ABSTRACT
Combretum lamprocarpum,is a plant used in tropical and sub Saharan parts of Africa in the treatment of wounds, stomach ache, diarrhoea, and vomiting. The dried powdered plant material(leaves) was extracted using a microwave-assisted extraction technique.Phytochemical and antimicrobial screening of the extracts were carried out using standard methods.The compound was purified using conventional chromatographic techniques and characterised using spectroscopic techniques. The phytochemical screening of the extracts revealed the presence of steroids, terpenes, alkaloids, carbohydrates, saponins, tannins, cardiac glycosides and flavonoids. The antimicrobial screening of the crude extracts showed that all the extracts had significant activity against microbes with the exception ofhexane extract. The diameter of the zone of inhibition ranged between 12 and 29 mm, the minimum inhibitory concentration ranges between 3.13 and 50.00 mg/ml. The minimum bactericidal concentration/minimum fungicidal concentration was between 6.25 and 50.00 mg/ml. A bioactive triterpene coded K1, was isolated as a white crystalline compoundfrom the ethyl acetate extract and characterised using FT-IR, 1H-NMR, 13C-NMR analysis as lupeol. The isolated compound from the ethyl acetate extract was found to be active againstStaphylococcus aureus(MBC=25.00μg/ml), Salmonella typhi(MBC=12.50μg/ml), Escherichia coli(MBC=6.25μg/ml),and Pseudomonas aeruginosa (MBC=12.50μg/ml). The structure of the compound was supported by comparing the experimental spectroscopic data with that of literature. In conclusion, thisstudy revealed that the leaves ofCombretum lamprocarpumhasa potent medicinal value. The chloroform and methanol extracts showedbioactivity and therefore, should be studied for possible bioactive compounds.
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TABLE OF CONTENTS
Cover page i
Title page ii
Declaration iii
Certification iv
Dedication v
Acknowledgement vi
Abstract vii
Table of Contents viii
List of Figures xiv
List of Tables xv
List of Plates xvi
Abbreviations and Symbols xvii
Appendix xviii
CHAPTER ONE 1
1.0 INTRODUCTION 1
1.1 Background of the Study 1
1.2 Aim and Objectives of the Research 4
1.2.1 Aim of the research 5
1.2.2 Objectives of the research 5
1.3 Justification of the Research 4
1.4 Scope and Limitation of the Research 6
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CHAPTER TWO 7
2.0 LITERATURE REVIEW 7
2.1 The family Combretaceae 7
2.1.1 The genus Combretum 7
2.2 Taxonomic Classification of Combretum lamprocarpum 9
2.3 Morphological Description of the Genus Combretum lamprocarpum 9
2.4 Ethno-medicinal uses of Combretum lamprocarpum and other species 12
2.5 Phytochemical Studies on Combretum species 13
2.6 Biological Activity of Combretum species 13
2.6.1 Anti-inflammatory activity 14
2.6.2 Anti-cancer activity 14
2.6.3 Antifungal activity 15
2.6.4 Antibacterial activity 15
2.7 Compounds Isolated from Combretaceae Family 15
2.8 Secondary Metabolites from Plants 18
2.8.1 Alkaloids 19
2.8.2 Saponins 22
2.8.3 Tannins 23
2.8.4 Cardiac glycosides 25
2.8.5 Steroids 26
2.8.6 Flavonoids 27
2.8.7 Anthraquinones 29
2.8.8 Terpenes and Terpenoids 31
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2.9 Some Bacterial/Fungal Agents and their Effect on Human 32
2.9.1 Aspergillus niger 32
2.9.2 Candida albicans 33
2.9.3 Escherichia coli 33
2.9.4 Pseudomonas aeruginosa 34
2.9.5 Staphylococcus aureus 34
2.9.6 Salmonella typhi 35
CHAPTER THREE 36
3.0 MATERIALS AND METHODS 36
3.1 Materials 36
3.1.1 Equipment 36
3.1.2 Reagents 36
3.1.3 Solvents 37
3.1.4 Culture media 37
3.1.5 Test microorganisms 37
3.2 Methods 38
3.2.1 Collection of plant material 38
3.2.2 Extraction of the plant material 38
3.2.3 Preparation of stock solution for antimicrobial studies 39
3.3 Phytochemical Screening 39
3.3.1 Test for carbohydrates 40
3.3.2 Test for anthraquinones glycosides 40
3.3.3 Test for saponins glycosides 40
3.3.4 Test for steroids and terpenoids 41
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3.3.5 Test for tannins 41
3.3.6 Test for cardiac glycosides (Keller-Killiani test) 41
3.3.7 Test for flavonoids 42
3.3.8 Test for alkaloids 42
3.4 Antimicrobial Screening 42
3.4.1 Cultivation and standardisation of test organisms 42
3.4.2 Preparation of culture media 43
3.4.3 Antimicrobial profile (susceptibility test) 43
3.4.4 Screening for Antifungal Assay using Agar Well Diffusion Method 42
3.4.5 Determination of Minimum Inhibitory Concentration (MIC) 43
3.4.6 Determination of Minimum Bactericidal and Fungicidal Concentration (MBC)/(MFC) 44
3.5 Chromatographic Separation of Extracts 44
3.5.1 Thin layer chromatography (TLC) 45
3.5.2 Column chromatography (CC) 45
3.5.3 Solvent system 46
3.5.4 Analytical thin layer chromatography of the ethyl acetate extract 46
3.5.5 Column Chromatography of ethyl acetate extract 46
3.6 Melting Point Determination 47
CHAPTER FOUR 48
4.0 RESULTS 48
4.1 Extraction of plant material 48
4.2 Result of Preliminary Phytochemical Analysis 49
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4.3 Result of Zone of Inhibition studies 50
4.4 Result of Minimum Inhibitory Concentration (MIC) 51
4.5 Result of Minimum Bactericidal/Fungicidal Concentration(MBC/MFC) 52
4.6 Analysis of isolated compound K1 53
4.7 Result of 1D NMR Analysis of Compound K1 54
4.7.1 1H-NMR spectrum of compound K1 (δ ppm, 400 MHz, CDCl3) 54
4.7.2 13C-NMR spectrum of compound K1 55
4.8 Result of Zone of Inhibition of the Isolated Compound K1 58
4.9 Result of Minimum Inhibitory Concentration of the Isolated Compound K1 59
4.10 Result of MBC/MFC of the Isolated Compound K1 60
4.11 Result of TLC and Melting Point of Compound K1 61
CHAPTER FIVE 62
5.0 DISCUSSION 62
5.1 Extraction 62
5.2 Phytochemical Screening 62
5.3 Antimicrobial Test 62
5.4 Isolation and Characterisation of Compound K1 63
5.5 Biological Activity of Compound K1 64
CHAPTER SIX 65
6.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS 65
6.1 Summary 65
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6.2 Conclusion 65
6.3 Recommendations 66
REFERENCES 67
APPENDIX 80
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of the Study
The use of medicinal plants in the treatment of diseases is as old as mankind. A medicinal plant is defined as a plant that is useful in therapeutics because it contains active constituents (Sher, 2004). These constituents are mostly secondary metabolites such as alkaloids, essential oils, flavonoids, glycosides, resins, saponins, tannins, and terpenoids (Cowan, 1999).Plants remain a rich source of secondary metabolites that have been widely used as bioactive constituents in therapeutically effective medicines for several ailments (Hoareau and Da Silva, 1999). Fossil records revealed that the use of plants as medicines by human may be traced back to about 60,000 years ago (Fabricant and Farnsworth, 2001). The search for plants that for remedy against diseases and ailments have been documented since ancient times in the form of traditional medicine. Historically,the beginning of the nineteenth century marked the era of “modern” drugs (Joo, 2014). In the year 1805, morphine, the first pharmacologically active compound was isolated from the opium plant by a German pharmacist, Friedrich Sertürner (Hamilton and Baskett, 2000).
Despite the breakthrough made by mankind in the synthesis of pharmaceutical drugs, the decreasing efficacy of synthetic drugs and the increasing contraindications of their usage, make the continueduse of plants as important sources of medicinal agents (Petrovska, 2012). According to the World Health Organization, traditional medicine is the sum total of the knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health, as well as in the prevention, diagnosis, improvement or treatment of physical and mental illnesses (WHO, 2005). Traditional medicines could not be risk-free, and due to
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their rampant use, it is important that the population be informed of risks that may be linked to their use. One of such risks is associated with the contamination of these herbal remedies with potential toxins such as heavy metals and organic pollutants (Awodele et al., 2013). Traditional medicinal practice has been documented for centuries in many parts of the world. Several plants and herbs are used worldwide by traditional medical practitioners. Extracts from the various plant parts (leaves, stem bark and roots) of various higher plants are used in herbal medicine prescription (Sofowora, 1993). Crude extracts from plants are usually administered as concoctions for the treatment of various diseases. However, about 75% of the world population depends on these various forms of concoctions and herbal decoctions for the treatment of infectious diseases (Robinson and Zhang, 2011). Natural products, which constitute the secondary metabolites, have been used since ancient times in traditional medicine for the treatment of many diseases and illnesses (Diaset al., 2012). In Nigeria, the use of potent medicinal plants in management of different diseases has been reported (Ogunshe et al., 2008; Weintritt, 2007; Aiyeloja and Bello, 2006; Blench and Dendo, 2003). Phytochemicals are the sources of basic raw-materialfor the establishment of pharmaceutical industries (Mothana and Lindequist,2005). Phytochemical screening plays a vital role in identifying new sources of pharmacologicallyactive compounds such as alkaloids, anthraquinones, flavonoids, phenolic compounds, saponins, steroids, tannins and terpenoids (Akindele and Adeyemi, 2007). Most of these phytochemical constituents are potent bioactive compounds found in parts of the medicinal plant which serve as lead for modern drugs (Sofowora, 1993).
Phytochemistry has been developed in recent years as a separate discipline. It is concerned with the enormous variety of organic substances that are biosynthesized and
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stored by the plants.They have been known to reduce the risk of many human diseases including cardiovascular disease, hepatorenal diseases, diabetes, cancers and neurodegenerative disorders (Modak et al., 2007; Shakya and Shukla, 2011). Phytochemicals can be classified on the basis of their chemical composition or functional group(s).These include alkaloids, glycosides, flavonoids, tannins, saponins, terpenes, steroids, anthraquinones (Shakya, 2016). Alkaloids are one of the major classes of natural products that exhibit antimalarial activity. The first antimalarial drug -quinine, isolated from the bark of cinchona specie (Rubiaceae) belongs to this class. However, over 100 alkaloids from higher plants were reported to exhibit significant antimalarial activity (Saxena et al., 2003). Taxol a plant alkaloid (generic name paclitaxel) is a microtubule-stabilising drug that is approved by the Food and Drug Administration for the treatment of patientswith ovarian, breast, and lung cancer as well as Kaposi’s sarcoma (Shoeb, 2006). Taxol® is considered the most efficacious developed anti-cancer drug in the past five decades and represents taxane family of drugs with worldwide sales of $1.5 billion in the year 1999 (Isah, 2015). Alkaloids have wide range of pharmacological activities including antispasmodic, antimalarial, analgesic, diuretic activities (Kittakoopet al., 2014). Terpenes are a large and diverse class of organic compounds.They can occur as monoterpenes, diterpenes, sesquiterpenes, triterpenes, and tetraterpenes (C10, C15, C20, C30 and C40 respectively). A number of terpenes or terpenoids are reported to be active against fungi (Arif et al., 2009). The oxygen containing derivatives i.e.terpenoids are known for their antiviral, anthelmintic, antibacterial, anti-cancer, antimalarial, and anti-inflammatory properties (Chen et al., 2012).
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Flavonoids are phenolic compounds that continue to draw the interests of the scientific community because of its wide range of bioactivity. Flavonoids have an antioxidant, anti-allergic, antibacterial properties among others (Xia et al., 2014; Nobakht et al., 2017) and saponins are reported to have anti-inflammatory and antiviral activities (Chopra and Doiphode 2002; Maurya et al., 2008). Anthraquinones(9, 10-dioxoanthracenes) constitute a vital class of natural products with numerous applications. Apart from their use as colourants, anthraquinone derivatives have been used over the centuries for medicinal purposes such as laxatives, antimicrobial, and anti-inflammatory agents (Malik and Muller, 2016). The example of antifungal anthraquinone from medicinal species includes a new 1,3-dihydroxy-2-methyl-5,6-dimethoxy-anthraquinone (Fatope et al., 2003).
1.2 Statement of the Research Problem
About fifty percent of the number of death recorded in the tropical countries are largely due to infectious diseases (Iwu et al., 1999). This can be linked to the increasing bacterial resistance to antibacterial drugs (Fair and Tor, 2014).The increasing occurrenceof resistance to antimicrobial drugs is attributed to the indiscriminate and abuse of the modern antimicrobial drugs (Usha et al., 2010). However, the ethno medicinal uses of the leaves of C. lamprocarpum in Nigeria for the treatment of ailments such diarrhoea, stomach-ache, healing of woundshave not been scientifically verified. It is against this backgroundthat there is need to develop a more convenient and very active therapeutic antimicrobial agent.
1.3 Justification for the Research
This research was conducted in order to search for newer and more effective antimicrobial agents because of the alarming increase in multidrug resistance. The use of
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antimicrobial drugs from medicinal plants, have many advantages such as less side effects, cheaper, acceptance due to long history of use, and being renewable in nature (Gur et al., 2006). To the best of our knowledge, there is no report on the pharmacological activity or isolation and characterization of bioactive compounds fromthe plant. Therefore, there is need to validate the claims of the ethnomedicinal practices and to identify the active components of the leaf of Combretum lamprocarpumwhich are responsible for the treatment of numerous ailments such as diarrhoea, stomach-ache, healing of wounds etc.
1.4 Aim and Objectives of the Research
1.4.1 Aimof the research
The aim of this research was to validate the ethnomedicinal claim on Combretum lamprocarpumleaf by isolation and characterisation of bioactive constituents responsible for the ethnomedicinalclaims on the plant.
1.4.2 Objectives of the research
The aim of the research stated above will be achieved through the following objectives:
i. Preliminary phytochemical screening of the crude extracts.
ii. Antimicrobial screening of the crude extracts.
iii. Purification of the extracts and isolation of bioactive compound(s) using chromatographic techniques
iv. Structure elucidation of the isolated compound(s) using spectroscopic techniques (1H,13C NMR and IR).
v. Determination of the antimicrobial activity of the isolated compound(s).
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1.5 Scope and Limitation of the Research
This research work is expected to cover extraction of the leaf of C. lamprocarpum, phytochemical screening of the plant extracts, antimicrobial screening of the crude extracts and any isolated compound(s), purification of the extracts, isolation of bioactive compound(s), and characterisation of the isolated compound(s).
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