ABSTRACT
The Stembark of the Erythrophleum africanum plant (Welw. ex Benth) collected from Niger State, Nigeria was air-dried, pulverized and exhaustively extracted by maceration using ethanol. The phytochemical screening of the extract revealed the presence of secondary metabolites which include cardiac glycosides, flavonoids, terpenoids, saponins, alkaloids and steroids. The antimicrobial sensitivity test showed that the extract was sensitive to these organisms (with zone of inhibition indicated): S. aureus (25 mm), S. faecalis (27 mm), E. cloacae (29 mm), P. mirabilis (22 mm) and C. stellatoidea (24 mm) while E. coli, P. aeruginosa and C. albicans were resistant. The minimum inhibitory concentration (MIC) results revealed that the extract inhibited the growth of five (5) out of eight (8) tested organisms at 5 mg/ml for S. aureus, S. faecalis, E. cloacae, P. mirabilis and C. stellatoidea. P. aeruginosa, E. coli, C. ulcerans and C. albicans are not inhibited even at 20 mg/ml. The minimum bactericidal/fungicidal concentration (MBC/MFC) of the extract was 20 mg/ml against S. aureus and P. mirabilis, and 10 mg/ml for S. faecalis, E. cloacae and C. stellatoidea, while P. aeruginosa, E. coli, C. ulcerans and C. albicans proved to be resistant. The crude ethanol extract was dissolved in distilled water and then partitioned with petroleum ether, ethyl acetate, chloroform and n-butanol respectively. Preparative TLC on the n-butanol fraction developed with ethyl acetate/methanol (7:3 v/v) led to the isolation of the compound coded, NA. The isolated compound NA was screened for antimicrobial properties with the zone of inhibition found to be higher than that of crude ethanol extract. For the isolated compound NA, the zone of inhibition are S. aureus (26 mm), S. faecalis (29 mm), E. cloacae (30 mm), P. mirabilis (26 mm), and C. stellatoidea (29 mm). The minimum inhibitory concentration of the isolated compound NA against S. aureus and P. mirabilis was observed at 5 mg/ml and for S. faecalis, E. cloacae and C. stellatoidea at 2.5 mg/ml while P.
aeruginosa, E. coli, C. ulcerans and C. albicans are not inhibited even at 20 mg/ml. The minimum bactericidal/fungicidal concentration of the isolate NA against S. aureus, P. mirabilis, and C. stellatoidea was observed at 10 mg/ml while S. faecalis and E. cloacae was observed at 5 mg/ml, and the organisms showed moderate to very heavy colony growth at 2.5 to 1.25 mg/ml. The isolated compound NA was subjected to NMR analysis i.e 1HNMR, 13CNMR, DEPT, COSY, NOESY, HMBC and HSQC from which the structure of the isolated compound NA was elucidated and found to be ipolamiide which was confirmed from spectra data reported in the literature.
TABLE OF CONTENTS
Declaration i Certification ii Dedication iii Acknowledgement iv Abstract v Table of content vi List of Tables vii List of Figures viii Abbreviations ix CHAPTER ONE
1.0 INTRODUCTION 1
1.2 Aim and Objectives of the Research 8 1.2.1 Aim of the research 8 1.2.2 Objectives of the research 8 1.3 Statement of the Problem 8 1.4 Justification of the Research 9
1. Scope and Limitation of the Research 9 CHAPTER TWO 2.0 LITERATURE REVIEW 10 2.1 The Taxonomical Description of the Plant Erythrophleum africanum (Welw. ex Benth) Harms 10 2.1.1 Origin and geographical distribution 10 2.1.2 Traditional uses 10 2.1.3 Botany 11 2.1.4 Fruits 12 2.1.5 Ecology 12 2.1.6 Management (Morphology) 13 2.1.7 Genetic resources and breeding 13 2.1.8 Pest species 13 2.1.9 Prospects 13 2.2 Chemical Constituents and Antimicrobial Studies on the Phylum, Family and Species of E. afranum 14 2.2.1 Chemical constituents and antimicrobial studies on phylum
(Magnoliophyta) of E. afranum 14 2.2.2 Chemical constituents and antimicrobial studies on family (Leguminosae) of E. afranum 16 2.2.3 Chemical constituents and antimicrobial studies on species (Magnoliophyta) of E. afranum 18 2.2.4 Chemical constituents and antimicrobial studies on E. africanum 24 CHAPTER THREE 3.0 MATERIALS AND METHODS 26 3.1 Materials for Extraction and Phytochemical Test 26 3.1.1 Reagents for extraction and phytochemical screening 26 3.2 Materials for Chromatography (TLC) 27 3.3 Methodology 27 3.3.1 Preparation of plant material 27 3.3.2 Extraction 28 3.3.3 Extraction procedure 28 3.4 Phytochemical Screening 30 3.4.1 Test for carbohydrates 30
3.4.2 Test for flavonoids 31 3.4.3 Test for tannins 31 3.4.4 Test for saponins 32 3.4.5 Test for alkaloids 32 3.4.6 Test for steroids 33 3.4.7 Test for glycosides 32 3.5 Microbial Media and Test Organisms for Antimicrobial Screening 34 3.5.1 Microbial media for antimicrobial screening 34 3.5.2 Test organisms for antimicrobial screening 34 3.6 Antimicrobial Studies 34 3.6.1 Test microorganisms 35 3.6.2 Zone of inhibition of the crude ethanol extract 35 3.6.3 Minimum inhibition concentration of the crude ethanol extract 35 3.6.4 MBC/MFC of the crude ethanol extract 36 3.7 Chromatography Techniques 37 3.7.1 Thin Layer Chromatography 37 3.7.2 Preparation of preparative TLC plates 37
3.7.3 Isolation of compound NA from n-Butanol fraction 38 3.8 Principle behind the Operation of the NMR machine 38 3.9 Spectra Analysis 39 CHAPTER FOUR 4.0 RESULTS 40 4.1 Result of Extraction 40 4.1.1 Percentage (%) extractable estimation of fractions from crude Ethanols extract 40 4.2 Phytochemical Screening of the Crude Ethanol Extract 42 4.3 Result of Antimicrobial Studies 44 4.3.1 Antimicrobial sensitivity studies of the crude ethanol extract 44 4.3.2 Zone of inhibition of the crude ethanol extract 46 4.3.3 Minimum inhibitory concentration of the crude ethanol extract 48 4.3.4 Minimum Bactericidal/Fungicidal Concentration of the crude ethanol extract 50 4.3.5 The antimicrobial activities of the isolated compound NA and antibiotic drug 52 4.3.6 Measurement of zone of inhibition of the isolated compound NA against Test organisms 54
4.3.7 MIC of the isolated compound NA against the test organisms 56 4.3.8 MBC/MFC of the isolated compound NA against the test organisms 58 4.4 Results of Spectral Analysis of the Isolated Compound NA 60 4.4.1 The Proton 1HNMR spectrum of isolated compound NA 60 4.4.2 The 13CNMR spectrum of isolated compound NA 62 4.4.3 The DEPT spectrum of isolated compound NA 64 4.4.4 The H-H Correlation Spectroscopy (COSY) spectrum of isolated compound NA 66 4.4.5 The HMBC spectrum of isolated compound NA 68 4.4.6 The HSQC spectrum of isolated compound NA 70 4.4.7 The NOESY spectrum of isolated compound NA 72 4.4.8 Comparison of the 13CNMR and 1HNMR data of the isolated compound NA With Ipolamiide 74 CHAPTER FIVE 5.0 DISCUSSION 75 5.1 Phytochemical Screening of the Crude Ethanol Extract 75 5.2 Antimicrobial Screening Result 77 5.3 Spectroscopic Analysis 80
5.3.1 The 1HNMR Analysis 80
5.3.2 The 13CNMR Analysis 80 CHAPTER SIX 6.0 SUMMARY, CONCLUSION AND RECOMMENDATION 83 6.1 Summary 83 6.2 Conclusion 84 6.3 Recommendation 86 References
CHAPTER ONE
1.0 INTRODUCTION
Traditional medicine is an important part of human health care. It is the sum total of knowledge, skill and practice based on the theory, belief and experience used in maintaining health as well as curing diseases using medicinal plants. The early man lived and depended on plant for his food, fuel, shelter and medicare. In the course of doing this, they notice that some plants when consumed gave them some relief from symptoms of discomfort. Some were just for nourishment, some elevated their mood through hallucinations and quite a number make them ill or even killed them (Thomson, 1978). However, going by this gradual proceeding of trial and error, primitive man was able to sort out flora they could eat, those they could not, those with healing properties and the knowledge concerning their medicinal usage. Hence, varieties of plants have been used medicinally and for this reason the plant kingdom was described as the sleeping giant of drug development by Verpoorte et al. (1987), while the forest is generally referred to as “God’s Own Pharmacy” (Treben, 1986). The medicinal plants are those in which one or more of its parts contain substances that can be used for therapeutic purposes or which are precursors for the synthesis of useful drugs (Sofowora, 1993). The medicinal property of plants depends on the presence of a variety of chemical substances known as secondary metabolites. These secondary metabolites are constituents synthesized by plants in addition to the primary metabolites, which may be concentrated in different parts of the plants (Evans, 1997).
Some of these compounds include saponins, glycosides, flavonoids, alkaloids, steroids, terpenoids, tannins, volatile oil, organic acids, e.t.c which differs from one plant to another at varying concentrations (Miller, 1973). The phytochemical examination of these plant constituents has been made possible and easier by improved methods of extraction, separation, isolation and characterization (Harbone, 1973).
The application and research for drugs, food and feed supplements obtained from plants have increased over the years (Frankič et al., 2009). Spices, herbs and their constituents are generally recognized to be safe, either because of their traditional use without any documented detrimental effect or because of much toxicological studies (Frankič et al., 2009). Plants being a rich source of secondary biomolecules which exhibit significant pharmacological effects, spices and herbs appeal to many consumers who question the safety of synthetic food additives (Craig, 1999). Plant extracts and essential oils have been used as alternatives to antibiotics due to their antimicrobial activities and the favorable effect on the animal intestinal system (Al-Kassien, 2009). Spices and herbs can have a great influence on the function and reactivity of the immune system of the farm animals (Craig, 1999). The growth promoting active feed supplements improve stability of feed and auspiciously impact the digestive micro population mostly through inhibition of pathogenic microorganism growth. In consideration of promoted health status of intestinal tract, farm animals are less exposed to the toxins produced by different microorganisms (Frankič et al., 2009). Windisch et al. (2008) reported that spices and herbs have beneficial effect on the stress resistance of the animals and amplify the absorption of essential nutrients. There is also, one other important advantage of using plant extracts of herbs and spices or their essential oils instead of synthetic drugs in feed stocks: synthetic drug residues in animal meat and eggs can cause health problems in people who consume them, especially due to increasing resistance of pathogens present in the human body as a result of prolonged use of synthetic drugs (Barbour et al., 2010).
Despite the tremendous progress in human medicine, infectious diseases caused by bacteria, fungi, virus and parasites are still a major threat to public health. Their impact is particularly large in developing countries due to relative unavailability of medicine and emergence of drug resistance (Zampini et al., 2009). During the last two decades, the resistance of microorganisms to synthetics drugs
as well as the appearance of undesirable side effects of certain antibiotics reported by Okemo et al. (2003), has led to the search for new biologically active compounds from natural sources as new antimicrobial agents with the view to discover new chemical compounds, which could overcome the above disadvantages (Meenakshi et al., 2001; Bouamama et al., 2006). The development of resistance to current antibiotics by disease causing microbes has also reinforced research for discovery of new ones. Current trends in drug development process are focused on natural sources, especially of plant origin due to some proven correlation between the folkloric medicinal uses of some of these plants to biological activity. Therefore, the use of plant materials to prevent and treat infectious diseases successfully over the years has continued to attract the attention of scientist’s worldwide (Osawa et al., 1990; Begum et al., 2002; Sophon et al., 2002).
In some parts of the world, the administration of decoction of plants is still in practice (Chopra, 1958; Said, 1969). Traditionally, this treasure of knowledge has been passed on orally from generation to generation without any written documentation (Perumal and Ignacimuthu, 2000). The importance of herbs in the management of human ailments cannot be overemphasized. It is clear that the plant kingdom harbors an inexhaustible source of active ingredients invaluable in the management of many intractable diseases. Furthermore, the active components of herbal plants have the advantage of being combined with many other substances that appear to be inactive. However, these complementary components give the plant as a whole a safety and efficiency much superior to that of its isolated and pure active components (Shariff, 2001). In the past century, remarkable progress in the discovery of antimicrobial drugs has been made and thus several numbers of synthetic drugs have also been synthesized. These new synthetic antimicrobial drugs play a vital role in treatment of various types of microbial infections and reducing the number of fatalities that are associated with infections caused by microbes. However, despite this remarkable progress, multiple drug resistance pathogenic
microorganisms have emerged due to indiscriminate use of the commercially available antimicrobial drugs in human (Cowan, 1999; Newman et al., 2000; Poole, 2005). Thus, the emergence of previously uncommon infections is considered as a serious medical problem by World Health Organization, governments, the public health and scientific communities as well as health care providers at large. In fact, approximately 25 % of prescribed medicines in industrialized countries are obtained directly or indirectly from plant sources (Newman et al., 2000). In addition, according to an estimate of the World Health Organization, approximately 70 to 80 % of the world populations presently use plants for medicinal purposes (Wills et al., 2000).
It is also worthy of note that in developing countries where medicines are quite expensive, investigation on antimicrobial activities from medicinal plants may still be needed. Nonetheless, plants used in folk medicine in most developing are still understudied, particularly in clinical microbiology (Kirby, 1996; Wills et al., 2000; Pavrez et al., 2005; Zakaria et al., 2007). Recently, several numbers of plant species have been investigated for their potential antimicrobial activity using various extraction methods (percolation, tincture, soxlet, maceration e.t.c) with water, methanol, ethanol, n-hexane, chloroform, butanol and acetone. These extracts have been assessed for their antimicrobial activity against several numbers of bacteria isolates by broth dilution (Bacteria are inoculated in liquid growth media), microtiter plate and/or disc diffusion methods(Bacteria are inoculated in agar media) (Scalbert et al., 2005; Zakaria et al., 2007; Lawrence et al., 2009). For example, Sweet Basil, stem bark of Distemonanthus benthamianus Baill, Muntingia calabura (L.) and Dicranopteris linearis (L.) and E. suaveolens, E guineense, E. ivorense, have been shown to exhibit antibacterial activity against some selected bacterial strains (Zakaria et al., 2007; Aiyegoro et al., 2007; Moghaddam et al., 2009). These studies demonstrated that plant extracts derived from these plant species could be a potential source of antibacterial agents for the treatment of normal infection caused by some bacteria such as H. pylori strains, S. aureus strains, E. coli and others. Additionally, several mechanisms of action have been
suggested with regards to the chemical compounds which might be present in these plant extracts,
particularly, flavonoids (1) (rutin and quercetin), tannins (2) and others (Aiyegoro et al., 2008).
O
Flavonoid (1)
OH
HO OH
Tannin (2) phloroglucinol
Furthermore, previous as well as recent investigators reported that phenols, polyphenol and
flavonoids are natural antioxidant plant products that have been found in various concentrations in
most medicinal plants as well as other plant kingdom (Rice-Evans et al., 1995; Middleton et al., 2000;
Scalbert et al., 2005; Abdel-Hameed, 2009). They also indicated that these compounds have been shown
to possess various therapeutic values such as antibacterial, anti-mutagenic, anti-carcinogenic and antiinflammatory,
anti-aging activities by reducing the oxidative damages or stresses that are commonly
induced by free radical species. Furthermore, the growing interest in the substitution of synthetic
antioxidants with natural ones has encouraged research on plant sources and screening of raw materials for identifying new ones. Plant-derived antioxidants such as ascorbic acid, β-carotene, α-tocopherol, phenolic acids and flavonoids, among others, are becoming increasingly known as a primary dietary factors for humans (Ferguson, 2001; Martin and Appel, 2010). The interest in these compounds is largely due to the growing evidence of their potential health benefits, particularly for their role in reduced risk for heart diseases, cancer and age-related diseases as well as protection against cellular damage caused by reactive oxygen species (free-radicals) (Ferguson, 2001; Abdel-Hameed, 2009; Martin and Appel, 2010). Reduced susceptibility to antibacterial drugs is continuously increasing which is attributed to indiscriminate use of broad-spectrum antibacterials and immunosuppressive agents (Dean and Burchard, 1996). This situation provided the impetus to search for new antimicrobial substances from various sources like medicinal plants reported by Cordell (2000) and screening of these plants may result in the discovery of new effective compounds and can raise the spectrum of untreatable bacterial infections and adds urgency to the search for new infection fighting strategies (Tomoko and Takashi, 2002). There is a need to search for new infection-fighting strategies to control microbial infections. Several studies have been carried out on various medicinal plants screening for their antimicrobial activity (Parekh and Chanda, 2006; Firas and Hassan, 2008). For the past two decades, antibacterial properties of various plants and plant parts like root, stem, leaves, seeds and flowers have been well documented for some of the medicinal plants (Nandagopal and Kumari, 2007).
The acceptance of traditional medicine using medicinal plants as an alternative form of health care and the development of microbial resistance to the available antibiotics has led researchers to investigate the antimicrobial herbal extracts (Bisignano et al., 2000; Hammer et al., 1999). Plants containing flavonoids, terpenoids, steroids, phenolic compounds and alkaloids have been reported to
have antimicrobial activity (Hostettmann et al., 1977). The medicinal plant plays an important role in the healthcare system of the third world population (Akerele et al., 1991). Africa, particularly Nigeria is rich with indigenous plants which are used in herbal medicine to cure diseases and to heal injuries. Investigators have implicated alkaloids to be directly involved in eliciting diverse biochemical and pharmaceutical characteristics on biological systems (Clarke, 1970). In the Savannah regions of Nigeria, the cattle herders always prevent their animals from grazing along the routes where these species are known to grow (Nwude and Chineme, 1981). Other investigators have reported cases of accidental human poisoning after the use of the bark of Erythrophleum species for traditional uses (Nwude, 1981). 1.2 Aim and Objectives of the research 1.2.1 Aim of the research To isolate and characterize the chemical components of the stembark extract of E. africanum and investigate its antimicrobial activities as well as ethnomedicinal/cultural claims for its use as medicinal plant. 1.2.2 Objectives of the research The objectives of the study are:
1. Collection, identification, drying, grinding of plant material and extraction of powdered plant with ethanol to exhaustion.
2. Partitioning of crude ethanol extract with petroleum-ether, ethyl acetate, chloroform and n-butanol.
3. Phytochemical screening of the plant extract.
4. Antimicrobial studies of the plant extract
5. Isolation and structural elucidation of the isolated compound(s) using spectral techniques e.g 1HNMR, 13CNMR, DEPT, COSY, NOESY, HMBC and HSQC.
1.3 Statement of the Problem
Many active compounds are known which are use in the treatment of different types of diseases, though only few were identify by the scientists, thus there is need to search for more active compounds which can be use to cure disease.
1.4 Justification of the Research
The uses of Erythrophleum africanum as claimed by traditional healers in the treatment of various ailments has necessitated the need for a scientific study to ascertain its antimicrobial properties and the active phytochemical components responsible for curing the said disease conditions. 1.5 Scope and Limitation of the Research The scope of the study is phytochemical and antimicrobial screening, isolation and structural elucidation of active compounds of E. africanum. To investigate if any isolated compound could account for the use of the plant as claimed by traditional healers, the isolated compound must be tested for antimicrobial activities against suspected microbes that can causes the said disease condition.
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