PHARMACOGNOSTIC AND ANTIMICROBIAL STUDIES ON THE STEM-BARK OF FICUS KAMERUNENSIS WARB. (MORACEAE)


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TABLE OF CONTENT

Title Page
Abstract
Table of Content
List of Abbreviations and Acronyms

CHAPTER ONE
1.0       Introduction
1.1       Pharmacognosy
1.2       Pharmacognostic Studies
1.3       Medicinal Plants
1.4       Phytochemistry
1.5       Antimicrobial Activities of Medicinal Plants
1.6       Antimicrobial Agents of Plant Origin
1.7       Statement of research problem
1.8       Justification of the research
1.9       Hypothesis
1.10     Aim
1.11     Objectives

CHAPTER TWO
2.0       LITERATURE REVIEW
2.1       Description of the Moraceae family
2.2       Description of the Genus Ficus
2.3       Nutritional Properties of Ficus
2.4       Some Medicinal properties and uses of Genus Ficus
2.5       Pytochemical constituents of the Genus Ficus
2.6       Description of the Ficus kamerunensis Species
2.6.1Botanical Description of Ficus kamerunensis
2.6.2 Scientific Classification of Ficus kamerunensis

CHAPTER THREE
3.0       MATERIALS AND METHODS
3.1       List of Chemicals and Reagents
3.2       List of Equipments
3.3       Collection and Identification of Ficus kamerunensis
3.4       Preparation of the Stem-bark of Ficus kamerunensis
3.5       Pharmacognosic Studies on Ficus kamerunensis
3.5.1    Macroscopic Examination on the stem-bark of Ficus kamerunensis
            Microscopical examination of the stem-bark of Ficus kamerunensis
3.5.3    Determination of physicochemical parameters of the powdered Stem-bark of Ficus kamerunensis
3.6       Preliminary Phytochemical Screening of the Stem-bark of f Ficus kamerunensis
3.6.1    Tests for Carbourhydrates
3.6.2    Tests for Anthraquinones
3.6.3    Tests for Cardiac glycosides
3.6.4    Tests for Saponin Glycosides
3.6.5    Tests for Steroids and or Triterpenoids
3.6.6    Tests for Flavonoids
3.6.7    Tests for Tannins
3.6.8    Tests for Alkaloids
3.7 Phytochemical Analysis of the Stem-bark of Ficus kamerunensis
3.7.1    Extraction Process of the Stem-bark Ficus kamerunensis
3.7.2    Fractionation of the methanol extract of the Stem-bark of Ficus kamerunensis
3.8 Thin layer Chromatography (TLC) of the Diethyl ether (EXT 1) partitioned fraction
3.8.1    Preparation of Solvent system for TLC
3.8.2    Spotting of the sample
3.8.3    Documentation
3.9 Column Chromatography of the Diethyl ether fraction (EXT 1)
3.9.1 Purification of the EXT 1
3.10     Analysis of the isolated Compound
3.10.1 Physiochemical Analysis
3.10.2 Structure Elucidation of the Isolated Compound
3.11     Antimicrobial Studies of Ficus kamerunensis
3.11.1  Collection of Clinical Isolates
3.11.2  Preparation of Stock Solution
3.11.3  Mc-Farland Barium Sulphate Turbidity Standard
3.11.4  Preparation of Culture Media
3.11.5  Determination of Zone of Inhibition
3.11.6  Determination of Minimum Inhibitory Concentration (MIC)
3.11.7  Determination of minimum Bactericidal Fungicidal Concentrations (MBC/MFC)
3.11 Biological Studies of Ficus kamerunensis
3.11.1 Median Lethal Dose (LD50) Determination of the Methanol Extract of Ficus kamerunensis
3.12 Statistical Analysis

CHAPER FOUR
4.0 RESULTS
4.2 Pharmacognostic Studies on Ficus kamerunensis
4.2.1    Macroscopical Features of the stem-bark of Ficus kamerunensis
4.2.2    Microscopical Features of the leaf of Ficus kamerunensis
4.3.0    Physicochemical Constants of powdered Stem-bark of Ficus kamerunensis
4.3.1    Moisture Content (Loss on drying) of the powdered Stem-bark of Ficus kamerunensis
4.3.2    Ash Values of the powdered Stem-bark of Ficus kamerunensis
4.3.3    Extractive Values of the powdered Stem-bark of Ficus kamerunensis
4.4       Preliminary Phytochemical Screening of the Stem-bark of Ficus kamerunensis
4.5       Phytochemical Analysis of the Powdered Stem-bark of Ficus kamerunensis
4.5.1    Extraction of the Powdered Stem-bark of Ficus kamerunensis
4.5.2    Fractionation of the Methanol Extract of the Stem-bark of Ficus kamerunensis
4.5.3    Thin layer Chromatography of the diethyl ether fraction (EXT 1) of Ficus kamerunensis
4.5.4    Column Chromatography for the Isolation of Compound from EXT 1
4.5.5    Preliminary Studies on the Isolated Compound
4.5.6    Structure of the Isolated Compound
4.7 Antimicrobial Studies of Ficus kamerunensis
4.7.1    Minimum Inhibitory Concentrations of the Plant Extract and the Column Fraction
4.7.2    Minimum Bactericidal/Fungicidal concentration (MBC/MFC) of Plant Extract Column Fraction
4.6 Median Lethal Dose of the Methanol Extract of Ficus kamerunensis

CHAPTER FIVE
5.0 DISCUSSION

CHAPTER SIX
6.0 CHAPTER VI SUMMARY, CONCLUSION AND RECOMMENDATION
6.1 Summary
6.2 Conclusion
6.3 Recommendation
References
Appendices



ABSTRACT

Ficus kamerunensis is an epiphytic shrub or tree growing up to 20 m high. The plant used in ethnomedicine to treat microbial infections such as sexually transmitted infections. Pharmacognostic studies were conducted on the fresh and dried whole stem-bark of F. kamerunensis. The quantitative physical standards were carried out. Preliminary phytochemical screenings of the powdered stem bark of the plant were conducted using appropriate chemical reagents. The powdered stem bark (1 kg) of the plant was extracted using cold maceration technique with 3 L of 95% methanol in a glass jar for 3 days (72 hours) at room temperature, then partitioned with diethylether, ethyl acetate and n-butanol. Thin-layer chromatography was carried out using diethyl ether fraction to obtain the best solvent system to be used for column chromatography. A combination of column chromatography and preparative thin layer Chromatography using Column chromatography were carried out using the best solvent system obtained (Hexane: Ethyl acetate 7:3) for compound isolation. The structure of the isolated compound was determined by the analysis of 1H NMR, 13C NMR, and 2D NMR spectral data, as well as comparison with reported data. Antimicrobial evaluations of the stem-bark of the plant were carried out for diethyl ether, ethyl acetate, n-butanol and 95% methanol extract at different concentrations (50, 25, 12.5, 6.25 and 3.125 mg/ml) and tested against four Gram-positive bacteria—Staphylococcus aureus, Streptococcus pyrogenes, MRSA, and Neisseria gonorrheae, and four Gram-negative bacteria—Corynebacterium ulcerans, E. coli, Pseudomonas aeroginosa and Proteusmirabilis as well as four fungi— Candida albicans, Candida krusei, Candida stellatoidea and Candida tropicalis. Mc-farland’s turbidity standard scale (number 0.5) was used to standardize the test micro organisms. Agar Diffusion method was used to screen the extracts while the minimum inhibitory concentrations (MIC) as well as minimum bactericidal and fungicidal concentrations (MBC/MFC) were determined using broth dilution method.The acute toxicity studies (LD50) was carried out using Lorke method. Phytochemical screening of the powdered stem-bark of Ficus kamerunensis reveals the presence of carbohydrates, anthraquinones, saponins, cardiac glycosides, terpenoids, flavonoids, tannins and alkaloids. Physicochemical studies showed moisture content, total ash acid insoluble ash, water soluble ash, alcohol extractive and water extractive values of 11.7, 8.8 1.7, 3.7, 10.4 and 18.4 % respectively. Antimicrobial evaluation shows that the extracts have activity against E. coli,


Candida albican, Candida stellatodea, Corynebacterium ulcerans, MRSA, Neisseria gonorrhea,

Pseudomonas  aeroginosa  Staphylococcus  aureus  while  Candida  tropicalis,  Candida  krusei,

Proteus mirabilis and Streptococcus pyrogens were resistant to the tested extracts. Known compound (pentacyclic triterpene) α-amyrin acetate was isolated and characterized from the diethyl ether fraction of the methanol extract of the plant material.





CHAPTER ONE

1.0 INTRODUCTION


The World Health Organization (WHO) estimates that up to 80% of the population in Africa make use of traditional medicine as well as about 65% of the world's population (Fabricant and Farnsworth, 2011). Plants used in traditional medicine, also called phytomedicine are plant-derived medicines that contain chemicals, more usually, mixtures of chemical compounds that act individually or in combination on the human body to prevent disorders and to restore or maintain health (Van- Wyk, et al,1997). About 3.4 billion people in the developing world depend on plant based traditional medicines. This represents about 88 percent of the world’s inhabitants who relied mainly on traditional medicine for their primary health care (WHO, 1999). Owing to poverty, unawareness and unavailability of contemporary health facilities, most people, especially rural people are still compelled to practise traditional medications for the treatment of their day to day illnesses (Khan, et. al, 1993).


Natural products (secondary metabolites) have been the most successful source of potential drug leads. However, their recent implementation in drug discovery and development efforts have somewhat demonstrated a decline in interest (Mishra et.al, 2012). Nevertheless, natural products continue to provide unique structural diversity in comparison to standard combinatorial chemistry, which presents opportunities for discovering mainly novel low molecular weight lead compounds. Since less than 10% of the world’s biodiversity has been evaluated for potential biological activity, many more useful natural lead compounds await discovery with the challenge being how to access this natural chemical diversity (Cragg and Newman, 2005).....


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