ABSTRACT
In an attempt to explain the scientific basis for the medicinal and nutritional benefits of the leafy vegetables of O. gratissimum L. cultivated within Nsukka area of Enugu state, in the South Eastern region of Nigeria, the chemical constituents, the antimicrobial properties and the synergistic potential (with standard antibiotics) of the leaves’ hydro distilled essential oil were evaluated in vitro against 146 clinical isolates of Gram positive (Staphylococcus aureus, Streptococcus spp., Coagulase Negative Staphylococcus spp.), Gram negative (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumonia, Proteus mirabilis) bacteria and Candida albicans using standard methods.
Hydro distillation of the fresh leaves yielded 0.97% (v/w) oil, while GC-MS analysis of the oil identified 27 constituents with Thymol(26.6%), γ-Terpinene (14.4%),β-trans-Ocimene (10.1%), and α-Selinene (6.4%) as the major components. An Antibiogram conducted on the isolates using some commonly used antibiotics showed that with the exception of P. aeruginosa isolates all the other bacterial isolates were more susceptible to the essential oil than the conventional antibiotics. The preliminary antimicrobial screening of the essential oil also demonstrated a concentration dependent activity against all the isolates. The minimum inhibitory concentration (MIC) of the oil on the isolates determined by the agar dilution method ranged between 0.02 and 2.50%v/v; the MIC of ciprofloxacin on bacterial isolates ranged between 0.006 and 25.00 µg/ml, while that of fluconazole on Candida albicans isolates ranged between 9.77 and 1,250µg/ml.Pseudomonas aeruginosa isolates were the least susceptible to the oil and the most susceptible to ciprofloxacin.Out of the 180 varied combinations of the agents in the interaction study, 62% showed Synergism, 31% showed Additivity, while 7% showed Indifference and there was no case of Antagonism.
The result revealed that the O. gratissimum L. cultivatedwithin the region are of thymol chemotype. It also validated the folkloric use of the plant in herbal medicinal practices and proved that the concomitant administration of these standard antibiotics with food containing the leaves of O. gratissimum L. will not antagonize the activity of the drug.
CHAPTER ONE
INTRODUCTION
1.1 General Introduction
The history of the use of herbs dates back to the time of the early man who used herbs to keep fit (Kafaru, 1994). The earliest evidence of humans’ use of plants for healing dates back to the Neanderthal period (Kleiner, 1995). This made botanic medicine one of the oldest practiced professions by mankind (Kambizi and Afolayan, 2001).
Herbal medicine practice flourished until the 17th century when more “scientific pharmacological remedies were favoured (Trevelyan, 1993). In the early 19th century, scientific methods became more advanced and preferred, and the practice of botanical healing was dismissed as quackery (Aiyegoro et al., 2010). Before then the plant kingdom had served as an inexhaustible source of useful drugs, foods, additives, flavouring agents, lubricants, colouring agents and gums from time immemorial (Parikh et al., 2005). Different medicinal plants have different medicinal properties and no one herb is found to be used for just one purpose (Kafaru, 1994).
Recently, researchers’ interest on the role of complementary and alternative medicines for the treatment of various acute and chronic diseases is on the increase (Schafer and Wink, 2009). Presently, it is estimated that over 80% of the developing world’s population still depend on herbal medicines, while half of the population in the industrialized countries, also use herbal formulations for health care (Akerele, 1993;WHO, 1998).
Plants’ vegetables and spices used in folk and traditional medicine have gained wide acceptance as one of the main sources of prophylactic and chemo-preventive drug discovery and development (Schafer and Wink, 2009). Worldwide, herbal use again became popular and in 1974 the World Health Organization (WHO, Geneva, Switzerland) encouraged developing countries to use traditional plant medicines to “fulfil a need unmet by modern systems” (Trevelyan, 1993). Hence, the practice of herbalism has become main stream throughout the world within the last century. Moreover, in spite of the great advances observed in modern medicine, plants still make an important contribution to health care and it is estimated that about 30% of the drugs in the modern pharmacopeia were derived from plants and many others, which are synthetic analogues, were built on prototype compounds isolated from plants (Farnsworth, NR et al., 1985; De Silva, 2005; Kim, 2005) and over the years there have been increasing interests in the use of herbal therapeutics worldwide. This is due in part to the recognition of the value of traditional medical systems, particularly of Asian origin, and the identification of medicinal plants from indigenous pharmacopoeias, which have significant healing power. Medicinal plants are distributed worldwide, but they are most abundant in tropical countries (Calixto 2000, Lewis 2001). In Brazil, around 80,000 species of higher plants were described, which offer enormous prospects for discovering new compounds with therapeutic properties. There has recently been a renewed interest in the antimicrobial effects of natural compounds which were commonly used as health remedies in the Western world until the advent of antibiotic drugs in the 1940s and 50s.
1.1.1 Justification for the renewed interest in herbal medicine
After the emergence of antibiotics many previously fatal infections and infectious diseases were brought under control and millions of lives were saved. Due to the dramatic effect of the new synthetic drugs, some health professionals even believed that the threat to mankind of pathogenic microorganisms had finally been eliminated (Halldor, 2011).
The success of antibiotic drugs is due to the fact that our knowledge of their actions is based on a solid scientific ground. Their actions are in most cases predictable and their side effects known, because they have undergone a thorough scientific scrutiny, for both safety and activity, before being applied to the general public. In contrast, the use of natural health remedies was for a long time mainly based on anecdotal evidence and on accumulated experience of their beneficial effects obtained over centuries. The knowledge was mostly empirical. However,recently, and mostly during the past few decades, the antimicrobial actions of the natural compounds, which originate in both the animal and the plant kingdom, have been studied by modern scientific methods similar to those applied in the study of synthetic drugs (Halldor, 2011).
The great success of chemotherapy, using synthetic antibiotics against bacterial and fungal infections and nucleoside analogues against viral infections, discouraged researchers and the pharmaceutical industry from making serious efforts to develop drugs containing simple natural compounds. Recently, there is an increased attention on extracts and biologically active compounds isolated from plant species used in herbal medicine due to the side effects and the resistance that pathogenic micro-organisms build against conventional antibiotics (Essawi and Srour, 1999). The primary benefits of using plant-derived medicines are that they are relatively safer than synthetic alternatives, offering profound therapeutic benefits and more affordable treatment (Van Wyk and Gericke, 2000). Eloff (1999) stated that the antimicrobial compounds from plants may inhibit bacteria by a different mechanism than the presently used antibiotics and may have clinical value in the treatment of resistant microbial strains.
The clinically useful antibiotics now in use have major setbacks. Apart from the narrow spectrum of antimicrobial activity many of them have been found to be neurotoxic, nephrotoxic, ototoxic or hypertensive and few others cause severe damage to the liver and cause bone marrow depression (Chong and Pagano, 1997) and importantly; infectious pathogens have developed resistance to all known antibiotics (Aiyegoro and Okoh, 2009).
1.1.2 Justification for the use of plants in Combination Therapy
Several reasons have been advanced to justify the use of combination of two or more antibiotics in treatment (Esimone et al., 2006; Ibezim et al., 2006).
As certain antibiotic treatments lead to the development of multi resistant organisms, it is now a standard clinical practice to use a combination of two or more antibiotics with different mechanisms of action in an attempt to prevent the early development of antibiotic resistance and improve the outcome of therapy (Beringer, 1999). Combination therapy can be used to expand the antimicrobial spectrum, to prevent the emergence of resistant mutants, to minimize toxicity and to obtain synergistic antimicrobial activity, it could be an alternative to monotherapy for patients with invasive infections that are difficult to treat, such as those due to multi-resistant species and for those who fail to respond to standard treatment (Kamatou et al., 2006).
Synergism is a positive interaction created when two agents combine and exert an inhibitory effect that is greater than the sum of their individual effects. Antimicrobial compounds used in combination might promote the effectiveness of each agent, with efficacy being achieved using a lower dose of each drug. Pharmacological benefits would accrue, with one drug clearing infection from one body system while the other clears it from a different site (Williamson, 2001). In addition, synergism in antimicrobials could be utilized in an attempt to prevent or delay the emergence in vivo of resistant populations of the pathogenic organisms (Lupetti et al., 2002).
Betoni et al. (2006) demonstrated that plants either contain antimicrobials that can operate in synergy with antibiotics or possess compounds that have no intrinsic antibacterial activity but are able to sensitize the pathogen to a previously ineffective antibiotic. Various reports have documented the enhanced antimicrobial activities (i.e. synergistic potentials) of standard antibiotics in combinations with plant extracts even when the organisms are no more susceptible to the drug (Van Wyk and Gericke, 2000). It has been proven that, in addition to the production of intrinsic antimicrobial compounds, plants also produce Multi-Drug Resistance (MDR) inhibitors which enhance the activity of the antimicrobial compounds (Stermitz et al., 2000a). The synergistic effect of the association of antibiotic and plant extracts against resistant pathogens leads to new choices for the treatment of infectious diseases. Also synergy between bioactive plant product and antibiotics will confront the problems of toxicity and overdose since lesser concentrations of two agents in combination are required; due to these reasons, there is need therefore, for continuous exploration of multidrug resistance modulating principles from plants sources (Aiyegoro et al., 2008a).
