ABSTRACT
The plant Adenodolichos paniculatus which is used in ethno – medicine for the treatment
of blennorrhoea, diarrhea, dysentery, small pox, heart burn and wound dressing was
investigated for its chemical constituents and antimicrobial activity. The leaves of this
plant were subjected to microwave assisted extraction to obtain n – hexane, chloroform,
ethyl acetate and methanol extracts. The extracts were then subjected to preliminary
phytochemical screening which revealed the presence of alkaloids, anthraquinones,
flavonoids, reducing sugars, saponins, tannins and steroids/terpenes. Purification through
various column chromatography, the ethyl acetate extract led to the isolation of compounds
X1 (mixture of stigmasterol and β – sitosterol) and X2 (nonanoic acid) after spectral
analysis IR, 1H NMR and 13C NMR. Determination of inhibitory activity (sensitivity test)
using agar well diffusion method showed activity i.e. minimum inhibitory concentration
(MIC) against Staphylococcus aureus, Salmonella typhi, and Candida albicans ranging
from 12 – 50 mg/ml, Minimum Bactericidal Concentration/ Minimum Fungicidal
Concentration (MBC/MFC) ranging from 25 – 50 mg/ml, which is found to be
bacteriostatic against Salmonella typhi. Escherichia coli, Pseudomonas aeruginosa,
Salmonella typhi, Bacillus subtili showed no MBC (Bacteriostatic) for the methanol, ethyl
acetate, chloroform and n –hexane extract.
TABLE OF CONTENTS
Title Page ii
Declaration iii
Certification iv
Dedication v
Acknowledgement vi
Abstract vii
Table of Contents viii
List of Tables xi
List of Figures xii
List of Plates xiii
Abreviations xiv
CHAPTER ONE 1
1.0 INTRODUCTION 1
1.2 Statement of the Problem 2
1.3 Aim and Objectives of the Research 3
1.3.1 Aim 3
1.3.2 Objectives of the research 3
1.4 Research Justification 2
CHAPTER TWO 4
2.0 LITERATURE REVIEW 4
2.1 The Family Fabaceae 4
2.1.1 The Genus adenodolichos 4
2.2. Common Names 5
2.2.1 Uses 5
2.2.2 Morphology (description) 5
2.3 Pharmacological Products in Plants 6
2.3.1 Alkaloids 6
2.3.2 Tannins 8
2.3.3 Steroids 9
2.3.4 Phenolic compounds 10
2.3.5 Terpenoids 11
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2.4 Bacterial Agents 12
2.4.1 Staphylococcus 12
2.4.2 Bacillus subtilis 12
2.4.3 Escherichia coli 13
2.4.4 Salmonella 14
2.4.5 Pseudomonas aeruginosa 14
2.4.6 Fungi 15
2.5 Infectious Diseases 16
2.6 Phytochemistry of Adenodolichos paniculatus and other species in the genus. 17
2.7. Biological Activity of Adenodolichos paniculatus and other species in the genus 20
2.8. Microwave-assisted Extraction (MAE) 20
CHAPTER THREE 21
3.0 MATERIALS AND METHODS 21
3.1 Materials 21
3.1.1 Chemicals/Reagents 21
3.1.2 Equipment 21
3.2 Methods 21
3.2.1 Plant collection 21
3.2.2 Extraction 22
3.3.1 Test for reducing sugar 23
3.3.2 Test for anthraquinone glycosides. 23
3.3.3 Test for saponin glycosides 24
3.3.4 Test for steroids and terpenoids 24
3.3.5 Test for tannins 24
3.3.6 Test for alkaloids 25
3.3.7 Test for cardiac glycosides 25
3.3.8 Test for flavonoids 26
3.4 Antimicrobial Screening 26
3.4.1 Cultivation and standardization of test organisms 26
3.4.2 Preparation of extracts solution for antibacterial activity assay 27
3.4.3 Determination of Inhibitory activity (Sensitivity Test) of the extract using Agar
well diffusion method 27
3.4.4 Determination of Minimum Inhibitory Concentration (MIC) 28
x
3.4.5 Determination of minimum bactericidal concentration/minimum fungicidal
concentration (MBC/MFC) 28
3.5. Purification Techniques 29
3.5.1 Thin layer chromatography (TLC) 29
CHAPTER FOUR 32
4.0 RESULTS 32
4.1 Extraction 32
4.2 Phytochemical Screening 33
4.4 Melting point of X1 and X2 38
4.4.1 FTIR Analysis of X1 39
4.4.2 1H NMR (δppm, 400 MHz, CDCl3 ) of Compound X1 40
4.4.3 13C NMR (δppm, 400MHz, CDCl3) Spectral Analysis of Compound X1 41
4.5.2 Antimicrobial screening 47
4.6 Proposed Structures of Compound X1 and X2 51
CHAPTER FIVE 52
5.0 DISCUSSION 52
CHAPTER SIX 55
6.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS 56
6.1 Summary 56
6.2 Conclusion 56
6.3 Recommendations 57
References 58
Appendix 66
CHAPTER ONE
1.0 INTRODUCTION
There are a large number of medicinal plants whose scientific importance has not been
explored. All over the world, plants have served as the richest source of raw materials for
traditional as well as modern medicine, particularly in Africa and Asia (Tsakala et al.,
2006). Medicinal plants contain some compounds which have definite physiological
actions on animals and humans. These are called secondary metabolite, and include
tannins, alkaloids, carbohydrates, terpenoids, steroids and flavonoids (Doge et al., 2005).
Medicinal plants are useful for healing as well as for curing of human diseases because of
the presence of phytochemical constituents (Ododo et al., 2016). Phytochemicals are
naturally occurring in medicinal plants leaves, stem bark, fruits and roots where they serve
as defense mechanism and protect from various diseases (Ahmad et al., 2006). Traditional
medicine by means of plant extracts continues to provide health treatment for over 80% of
the world‘s population, especially in the developing world (WHO, 2002). The rapid
increase of bacterial drug resistance is becoming a worldwide problem. There is an urgent
need to develop new antimicrobial drugs with better/improved activity in order to
overcome bacterial drug resistance (Afolayan et al., 2008). The continual search for, and
the interest in natural plant products, for use as medicines has acted as the catalyst for
exploring methodologies involved in obtaining the required plant materials and hence
searching into their constituents for possible medicinal usage (Ibrahim et al., 2013). The
identification and isolation of such active compounds makes it a more effective therapeutic
application. It prevents consumers from taking certain plants that have no medicinal value
or are poisonous to them (Ozolua et al., 2009). The use of plants remains undisputed
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despite the advent of the Pharmaceutical Chemistry during the early twentieth century,
which brought with it the ability to synthesize an enormous variety of drug molecules and
there by allowed the treatment of previously incurable and/or life-threatening diseases
(Dutta, 2013). Although chemically synthesized drug have also gained popularity and
became the basis of Pharmaceutical industries. Over the years, however, synthetic drugs
have been hijacked by unwanted side-effects, toxicity, high cost and inefficiency, among
other problems. Also the search for new drugs against a variety of illnesses through
chemical synthesis and other modern approaches has not been too successful. These
factors, as well as the emergence of new infectious diseases, the proliferation of disorders
such as cancer and growing multidrug resistance in pathogenic microorganisms, have
prompted renewed interest in the research and discovery of potential drug molecules from
medicinal plants (Arjun Patra, et. al, 2010).
1.2 Statement of the Problem
The rapid increase of bacterial drug resistance is becoming a worldwide problem. There is
an urgent need to develop new antimicrobial drugs with better/improved activity in order
to overcome bacterial drug resistance (Afolayan et al., 2008). There are a large number of
medicinal plants whose scientific importance has not been explored. All over the world,
plants have served as the richest source of raw materials for traditional as well as modern
medicine, particularly in Africa and Asia (Tsakala et al., 2006).
1.3 Research Justification
This work was conducted because of the need to search for newer and more potent
antimicrobial agents to whom there is lesser resistance in a world where pathogenic
microorganisms are building and increasing resistance to drugs. The need to scientifically
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establish or otherwise comfirm the ethnomedicinal claims on the plant. The World Health
Organization (WHO, 2005) estimates that 4 billion people, or 80 percent of the world‘s
population, use herbal medicine for some aspect of primary health care. It is against this
background that, Adenodolichos paniculatus which is a plant used extensively as herbal
remedy in some parts of Nigeria and West Africa, is being investigated. To the best of our
knowledge, there is no reported work on the isolation and characterization of some
bioactive compounds from the plant.
1.4 Aim and Objectives of the Research
1.4.1 Aim
The aim of this project work is to isolate and characterize some bioactive compounds from
the leaves extract of Adenodolichos paniculatus and to evaluate their biological activity
1.4.2 Objectives of the research
This aim will be achieved through the following objectives:
i. Phytochemical and antimicrobial screening of the crude extracts.
ii. Separation, purification and isolation of the bioactive constituents using
chromatographic techniques ( TLC, column chromatography)
iii. Spectroscopic analysis ( NMR FTIR)
iv. Characterization and structural elucidation of the isolated compound(s) using
spectroscopic techniques.
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