ABSTRACT
The plant Spermacoce verticillata is used in ethnomedicine for the treatment of itches, diarrhoea, sores and other ailments. The hexane soluble fraction of acetone extract of the whole plant Spermacoce verticillata was subjected to phytochemical and antimicrobial screening. The phytochemical techniques employed were dry column vacuum chromatography and preparative thin layer chromatography. The antimicrobial activity was studied using agar diffusion, and broth dilution methods. The result of preliminary phytochemical screening carried out on the crude acetone extract revealed the presence of carbohydrates, Flavonoids, Anthraquinones, terpernoids, Tannis and Steroids; Tannins flavonoids, and Steroids/terpenes were also found to be present in the hexane fraction. Extensive chromatographic separation of the hexane soluble fraction using silica gel, dry column chromatography, followed by preparative thin layer chromatography led to the isolation of stigmasterol. The structure of this compound was established by spectral analysis including 1D and 2D NMR. The result suggest that the hexane fraction of Spermacoce verticillata plant possess phytochemical constituents that may be useful in the management of microbial diseases.
CHAPTER ONE
INTRODUCTION
1.0 General Introduction
The use of plants and their preparations to treat infectious diseases is an age‐old practice and in the past possibly the only method available (Peach, 1995). However, the systemic study of plants for detecting antimicrobial activity is of comparatively recent origin. These investigations have been triggered by the emergence and spread of antibiotic resistant microorganisms causing the effective life‐span of existing antibiotics limited. Hence the plant kingdom is being screened for newer and effective chemotherapeutic agents. Higher plants can serve both as potential antimicrobial crude drugs as well as a source of new anti‐infective agents (Peach, 1995).
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 diseases, natural products remain a crucial, cheap and uncontroversial component of the comprehensive health care strategy for the future (Patwadhan, 2005). Natural products such as plant extracts, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drugs discoveries because of the unmatched availability of chemical diversity (Cosa et al., 2006). In the last decades, various studies have demonstrated that plants serve as reservoirs for innumerable microorganisms known as endophytes; these microorganisms live in the host plant at least for one period of their life cycle, without causing apparent harm to them (Petrini, 1991; Bacon and White, 2000). Even with the existence of uncountable epiphytic and soil microorganism, diverse studies have shown the potential of endophytes as a promising source of natural products for the discovery of a variety of different classes of bioactive molecules to be applied in medicine, industry and Agriculture (Schulz et al., 2002; Joseph and Priya, 2011). Investigations have shown that the search for microorganisms of biotechnological interest based in the ethno botanical pharmacology represents an alternative to discover new microorganisms and bioactive molecules (Li et al., 2005, Qin et al; 2011). Based on these principles, the bio prospection of microorganisms isolated from new ecological niches constitutes an important strategy for obtaining more efficient and less toxic antibiotics, which may ultimately control pathogenic bacteria that are resistant to the diverse antibiotics commonly used in current time (Appelbaum and Jacobs, 2005). The use of the plant to treat diseases continue to play a key role in health care. According to the World Health Organization (WHO), 80% of the developing world's rural population depends on traditional medicines for its primary healthcare needs (Bannerman et al; 1993). In some African countries such as Ghana, Mali, Nigeria and Zambia, the first line treatment for 60% of the children with high fever resulting from malaria is the use of herbal medicines at home (WHO, 2003). In these societies, the tradition of collecting, processing, and applying plants and plant-based medications have been handed down from generation to generation. Traditional Medicine, with medicinal plants as their most important components are sold in market places and prescribed by traditional healers in their homes (VonMaydell, 1996).
The development of resistance to most of the available antimicrobial agents and the high costs of treatment consequent upon this resistance has necessitated a search for new, safe, efficient and affective agents for the management of infection (Okwu and Uchenna, 2009). These research for new effective agents against infectious diseases and other diseases such as cancers, diabetes, cardio-vascular, neurological, respiratory disorders, among others; has led to increased interest in existing information about the remedies of these diseases from natural sources, principally the plants (Karou et al.,2007;Ouattara et al., 2007). Because of this strong dependence on plants as medicines, ethno pharmacological studies have been conducted to determine their safety and efficacy and on the other hand to find out new active principles from plants (Zongo et al., 2010; Ouattara et al., 2011).
Herbal medicine is now globally accepted as a valid alternative system of therapy in the form of pharmaceuticals, functional foods, a trend recognised and advocated by the World Health Organisation (WHO, 2003). Various studies around the world, especially in Europe, have been initiated to develop scientific evidence-based rational herbal therapies. Though ancient medical treatises have remained largely undocumented as the knowledge of their use are mainly being passed down from generation by word of mouth. New plants sources of medicine are also being discovered (WHO, 2003).
Herbal medicine can be valuable to modern medicine in many ways. Plants in the tropics are often used as direct source of drugs, e.g. the alkaloid tubercurarine (I), is used as muscle relaxant in surgery, and till date chemists are unable to produce it synthetically, although its analogue atracurium besylate (II) was synthesed (WHO, 2003).
In medicine, trees of the genus Cinchona are of great interest because of their alkaloids, the most familiar being quinine, (III) the first effective agent in treating malaria (WHO, 2003).
The search for biologically active molecules from marine organisms is a relatively new but rapidly expanding branch of natural products chemistry. It was estimated that about 3000 novel natural products were isolated in the last 30 years alone. These were isolated from sponges, corals, and sea-dwelling microorganisms. Example of bioactive compounds isolated from marine source is manoalide (IV) from a sponge, Litffariella variabilis. It is a 25-carbon marine natural product with anti-inflammatory activity which acts by selectively inhibiting cyclooxygenase (COX)-enzyme (DeRosa et al., 1998).
Plant kingdom has been haphazardly investigated; some families have been relatively well studied while others were almost completely overlooked. In general, of the estimated 250,000 higher plant species discovered, only about 6% have been screened for biologic activity and a reported 15% have been evaluated phytochemically (Fabricant and Farnsworth, 2001). A major challenge to herbal medicine and development of drugs from herbal medicine is the rapid rate of extinction among the global flora; the world genetic flora is rapidly diminishing. Tropical rain forest plants, which are found exclusively in developing countries, are being destroyed at the rate that many scientists believe to be unjustifiable from both economic and ecological perspectives (Principe, 1991). According to International Union for Conservation of Nature and Natural Resources (IUCN) and the World Wide Fund for nature (WWF), 60,000 higher plant species could become extinct or near extinct by the middle of this century if the present trend continues. The main causes of deforestation and subsequent extinction of plant species are: outgrowth of population and rural poverty shifting cultivation, agricultural conservation, fuel wood gathering and impact of large development projects (Allen and Bernes, 1985). Higher plants have been described as chemical factories that are capable of synthesizing unli-mited numbers of highly complex and unusual chemical substances whose structure could escape the imagination of synthetic chemist forever; considering that many of these unique gene sources may be lost forever through extinction and that plants have a great potential for producing new drugs of great benefit to mankind, some action need to be taken to reverse the current apathy (Farnsworth, 1988). The validation of the folkloric claims of these medicinal plants will provide scientific basis for the conservation of tropical medicinal resources, the deployment of the beneficial ones as phy-tomedicine in the primary health care and the development of potential bioactive constituents. These could provide novel lead compounds or precursors in drug development, and utilization of isolated compounds as investigative, evaluative and other research tools in drug development and testing processes. The approved and regulated chemical armamentarium available today is failing to provide effective health care for even one-third of human population ( Henry, 2000). In addition, a number of significant global disease, including cancer, malaria, tuberculosis and certain viral, fungal and bacterial infections are showing significant pattern of drug resistance to the known therapeutic agents, and the time and cost of developing a new drug from initial discovery to final approval are both too long and too expensive ( Cordell et al., 2001), because of the affirmation reasons it is obtained that drug discovery is necessary in many diseases such as cancer, heart diseases, lung diseases, HJV-infection, alzheimers diseases, arthritis and resistant fungal, bacterial and viral infections. Others are malaria, infantile diarrhea and tuberculosis (Cordell et al., 2001).
1.1 Bacterial infections
Infectious disease is one of the leading causes of death world-wide and accounts for approximately 50% of death in tropical countries (Iwu et al., 1999). Long before the discovery of the existence of microbes, the idea that certain plants had healing potential, indeed, that they contained what we would currently characterize as antimicrobial principles was well accepted. Since antiquity, humans have used plants to treat common infectious diseases, and some of these traditional medicines are still included as part of the habitual treatment of various maladies. For example, the use of bearberry Arctostaphylos uvaursi and cranberry juice Vaccinium macrocarpori to treat urinary tract infection is reported in different manuals of phytotherapy, while species such as lemon balm Melissa officinalis\ garlic Allium sativum, and tee tree Melalenca alternifolia are described as broad-spectrum antimi- crobial agents (Mendonca-Filho, 2006). That being said, it has generally been the essential oils of these plants rather than their extracts that have had the greatest use in the treatment of infectious pa-thologies in the respiratory system, urinary tract, gastrointestinal, and biliary systems, as well as on the skin. In the case of Melaleuca alternifolia, for example, the use of the essential oil (tee tree oil) is a common therapeutic tool to treat acne and other infectious troubles of the skin (Mendonca-Filho, 2006). Antimicrobial resistance is one of the biggest challenges facing global public health. Although antimicrobial drugs have saved many lives and eased the suffering of many millions, poverty, ignor-ance, poor sanitation, hunger and malnutrition, inadequate access to drugs, poor and inadequate health care systems, civil conflicts and bad governance in developing countries have tremendously limited the benefits of these drugs in controlling infectious diseases. The development of resistance to the pathogens has worsened the situation, often with very limited resources to investigate and provide reliable susceptibility data on which rational treatments can be based as well as the means to optimize the use of antimicrobial agents (Mendonca-Filho, 2006).The emergence of multidrug-resistant isolates in tuberculosis, acute respiratory tract infections, and diarrhea, often referred to as the diseases of poverty, has had its greatest toll in developing countries. The epi-demic of HIV/AIDS, with over 30 million cases in developing countries, has greatly enlarged the population of immune compromised patients. The disease has left these patients at great risk of numerous infections and even greater risk of acquiring highly resistant organisms during long periods of hospitalization (Mendonca-Filho, 2006). Antibiotic resistance can occur via three general mechanisms: prevention of interaction of the drug with target, efflux of the antibiotic from the cell, and direct destruction or modification of the compound. The emergence of multidrug resistance in human and animal pathogenic bacteria as well as undesirable side-effects of certain antibiotics has triggered immense interest in the search for new antimicrobial drugs of plant origin (Ahmad and Beg, 2001). Many plants are used in treating bacterial related diseases. Only a small fraction of these plants has been investigated (Cart and Rogers, 1987). Several compounds were isolated and have demonstrated good antimi-crobial activity, and these include: Asiaticode, an anti-microbial compound isolated from Centel-laasiallca (used traditionally in skin diseases and leprosy), has been studied in normal as well as delayed-type wound healing. The results indicated significant wound healing in both models. Another compound, cryptolepine, isolated from Crytolepis sanguinolenta which was found to be active against Campylobacter species, has been used traditionally in Guinea Bissau in the treat-ment of hepatitis and in Ghana for the treatment of urinary and upper respiratory tract infections and malaria (Angeh, 2006). The development of organic chemistry took place along with the study of plants, mainly in the 19th century, when the first studies on plants were scientifically recorded. This ended up in the isolation of some active compounds from plants (Wheelwring, 1974). It is the vegetal kingdom that has contributed in a more meaningful way, to the supply of useful substances for the treatment of human diseases (Galhiane, 2006).
1.2 Statement of Research Problem.
Infectious diseases are the number one cause of death world-wide, and in tropical countries it accounts for approximately 50% of death (Iwuet al., 1999). Apart from resistance, some antibiotics have serious undesirable side effects which limit their applications. Therefore, there is a serious need to develop new antimicrobial agents that are very effective with minimal side effects. Higher plants represent a potential source of novel antibiotic, prototypes (Maureer-Grims et al., 1996)
1.3 Justification of the Study.
Spermacoce verticillata has been used traditionally in the management of microbial infections. Literature search in scientific data base showed that no much phytochemical work was reported on the plant. Many plant extracts from other species of the family have been tested and found to have antimicrobial activities. This makes it necessary to search for drugs that may be more potent. This will go a long way to proposing a template or lead that can be used for designing other drugs.
Aims of the study:
The aim of the research is to carry out phytochemical studies on Spermacoce verticillata, and evaluate its anti-microbial activities.
1.4 Objectives of the Study.
To carry out preliminary phytochemical screening of S. verticillata using standard procedures. To isolate some of the biologically active compounds from the plants
To elucidate the structure of the isolated compounds using spectroscopic analysis To validate the anti-microbial activities of the plant
1.5 Statement of Research Hypothesis.
The whole plant of Spermacoce verticillata contains phytochemical constituents with antimicrobial activity.
