ABSTRACT
The petroleum ether, chloroform, ethyl acetate and methanol extracts of the aerial plant parts of Maesobotrya barteri were screened for their antimicrobial activity and their chemical constituents. The preliminary phytochemical screening of the various extracts showed the presence of carbohydrates, saponnins, triterpenes, steroids, tannins, flavonoids, and cardiac glycosides. The result of the antimicrobial screening showed that all the extracts had significant activity against most of the microorganisms tested. The minimum inhibitory concentration (MIC), minimum bactericidal concentration and minimum fungicidal concentration (MBC/MFC) of the extracts showed that almost all the organisms tested were responsive to the extracts at a given concentration. It also showed that, ethyl acetate and chloroform crude extracts exhibited the highest activity against the bacterial Salmonellae typhi and Shigella dysenteriae and Candida krusei and Candida stellatoidea for fungi. The controls used were Sparlfloxacin 2mg/ml for the bacteria and Fluconazole 5mg/ml for the fungi. Chromatographic separation of the ethyl acetate and petroleum ether extracts of the aerial plant parts of Maesobotrya barteri afforded two compounds labelled as 1 and 2. These compounds (1 and 2) were characterised by 1D and 2D NMR spectroscopy and identified as β-amyrin and an isomer of betulinic and oleanolic acid. They were obtained from the ethyl acetate and petroleum ether fractions, a white crystalline and yellowish crystalline compound respectively. They showed significant antimicrobial activities. Compound (1) (β-Amyrin) from the ethyl acetate fraction was found to be active against most of the tested microbes and gave a zone of inhibition range between 26 – 32 mm. The MIC value for the most sensitive organisms were recorded at a concentration of 6.25 μg/ml and the other microbes were sensitive at 12.5 μg/ml with a corresponding MBC/MFC recorded at 25 μg/ml for some of the test microbes and 50 μg/ml for the others. Antimicrobial
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result for compound (2), betulinic acid and oleanolic acid (as isomer) from petroleum ether fraction showed a zone of inhibition range between 24 – 30 mm. The MIC values for the most sensitive organisms were recorded to at a concentration of 6.25 μg/ml and the other microbes were sensitive at 12.5 μg/ml. The activity recorded by the crude and pure isolates on the organisms are in line with the ethnomedicinal claims of the plant based on their zones of inhibition as compared with those of standard drugs.
TABLE OF CONTENTS
Cover Page i
Flyleaf ii
Title Page iii
Declaration iv
Certification v
Dedication vi
Acknowledgements vii
Abstract ix
Table of contents xi
List of Tables xvii
List of Figures xviii
List of Structures xix
List of Abbreviations xx
CHAPTER ONE
1.0 INTRODUCTION 1
1.1 Medicinal Plants 1
1.2 Background of the Use of Natural Product as Therapeutic Agents 1
1.3 The Medicinal Uses of Plants 3
1.4 Justification of the Research 4
1.5 Aim of the Research 5
1.6 Objectives of the Research 5
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CHAPTER TWO
2.0 LITERATURE REVIEW 6
2.1 Botanical Characteristics of Maesobotrya Species 6
2.1.1 Description 7
2.1.2 Maesobotrya pynaertii 9
2.1.3 Maesobotrya floribunda 9
2.2 Medicinal and Nutritive Applications of Maesobotrya Species 9
2.3 The Main Groups of Active Ingredients of Medicinal Plants 11
2.3.1 Flavonoids 11
2.3.2 Tannins 12
2.3.3 Alkaloids 13
2.3.4 Phenols 14
2.3.5 Saponins 15
2.3.6 Steroids 15
2.3.7. Glycosides 16
2.4 Some Microorganisms and their Effects on the Human Body 22
2.4.1 Enterobacteriaceae 22
CHAPTER THREE
3.0 MATERIALS AND METHODS 27
3.1 Materials 27
3.1.1 Reagents 27
3.1.2 Solvents for extraction 28
3.1.3 Materials for chromatographic separations 28
3.1.4 Materials for antimicrobial test 28
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3.2 Methodology 28
3.2.1 Collection of plant material 28
3.2.1 Extraction 29
3.3 Phytochemical Screening 29
3.3.1 Test for carbohydrate 29
3.3.2 Test for flavonoids 30
3.3.3 Test for alkaloids 30
3.3.4 Test for tannins 30
3.3.5 Test for saponins 31
3.3.6 Test for glycoside 31
3.3.7 Test for steroids 31
3.3.8 Test for terpenoids 32
3.4 Antimicrobial Screening 32
3.4.1 The test organisms 32
3.4.2 Culture media 32
3.4.3 Preparation of inoculums of test organisms 32
3.4.4 Sensitivity test of the crude extracts 33
3.4.5 Minimum inhibitory concentration 33
3.4.6 Minimum bactericidal and fungicidal concentration 34
3.5 Methods of Isolation/Purification 34
3.5.1 Thin Layer Chromatography (TLC) 34
3.5.2 Column Chromatography (CC) 35
3.6 Column Chromatography of Ethyl acetate extract 35
3.7 Column Chromatography of Petroleum Ether Extract 36
3.8 Spectral Analysis 37
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CHAPTER FOUR
4.0 RESULTS 38
4.1 Result of Extraction of Plant Material 38
4.2 Result of Phytochemical Screening 39
4.3 Result of the Zones of Inhibition of the Crude Extracts 40
4.4 Result of the MIC of the Crude Extracts 41
4.5 Result of the MBC/MFC of the Crude Extracts 42
4.6 Result of the Zones of Inhibition of the Isolates 43
4.7 Results of the MIC of the Isolates 44
4.8 Results of MBC/MFC of the Isolates 45
4.9 Result of 1D and 2D NMR of Compound 1 46
4.9.1 1H NMR of compound 1 46
4.9.2 13C NMR of compound 1 47
4.9.3 DEPT experiment of compound 1 48
4.9.4 2D 1H-1H COSY correlation of compound 1 49
4.9.5 NOESY experiment of compound 1 50
4.9.5 HSQC experiment of compound 1 51
4.9.6 HMBC experiment of compound 1 52
4.10 Result of 1D and 2D NMR of Compound 2 54
4.10.1 1H NMR of compound 2 54
4.10.2 13C NMR of compound 2 55
4.10.3 DEPT experiment of compound 2 56
4.10.4 2D 1H-1H COSY correlation of compound 2 57
4.10.5 NOESY experiment of compound 2 58
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4.10.5 HSQC experiment of compound 2 59
4.10.6 HMBC experiment of compound 2 60
CHAPTER FIVE
5.0 DISCUSSION 62
CHAPTER SIX
6.0 CONCLUSION, SUMMARY AND RECOMMENDATION 66
6.1 Summary 66
6.2 Conclusion 66
6.3 Recommendations 67
REFERENCES 68
CHAPTER ONE
1.0 INTRODUCTION
1.1 Medicinal Plants
A medicinal plant is any plant which in one or more of its organs contains substances that can be utilized for therapeutic purposes or which are precursors for the synthesis of useful drugs (Sofowora, 2008). Plants have been used in traditional medicines for many years, but no sufficient scientific data exist to confirm their efficacy (Sofowora, 2008). In Africa and most developing countries of the world, there is an interest in the study of medicinal plants and their curative properties and almost all the ingredients used in the formulation of medicinal remedies in Africa come from plant sources (Harborne et al., 1993). There is increasing scientific interest in the extraction and isolation of secondary metabolites from plants, as well as of biosynthetic, biochemical, pharmacological, chemotaxonomical, ecological, physiochemical and plant tissue culture studies as a result of modern isolation and pharmacological testing of purified substances rather than in the form of older galenical preparation (Silvia et al.,1998).
1.2 Background of the Use of Natural Product as Therapeutic Agent
Natural products are generally either of pre-biotic origin or originate from microbes, plants or animal sources (Nakanishi, 1999). As chemicals, natural products include such classes of compounds as terpenoids, polyketides, amino acids, peptides, proteins, carbohydrates, lipids, nucleic acids-bases, ribonucleic acids, deoxyribonucleic acids etc. Natural products are not just accidents or products of convenience of nature; they are a natural expression of the increase in complexity of organisms (Jarvis, 2000). Increase in
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natural sources to provide treatments for pain, palliatives, or curatives for a variety of maladies or recreational use reaches back to the earliest point of history.
Nature has provided many things for humankind over the years, including the tools for the first attempt at therapeutic intervention (Nakanishi, 1999). Neanderthal remains have been found to contain the remnants of medicinal herbs (Holt and Chandra, 2002). The Nei Ching is one of the earliest health science anthologist ever produced and dates back to the thirtieth century BC (Nakanishi, 1999). Some of the first records on the use of natural product in medicine were written in cuneiform in Mesopotamia on clay tablets and date to approximately 2600 BC (Cragg and Newman, 2001a). Indeed many of these agents continue to exist in one form or another to this day as treatment for inflammation, influenza, cough and parasitic infestation. Chinese herbs guide document the use of herbaceous plants as far back in time as 2000 BC (Holt and Chandra, 2002). For a variety of reasons, the interest in natural products continues to this very day. The first commercial pure natural product introduced for therapeutic use is generally considered to be the narcotic morphine, marketed by Merck in 1826 (Newman et al., 2000).
In recent years, a significant revival of interest in natural products as a potential source for new medicines has been observed among academia as well as pharmaceutical companies. Several modern drugs (~40% of the modern drugs in use) have been developed from natural products. More precisely, according to Cragg et al., 1997, 39% of the 520 new approved drugs between 1983 and 1994 were natural products or their derivatives, and 60–80% of antibacterial and anticancer drugs were from natural origins. In the year 2000, approximately 60% of all drugs in clinical trials for the multiplicity of
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cancers had natural origins. However, in most cases the term natural products refers to secondary metabolites, small molecules (mol wt <2000 amu) produced by an organism that are not strictly necessary for the survival of the organism. The study of botany in the area of medicine today is recognized as the most viable method of identifying new medicinal plants or refocusing on those earlier reports of bioactive constituents (Adjanohoun et al., 1991; Farnsworth, 1990). Natural products certainly play a crucial role in meeting this demand through the continued investigation of the world’s biodiversity, much of which remains unexplored (Cragg and Newman, 2001a).
Sometimes, however, a straightforward natural product isolation route, irrespective of bioactivity, is also applied, which results in the isolation of a number of natural compounds suitable for undergoing any bioactivity screening (Sarker and Lutfun, 2007). The most extensively used plant components can be classified into alkaloids, tannins, steroids, phenolic compounds, terpenoids and carbohydrates (Hill, 1952).
Environmental samples from soil and marine habitats will offer access to an untapped reservoir of genetic and metabolic diversity (Cragg and Newman, 2001b). China has emerged as a major producer of a number of quality medicinal plant products including coumarins, eucalyptus, peppermint, spearmint, sassafras and valerian in addition to its established listings (Trease and Evans, 2009). This is also true for nucleic acids isolated from symbiotic and endophytic microbes associated with terrestrial and marine macro organisms.
1.3 The Medicinal Uses of Plants
Diverse people and cultures throughout the world have employed herbal medicine to treat diseases throughout the history of mankind. This situation still persists in most developing countries where two-third of the people have no access to modern medicine.
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In the industrialized countries also, there is increase in the use of herbal drugs; in fact 25% of all prescribed drugs are substances derived from higher plants (Fransworth et al., 1985). Traditional medicine is now recognized by the World Health Organization (WHO) as a building block for primary health care (Akerele, 1998).
The medicinal use of herbs represents an important aspect of the history of medicine and has contributed immensely to the development of modern therapeutics (Akerele, 1998). Many modern day drugs are derived from plants; examples are reserpine, quinine, digoxine, morphine, codeine and host of others (Cannell, 2000).
1.4 Justification of the Research
The screening of plant extracts has had an impressive history of identifying active agents. The choice of the aerial part of Maesobotrya barteri as the plant parts of interest in this work was based on its vast medicinal importance among traditional medicine practitioners in Orokam, Benue state of Nigeria, other parts of the tropics, including West Africa. Maesobotrya barteri is used in these places to treat various ailments such as in the treatment of bruised or erupted skin, malaria, catarrh, vomiting, diarrhea, typhoid and eye pain (Dalziel, 1956). Therefore, there is reason for a scientific study to ascertain the medicinal potentials of this plant, isolate its active ingredient(s) and characterize them using usual organic analytical techniques.
1.5 Aim of the Research
The aim of this work is to:
investigate the ethnomedicinal claims of the aerial parts of Maesobotrya barteri, isolate and characterize some compounds(s) that may be responsible for the claimed ethno medicinal application.
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1.6 Objectives of the Research
The objective of this research are:
i. Phytochemical screening using the crude extracts of the solvents above.
ii. Antibacterial screening of the extracts.
iii. Antifungal screening of the extracts.
iv. Analytical separations involving several consecutive steps of chromatographic and purification techniques.
v. Verification of the purity of the isolated compound(s) and testing for antibacterial antifungal activities.
vi. Structural elucidation and characterization of the isolated compounds using available spectral techniques.
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