ABSTRACT
Ampelocissus grantii (Baker and Planch), Cissus crinita (Planch) and Cissus sokodensis (Gilg and Brandt) are plants that belong to the family Vitaceae and are used in traditional medicine in northern Nigeria for the management of cancers and infectious diseases. The phytochemistry of these plants has not yet been reported. Therefore, the plants have been deemed desirable for phytochemical investigation to identify the possible active compounds that may be contributing to these medicinal therapies claimed by the people using them. The plants‟ rhizomes, after collection, identification, air-drying and pulverisation, were subjected to cold extraction (maceration) using hexane, dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH). Crude extracts obtained were phytochemically screened and chromatographed, mainly by column chromatography on silica gel. Standard antimicrobial method was employed to determine the minimum inhibitory concentration (MIC) of both the crude extracts and the isolates using clinical bacteria and fungi isolates: Staphylococcus aureus, Bacillus subtilis (Gram positive bacteria), Escherichia coli, Pseudomonas aeruginosa (Gram negative bacteria), Crytococcus neoformans,Candida albicans, Candida tropicalis, Aspergillus niger (fungi).The pure compounds were analysed for structure elucidations, using spectroscopic techniques, majorly 1D and 2D NMR experiments. Preliminary phytochemical screening of the crude plant extracts revealed the presence of steroids, terpenoids, flavonoids, tannins, saponins in both Cissus sokodensis and Cissus crinita. In addition to these compounds, anthraquinones were present in Ampelocissus grantii. Results of antimicrobial studies showed moderate to good activity against the organisms tested. Generally both the crude extracts and the pure compounds isolated recorded higher activities against the fungi. The MIC values of the crude extracts against the fungi ranged from 25 μg/ml to 200 μg/ml, 25 μg/ml to 100 μg/ml and 12.5 μg/ml to 100 μg/ml for Ampelocissus grantii, Cissus crinita and Cissus sokodensis respectively. The highest activity was recorded from Cissus
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sokodensis’s methanol extract against Candida albicans (MIC: 12.5 μg/ml).The respective MIC value against the bacteria for Ampelocissus grantii, Cissus crinita and Cissus sokodensis ranged from 25 μg/ml to 200 μg/ml, 100 μg/ml to 200 μg/ml and 100 μg/ml to 200 μg/ml. Highest MIC was observed from Ampelocissus grantii ethyl acetate extract against Escherichia coli (25 μg/ml). The pure compounds recorded higher activity against both the fungi and the bacteria compared to the crude extracts. The antifungal activity (MIC: 6.25-50 μg/ml) of the pure compounds was higher than the antibacterial activity (MIC: 12.5-50 μg/ml). Highest activity (MIC: 6.25 μg/ml) was recorded against Candida albicans and Aspergillus niger. Nine chemical compounds were isolated from the plants: β-sitosterol, betulin, 15-hydroxymethyl-16-methylheptadecylferulate and 3-hydroxymethyl-5-methyl-16-oxoheptadecylferulate from Ampelocissus grantii, β-sitosterol, oleanolic acid and 2,3,5-trihydroxymethylbenzoate-4-methylene-α-D-glucopyranose from Cissus sokodensis and two, β-sitosterol and 2,3,5-trihydroxymethylbenzoate-α-D-glucopyranose from Cissus crinita. Four out of these compounds: 15-hydroxymethyl-16-methylheptadecylferulate, 3-hydroxymethyl-5-methyl-16-oxoheptadecylferulate, 2, 3, 5-trihydroxymethylbenzoate-4-methylene-α-D-glucopyranose and 2, 3, 5-trihydroxymethylbenzoate-α-D-glucopyranose, are novel. Literature has shown that all the known compounds isolated have anticancer properties; further the newly isolated molecules have moieties in their structures with anticancer potentialities. Hence, the studied plants contain compounds that have anticancer property. The presence of the compounds isolated suggests that the plants contain chemical compounds that could possess both anticancer and antimicrobial properties. Therefore, these compounds may have been responsible for the anticancer and antimicrobial effects the plants have been said to possess in ethnomedicine and hence the folklore usage of the plants in the management of cancers and bacterial/fungi diseases are said to be validated.
TABLE OF CONTENTS
Title Page
i
Cover Page
ii
Declaration
iii
Certification
iv
Dedication
v
Acknowledgement
vi
Abstract
vii
Table of Contents
ix
List of Tables
xvi
List of Figures
xvii
List of Abbreviations and Acronyms
xxi
CHAPTER ONE
1.0
INTRODUCTION
1
1.1
Natural Products and Medicine
1
1.2
Statement of the Research Problem
2
1.3
Justification of the Study
3
1.4
Aim and Objectives of the Research
3
CHAPTER TWO
2.0
LITERATURE REVIEW
5
2.1
Phytochemicals and Medicinal Plants
5
x
2.1
Description of Plant Family: Vitaceae
7
2.2
Chemical Constituents of Some Species of Plant Family: Vitaceae
9
2.2.1
Parthenocissus tricuspidata (Planch.)
9
2.2.2
Cissus cornifolia
9
2.2.3
Cissus quadrangularis (Linn)
9
2.2.4
Cissus adnata (Roxb.)
10
2.2.5
Cissus rheifolia (Planch.)
10
2.2.6
Cissus rheifolia (Planch.)
10
2.2.7
Vitis vinifera
10
2.2.8
Cissus populnea (Guill. & Perr.)
10
2.2.9
Cissus arnottiana
10
2.2.10
Ampelocissus species
11
2.2.11
Cissus crinita (Planch)
11
2.2.12
Cissus sokodensis (Gilg & Brandt)
11
2.2.13
Ampelocissus grantii (Baker) Planch
11
2.3
Pharmacological Activity of Some Viteceae Family Species (Vitis, Cissus and Ampelocissus Species)
11
2.3.1
Cissus cornifolia (Bak.) Planch
11
2.3.2
Cissus quadrangularis (Linn)
11
2.3.3
Vitis vinifera (L.)
12
2.3.4
Ampelocissus grantii (Baker) Planch
13
2.4
Local Uses of Some Vitaceae Species
13
xi
2.4.1
Vitis sylvestris.L
13
2.4.2
Cissus populnea Guill. & Perr
13
2.4.3
Cissus cornifolia (Bak.) Planch
13
2.4.4
Cissus quadrangularis (Linn)
13
2.4.5
Ampelocissus grantii (Bak.) Planch
14
2.4.6
Cissus crinita (Planch)
14
2.4.7
Cissus sokodensis (Gilg & Brandt)
14
2.4.8
Cissus aralioides (Welw. Ex Baker) Planch
14
2.4.9
Cissus integrifolia (Baker) Planch
14
2.4.10
Vitis sylvestris L.
15
2.4.11
Cissus populnea Guill. & Perr
15
2.5
Types of Natural Products
15
2.5.1
Natural products from microorganisms
15
2.5.2
Natural products from marine organisms
17
2.5.3
Natural products from animal sources
17
2.5.4
Natural products from plant sources
20
2.5.5
Plant-based anticancer drugs
20
CHAPTER THREE
3.0
MATERIALS AND METHODS
28
3.1
Chemicals/Reagents
28
3.1.1
Equipments/materials
28
3.1.2
Test organisms
28
3.2
Plant Material
28
3.3
Extraction
29
3.4
Preliminary Phytochemical Screening
29
xii
3.4.1
Test for sterols/terpenes
29
3.4.2
Test for flavonoids
30
3.4.3
Test for alkaloids
30
3.4.4
Test for tannins
31
3.4.5
Test for anthraquinones
31
3.4.6
Test for saponins
31
3.5
Chromatoghraphic Seperation, Purification and Characterisation of Pure Compounds
31
3.5.1
Isolation and purification of G1 and G2
32
3.5.2
Melting point determination of G1 and G2
32
3.5.3
Spectroscopic analyses of G1 and G2
32
3.5.4
Isolation and purification of G3
33
3.5.5
Melting point of determination G3
33
3.5.6
Spectroscopic analyses of G3
33
3.5.7
Isolation and purification of G4
33
3.5.8
Melting point determination of G4
34
3.5.9
Spectroscopic analyses of G4
34
3.5.10
Isolation and purification of S1 and S2
34
3.5.11
Melting point determination of S1 and S2
35
3.5.12
Spectroscopic analyses of S1 and S2
35
3.5.13
Isolation and purification of S3
35
3.5.14
Melting point determination of S3
36
3.5.15
Spectroscopic analyses of S3
36
3.5.16
Isolation and purification of C1 and C2
36
3.5.17
Melting point determination of C1 and C2
37
xiii
3.5.18
Spectroscopic analyses of C1 and C2
37
3.6
Antimicrobial Studies of Extracts and Pure Compounds
37
3.6.1
Test organisms
38
3.6.2
Preparation of medium
38
3.6.3
Preparation of the plant extracts and pure compounds stock solution and working standard
38
3.6.4
Preparation of inoculums
38
3.6.5
Determination of minimum inhibitory concentration
39
CHAPTER FOUR
4.0
RESULTS
40
4.1
Extraction
40
4.2
Phytochemical Screening
40
4.3
Isolation and purification of G1 and G2
40
4.3.1
Nuclear magnetic resonance spectra Data of G1
40
4.3.2
Nuclear magnetic resonance spectra Data of G2
41
4.3.3
Infra red spectrum data of G1
41
4.3.4
Infra red spectrum data of G2
41
4.4
Isolation and Purification of G3
41
4.4.1
Nuclear magnetic resonance spectra data of G3
41
4.4.2
Infra red spectrum data of G3
42
4.5
Isolation and Purification of G4
42
4.5.1
Nuclear magnetic resonance spectra data of G4
42
4.5.2
Infra red spectrum data of G4
42
4.6
Isolation and Purification of S1 and S2
42
4.6.1
Nuclear magnetic resonance spectra data of S1
43
xiv
5.0
DISCUSSION
128
5.1
Extraction of the Plant Rhizomes
128
5.2
Phytochemical Screening of the Plants
128
5.3
Isolation and Characterization of Pure Compounds from A. grantii Rhizome
129
5.3.1
Isolation and characterization of G1
129
5.3.2
Isolation and characterization of G2
133
5.3.3
Isolation and characterization of G3
137
5.3.4
Isolation and characterization of G4
142
5.4
Isolation Characterization of Pure Compounds from C. sokodensis Rhizome
147
5.4.1
Isolation and characterization of S1
147
4.6.2
Nuclear magnetic resonance spectra data of S2
43
4.6.3
Infra red spectrum data of S1
43
4.6.4
Infra red spectrum data of S2
43
4.7
Isolation and Purification of S3
44
4.7.1
Nuclear magnetic resonance spectra data of S3
44
4.7.2
Infra red spectrum data of S3
44
4.8
Isolation and Purification of C1 and C2
44
4.8.1
Nuclear magnetic resonance spectra data of C1
44
4.8.2
Nuclear magnetic resonance spectra data of C2
45
4.8.3
Infra red spectrum data of C1
45
4.8.4
Infra red spectrum data of C2
45
4.9
Results of Antimicrobial Studies of the Plant Extracts and the Pure Compounds
45
CHAPTER FIVE
xv
5.4.2
Isolation and characterization of S2
149
5.4.3
Isolation and characterization of S3
152
5.5
Isolation, Purification and Characterization of Pure Compounds from
C. crinita Rhizome
157
5.5.1
Isolation and characterization of C1
157
5.5.2
Isolation and characterization of C2
158
5.6
Antimicrobial Studies of the Plant Extracts and the Isolated Compounds
163
5.7
The Anticancer Potentialities of the Compounds Isolated from the Plants
166
CHAPTER SIX
6.0
SUMMARY, CONCLUSION AND RECOMMENDATIONS
169
6.1
Summary
169
6.2
Conclusion
171
6.3
Recommendations
171
REFERENCES
173
xvi
CHAPTER ONE
1.0 INTRODUCTION 1.1 Natural Products and Medicine Over the centuries, the use of medicinal herbs has become an important part of daily life of humans and despite the progress in modern system of medicine; plants are one of the most important sources of medicines (Mohd et al., 2012). This is because chemical compounds from natural sources (like plant) have been elaborated within living systems, thus are often perceived as showing more “drug-likeness and biological friendliness than totally synthetic molecules,” (Koehn, 2005a) making them good candidates for further drug developments (Balunas and Kinghorn, 2005; Drahl et al., 2005). Many people use herbal concoctions with the assumption that such is safe and effective. For people with chronic diseases, combining herbal medicine with conventional medicine is a common practice. It is expected that there may be synergistic effects between the conventional drugs and the herbal remedies. The use of indigenous medicinal plants is not based on science and is mostly unregulated and as such varies considerably from place to place.There is often no standard dosage and the safety of such practice remains unclear (Nyika, 2009).
In traditional practice, several plants are often used in combinations and the activity may be a result of either additive or synergistic effects. Many medicinal plants are the focus of biological screening and the crude extracts are screened for in vitro or in vivo activity or both. In some cases single active constituents for a particular action have been successfully identified. Phytochemical and toxicological studies can identify toxic components in order to assess and possibly eliminate or reduce harmful side effects of the extracts (Nyika, 2009).There is still a great need for phytochemical and pharmacological research of
2
medicinal plants in order to improve the efficacy and reduce potentially harmful side effects that may be associated with it. Although there are many clinically approved anticancer and antimicrobial drugs, both of synthetic and natural (microbial and plant) origin (Fansworth, 1985; Chabner, 2005). However, much progress needs to be made to overcome the problems of resistance to and toxicity of existing cancer and pathogenic microbial chemotherapeutic agents. Medicinal plants have been shown to possess bioactive compounds such as polyphenolics, vinca alkaloids, podophytotoxin derivatives and taxol derivatives which reduce the incidence of cancers; and different classes of compounds for example: alkaloids, some flavonoids, quinones, xanthones and terpenes that serve as antimicrobial agents. It is in view of these that the three (3) plant candidates that have been used for the treatment of cancers (e.g leukemia and breast cancer) and microorganisms related diseases by the natives, especially in northern Nigeria, were selected for this research. Furthermore, this study seeks to isolate and identify novel substances responsible for the acclaimed efficacy in curing cancer and that could also serve as antimicrobial agents with less toxicity or side effects. 1.2 Statement of the Research Problem Developing countries like Nigeria are bedevilled by inadequacy of health care facilities, poor accessibility, high poverty level, lack of trained man power and often, low demand for conventional health care system. An estimated 80% of the populace relies on ethno-medicinal practices, most of which are herbal preparations (Omole-Ohonsi and Aiyedun, 2013) for their primary health care needs.
3
The emergence and wide spread of pathogenic microorganism resistant strains is on the increase on daily basis (Tijjani et al., 2011). Beside this, diseases like cancers have continued to be of great concerned to man. This is because there is no „total cure for cancer‟ up to now once it reaches malignant stage (Jemal, 2011). Furthermore, drugs used for the treatment of cancer and also serve as antimicrobial agents are associated in one way or the other with side effects or toxicity. These have carried the attention of scientists and made them continuously investigate for possible solutions to the stated menace. 1.3 Justification of the Study Medicinal plants have proven to be reservoirs for molecules such as alkaloids, flavonoids and saponins that showed wide range pharmacological activities.Therefore, there is continuous need to search for plants to source novel and cost effective molecules with less or no toxicity capable of bringing total cure for diseases with high mortality rate like cancer and ailments caused by pathogenic microorganisms. 1.4 Aim and Objectives of the Research The aim of the research is to seek scientific validation of the local use of the plant candidates (rhizomes of Ampelocissus grantii, Cissus crinita and Cissus sokodensis) as anticancer as well as antimicrobial agents in ethnomedicine and possible discovery of new medicinal entities from the natural sources. The stated aim will be achieved through the following objectives. I. Extraction of the chemical compounds from the plants using appropriate solvents. II. Phytochemical screening of the plants extracts.
III. Determination of the biological activities of the extracts of each of the species in line with the ethnomedicinal uses of the plants.
4
IV. Isolation of the novel chemical compounds from the selected plants. V. Identification of the chemical compounds that can serve as anticancer and antimicrobial agents. VI. Isolation of any other bioactive compound(s) from the plants and VII. Characterization and elucidation of the chemical structures of the compounds isolated.
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