PHYTOCHEMICAL AND ANTIMICROBIAL STUDIES ON THE STEM-BARK OF COMMIPHORA MOLLIS (OLIV. ) ENGL. (BURSERACAEA)
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ABSTRACT
Commiphora mollis is traditionally used in the treatment of fever (malaria and typhoid) wound healing, cancer, ulcer and rheumatic condition. The plant grows in Nigeria and across Africa. Phytochemical analysis of methanol extract of the stem-bark of Commiphora mollis showed the presence of flavonoids, saponins, tannins, terpenoids and alkaloids. Extensive Phytochemical studies of ethylacetate water soluble fraction of methanol extract resulted in
isolation (55 fractions, 100ml each were collected and pooled together based on similarities in their TLC profile to gave 8 major fractions and repeated gel filtration chromatography of Fraction 5 on Sephadex LH-20 packed column then preparative gave 7.5 mg of compound X1) and characterization using 1HNMR (9 proton signal 5 aromatic proton δ 7.0- 5.94 ppm, 2 oxymithine protons at 4.8 and 4.2 ppm and 2 methylene protons at 2.8 and 2.75 ppm), 13CNMR (15 carbon signals 12 in aromatic region, 2 oxymethine carbon and 1 methylene carbon.), DEPT (7 methine 1 methylene 1 quatenery ), 1H 1H COSY ( correlation between
protons at 7.0, H2’ // 6.83, H6’ 7.0, H2’ // 4.8, H2 6.83, H6’ // 4.8, H2 4.8, H2 // 4.2,
| H3 4.8, H2 // | 2.8, H4 4.2 , H3 // 2.8, H4 ), HSQC (correlation between proton and | ||
| carbon at | 7.0, H2’ // 114, C2’ | 6.83, H6’ // 118.10, C6’ 6.8, H5’ // 114.60, C5’ 5.8, H6 // | |
| 94, C6 | 4.82, | H2 // 78.48, C2 | 4.21, H3 // 66.10, C3 2.8, H4 and 2.7, H4 // 27.84, C4 ), |
HMBC and NOESY. The antimicrobial studies of the crude methanol extract, Chloroform and ethylacetate fractions were carried out using disc diffusion and broth agar dilution methods on clinical isolates of Corynebacterium ulcerans, Salmonella typhi, Proteus mirabilis, Bacillus sabtilis, Staphylococcus aureus, Streptococcus pyogens, Escherichia coli, Candida brusei, Shigella dysenteriae and Candida albicans. The crude methanol extract, chloroform and ethylacetate fractions showed strong inhibitory activity against all tested microoganisms with exception of Corynebacterium ulcerans, Salmonella typhi and Proteus mirabilis. Crude methanol extract was found to have MIC at 10 mg/ml for all organisms and variable value of MBC/MFC and that of chloroform and ethylacetate fractions have variable value for both MIC and MBC. The stem-bark of Commiphora mollis was rich in bioactive phytochemicals which have antimicrobial activity and could serve as a potential source of compounds effective against disease causing micro- organisms.
CHAPTER ONE
1.0 INTRODUCTION
Natural products are the chemical compounds found in nature that usually have a pharmacological or biological activity for use in pharmaceutical drug discovery and drug design (Samuel, 1999). According to the World Health Organization (WHO), about 80 % of the world’s population relies on traditional medicine for their primary health care need (WHO, 2002). For thousands of years natural products have played a very important role in health care and prevention of diseases. The ancient civilizations of the Chinese, Indians and North Africans provide written evidence for the use of natural sources for curing various diseases (Phillipson, 2001). Secondary metabolites are chemical compounds derived from living organisms. The study of natural products involves isolation in a pure form of these compounds and investigation of their structure. Secondary metabolites appear to function primarily in defense against predators and pathogens and in providing reproductive advantage as intraspecific and interspecific attractants. They may also act to create competitive advantage as poisons of rival species (Croteau, et al., 2000). Many plant terpenoids are toxins and feeding deterrents to herbivores or are attractants, and many possess pharmacological activity. Tannins, lignans, flavonoids, and some simple phenolic compounds serve as defenses against herbivores and pathogens, also many flavonoid pigments are important attractants for pollinators and seed dispersers.
The study of natural products has had a number of rewards. It has led to the discovery of a variety of useful drugs for the treatment of diverse ailments and contributed to the development of separation science and technology, spectroscopic methods of structure elucidation and synthetic methodologies that now make up the basics of analytical organic chemistry. One of the most important areas of application of natural products is in the treatment of human and veterinary ailments (Newman et al., 2000). Although the use of natural products as medicinal agents presumably predates the first recorded history as the earliest humans used various, but specific plants to treat illness, the treatment of diseases with pure pharmaceutical agents is a relatively modern phenomenon. For thousands of years medicine and natural products have been closely linked through the use of traditional medicines and natural poisons (Butler, 2004). Clinical, pharmacological, and chemical studies of these traditional medicines, which were derived predominantly from plants, were the basis of most early medicines such as aspirin (I), morphine (II), digitoxin (III) and pilocarpine (IV) (Butler, 2004). The discovery of antibacterial filtrate “penicillin” by Fleming in 1928, re-isolation and clinical studies by Chain, Florey, and co-workers in the early 1940s, and commercialization of synthetic penicillins evolutionized drug discovery research (Butler, 2004). Following the success of penicillin, drug companies and research groups soon assembled large microorganism culture collections in order to discover new antibiotics. The output from the early years of this antibiotic research was prolific and included examples such as streptomycin (V), chloramphenicol (VI), chlortetracycline (VII), cephalosporin C (VIII), erythromycin (IX), and vancomycin (X) (Butler, 2004). All of these compounds, or derivatives thereof, are still in use as drugs today.
H1.1 Traditional medicine (TM):
Traditional medicine is the sum total of the knowledge, skill, 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 illness (WHO, 2013). It is widely practiced, especially in developing countries. This is a result of primary health care facilities being unable to manage the number of patients requiring aid, the high cost of Western pharmaceuticals and health care, as well as the fact that traditional health care is highly sought after in terms of certain cultural elements in the lives of these individuals within these societies (Taylor et al., 2001). The World Health Organization estimates that 80% of the populations of Asia, Africa and Latin America use traditional medicine to meet their primary health care needs. For many people in these countries, particularly those living in rural areas, this is the only available, accessible and affordable source of health care (WHO, 2010).
Traditional medicine has also maintained its popularity in all regions of the developing world and its use is rapidly spreading in the industrialized countries. In China, for example, traditional herbal preparations account for 30%-50% of the total medicinal consumption (Bannerman et al., 1993). In Ghana, Mali, Nigeria and Zambia, the first line of treatment for 60% of children with high fever resulting from malaria is the use of herbal medicines at home (Bannerman et al., 1993). Practices of traditional medicine vary greatly from country to country, and from region to region, as they are influenced by factors such as culture, history, personal attitudes and philosophy. In many cases, their theory and application are quite different from those of conventional medicine. Long historical use of many practices of traditional medicine, including experience passed on from generation to generation, has demonstrated the safety and efficacy of traditional medicine. However, scientific research is needed to provide additional evidence of its safety and efficacy (WHO, 2000). In many parts of the world, policy-makers, health professionals and the public are wrestling with issues regarding the safety, effectiveness, quality, availability, preservation and regulation of traditional medicines. At the same time, interest in traditional medicine is expanding beyond products to focus on practices and practitioners (WHO, 2013). This vast usage of and great dependence on traditional plants as the preferred form of health care is aided by the fact that most of these plants are widely available and affordable, and additionally encompasses practices based on the socio-cultural norms and religious beliefs. It is evident that, even though scientific advances have been made in our quest to understand the physiology of the body, biotechnology and the treatment of disease, natural products remain a crucial component of the comprehensive health care strategy for the future (Patwardhan, 2005). The Greek physician Dioscorides (AD 70) compiled an extensive listing of medicinal herbs and their virtues. This was originally written in Greek, and later translated into Latin as De Materia Medica, and remained the authority in medicinal plants for over 1500 years (Mendonça-Filho, 2006). Another Greek physician, Galen (AD 129-200), devised the pharmacopoeia describing the appearance, properties and use of many plants of his time. It was the discovery of medicines that sparked an interest in the study of plants as medicinal agents; with the isolation of morphine from opium by Serturner (1805) being the start of natural product chemistry (Patwardhan et al., 2004). Currently It is clear, however, that there is a need to validate the information through an organised research for it to be used as an effective therapeutic means, either in conjunction with existing therapies, or as a tool in novel drug discovery. Traditional medicine utilises biological resources and the indigenous knowledge of traditional plant groups, the latter being conveyed verbally from generation to generation. This is closely linked to the conservation of biodiversity and the related intellectual property rights of indigenous people (Timmermans, 2003). Although it is these traditional medicines that provided 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).
1.2 Anti-microbial Agents
Anti-microbial agents are substances that kill micro organisms or inhibit their growth. They are widely employed to cure bacterial diseases. Antimicrobial agents that reversibly inhibit growth of bacteria are called bacteriostatic whereas those with irreversible lethal action on bacteria are known as bactericidal (Rajesh and Rattan, 2008). Ideally, antimicrobial agents disrupt microbial processes or structures that differ from those of the host. They may damage pathogens by hampering cell wall synthesis, inhibiting microbial protein and nucleic acid synthesis, disrupting microbial membrane structure and function, or blocking metabolic pathways through inhibition of key enzymes (Willey et al., 2008). Haslam et al. (1989) reported that plant extracts and their products are used in many parts of the world as the active principles in herb remedies. They are used locally in the treatment of infections, many centuries before scientific studies were discovered. Before an antimicrobial agent is accepted for use in human beings it must demonstrate most, if not all, of the following properties: selective toxicity (it should act on bacteria without damaging the host tissues); it should be bactericidal rather than bacteriostatic; it should be effective against a broad range of bacteria; it should not be allergic; it should remain active in plasma, body fluids etc.; it should be stable and preferably water soluble; desired levels should be reached rapidly and maintained for adequate period of time; it should not give rise to resistance in bacteria; it should have long shelf life; it should not be expensive (Rajesh and Rattan, 2008).
1.3 Statement of Reseach Problem
It has been reported that most bacteria are resistant to currently used antimicrobial agents (Truiti et al., 2003). Also the severe side effects of most antimicrobial agents have necessitated the search for new antimicrobial with little or no side effect. This phenomenon of increased drug resistance, combined with the multiplicity of side effects caused by existing agents and the emergence of diseases for which no treatment yet exists, makes the search for new antimicrobial agents a highly relevant and important subject for research. For centuries, plants have been used in the traditional treatment of microbial infections. This assembly of knowledge by indigenous peoples about plants and their products continue to play an essential role in health care of a great proportion of the population (Iwu et al., 1999). Traditionally Commiphora mollis has been used in the treatment of fever infection, malaria, typhoid and cancer, oxidant, ulcer, rheumatic condition. The resin is applied topically to aid wound healing. For this reason it was concluded to screen and chemically characterized the stem-bark of this important medicinal plant.
1.4 Justification
Traditionally Commiphora mollis has been claimed to have medicinal properties which include, treatment of fever (malaria and typhoid), wound healing, cancer, ulcer and rheumatic condition.
Despite this, literature survey showed that no work has been carried out on the phytochemical analysis and pharmacological activities of the Commiphora mollis. For this purpose, such studies were initiated as a basis for scientific verification regarding the traditional use of Commiphora mollis.
1.5 Aim of the Study
The aim of this reseach work is to carry out phytochemical studies of the stem bark of Commiphora mollis and justify the ethnomedicinal claims of the use of the plant in the treatment of antimicrobial infection.
1.6 Objective of the Study
The Objectives of this study is to:
- Carry out pyhtochemicals screening of the stem-bark of Commiphora mollis
- Isolate some of the bioactive compounds present in the stem-bark of Commiphora mollis
- Elucidate the structure of the compounds isolated from the stem-bark of Commiphora mollis
- Validate the medicinal use of the stem-bark of the plant to treat infectious diseases.
1.7 Research Hypothesis
The stem-bark of Commiphora mollis contains phytochemical constituents with antimicrobial activities.