ABSTRACT
Phytochemical studies, antimicrobial and antituberculosis screenings of extracts from the leafs of Clerodendrum capitatum, Heeria insignis and stem bark of Psorospermum senegalense were carried out. The phytochemical studies of the three plants revealed the presence of carbohydrates, cardiac glycosides, glycoside, saponins, streroids, triterpenes,
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flavanoids and tannins. The antimicrobial screening of the hexane , dichloromethane, ethyl acetate and methanol extracts of the three plants showed that they were active against most of the test microorganisms namely Shigella dysenteriae, Salmonella typhi, Corynebacterium ulcerans, Klebsiella pneumoniae, Staphylococcus aureus, Methicillin resistant Staphylococcus aureus, Proteus mirabilis, Streptococcus pneumoniae, Proteus vulgaris, Vancomycin resistant enterococci, Bacillus subtillis, Escherichia coli, Pseudomonas flourescense, Streptococcus pyogenes, Enterobacter specie, Streptococcus feacalis, Pseudomonas aeruginosa, Proteus rettgeris, Candida tropicalis, Candida pseudotropicalis, Candida krusei, Candida albicans and Candida stellatoid. However, the ethyl acetate extract showed the highest activity of all the extracts. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) of the extracts were determined. Antituberculosis screening of the hexane, dichloromethane, ethyl acetate and methanol extracts against Mycobacteria bovis showed that dichloromethane extracts of C. capitatum and H. insignis were most active while only the ethyl acetate extract of P.senegalense was active. Heeria insignis was the most active of the three plants. Chromatographic separation of the dichloromethane extract of C. capitatum and H. insignis and ethyl acetate extract of P. senegalense yielded six chemical substances which were characterized using 1-D and 2-D NMR spectra to be 3-hydroxylanost-7-en-29-carboxylic acid (C1), Betulin (C2 & H1), 3-hydroxy-7-lanostene (H3), a yet to be identified compound (H4) and α-amyrin (P2). Antimicrobial studies of the isolated compounds revealed that they were active against most of the test microorganisms. S. dysenteriae was the most sensitive to all the isolated compounds with MIC of 62.5 μg/mL and MBC of 125 μg/mL. Antituberculosis evaluation of the compounds showed that they were all active against Mycobacteria bovis with H3 being the most active with MIC of 125 μg/mL. Findings from this work clearly shows that these plants have potentials that can be explored in the search for anti-TB drugs from
TABLE OF CONTENTS
Title Page
i
Cover Page
ii
Declaration
iii
Certification
iv
Dedication
v
Acknowledgement
vi
ix
Abstract
vii
Table of Contents
ix
List of Tables
xvi
List of Figures
xviii
List of Plates
xx
List of Abbreviations and Acronyms
xxi
CHAPTER ONE
1.0
INTRODUCTION
1
1.1
Medicinal Plants
1
1.2
Natural Products as Leads in Novel and Active Chemotypes
3
1.3
Statement of Research Problem
4
1.4 1.5
Justification Aim of the Research
4 5
1.6
Objectives of Research
5
CHAPTER TWO
2.0
LITERATURE REVIEW
6
2.1 2.1.1
Tuberculosis Symptoms of tuberculosis
6 6
2.1.2
Treatment of tuberculosis
6
2.1.3
Epidemiology
7
2.2
Anti-Tubercular Plants
8
2.3
The Verbenaceae Family
11
x
2.3.1
The genus Clerodendrum
11
2.3.2
Clerodendrum capitatum
12
2.3.3
Medicinal uses of Clerodendrum capitatum
13
2.3.4
Pharmacological investigation of members of Clerodendrum genus
13
2.3.5
Some compounds isolated from Clerodendrum genus
16
2.4
The Anacardiaceae Family
22
2.4.1
The genus Heeria
22
2.4.2
Heeria insignis
23
2.4.3
Medicinal uses of Heeria insignis
24
2.4.4
Pharmacological investigation of members of Heeria genus
24
2.4.5
Some compounds isolated from Heeria genus
26
2.5
The Guttiferae Family
28
2.5.1
The genus Psorospermum
28
2.5.2
Psorospermum senegalense Spach
29
2.5.3
Medicinal uses of Psorospermum senegalense
30
2.5.4
Pharmacological investigation of members of Psorospermum genus
30
2.5.5
Some compounds isolated from Psorospermum genus
32
CHAPTER THREE
3.0
MATERIALS AND METHODS
36
3.1
Materials
36
3.1.1
Solvents/Reagents
36
3.1.2
Equipments
36
3.1.3
Plant material
36
3.2
Extraction of Plant Material
37
xi
3.3
Preliminary Phytochemical Screening
37
3.3.1
Test for carbohydrates (Molischs’ test)
37
3.3.2
Test for tannins (ferric chloride test)
37
3.3.3
Test for flavonoids (Shinoda test)
38
3.3.4
Test for anthraquinones (free anthraquinones)
38
3.3.5
Test for saponins (frothing test)
39
3.3.6
Test for glycoside (FeCl3 test)
39
3.3.7
Test for cardiac glycoside (Kella-Killani test)
39
3.3.8
Test for steroids/terpenes (Liebermann-Buchard test)
39
3.3.9
Test for alkaloids
40
3.4
Antimicrobial Activity Studies on Extracts and Isolates
40
3.4.1
Test organisms
40
3.4.2
Preparation of the plants extracts
41
3.4.3
Preparation of culture media
41
3.4.4
Antimicrobial sensitivity testing
41
3.4.5
Minimum inhibitory concentration (MIC)
42
3.4.6
Minimum bactericidal concentration & fungicidal concentration (MBC/MFC)
42
3.5
Antibacterial Assay
43
3.5.1
Extract preparation
43
3.5.2
Preparation of Mycobacterium bovis (BCG)
43
3.5.3
Antituberculosis screening
43
3.6
Chromatographic Procedure
44
3.6.1
Thin layer chromatography (TLC)
44
3.6.2
Column chromatography
44
xii
3.7
Chromatographic Separation
45
3.7.1
Column chromatography of dichloromethane extract of H. insignis
45
3.7.2
Preparative thin layer chromatography of fraction HF417-47
45
3.7.3
Column chromatography of dichloromethane extract of C. capitatum
45
3.7.4
Preparative thin layer chromatography of fraction CF38-15
45
3.7.5
Preparative thin layer chromatography of Fraction CF416-18
46
3.7.6
Column chromatography of dichloromethane fraction of P. senegalense
46
3.7.7
Preparative thin layer chromatography of fraction PF6
47
3.8
Melting Point Determination
47
3.9
Spectral Analyses
47
CHAPTER FOUR
48
4.0
RESULTS
48
4.1
Result of Extraction
52
4.2
Result of Phytochemical Screening
53
4.3
Result of Antimicrobial Activity of the Plant Extracts
54
4.3.1
Result of zones of inhibition
54
4.3.2
Result of minimum inhibitory concentration (MIC)
55
4.3.3
Result of minimum bactericidal/fungicidal concentration (MBC)/(MFC)
56
4.4
Result of Antituberculosis Activity
57
4.5
Result of Chromatographic Separation
58
4.5.1
Thin layer chromatography of the dichloromethane extract of C. capitatum
58
4.5.2
Column chromatography of dichloromethane extract of C. capitatum
59
4.5.3
Thin layer chromatography of the dichloromethane extract of H. insignis
60
xiii
5.0
DISCUSSION
106
5.1
Extraction of the Leaves of H. insignis, C. capitatum and Stem Bark of P.senegalense
106
5.2
Phytochemical Screening of the Leaves of H. insignis, C. capitatum and Stem Bark of P. Senegalense
106
5.3
Antimicrobial Screening of the Leaves of H. insignis, C. capitatum and Stem bark of P. senegalense
107
5.3.1
Antimicrobial screening of the leaves of C. capitatum
107
5.3.2
Antimicrobial screening of the leaves of H. insignis
108
5.3.3
Antimicrobial screening of stem bark of P. Senegalense
108
5.4
Antituberculosis Screening of Leaves of H. insignis, C. capitatum and Stem bark of P. Senegalense
109
5.5 5.5.1 5.5.2
Isolation, Purification and Characterization of Isolates Isolation, purification and characterisation of isolates from C. Capitatum Isolation and characterisation of C1
109 110 110
5.5.3
Isolation and characterisation of C2
110
4.5.4
Column chromatography of dichloromethane extract of H. insignis
61
4.5.5
Thin layer chromatography of the ethyl acetate extract of P. senegalenses
62
4.5.6
Column chromatography of ethyl acetate extract of P. senegalenses
63
4.6
TLC Analysis of Isolated Compounds
64
4.7
Chemical Test on the Compounds
73
4.7.1
Melting point determination of the compounds
73
4.8
Result of Spectral Analyses
73
CHAPTER FIVE
xiv
5.5.4
Isolation, and characterisation of isolates from H. insignis
111
5.5.5
Isolation and characterisation of H1
111
5.5.6
Isolation and characterisation of H3
112
5.5.7
Isolation of H4
112
5.5.8
Isolation, and characterisation of isolates from P. Senegalense
113
5.5.9
Isolation and characterisation of P2
113
CHAPTER SIX
113
6.0
SUMMARY, CONCLUSION AND RECOMMENDATIONS
115
6.1
Summary
115
6.2
Conclusion
116
6.3
Recommendations
116
REFERENCES
116
CHAPTER ONE
1.0 Introduction
1.1 Medicinal Plants Medicinal plants are natural sources of compounds that can be used against many diseases (Kubmarawa et al., 2007). The medicinal values of these plants lie in bioactive compounds that produce definite physiological actions on the human body (Krishnaiah et al., 2009). Medicinal plants have been shown by many studies as sources of diverse nutrients and non-nutrient compounds. Many of the medicinal plants display antioxidant and antimicrobial properties which can protect the human body against both cellular oxidation reactions and pathogens. Thus, it is important to characterize different types of medicinal plants for their antioxidant and antimicrobial potentials (Bajpai et al., 2005; Mothana and Lindequist, 2005; Wojdylo et al., 2007). In Africa and developing countries, it is estimated that 70 to 80% of people rely on traditional healers and herbal practitioners for their health needs (Agyare et al., 2006) and medicinal plants are the main sources of remedies used in this therapy. Some of these medicinal plants have been used for the management of different disease conditions such as bacterial infections, parasitic infections, skin diseases, hypertension, pains and inflammation such as rheumatoid arthritis (Muthu et al., 2006).
The World Health Organization (2000) defines traditional medicine as “the diverse health practices, approaches, knowledge and beliefs incorporating plant, animal and/or mineral based medicines, spiritual therapies, manual techniques and exercises applied singularly or in combination to maintain well-being, as well as to treat, diagnose or prevent illness”. Traditional medicine utilizes biological resources and the indigenous knowledge of plant groups conveyed verbally through generations. This is closely linked to the conservation of biodiversity and the related intellectual property rights of indigenous people (Timmermans, 2003). It is however necessary to validate the information through an organized infrastructure
2
for it to be used as an effective therapeutic means, either in conjunction with existing therapies or as a tool in novel drug discovery. Although it is these traditional medicines that provide the link between medicine and natural products, it was not until the 19th century that active compounds were isolated and principles of medicinal plants identified (Phillipson, 2001). The isolation of morphine from opium by Serturner (1805) started the chemistry of natural products (Patwardhan et al., 2004). Despite the discovery of natural products from higher plants, the interest of chemists and pharmaceutical scientists shifted to production of synthetic compounds and modification of natural products to enhance biological activity, increase selectivity and to decrease toxicity and side effect. Aspirin is one such example and was the earliest of these modified natural products. In more recent years, industry has once again turned its interest to natural product research (Phillipson, 2001). This is as a result of drug-resistant microorganisms, side effect of modern drugs and emerging diseases for which no medicine is available (Phillipson, 2001). Some classes of chemical compounds that have been isolated from natural products include; terpenoids, alkaloids, flavonoids and glycosides. These are the major classes of components of natural products (Kaisar et al., 2011).
a) Terpenoids: These are modified terpenes, where methyl groups are moved or removed or oxygen atoms added. They represent the oldest group of small molecular products synthesized by plant and are probably the most widespread groups of natural products (Kaisar et al., 2011).
b) Alkaloids: These are a group of naturally occurring chemical compounds that contain a basic nitrogen atom. They have a bitter taste and are generally white solids (Kaisar et al., 2011).
3
c) Flavonoids: These are a class of plant secondary metabolites. They are polyphenolic compounds that are ubiquitous in nature and are categorized according to chemical structure into flavonols, flavones, and flavonones, isoflavones, catechins, anthocyanidins and chalcones. They have aroused considerable interest recently because of their potential beneficial effects on human health. They have been reported to have antiviral, anti-allergic, anti-platelet, anti-inflammatory, anti-tumor and antioxidant activities (Kaisar et al., 2011).
d) Glycosides: These are molecules in which a sugar is bound to a non-carbohydrate moiety, usually as small organic molecule. Many plants store chemicals in the form of inactive glycosides (Kaisar et al., 2011).
1.2 Natural products as leads in novel and active chemotypes
There is an urgent need to identify novel and active chemotypes as leads for effective drug development. Natural products including plants, animals and minerals have been the basis of treatment of human diseases. The current accepted modern medicine or allopathy has gradually developed over the years by scientific and observational efforts of scientists (Vickrant et al., 2011). Natural products as crude materials with efficacy against various diseases have been selected by humans over many generations of practical experience. Such experiential evaluation is different from the scientific evaluation of orthodox medicine and is underestimated sometimes. In 2001, researchers identified 122 compounds used in modern medicine which were derived from ethnomedical sources, 80% of these have had an ethnomedical use identical or related to the current use of the active elements of the plant (Fabricant and Farnsworth, 2001). Most of the pharmaceuticals currently available to physicians such as aspirin, digitalis, quinine, opium, morphine, atropine, ephedrine, and digitoxin were developed from natural products (Vickrant et al., 2011).
4
The substances that can inhibit pathogens and have little toxicity to host cells are considered candidates for developing new antimicrobial drugs. On the other hand, indiscriminate use of commercial antimicrobial drugs in the treatment of infectious diseases has resulted in multiple-drug resistance of many human pathogenic microorganisms. This situation has necessitated a more radical approach to the search for new antimicrobial substances from various sources which could be used as novel antimicrobial chemotherapeutic agents (Navarro et al., 1996). The search for new antibiotics includes various sources such as the synthetic compounds, bioactive agents from aquatic microorganisms, and natural products including medicinal plants. 1.3 Statement of research problem Infectious diseases are the number one cause of deaths world-wide and in tropical countries it accounts for approximately 50% of deaths (Iwu et al., 1999).This is partly due to increasing incidence of multiple drug resistance. Bacterial resistance to almost all antibacterial agents have been reported (Ojiako, 2014) and this is largely due to indiscriminate use of antimicrobial drugs used in the treatment of infectious diseases. Apart from resistance, high costs, long periods of medication and undesirable side effects, some antibiotics have limited applications, hence there is serious need to develop safer and more active chemotherapeutic agents that will act over a short period of time. 1.4 Justification
Heeria insignis (DEL), Psorospermum senegalense (SPACH) and Clerodendrum capitatum (WILD) have been used in traditional medicine in many communities in northern Nigeria, for the treatment of various infectious diseases including tuberculosis. These plants are among plants claimed to treat tuberculosis and to the best of our knowledge, there is no reported
5
work on the antituberculosis activities of these, hence there is a need to justify scientifically the ethnomedicinal application of these plants. 1.5 Aim of the research The aim of the research work is to validate the antituberculosis claim by traditional medicine practitioners associated with local uses of the stem bark of Psorospermum senegalense (Spach), leaves of Heeria insignis (Del) and Clerodendrum capitatum (Wild) and to isolate and characterise any possible antituberculosis compound(s) from these plants. 1.6 Objectives of the research The aim stated above will be achieved through the following objectives:
i. collection and identification of the three plants parts,
ii. air drying, pulverising and extraction using known techniques,
iii. phytochemical screening of the crude plant materials,
iv. antimicrobial screening of the crude extracts of the plants,
v. antituberculosis screening of the crude extracts of the plant materials,
vi. isolation of phytochemicals present in the extracts and
vii. testing and characterisation of the isolated compounds for activities.
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