ABSTRACT
Aeschynomene schimperi is a medicinal plant and used as animal food which belongs to the Fabaceae family. The whole plant of Aeschynomene schimperi was first extracted exhaustively with petroleum ether (60-80 °C) using a soxhlet extractor. The procedure was sequentially repeated using chloroform, ethyl acetate and methanol. The phytochemical screening revealed the presence of carbohydrates, reducing sugar, glycosides, cardiac glycosides, Triterpenes, flavanoids, steroids, tannins, saponins (hydrolysable), glycosides, alkaloids and combined anthraquinone in the plant. The antimicrobial sensitivity test of the extracts were carried out using nine pathogenic microorganisms; Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Shigella dysenteriae, Aspergillus nigre, Candida krusei and Candida albicans. Streptococcus pyogenes, Staphylococcus aureus, Shigella dysenteriae and Candida krusei were sensitive to all the plant extracts while Proteus mirabilis, Pseudomonas aeruginosa and Aspergillus nigre were resistant to all the plant extracts. Escherichia coli and Candida albicans were resistant to petroleum ether extract. The petroleum ether extract showed diameters of zones of inhibition of 12-17mm against four microorganisms. Chloroform extract showed diameters of zones of inhibition of 20-27mm against six microorganisms. Ethyl acetate extract showed diameters of zones of inhibition of 20-23mm against the same organisms as in chloroform extract. Methanolic extract showed diameters of zones of inhibition of 17-21mm against the same organisms as in chloroform and ethyl acetate extracts. Chloroform extract had the highest antimicrobial activity of the four crude extracts recording 27mm in diameters of zones of inhibition in Bacteria. Petroleum ether extract recorded the lowest inhibition zone (12mm in diameter). Candida krusei. Proteus mirabilis, Pseudomonas
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aeruginosa and Aspergillus nigre could not respond at all to all the extracts. In the petroleum extract, the Minimum Inhibitory Concentration (MIC) was 5 mg/ml. In the chlororm extract, the MIC was 2.5 mg/ml, in the ethyl acetate extract, 2.5 mg/ml was the MIC, while in the methanolic extract, the MIC was 5 mg/ml for Candida albicans and Candida krusei and 2.5 mg/ml for the remaining microorganisms that responded. In the fungi that responded to the test, the MFC was 10 mg/ml, in the methanolic extract, the MBC was 10 mg/ml while in the remaining extracts ,there were variations in the MBC of the extracts against the bacteria. Isolation of the compound was from chloroform extract using series of chromatographic processes. By comparison of the spectral data of the isolated compound with those reported in the literature, Stigmast-5-en-3β-ol (C29H50O) also known as beta-sitosterol was proposed to be the isolated compound which is one of the possible bioactive constituents of the plant responsible for various pharmacological activities of the
TABLE OF CONTENTS
Title Page – – – – – – – – – – i Declaration – – – – – – – – – – ii Certification – – – – – – – – – – iii Acknowledgement – – – – – – – – – iv Abstract – – – – – – – – – – v Table of contents – – – – – – – – – vii List of Tables – – – – – – – – – – xii List of figures – – – – – – – – – – xiii List of Appendices – – – – – – – – – xiv List of Abbreviations – – – – – – – – – xv CHAPTER ONE 1.0 Introduction – – – – – – – – – 1 1.1 Phytochemicals – – – – – – – – 1 1.2 Some Uses of Phytochemicals – – – – – – 2 1.3 Herbal Medication – – – – – – – – 4 1.4 Aim and Objectives of the study – – – – – – 5 1.5 Justification for Research – – – – – – – 6 1.6 Scope of the Research Work – – – – – – – 6 1.7 Limitation – – – – – – – – – 6 CHAPTER TWO 2.0 Literature Review – – – – – – – – 7 2.1 Fabaceae – – – – – – – – – 7
2.2 Etymology of Fabaceae – – – – – – – 7
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2.3 Distribution – – – – – – – – – 8 2.4 Taxonomy – – – – – – – – – 8 2.5 Description – – – – – – – – – 8 2.6 Agricultural and Economic Importance of Legumes – – – – 8 2.7.0 Medicinal Values of Legumes – – – – – – 9 2.7.1 HIV Inhibition – – – – – – – – 9 2.8 Nutritional Values – – – – – – – – 10 2.9.0 The Genus Aeschynomene – – – – – – – 11 2.9.1 Examples of Some Species of the Genus – – – – – 11 2.9.2 The Specie- Aeschynomene schimperi – – – – – 11 2.9.3 Reported information on Some Species of the Genus – – – 12 2.9.3.1 Aeschynomene grandiflora- – – – – – – – 12 2.9.3.2 Aeschynomene fluminensis – – – – – – 13 2.9.3.3 Aeschynomene indica – – – – – – – – 13 2.9.3.4 Aeschynomene aspera – – – – – – – – 14 2.10 Phytochemical and Pharmacological Importance of some isolated compounds from the Fabaceae (legumes) family – – – – 15
2.10.1 Protostane and fusidane – – – – – – – – 15 2.10.2 Isoflavones – – – – – – – – – 16 2.10.3 Steroidal Lactone – – – – – – – – 18 2.10.4 Betulinic and Ursolic acids – – – – – – – 19 2.10.5 Stigmasterol tritriacontanate – – – – – – – 20
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2.10.6 Coumestrol – – – – – – – – – 20 2.10.7 Resveratrol – – – – – – – – – 21 CHAPTER THREE 3.0 Experimental – – – – – – – – – 23 3.1 Materials and methods- – – – – – – – 23 3.2 Apparatus and equipment used for extraction – – – – 23 3.3 Solvents for extraction of the plant material – – – – – 23 3.4 Reagents for phytochemical screening – – – – – 24 3.5 Equipment /materials used for antimicrobial screening – – – 25 3.6 Microorganisms used for antimicrobial screening – – – – 26 3.7 The plant material – – – – – – – – 26 3.8 Extraction of the plant material – – – – – – 27 3.9 Phytochemical screening – – – – – – – 27 3.9.1 Test for glycosides – – – – – – – – 27 Fehling’s solution test – – – – – – – 27 Ferric chloride test – – – – – – – – 27 3.9.2 Test for tannins – – – – – – – – 28 3.9.3 Test for Anthraquinones – – – – – – – 28 Free Anthraquinones – – – – – – – – 28 Combined Anthraquinones – – – – – – – 28 3.9.4 Test for alkaloids – – – – – – – – 28
3.9.5 Test for cardiac glycoside – – – – – – – 29
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Salkowski’s test – – – – – – – – 29 3.9.6 Reducing sugar – – – – – – – – 29 3.9.7 Test for flavonoids – – – – – – – – 29 Ferric chloride test – – – – – – – – – 29 Shinoda’s test – – – – – – – – – 29 3.9.8 Test for steroids/terpenoids – – – – – – – 29 Liebermann- Buschard’s test – – – – – – – – 29 Salkowski’s Test – – – – – – – – 30 3.9.9 Test for saponins – – – – – – – – 30 3.9.10 Test for carbohydrates- – – – – – – – 30 Molisch’s Test – – – – – – – – 30
3.10 Preparations of the Culture Media, Test Microorganisms and
Concentration of the Plant extracts – – – – – – 31 3.10.1 Preparation of the Culture media – – – – – – 31 3.10.2 Preparation of test microorganisms – – – – – – 31 3.10.3 Preparation of Concentration of plant extracts – – – – 31 3.11 Antimicrobial screening – – – – – – – 31 3.11.1 Minimum inhibitory concentration (MIC) – – – – – 32 3.11.2 Minimum bactericidal concentration (MBC)/ minimum fungicidal concentration (MFC) of the crude extracts – – – – – 33 3.12 Isolation of pure component – – – – – – – 33 3.12.1 Thin layer chromatography (TLC) – – – – – – 33
3.12.2 Column chromatography – – – – – – – 34
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Preparation of column – – – – – – – 34 Loading of the extract – – – – – – – 34 Collection of fractions- – – – – – – – 34 CHAPTER FOUR 4.0 Results and Discussions – – – – – – – 35 4.1 Extraction – – – – – – – – – 35 4.2 Phytochemical screening – – – – – – – 36 4.3 Antimicrobial tests – – – – – – – – 38 4.4 Zone of inhibition of the plant extracts against the test microorganisms – – – – – – – – 40 4.5 Minimum inhibitory concentration (MIC) of the extracts of the whole plant of Aeschynomene schimperi – – – – – 42 4.6 Minimum bactericidal and fungicidal concentrations (MBC/MFC) of the extracts against the test microorganisms – – – – – 43 4.7 Spectral result – – – – – – – – 43 4.7.1 Discussion on the spectral result – – – – – – 46 CHAPTER FIVE 5.0 Conclusion and recommendation – – – – – – 50 5.1 Conclusion – – – – – – – – – 50 5.2 Recommendation – – – – – – – – 51 References – – – – – – – – – 52
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CHAPTER ONE
1.0 INTRODUCTION 1.1 PHYTOCHEMICALS
Phytochemicals are chemical compounds that occur naturally in plants (phyto means “plant” in Greek). Some are responsible for color and other organoleptic properties, such as the deep purple of blueberries and the smell of garlic. The term is generally used to refer to those chemicals that may have biological significance, for example antioxidants, but are not established as essential nutrient. Scientists estimate that there may be as many as ten thousand different phytochemicals having the potentials to affect diseases such as cancer, stroke or other ailments (Neuwinger, 2000). Without specific knowledge of their cellular actions or mechanisms, phytochemicals have been considered as drugs for millennia. For example, Hippocrates may have prescribed willow tree leaves to abate fever. Salicin, having anti-inflammatory and pain-relieving properties, was originally extracted from the bark of the white willow tree and later, synthetically produced, became the staple over-the-counter drug aspirin(Brown and Arthur, 2001).Sometimes they can be harmful and sometimes they can be very helpful. There is evidence from laboratory studies that phytochemicals in fruits and vegetables may reduce the risk of cancer, possibly due to dietary fibers, polyphenol antioxidants and anti-inflammatory effects (Neuwinger, 2000). Specific phytochemicals, such as fermentable dietary, are allowed limited health claims by the US Food and Drug Administration. An important cancer drug, Taxol (paclitaxel), is a phytochemical initially extracted and purified from the Pacific yew tree(Brown and Arthur, 2001).Some phytochemicals with physiological properties may be elements rather than complex organic molecules. For example, selenium,
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which is abundant in many fruits and vegetables, is involved in major metabolic pathways, including thyroid hormone metabolism and immune function. Particularly, it is an essential nutrient and cofactor for the enzymatic synthesis of glutathione, an endogenous antioxidant (Brown and Arthur, 2001). 1.2 SOME USES OF PHYTOCHEMICALS
Phytochemicals exist in various foods and in different concentrations. Broccoli, pumpkin, spinach, squash, yams, and sweet potatoes all contain important carotenoids, an important phytochemical. Flavonoids can be found in many different natural foods, including: berries, soybeans, carrots, tomatoes, cabbage, parsley, and cucumber. Legumes, peas, and beans contain healthy isoflavones. Indole, another phytochemical, is available in cabbage, bokchoy, turnips, brussel’s sprouts, kale, and cauliflower. Flaxseeds and walnuts contain Lignans, as well as many other kinds of seeds and nuts. Fatty acids, or lipids, are seen in flaxseeds, hemp oil and hemp seeds, and walnuts (Damery et al., 2011).Eggplant, hemp seeds and oil, peppers, squash, cabbage, cucumber, tomatoes, broccoli, cabbage, and soybeans, contain plant sterols. There are tons of nutrients that are contained by the entire phytochemical family, and these are perfect for any diet. Health problems and diseases can be fought in many ways through phytochemicals, since they readily help your body in several ways. When carcinogens try to enter into the cell walls, phytochemicals help block them. Malignant changes that have already started in the cells by carcinogens are battled. Phytochemicals seem to increase the benefits of the many protective enzymes that are eaten in the diet, by raising enzyme activity. The damage that the body can receive from free radicals is scavenged by phytochemicals that mix with different vitamins to
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boost antioxidants (Chantel, 2009). This is done before damage can be caused to the body. Bad cholesterol levels have been shown to be reduced as well. By maintaining a diet that is rich in a variety of vegetables, fruits, and nuts, the benefits of the phytochemicals and vitamins go far in helping one reach maximum health. Knowing the names and benefits of the varied range of existing phytochemicals is not important, but understanding how eating a diet rich in these foods can help your body, is of great importance. One food source that is often overlooked that contains essential phytochemicals is hemp seeds which are abundant in essential fatty acid (Chantel, 2009). There are many phytochemicals and each works differently. These are some possible actions of some of them. Antioxidant – Most Phytochemicals have antioxidant activities and protect our cells against oxidative damage and reduce the risk of developing certain types of cancer. Some phytochemicals with antioxidant activities are: allyl sulfides (onions, leeks, and garlic), carotenoids (fruits, carrots), flavanoids (fruits, vegetables), polyphones (tea, grapes) (Dragland, 2003). Hormonal action – Isoflavones found in soy bean plant, imitate human estrogens and help to reduce menopausal symptoms and osteoporosis (Neuwinger, 2000). Stimulation of enzymes – Indoles which are found in cabbages, stimulate enzymes that make the estrogen less effective and could reduce the risk for breast cancer. Other phytochemicals which interfere with enzymes are protease inhibitors (soy and beans), terpenes (citrus fruits and cherries) (Chantel, 2009).
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Interference with DNA replication – Saponins found in beans interfere with the replication of cell DNA, thereby preventing the multiplication of cancer cells. Capsaicin, found in hot peppers, protects DNA from carcinogens (Dragland, 2003). Anti-bacterial effect – The phytochemical, allicin from garlic, has anti-bacterial properties (Brown and Arthur, 2001).
Physical action – Some phytochemicals bind physically to cell walls thereby preventing the adhesion of pathogens to human cell walls. Proanthocyanidins are responsible for the anti-adhesion properties of cranberry. Consumption of cranberries will reduce the risk of urinary tract infections and will improve dental health (Damery et al., 2011). 1.3 HERBAL MEDICATION Herbal medicine is also called botanical medicine or phytomedicine which refers to using plants’ parts for medicinal purposes (Chantel, 2009). Herbalism has a long tradition of use outside of conventional medicine. It is becoming more mainstream as improvements in analysis and quality control along with advances in clinical research show the value of herbal medicine in treating and preventing diseases (Izzo and Ernst, 2009). Plants had been used for medicinal purposes long before recorded history. Ancient Chinese and Egyptian papyrus writings describe medicinal uses for plants as early as 3,000 BC. Indigenous cultures (such as African and Native American) used herbs in their healing rituals, while others developed traditional medical systems (such as Ayurveda and Traditional Chinese Medicine) in which herbal therapies were used. Researchers found that people in different parts of the world tended to use the same or similar plants for the same purposes (Abeloff, 2008). In the early 19th century, when chemical
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analysis first became available, scientists began to extract and modify the active ingredients from plants. Later, chemists began making their own version of plant compounds and, over time, the use of herbal medicines declined in favor of drugs. Almost one fourth of pharmaceutical drugs are derived from botanical sources. Recently, the World Health Organization (2009) estimated that 80% of people worldwide rely on herbal medicines for some part of their primary health care (Damery et al., 2011). In Germany, about 600 – 700 plant based medicines are available and are prescribed by some 70% of German physicians. In the past 20 years in the United States, public dissatisfaction with the cost of prescription medications, combined with an interest in returning to natural or organic remedies, has led to an increase in herbal medicinal use (Abeloff, 2008). Herbal medicine is used to treat many conditions, such as asthma, eczema, premenstrual syndrome, rheumatoid arthritis, migraine, menopausal symptoms, chronic fatigue, irritable bowel syndrome and cancer, among others. Herbal supplements are best taken under the guidance of a trained health care provider. For example, one study found that 90% of arthritic patients use alternative therapies, such as herbal medicine (Damery et al., 2011). 1.4 AIM AND OBJECTIVES OF THE STUDY The aim of the research is to screen the plant-Aeschynomene schimperi for phytochemical and antimicrobial activities. The objective of the study is to carry out the structural elucidation of possible isolate(s) from the plant.
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1.5 JUSTIFICATION FOR RESEARCH The frequently mentioned effects of the herbs in the Fabaceae family from the traditional perspective are: dispelling heat, cleaning toxins, resolving swellings; removing water accumulation; opening meridians, vitalizing blood, controlling pain; controlling bleeding; and resolving phlegm accumulation, cough, and lung disorders (Hu, 1980). The bark, leaves, flowers and fruits of Aeschynomene schimperi are used in traditional medicine to treat multifactorial diseases like leprosy, gout, acute rheumatic fever, sores, boils, bloody diarrhea, and vaginitis (Joshi, 2000). A number of the herbs in this family have traditional indications in common. Hence there is need for a scientific investigation of the plant for justification (Hu, 1980). 1.6 SCOPE OF THE RESEARCH WORK This research work would cover the following: i. Phytochemical screening ii. Antimicrobial screening iii. Isolation of active component(s) iv. Structural elucidation of the active component(s) 1.7 LIMITATION This research work would be limited to structural elucidation of active component(s) which may also be dependent on the availability of spectroscopic instrument.
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