ABSTRACT
The preliminary phytochemical screening of the aerial parts of Aeschynomene uniflora was carried out using standard method. The result of the phytochemical screening of crude petroleum ether, chloroform, ethyl acetate and methanol extracts revealed the presence of carbohydrate, cardiac glycoside, tannins, saponins flavonoids and anthraquinone The antimicrobial screening against Corynbacterium ulcerans, Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Candida albicans, Candida stellatoidea and Candida krusei was done using agar well diffusion method. The zone of inhibition of growth of microorganisms ranged from 16 – 18 mm for the petroleum ether extract, 22-27 mm for the chloroform extract, 22-26 mm for ethyl acetate extract and 19-22 mm for the methanol extract. The minimum inhibitory concentrations of the extracts were found to be between 15 mg/ml and 30 mg/ml while the minimum bactericidal /fungicidal concentration were found to be between 15 mg/ml and 60 mg/ml. The purification of the n-hexane fraction of the extract of Aeschynomene uniflora using a column of silica gel gave 3β, 22E-Stigmasta-5, 22-dien-3-ol (C29H48O, 417 g/mol) (stigmasterol). The structure of the isolated compound was established based on spectral data (IR, 1D- and 2D- NMR) and comparison with literature. The compound is reported for the first time in this plant.
8
TABLE OF CONTENTS
Certification ii Declaration ii Dedication iii Acknowledgement iv Abstract v Table of content vi List of Tables xii List of Figures xiii Abbreviation xix 1.0 INTRODUCTION 1 1.1 Biological Background of Medicinal Plants 2 1.2 History of Herbal Medicine 4 1.2.1 Herbal medicine today 5 1.3 Justification of the Research 7 1.4 Scope and Limitation of This Study 8
1.5 Aims and Objective of the Research 8
9
1.5.1 Aim 8 1.5.2 Objectives 8 2.0 LITERATURE REVIEW 9 2.1 Morphological Description of Aeschynomene uniflora 9 2.2 Traditional uses of Aeschynomene uniflora 9 2.3 The Aeschynomene Species 10 2.3.1 Aeschynomene virginica 10 2.3.2 Aeschynomene aspera 12 2.3.3 Aechynomene indica 13 2.3.4 Sesbania grandiflora 14 2.4 Phytoconstituents and Medicinal Uses of the Aeschynomene Species 15 2.4.1 General medicinal properties of the Aeschynomene species 15 2.4.2 Phytochemical properties of the Aechynomene species 16 2.4.3 Antimicrobial properties of the Aeschynomene species 16
2.5 The Fabaceae Family 20
2.5.1 Phytochemical constituents 24
10
2.5.2 Antidiabetic activity 31
2.5.3 Antimicrobial activity 31
2.5.4 Anticonvulsant activity 31 2.5.5 Antibacterial activity 32 2.5.6 Anticancer activity 32 2.5.7 Antioxidant activity 32 2.5.8 Antimicrobial activity 33 2.5.9 Anti-cancer activity 33 3.0 MATERIALS AND METHODS 34 3.1 Materials 34 3.1.1 Reagents 34 3.1.2 Equipment 34 3.1.3 Collection and identification of plant materials 34 3.1.4 Microbial media 35 3.2 Methods 35 3.2.1 Extraction 35 3.2.2 Phytochemical screening 35
11
3.2.3 Antimicrobial Screening 37 3.2.4 Chromatographic Purification of Extracts 40 4.0 RESULTS 42 4.2 Result of phytochemical screening 42 4.2 Result of Anti-Microbial Screening of Extracts 42 4.3 Result of Antimicrobial Screening of AEPE 43 4.4 Result of FTIR spectrum f AEPE 44 4.5 Chemical test on AEPE 54 4.6 Melting Point Determination 54 4.7 Result of the 11HNMR spectra of AEPE 54 4.8 Result of the 13CNMR spectra of AEPE 54 4.9 Result of the COSY spectra of AEPE 55 4.10 Result of the DEPT spectral of AEPE 55 4.11 Result of the HMBC spectral of AEPE 55 4.12 Result of the HSQC spectral of AEPE 55 5.0 DISCUSSION 71
12
6.0 CONCLUSION AND RECOMMENDATION 77 6.1Conclusion 77 6.2 Recommendation 77 References 78
13
CHAPTER ONE
1.0 INTRODUCTION
There is a growing demand for herbal remedies for the world market despite the growth of the synthetic medicine production. At the same time, in the last decades there has been a sharp increase in the collection of some rare and endangered herbs from wild populations in many regions of the world (Manukyan, 2011). Natural products from medicinal plants, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drugs because of the unmatched availability of the chemical diversity. Due to an increasing demand for chemical diversity, seeking therapeutic drugs from natural products, with interest particularly in edible plants has grown throughout the world. Botanicals and herbal preparations for medicinal usage contain various types of bioactive compounds (Sasidharan et al., 2010). World Health Organisation (WHO) encourages the traditional drugs because of its fewer side effects and most of the European countries are expanding towards traditional medicines. Since ancient times, people have been exploring nature, particularly plants, in search of new drugs (Savithramma et al., 2011). Plant materials have been used for the treatment of various diseases throughout the world before the advent of modern clinical drugs. The use of medicinal plants still plays an important role to cover the basic health needs in the developing countries and the industrialized societies had been traced to the extraction and development of several drugs from these plants as well as from traditionally used folk medicines (Shrikumar and Ravi, 2007). Various medicinal properties have been attributed to natural herbs; medicinal plants constitute the main source of new pharmaceuticals and health care products (Savithramma et al., 2011).
Previously crude drugs were identified by comparison only with the standard descriptions available, but recently due to advancement in the field of pharmacognosy various techniques
17
have been used for the standardization of crude drugs (Savithramma et al., 2010). Plant products have been part of phytomedicines since time immemorial. These can be derived from any part of the plant like bark, leaves, flowers, seeds, etc i.e., any part of the plant may contain active components (Cragg and David 2007). The World Health Organization (WHO) has described traditional medicine as one of the surest means to achieve total health care coverage of the world’s population. In spite of the marginalization of traditional medicine practiced in the past, the attention currently given by governments to widespread health-care application has given a new impetus to research, investment and design of programmes in this field in several developing countries in Africa and elsewhere. Traditionally, rural African communities have relied upon the spiritual and practical skills of the TMPs (traditional medicinal practitioners) whose botanical knowledge of plant species and their ecology and scarcity are invaluable. Throughout Africa, the gathering of medicinal plants was traditionally restricted to TMPs or to their trainees. Knowledge of many species was limited to this group through spiritual calling, ritual, religious controls and, in southern Africa, the use of alternative names not known to outsiders (Cunningham, 1993). Medicinal plants contain some organic compounds which produce definite physiological actions on the human body and these bioactive substances include tannins, alkaloids, carbohydrates, terpenoids, steroids and flavonoids (Edeoga et al., 2005). Medicinal plants are of great importance to the health of individuals and communities (Edeoga et al., 2005). Many of these indigenous medicinal plants are used as spices and food plants. They are sometimes added to foods meant for pregnant and nursing mothers for medicinal purposes (Okwu, 2001).
18
1.1 Biological Background of Medicinal Plants.
All plants produce chemical compounds as part of their normal metabolic activities. These are arbitrarily divided into primary metabolites such as sugars and fats found in all plants, and secondary metabolites which are compounds that are not essential for basic functions. The secondary metabolites are found in a smaller range of plants or some useful ones found only in a particular genus or species (Patrick, 2005). The functions of secondary metabolites are varied. For example, some secondary metabolites are toxins to deter predators and others are pheromones used to attract insects for pollination. Phytoalexins protect plant against bacterial and fungal invasions, while allelochemicals inhibit rival plants that are competing for soil and light (Patrick, 2005). Plants regulate their biochemical paths in response to the local mix of herbivores, pollinators and microorganisms. The chemical profile of a single plant may vary over time as it reacts to changing conditions. It is the secondary metabolites and pigments that can have therapeutic actions in human which can be refined to produce drugs (Patrick, 2005). Alkaloids and other nitrogen containing cyclic compounds are plant poisons and drugs. Phenolics are benzene hydroxyl groups for example, anthocyanins, isoflavones and tannins. Terpenoids are built up from five –carbon isoprene units and so can also be called isoprenoids if it contains oxygen and is present in many spices, which are fragrances (Cunningham, 1993). The word ―drug‖ comes from the Dutch word ―droog‖ (from the French word Drogue) which means ―dried plant‖. Some examples are insulin from the roots of dahlias, quinine from the cinchona, morphine and codeine from the poppy. Medicinal plants are plants whose extracts or parts can be used directly or indirectly for the treatment of different ailments. Therefore, the use of traditional medicine and medicinal plants in most developing countries as a basis for the maintenance of good health has been widely observed (Edward, 2001). Scientists throughout the
19
world are trying to explore the precious assets of medicinal plants to help the suffering humanity. Furthermore, today more than 30% of the pharmaceutical preparations are based on plants (Shinwari and Khan, 1998). However, an increasing reliance on the use of medicinal plants in the industrialized societies has been traced to the extraction and development of several drugs and chemotherapeutics from these plants. The use of medicines from plants in the form of local medicine dates back to 4000-5000 B.C, while the medicinal values of these plants are due to the presence of some active compounds which produce physiological actions in the human and animal body (Shrikumar and Ravi, 2007 ). Some of the important bioactive compounds found in medicinal plants are alkaloids, glycosides, resins, gums, mucilages etc. (Sack and Forehlich, 1982). It was observed that developed countries mostly import raw materials of valuable medicinal plants from developing countries. They are then screened, analyzed and used in drug preparations, and returned as high priced medicines to developing countries (Shinawie, 2002). In Pakistan there are about 2000 estimated species of medicinal plants out of which 400 are extensively used in traditional medicine. Pakistan has variety in climate and therefore rich in medicinal plants, but no systematic attempt has been made to work and utilize natural resources of this country. Interest was revived recently in the investigation of medicinal plants to identify novel active phytochemicals that might lead to drug development. Nature has generated such substances for millennia — before modern synthetic chemistry developed in the mid-19th century. Because these substances arise from a more or less hostile environment, the percentage of biologically active natural substances is relatively high in comparison with substances from artificial sources. Currently, more than 50% of drugs in clinical use have a natural-product origin, and about half of the world’s 25 best-selling pharmaceutical agents are natural-product derived (Jin et al., 2011).
20
1.2 History of Herbal Medicine Herbal medicine sometimes referred to as botanical medicine or herbalism, involves the use of plants or parts of plants to treat injuries or illnesses. This field also covers the use of herbs or botanicals to improve overall health and wellness. Herbalist, herbal medicine practitioners, traditional medicine practitioners, ayurvedic, homeopathic, and naturopathic healers all use herbal remedies in their practices. Seeds, leaves, stems, bark, roots, flowers, and extracts of all of these have been used in herbal medicine over the millennia. These supplemental treatments have been delivered raw, in teas and tinctures, as topical applications, in liquid forms, and in pills and capsules. In the beginning the plants were consumed raw or combined with hot water as a soup or tea. Later, the plants were dried and crushed for other uses. The plants were found in the wild and uses were often based on superstitious or visual clues. Plants were often used to treat body systems because they were shaped like that body part or because they grew in a particular area. As science began to take a closer look at herbal remedies, their use became more refined. Herbs, and other plants, are actually the precursors to many of today’s medicinal drugs. Some of the pharmaceutical medications on the market are extracts of some of these traditional herbs (The National Center for the Preservation of Medicinal Herbs 1998). Today, many modern and Western medicine practitioners are beginning to look at herbal remedies for some common and not-so common, disorders. The lower cost, and often safer use, has attracted many medical professionals. Some physicians use herbs to off-set the side effects of pharmaceuticals.
21
1.2.1 Herbal medicine today
Herbal medicines are still in use today. In some respects they have gained a new momentum in the medical field. As many people seek alternative treatments and begin to check out traditional and Eastern medicine, herbs are becoming more popular. As physicians seek new treatments for many common illnesses they are beginning to revisit the traditional remedies using herbal medicines. Pharmaceutical medications with their potentials for harmful side effects and addiction are becoming less popular. People are seeking alternatives to the modern medical interventions. Improving and maintaining health naturally is a very popular approach to overall wellness. The herbs used today are generally cultivated for those purposes. Very few herbs are harvested from the wild, with the exception of a few still found in the rainforests and higher elevations. The cultivation of herbs for medicinal uses is a large field and more people are beginning to plant their own herb gardens. Many monasteries continue to grow large herbal gardens within their walls. Elderly people also metabolize medications differently, and generally are on more medications, and therefore must also exercise caution when trying new herbal treatments. Underlying ailments that may affect the body’s ability to process or absorb medications are also an issue (The National Center for the Preservation of Medicinal Herbs 1998). Herbal medicine has enjoyed a long, and colorful, history. From the early Chinese Empires to modern physicians’ offices, herbal medicines have continued to be a part of the medical field. Herbal treatments have matured throughout history, along with the methods of delivering them. In the beginning, the herbs were used in a hit or miss method and required major events to change their use. Research and clinical trials have helped to shape the field of medicine, and the future for herbal medicine looks bright (The National Center for the Preservation of Medicinal Herbs 1998). The widespread use of herbal remedies and healthcare
22
preparations has been traced to the occurrence of natural products with medicinal properties (Gyawali, 2010). Increasing reliance on the use of medicinal plants in the industrialized societies has been traced to the extraction and development of several drugs and chemotherapeutics from these plants as well as from traditionally used rural herbal remedies (UNESCO, 1998). The percentage composition of the volatile components and characteristics of Volatile Organic Compounds (VOCs) provide an important parameter for the characterization of the plant (Paula et al.,2001). Careful identification of VOCs for fragrance and pharmacologically active ingredients will show the presence of numerous useful compounds. They are gaining increasing interest because of their relatively safe status and their exploitation for potential multi-purpose functional use. Most of the constituents are terpenoids, generally monoterpenes and sesquiterpenes, as well as sometime diterpenes and aromatic compounds derivatives. The VOCs present in essential oils has been reported for their anti-spasmodic, restraining, diuretic, anti-biotical, antimicrobial, antifungal, insecticidal, and anthelmintic efficiency (Singh, 2004). Due to species climatic and geographical conditions, temperate and alpine plants of the Himalaya offer greater possibilities of having novel molecules and even largest quantities of the active compounds (Rajbhandari et al., 2001) 1.3 Justification of the Research The choice of Aeschynomene uniflora as the plant of interest in this work is based on its claimed ethno medicinal uses among traditional medicine practitioners in the tropics, including West Africa. Therefore, there is the need for a scientific study to justify or otherwise the medicinal potentials of this plant and identify the active ingredients
23
1.4 Scope and Limitation of This Study The research thesis is limited to phytochemical screening, antimicrobial screening, isolation, characterization and structural elucidation of the active principle of Aeschynomene uniflora
24
1.5 Aim and Objectives
1.5.1 Aim To determine the phytochemicals properties and the antimicrobial effects of crude ectracts of Aeschynomene uniflora .Mey crude extracts on some pathogens. 1.5.2 Objectives The objectives of this study are:
(i) Extraction of the ground plant material using different solvents, from non-polar to polar ones
(ii) Phytochemical screening of the crude extracts
(iii) Antimicrobial screening of the extracts against Staphylococcus aureus, Streptococcus pyogenes, Corynebaterium ulcerans, Bacillus subtlis, Escherichia coli, Klebsiella pneumonia, Proteus mirabilis, Pseudomonas aeruginosa, Proteus vulgaris, Candida albicans, Candida stellatoideaa and Candida krrusei
(iv) Chromatographic purification of the extracts
(v) Structural elucidation and characterization of the isolated compounds using available spectral techniques
(vi) Testing the potency of the isolated components against the selected microorganisms.
25
IF YOU CAN'T FIND YOUR TOPIC, CLICK HERE TO HIRE A WRITER»
