ISOLATION AND CHARACTERIZATION OF ANTIMICROBIAL COMPOUNDS FROM BERRIES OF HARRISONIA ABYSSINICA (SIMAROUBACEAE)

ABSTRACT
Plants are a potential source of antimicrobial compounds. In this study, a plant from the family Simaroubaceae, Harrisonia abyssinica, traditionally used to treat a number of diseases was used. Research has been carried out on its stem bark, root bark and leaves and a number of chemical constituents have been isolated but no research has been carried out on its berries. Therefore the berries of this plant were chosen in order to investigate for antimicrobial compounds. The berries of the plants were collected, defatted using hexane and extracted using a mixture of methanol and dichloromethane solvents in a ratio of 1:1. The extract was screened for antimicrobial activity (agar diffusion assay) and it was found to have significant activity against tested microorganisms. Fractionation by column chromatography yielded 35 fractions that were pooled to three main fractions by the aid of TLC analysis. The three were further purified by repeated column chromatography and gel filtration method. The pure compounds were coded HRF1, HRF2 and HRF3 and they were subjected to antimicrobial (agar diffusion assay) to confirm their antimicrobial activity. Compound HRF1 showed substantial activity and therefore was chosen and screened for its antimicrobial activity using serial dilution assay. The compound was found to have a minimum inhibitory concentration (MIC) of 5 µg/ml against Candida albicans, 6 µg/ml against Bacillus cereus and >20 µg/ml against other test microorganisms. The compounds were subjected to spectroscopic techniques; 1D NMR (1H NMR, 13C NMR), 2D NMR (COSY, DEPT, HSQC, HMBC and NOESY). HRF2 and HRF3 were found to obtain similar spectral features a part from the noise signals in HRF2 hence was found to be the same compound. The structures were confirmed using HRESIMS spectrometry. Compound HRF1 was named harronin I and HRF3 as harronin II, these compounds belong to a class of acetophenones and they are being isolated for the first time.

CHAPTER ONE
INTRODUCTION
Background information
For many years people have developed a store for empirical information concerning therapeutic values of local plants. In the African continent long before colonization and arrival of Western values, plants were used for medicinal purposes. Extraction of medicine from herbs was indeed an integral part of the lifestyles of the people of Africa (Rukangira, 2002).

Herbal remedies have been applied for treatment of many ailments since ancient time all over the world and about 25% of current drugs are derived from plants (Wanyoike, 2004). The WHO estimates that four billion people, 80% of the world population presently use herbal medicine for some aspects of primary healthcare (Akerere, 1992). In Africa, an estimated 85% of the populations rely totally or partially on traditional medicine for their healthcare needs. WHO defines traditional medicine as, “the sum total of all the knowledge and practices whether explicable or not, used in diagnosis, prevention and elimination of physical, mental or social imbalance and relying exclusively on practical experience and observation handed down from generation to generation whether verbally or in writing” (Rukangira, 2002).

Growing resistance of pathogens to therapeutic agents has given the urgency to search for both better and safer compounds, and delivery systems. The need to treat the target more precisely has provided additional opportunities for the use of natural products (Jasper et al., 1998). With the renewed interest from Western countries in herbal remedies, and the increasingly urgent need to develop new effective drugs, traditionally used medicinal plants have recently received the attention of the pharmaceutical and scientific communities (Cordell, 1981). This involves the isolation and identification of the secondary metabolites produced by the plants and used as the active principles in herbal preparations. Therefore search for appropriate medicine to counter the numerous diseases that endanger the life of man continues.

More than 50 years have passed since the modern antibiotic era opened with the clinical trial of penicillin in early 1940s. It is estimated that between 5,000 and 10,000 natural antibiotics have been isolated and characterized and at least 50,000 to 100,000 analogues have been synthesized, but clearly the vast majority fails to find medicinal use (Berdy, 1980). Most of the natural antibiotics have been isolated from soil microorganisms through intensive screening. The phytochemical screening of plant species especially ethnopharmaceutically provides a valuable baseline information in search for new pharmaceuticals (Farnsworth et al., 1985). The development of resistance by pathogens to many of the commonly used antibiotics provides sufficient impetus for search for more antimicrobial agents to combat infection and overcome the problem of resistance.

Plants produce diverse array of secondary metabolites, many of which have antimicrobial activity. Some of these compounds are constitutive, existing in healthy plants in their biologically active form; others such as cyanogenic glycosides occur as active precursors and are activated in response to tissue damage or pathogenic attack (Van et al., 1995). Plants are the oldest source of pharmacologically active compounds, and have provided humankind with many medically useful compounds for centuries (Bennett and wellsgrove, 1994). It is estimated that more than two thirds of the population relies on plant derived drugs; some 7000 medicinal compounds used in western pharmacopoeia are derived from plants. The search for natural products to cure diseases represents an area of great interest in which plants have been the important source. Thus, phytochemical screening of plant species, especially of ethnopharmaceutical use, will provide valuable information in search for novel pharmaceuticals (Haslam et al., 1989).

The use of shrub Harrisonia abyssinica particularly in East Africa to treat numerous diseases has been used as a basis for this study in order to investigate for the medicinal compounds of its berries. In the previous research done on its leaves, roots and stem, a number of chemical constituents that have shown to exhibit biological activity have been isolated and structurally elucidated (Okorie, 1982). The shrub indeed has so far been known to treat a number of diseases like, fever, tuberculosis, malaria, bubonic plague, haemorrhoids, snakebite, dysentery, herpes simplex and other uses (Kokwaro, 1993). This research project was conducted on the berries of H. abyssinica to investigate for antimicrobial chemical compounds.

Statement of the problem
H. abyssinica, is traditionally widely used to treat numerous diseases. A number of chemical constituents have been isolated from the root bark, stem bark, and leaves but no research has been done on its berries despite the fact that they are suspected to harbour a lot of secondary metabolites in addition to the germplasm and storage of carbohydrates. Therefore this has prompted this study on these berries in order to investigate for antimicrobial compounds.

Objectives
The main objectives are;

1. To extract dried berries of H. abyssinica shrub.

2. To screen for antimicrobial activity of the crude extract.

3. To isolate and purify compounds from crude extract.

4. To carry out chemical characterization of isolated compounds.

Justification
Secondary metabolites have proved to be biologically active molecules, in higher plants secondary metabolites serve as defence agents against invading microorganisms Chemical constituents for instance limonoids, chromones, and quassinoids responsible for antimicrobial, insect antifeedant, antiplasmodial and plant growth inhibitory activities have been isolated from the root bark, stem bark and leaves of the plant H. abyssinica. The scientific prospects of getting a new and novel chemical compound(s) will increase the number of isolated chemical constituents and hence support the role of this herbal medicine in managing microbial pathogens therefore validate the role of traditional medicine in provision of primary health care.

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Item Type: Kenyan Material  |  Attribute: 44 pages  |  Chapters: 1-5
Format: MS Word  |  Price: KSh900  |  Delivery: Within 30Mins.
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