic properties. Catechin is an example of non hydrolysable tannins.
1.1.6 Flavanoids
Flavonoids comprise a large group of plants secondary metabolites characterized by a diphenylpropane structure (C6-C3-C6). They are widely distributed throughout the plant kingdom and are commonly found in fruits, vegetables and certain beverages. Flavonoids are pigments that are largely responsible for colour of many fruits, flowers and vegetables. They provide many health–promoting benefit. They act as antihistamine, which is useful in reducing allergy sympton, and help to reduce inflammations associated with various forms of arthritis (Guardia et al., 2001)
Numerous preclinical and some clinical studies suggest that flavonoids have potentials for the prevention and treatment of several diseases. Some epidemiological studies support a protective role of diets rich in foods with flavonoids and a reduced risk of developing cancer and cardiovascular diseases (Lopez-Lizaro, 2009). Preclinical in vitro and in vivo investigations have shown plausible mechanisms by which flavonoids may control cancer and cardiovascular protection (Middleton et al., 2000). In addition to their preventive potential, certain flavonoids may be useful in the treatment of several diseases. Some evidence supporting the therapeutic potential of flavonoids comes from the study of plants used in traditional medicine to treat a wide range of diseases, which has shown that flavonoids are common bioactive constituents of these plants (Middleton et al., 2000; Ren et al., 2003; Wang, 2000; Lopez-Lizaro, 2002).
1.1.7 Saponins
Saponin has been reported to have anti microbial effects (Mahato et al., 1988, 1992) and could serve as precusors of steroidal substances with a wide range of physiological
activities (Madusolomo et al., 1999). Many saponins and their aglycone have been reported to have varied uses as antiulcerogenic, anti-inflammatory fibrinolytic, antipyretic, analgesic and anti-edmatous in action (Ndukwe et al., 2005). Just et al., (1998) revealed the inhibitory effect of saponins on inflammed cells.
People have been exploring nature, particularly plants, in search of new drugs with healing powers. The search for new drugs which are plant-derived has been receiving renewed interests among researchers globally in view of discovering new drugs that possess potency to combat the menace of drug resistant pathogenic microorganisms, antitumor and anticancer agents (Mirza, 2007; Pimenta et al., 2003). Also, the increased incidence of diseases for which there is yet an effective remedy for. Diseases like tuberculosis, pneumonia, typhoid fever, rheumatic fever and meningitis (Greenwood et al., 1992) still pose a major challenge to modern chemotherapeutic agents. In the constant effort to improve the efficacy of orthodox medicine, researchers are increasingly turning their attention to the chemistry of natural products which has its roots in the empirical knowledge of ancient medicine and finds its continuity in folk medicine today (Kan, 1995).
1.2 Historically Important Natural Products
Some well known and important drugs have originated from natural sources. A few examples are given to indicate what an important impact these drugs have had on medical treatment and disease control. Of note is the fact that these drugs were isolated from natural sources many of which have been used by various cultures throughout history.Some of them include: morphine, quinine, etc.
1.2.1 Morphine
Morphine was first isolated from the plant opium poppy (Papaver somniferum) in 1861 (Cragg and Newmann, 2001). In ancient Mesopotamia the oils of P. somniferum were used as an analgesic (Cragg and Newmann, 2001). The discovery and isolation of morphine led to an increased interest in alkaloid chemistry and resulted in the development of other analgesic agents (Cragg and Newmann, 2001). Morphine was the first commercially available pure natural product, marketed in 1826 (Newmann et al., 2000).
1.2.2 Quinine
Malaria has been and continues to be a problem in many areas around the world including Nigeria. The native Amerindians of the Amazon region used the bark of the Cinchona tree to treat malaria (Clark, 1996). Quinine, the active component of Cinchona bark, was isolated in 1820 from C. officinalis (Cragg, 2002). Quinine was the first effective anti-malarial drug to be isolated (Phillipson, 2001). Other anti-malarial drugs such as chloroquine and mefloquine were synthetically modified version of quinine (Guza, 2004).
1.2.3 Penicillin
Penicillin was first discovered in 1929 by Fleming from the fungus, Penicillium notatum (Cragg and Newmann, 2001). This discovery was important to the development of antibiotics and changed medicine forever (Clark, 1996). The discovery of this revolutionary drug from a natural source prompted the investigation of nature for other novel compounds (Cragg, 2002).
1.2.4 Vinblastine and Vincristine
The plant Catharanthus roseus, commonly known as the Madagascar periwinkle, was used in some cultures as a folk remedy to treat diabetes (Cragg and Newmann, 2001). However, when C. roseus was evaluated for hypoglycemic compounds but no such compounds were found (Clark, 1996). Instead, the results led to the hypothesis that the plant extract contained a compound potentially useful for the treatment of cancer (Clark, 1996). Upon further investigation of C. roseus for anti-cancer agents, vinblastine and vincristine were isolated in 1954 (Noble, 1990). These drugs were developed by Eli Lily and are important agents in the treatment of cancer (Guza, 2004).
1.2.5 Paclitaxel
Random collection of plants by the United States Department of Agriculture (USDA) for the National Cancer Institute (NCI) yielded an extract with anti-cancer activity (Cragg and Newmann, 2001). The extract was from the Pacific yew tree (Taxus brevifolia) and the active compound, paclitaxel, was isolated in 1969 (Cragg, 2002). Several Native American tribes use various parts of Taxus trees for the treatment of a wide range of non-cancerous ailments notatum (Cragg and Newmann, 2001). Paclitaxel is an important anti-cancer drug in use today (Guza, 2004).
1.3 Importance of natural products to drug discovery
For many decades, synthetic chemicals as drugs have been effective in the treatment of most diseases. The pharmaceutical industry has synthesized over 3 million new chemicals in their effort to produce new drugs (Eba, 2005). Despite their successes in
developing drugs to treat or cure many diseases, the treatment of certain diseases such as cancer, AIDS, heart disease and diabetes has not been a complete success due to the complexity of these diseases (Eba, 2005).
Over the centuries, people have been living in close association with the environment and relying on its flora and fauna as a source of food and medicine. As a result, many societies have their own rich plant pharmacopeias. In developing countries, due to economic factors, nearly 80% of the population still depends on the use of plant extracts as a source of medicine (Akindele and Adeyemi, 2007).
Natural products also play important roles in the health care system in developed countries. The isolation of the analgesic morphine from the opium poppy (Papaver somniferum) in 1816 led to the development of many highly effective pain relievers (Benyhe, 1994). The discovery of penicillin from the filamentous fungus Penicillium notatum by Fleming in 1929 had a great impact on the investigation of nature as a source of new bioactive agents (Bennett and Chung, 2001).
Natural products can also be used as starting materials for semisynthetic drugs. The main examples are plant steroids, which led to the manufacture of oral contraceptives and other steroidal hormones. Today, almost every pharmacological class of drugs contain a natural product or natural product analog (Eba, 2005). The investigation of higher plants has led to the discovery of many new drugs. So far only a small portion of higher plants has been investigated. In Nigeria, only a few plant species present in the country have been subjected to scientific evaluation for their potential chemoterapeutic values. As such, plants still remain a big reservoir of useful chemical compounds not only as drugs, but also as templates for synthetic analogues (Eba, 2005).
1.4 Aims and objective of the research
1.4.1 Aims
This research is aimed at establishing the medicinal potencials and claims of the plant Peucedanum winkleri H. Wollf and also isolate and characterize some compounds that may be responsible for the claimed ethnomedicinal values using organic analytical techniques.
1.4.2 Objectives
The objectives of this work includes:
i. Collection, proper boatanical identification, drying and grinding of plant material.
ii. Phytochemical screening of the plant material.
iii.Total extraction of ground plant material using methanol and then partitioning of dry methanol extract using petroleum ether, chloroform and ethyl acetate.
iv. Antimicrobial screening of the extracts.
v. Analytical separations and purification involving several chromatographic techniques.
vi. Verification of the purity of the isolated compound(s).
vii. Testing the potency of the isolated compound(s) on some microorganisms.
1.4.3 Justification of the research
The choice of Peucedanum winkleri as the plant of interest in this work is based on its claimed vast ethnomedicinal uses among traditional medicine practitioners in the tropics including Nigeria. It is therefore essential to scietifically study the plant in order to
ascertain its medicinal potentials and also isolate and characterize the medicinally active components of the plant with the help of analytical organic techniques.
1.4.4 Scope and limitation of the research
This research thesis is limited to phytochemical screening, antimicrobial screening, isolation, characterization and structural elucidation. But due to limited laboratory facilities, not all the active components may be completely elucidated. However, this challenge will not in anyway act as constraints towards progress and production of a sound work as relevant and updated materials will be used.
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