ABSTRACT
Microorganisms have developed and are still developing resistance to most antimicrobial agents in use. Thus there is a need to search for new drugs that are of organic origin from medicinal plants, because they are relatively safer than synthetic alternatives, offering profound therapeutic benefits at affordable costs, and serve as a primary source of new medicine and lead compounds for the development of new drugs against various diseases. The percentage recovery of 3.67 % for n-butanol extract (MBS) from the microwave-assisted extraction was recorded whereas the extracts obtained from the target extraction; MBC1, MBC2 and MBC3 were found to have the percentage recovery of 0.15%, 0.28% and 0.20 %, respectively. Phytochemical investigation of n-butanol extract from the root bark of Ficus sycomorus showed the occurrence of steroids, triterpenes, flavonoids, alkaloids, and tannins. Detailed chromatographic techniques performed on the n-butanol extract led to the successful isolation of two triterpenoid compounds; lupeol (MB01) and lupeol acetate (MB03). Compound (MB01) was isolated as a white crystalline solid with melting point of 213 – 215°C and compound (MB03) was isolated as an off-white powder with melting point of 216 – 218°C. Antimicrobial activity study of the isolated compounds using agar well diffusion method, against Escherichia coli, Samonella typhi, Bacillus subtilis and Staphylococcus aureus, with inhibition zones ranging from 11-18 mm as compared with ciprofloxacin (standard drug) with inhibition zones ranging from 26-31 mm, showed that the compounds are potential source of antibacterial agents.
TABLE OF CONTENTS
er page i
Fly leaf ii
Title page iii
Declaration iv
Certification v
Dedication vi
Acknowledgement vii
Abstract viii
Table of Contents ix
List of Tables xiii
List of Figures xiv
List of Plates xv
List of Appendices xvi
List of Abbreviations xvii
CHAPTER ONE 1
1.0 INTRODUCTION 1
1.1 Statement of Research Problem 2
1.2 Justification 3
1.3 Aim 3
1.4 Objectives of the Study 4
CHAPTER TWO 5
2.0 LITERATURE REVIEW 5
2.1 Extraction 5
2.2 The Genus Ficus 7
2.2.1 Ethnomedical uses of the genus Ficus 7
2.2.2 Biological activities found in the genus Ficus 9
2.2.3 Phytochemistry of the genus Ficus 9
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2.3 Ficus sycomor 16
2.3.1 Ethnomedicinal uses of Ficus sycomorus 16
2.3.2 Pharmacological activities of Ficus sycomorus 16
2.3.3 Phytochemistry of Ficus sycomorus 17
2.3.4 Taxonomic description 20
2.4 Microorganisms and their Association with Human Host 22
2.4.1 Escherichia coli 22
2.4.2 Salmonellae 22
2.4.3 Staphylococci 23
2.4.4 Bacillaceae 24
CHAPTER THREE 25
3.0 MATERIALS AND METHODS 25
3.1 Materials 25
3.1.2 Equipment 25
3.1.3 Test organisms for antimicrobial study 25
3.2 Methodology 26
3.2.1 Collection of Plant Material 26
3.2.2 Extraction 26
3.2.3 Preliminary Phytochemical Screening of the Plant Extract 29
3.2.4 Methods of isolation/purification of bioactive compound 30
3.2.5 Determination of Antimicrobial Activity of the plant extract and the isolated compound 33
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CHAPTER FOUR 36
4.0 RESULT 36
4.1 Result of Extraction of Plant Material 37
4.2 Result of Phytochemical Screening of the Extract/fractions 38
4.3 Result of Chromatographic Separation 39
4.3.1 TLC Analysis of the column fractions and the isolated compounds 39
4.3.2: Chemical test on the Isolated Compound 43
4.3.3: Physical properties of the Isolated Compounds 44
4.3.4 Spectroscopic Analysis of the Isolated Compounds 45
4.3.5: Summary of Spectral Data for the Isolated Compounds and Comparison with Literature Data 51
4.4 Result of Antimicrobial Activity of the Plant Extract/Fractions and the Isolated Compounds 53
4.4.1 Result of zones of Inhibition of the extract/fractions 53
4.4.2 Result of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the extract/fractions 54
4.4.3 Result of zones of Inhibition of the Isolated Compound 55
4.4.4: Result of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the isolated compound 57
CHAPTER FIVE 58
5.0 DISCUSSION 58
5.1 Plant Extraction 58
5.2 Phytochemical Profiling 58
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5.3 Isolation, Purification, Characterization and Biological Activity of the Isolated Compounds 58
5.4 Antimicrobial Screening of the Extract/Fraction and the Isolated Compounds 60
CHAPTER SIX 63
6.0 CONCLUSION AND RECOMMENDATIONS 63
6.1 Conclusion 63
6.2 Recommendations 63
References 65
Appendices 77
xiii
CHAPTER ONE
1.0 INTRODUCTION
Natural products, for ages, have been used as medicines and today, they continue to be a reservoir of potential drugs (Lamottke et al., 2011). Medicinal plants are essential and resorted to by more than 70 % of the world’s population that do not have access to Western medicine (WHO, 2008). The primary benefits of using plant-derived medicine are that they are relatively safer than synthetic alternatives and offer profound therapeutic benefits at affordable costs (Iwu, et al., 1999). There is an increasing awareness towards the use of herbal drugs and medicinal plant in the world. Germany, for example, is an active country among developed countries on herbal drug research, and France, where herbal extracts are sold as prescription drugs (Subramoniam, 2014).
On the other hand, natural products provide new medicine and lead compounds for the development of new drugs against various ailments. Out of the 1,135 new drugs approved from 1981 to 2010, 50 % were of natural product origin (natural, derivatives and analogues) (Cragg 2007; Newman and Cragg 2012). A more recent example is the development of artesunate (antimalarial and profound anticancer agent) a derivative of artemisinin from the Chinese medicinal plant Artemisia annua L. (Krusche et al., 2013) and the widely used breast cancer drug, paclitaxel (Taxol), isolated from the bark of the Pacific Yew, Taxus brevifolia (Dewick, 2009).
The presence of secondary metabolites in natural products accounts for their contribution towards drug development. There is wide consensus that the potential for new natural products is not exhausted, therefore natural products remain an important source for the lead in drug discovery (Chin et al., 2006; Zaku et al., 2009).
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The extraction method is one of the most important steps of any medicinal plant study in the processing of the bioactive constituents from the plant materials (Azwanida, 2015). The complete phytochemical profile of a given plant species can be investigated by fractionating the crude extract in order to separate the main classes of constituent from each other, known as target extraction, prior to chromatographic analysis.
The microwave-assisted extraction (MAE) is considered a selective method that favours polar molecules and solvents with high dielectric constants, for extracting bioactive constituents from the plant materials using microwave energy (Pare et al., 1994).
1.1 Statement of Research Problem
Plant materials have been the primary sources of medicine since early civilization (Subramoniam, 2014) and they continue to provide new medicine and lead compounds for the development of new drugs against various ailments (Parekh and Chanda, 2007). However, since these traditional herbal medicines were commonly prepared from crude materials, there are many questions concerning their specific medicinal effects and reproducibility, mechanism of action, and the identity of the active ingredients (Kim et al., 2010). Therefore, current some researches have focused on the specific components of an active herb rather than on the herb in its entirety. Several bioactive chemical constituents such as alkaloids, tannins, flavonoids and phenolic compounds have been reported in Ficus sycomorus (Sandabe et al., 2006; Mudi et al., 2015) and it is an important medicinal plant used traditionally for the treatment of different conditions including gastrointestinal, respiratory, cardiovascular disorders and inflammation (Hedberg and Staugard, 1989; Arnold and Gulumian, 2002; Sandabe et al., 2006; Hassan et al., 2007). However, research on this plant is scanty (Galil and Esiskowitch,
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1974; Sandabe et al., 2006) and to the best of our knowledge, no phytochemicals have been characterized from the root bark of this plant before now.
1.2 Justification
Most of the targeted microorganisms have developed resistance to most of the antimicrobial agents in use, thus there is need for search for new ones that are of organic origin (Larhsini et al., 2001). There are many advantages of using antimicrobial compounds from medicinal plants such as being safer and cheaper compared to the synthetic alternatives (Iwu, et al., 1999), acceptance due to long history of use, and being renewable in nature (Gur et al., 2006) and also higher plants represent a potential source of novel antibiotic prototypes (Parekh and Chanda, 2007). To the best of our knowledge, there is no reported work on the establishment of the antimicrobial study based on the individual classes of phytochemical constituents of the plant, Ficus Sycomorus, as well as characterization of bioactive compounds from the part of the plant under study.
1.3 Aim
The aim of this research is to carryout MAE, isolate, characterize and investigate the biological activity of phytochemical components from the root bark of Ficus sycomorus.
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1.4 Objectives of the Study
The aim will be achieved through the following objectives:
i. MAE and Target extraction on the plant material.
ii. Preliminary Phytochemical screening of the extracts.
iii. Separation, purification and isolation of the bioactive constituents using chromatographic techniques.
iv. Characterization and structural elucidation of the isolated compound(s) using spectroscopic techniques such as:
– Infrared spectroscopy (IR);
– Nuclear Magnetic Resonance (1H NMR, 13C NMR and 2D NMR)
v. Determination of the antimicrobial activity of the plant extracts and the isolated compound.
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