ABSTRACT
The root-bark of the plant Acacia ataxacantha DC of the family Fabaceae was investigated for medicinal values. The Phytochemical screening gave positive results for the presence of flavonoids, carbohydrates, glycosides, saponins, steroids/triterpenes, tannins and alkaloids. The antimicrobial screening of the crude methanol, ethyl acetate, chloroform and petroleum ether extracts showed that the plant roots could inhibit the growths of Bacillus subtilis, Streptococcus pneumonia, Streptococcus pyogenes, Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhi, Escherichia coli, Candida albicans and Candida krusei but were not active on Corynebacterium ulcerans, Streptococcus faecalis, Proteus mirabilis and Candida tropicalis. Ethyl acetate extract had the widest diameter of zone of inhibition of 30 mm, followed by chloroform with 25 mm, then methanol with 24 mm and petroleum ether with 19 mm against the various micro-organisms. The Minimum Inhibitory Concentration (MIC) of the extracts was determined for the organisms whose growths were inhibited. Methanol and chloroform fractions had MIC values of 5 mg/ml and petroleum ether had 10 mg/ml for all the test organisms, while ethyl acetate was the most active with 2.5 mg/ml for Bacillus subtilis, Escherichia coli, Salmonella typhi and Klebsiella pneumonia. The Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) showed that the ethyl acetate extract had cidal effect against Bacillus subtilis, Escherichia coli and Klebsiella pneumonae at a concentration of 5 mg/ml. The
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Ethyl acetate extract was purified using chromatographic techniques and two pure compounds (ABA and ABA 1) were isolated and characterized using spectral techniques. Based on available spectral data and comparison with existing data bank, the compounds were established to be α- amyrenol and lupeol respectively. The antimicrobial activity of α- amyrenol was determined with the same test organisms. The MIC and MBC/MFC were found to be 12.5 and 25 μg/ml respectively against B.subtilis, E.coli and S.typhi, thus justifying the numerous folkloric uses of the plant.
TABLE OF CONTENTS
Cover Page . . . . . . . . . i Title Page. . . . . . . . . . ii Declaration . . . . . . . . . iii Certification . . . . . . . . . iv Dedication . . . . . . . . . v Acknowledgement . . . . . . . . vi Abstract . . . . . . . . . vii Table of Contents . . . . . . . . ix List of Tables . . . . . . . . . xiv List of Figures . . . . . . . . . xv 1.0 INTRODUCTION . . . . . . . 1 1.1 Aim and Objectives of the Research . . . . 3 1.1.1 Aim of the Research . . . . . . . 3 1.1.2 Objectives of the Research . . . . . . 3 1.2 Justification of the Research . . . . . 4 1.3 Scope and Limitation of the Research . . . . 4
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2.0 LITERATURE REVIEW . . . . . . 5 2.1 General Botanical Features of the Fabaceae Family . . 5 2.1.1 Taxonomic Position of Family Fabaceae . . . . 6 2.1.2 Characteristic Features of Fabaceae Family . . . . 6
2.2 Acacia . . . . . . . . . 7
2.2.1 Species of the Genus Acacia. . . . . . 8
2.2.2 Medicinal Uses of Some Acacia Species. . . . . 8 2.2.3 Chemical Composition . . . . . . 10 2.3 Acacia ataxacantha DC . . . . . . 16 2.3.1 Scientific Classification . . . . . . 16
2.3.2 Botanical Description of Acacia ataxacantha DC . . . 16 2.3.3 Products and Uses . . . . . . . 17 2.3.4 Medicinal Uses of Acacia ataxacantha . . . . 17 2.4 Increasing Popularity of Medicinal Plants . . . 18 2.5 Plant Constituents of Pharmacological Importance. . . 20 2.5.1 Alkaloids . . . . . . . . 21 2.5.2 Flavonoids . . . . . . . . 23 2.5.3 Steroids . . . . . . . . 24 2.5.4 Glycosides . . . . . . . . 25 2.5.5 Saponins . . . . . . . . 26
2.5.6 Tannins . . . . . . . 26
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2.5.7 Terpenes . . . . . . . . 28 2.5.8 Carbohydrates . . . . . . . . 28 2.6 Test Micro-Organisms . . . . . . 31 3.0 MATERIALS AND METHODS . . . . . 35 3.1 Apparatus, Instruments and Reagents . . . . . 35 3.1.1 Apparatus and Instruments . . . . . . 35 3.1.2 Solvents . . . . . . . . 35 3.1.3 Micro Organisms . . . . . . . 36 3.2 Collection and Preparation of Plant Material . . . 36 3.2.1 Sample Collection and Identification . . . . . 36 3.2.2 Preparation of Pulverized Plant Material . . . . 37 3.3 Extraction . . . . . . . . 37 3.3.1 Fractionation . . . . . . . . 37 3.4 Phytochemical Screening . . . . . . 37 3.4.1 Test for Carbohydrates . . . . . . 38 3.4.2 Test for Glycosides . . . . . . . 38 3.4.3 Test for Cardiac Glycosides . . . . . . 38 3.4.4 Test for Free Anthracene Derivatives . . . . . 39 3.4.5 Test for Saponins . . . . . . . 39 3.4.6 Test for Tannins . . . . . . . 39
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3.4.7 Test for Flavonoids . . . . . . . 39
3.4.8 Test for Alkaloids . . . . . . . 39 3.4.9 Test for Steroids and Triterpenes . . . . . 40 3.5 Bioassay Analyses of the Plant Extracts . . . . 40 3.5.1 Antimicrobial Sensitivity Test . . . . . 41 3.5.2 Determination of Zone of Inhibition. . . . . . 41 3.5.3 Determination of MIC of the Extracts . . . . 42 3.5.4 Determination of MBC/MFC of the Extracts . . . . 42 3.6 Isolation of Active Components . . . . . 43 3.6.1 Thin Layer Chromatography (TLC) . . . . . 43 3.6.2 Column Chromatography . . . . . . 43 3.6.3 Preparative Thin Layer Chromatography . . . . 44 3.7 Spectroscopy . . . . . . . 44 3.8 Antimicrobial Screening of ABA . . . . . 44 4.0 RESULTS . . . . . . . 45 4.1 Extraction . . . . . . . . 45 4.2 Results of Phytochemical Screening Experiments . . . 45 4.3 Results of the Antimicrobial Screening Experiments . . 45 4.4 Antimicrobial Screening of Pure Compound (ABA) . . 45
4.5 Spectroscopy . . . . . . . . 45
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5.0 DISCUSSION . . . . . . . 72 5.1 Phytochemical Screening . . . . . 72 5.2 Antimicrobial Screening . . . . . . 73 5.2.1 Sensitivity Test of Extracts against selected Micro-organisms . 73 5.2.2 Diameter of Zones of Inhibition . . . . . 73 5.2.3 Minimum Inhibitory Concentration (MIC) . . . . 73 5.2.4 Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) . 74
5.3 Spectroscopy . . . . . . . 75
5.3.1 Spectral Analysis of ABA . . . . . . 75
5.3.2 Spectral Analysis of ABA 1 . . . . . 77
5.4 Antimicrobial Screening of Isolated Compound (ABA) . . 79 6.0 SUMMARY, CONCLUSION AND RECOMMENDATION . 81 6.1 Summary . . . . . . . . 81 6.2 Conclusion . . . . . . . . 82 6.3 Recommendation . . . . . . . 82 REFERENCES . . . . . . . 83
CHAPTER ONE
1.0 INTRODUCTION Ever since ancient times, people looked for drugs in nature in search for cure for their diseases. In view of the fact that at that time there was no sufficient information either concerning the reasons for the illnesses or concerning which plant and how it could be utilized as a cure, everything was based on experience. In time, the reasons for the usage of specific medicinal plants for treatment of certain diseases were being discovered; thus, medicinal plants’ usage gradually abandoned the empiric framework and became founded on explicatory facts (Petrovska, 2012). The use of the medicinal herbs for curing diseases has been documented in the history of all civilizations. The drugs were used in crude forms like expressed juice, powder, decoction or infusion (Amritpal, 2011). The World Health Organization (WHO) defined medicinal herbs as finished, labelled medicinal products that contain active ingredients aerial or underground parts of plants, or other plant material, or combinations thereof, whether in the crude state or as plant preparations. Herbal medicines may contain excipients [inert additives such as starch used to improve adhesive quality in order to prepare pills or tablets] in addition to the active ingredients (WHO, 1996).
Ethnobotany (the study of traditional human uses of plants) is recognized as an effective way to discover future medicines. In 2001, researchers identified 122 compounds used in modern medicine which were derived from “ethnomedical” plant sources; 80% of these have had an ethnomedical use identical or related to the current use of the active elements of the plant (Fabricant and Farnsworth, 2001). Many of the pharmaceuticals currently available to physicians have a long history of use as herbal remedies, including aspirin, digitalis, quinine, and opium. Numerous medicines in use today were extracted from plants. About 50 to 60%
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of pharmaceutical drugs are either of natural origin or obtained through use of natural products as starting points in their synthesis (Verlet, 1990; Balandrin et al., 1993).
Traditional medicine has a long history, it is the sum total of the knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health, as well as in the prevention, diagnosis, improvement or treatment of physical and mental illnesses (WHO, 2000). The widespread use of plants in folk medicine by traditional medicinal practitioners has continued to create awareness in the study of plants and medicinal plants. Plants have the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions, and to defend against attack from predators such as insects, fungi and herbivorousmammals. At least 12,000 such compounds have been isolated so far; a number estimated to be less than 10% of the total (Lai and Roy, 2004; Tapsell et al., 2006).
The world’s tropical rain forests are especially rich in biodiversity but there is rapid depletion of these natural resources worldwide, and in Nigeria in particular, the pressures from degradation, unsustainable arable land use, urbanization and industrialization are taking their toll as well (Obute and Osuji, 2002; Ayodele, 2005). The vegetable world comprises three main groups of plants; the Superior, Intermediary and the Inferior. These encompass bacteria, microscopic algae, mushrooms, ferns, brushes and trees among others (Sofowora, 1993). Medicinal plants and herbs contain substances known to modern and ancient civilization for their healing properties. Until the development of organic compounds in the nineteenth century, medicinal plants and herbs were the sole source of active principles capable of curing man’s ailments. Modern pharmaceuticals rely heavily on the same active principles, be they natural or synthetic. These active principles differ from plant to plant due to their biodiversity (Sofowora, 1993). Plants have continued to be major sources of medicine either in the form of traditional medicine preparations or as pure active principles. This has made it
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important to identify plants with useful therapeutic action for possible isolation and characterization of their active constituents (Ndukwe et al., 2007). The need to identify active chemical constituents in plant extracts requires phytochemical and analytical techniques. Different phytoconstituents have different degrees of solubility in different types of solvents depending upon their polarity and structure (El-Mahamood and Doughari, 2005). Phytochemical surveys are being seen as the first step towards the discovery and structural elucidation of useful natural organic constituents for medicinal applications (Hostettmann et al., 2000). Many plants are chemically different depending on the locality where they are found with some of the constituents occurring only at certain seasons of the year (Adelani, 2007). The continued investigation of their secondary metabolites has led to important breakthrough in pharmacology and has helped tremendously in the development of modern pharmaco-therapeutics in Africa and other parts of the world (Nwaogu et al., 2007). Plants are particularly interesting because they have the broadest spectrum of biosynthetic capability, and produce a wide variety of compounds. It is against this background that it has become necessary to investigate the active ingredient(s) of Acacia ataxacantha; which is a plant used extensively as herbal remedy in some parts of Nigeria and West Africa. 1.1 AIM AND OBJECTIVES OF THE RESEARCH 1.1.1 Aim of the Research The aim of the research is to screen the root-bark of Acacia ataxacantha of the family Fabaceae for bioactive constituents, together with the structural elucidation of such constituents. 1.1.2 Objectives of the Research
i. Collection, proper botanical identification, drying and pulverizing of the roots of the plant.
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ii. Extraction of the pulverized plant material using different solvents based on the eluotropic series i.e. from non-polar (petroleum ether 60-80oC) to polar (methanol).
iii. Phytochemical screening for bio-active compounds using the crude extracts.
iv. Antibacterial and Antifungal screening of the extracts.
v. Analytical separations involving several consecutive steps of chromatographic techniques and purification.
vi. Verification of the purity of the isolated compounds. Structural elucidation and characterization of the isolated compounds using available spectral techniques such as FTIR, 1H NMR, 13C NMR and DEPT
1.2 JUSTIFICATION OF THE RESEARCH The choice of Acacia ataxacanthaas the plant of interest in this work is based on its medicinal importance among traditional medicine practitioners. Although oral evidence indicates that the plant is implicated in thetreatment of chicken pox, headache, pneumonia, constipation, excessive cough, toothache, respiratory diseases, yellow fever and dysentery; and there is no documented evidence to support such uses.Therefore, there is need for a scientific validation of these claims. 1.3 SCOPE AND LIMITATION OF THE RESEARCH The scope of this research work involved the following:
i. Phytochemical screening.
ii. Antibacterial/antifungal screening
iii. Isolation
iv. Characterization and structural elucidation. However, not all the active components were elucidated due to limited laboratory equipment.
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