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PROJECT TOPIC- ANTIMICROBIAL ACTIVITY OF METHANOL EXTRACT AND FRACTIONS OF MORINGA OLEIFERA LAM. ROOT BARK ON CLINICAL ISOLATES OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS

PROJECT TOPIC- ANTIMICROBIAL ACTIVITY OF METHANOL EXTRACT AND FRACTIONS OF MORINGA OLEIFERA LAM. ROOT BARK ON CLINICAL ISOLATES OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS

 

ABSTRACT

Development of antimicrobial resistance by bacteria is now a world wide health issue, as infection is one of the leading causes of death in the world today. This fact is also as a result of the emergence of multiple antibiotic resistant bacteria known as methicillin resistant Staphylococcus aureus (MRSA) with potential of cross resistance to other antibiotics of choice like vancomycin. MRSA is often referred to as a potential killer and one of the tree top superbugs in hospitals multidrug resistant organisms (MDRO). The aim of this study was to evaluate the phytochemical components and antimicrobial activity of methanol extract and fractions of Moringa oleifera root bark as possible remedy for MRSA infections.
Staphylococcus aureus isolates from 3 different hospitals in South-east geopolitical region of Nigeria were confirmed by coagulase/staphylase test using Oxoid® reagents kits (DR0595A). The characterised S. aureus isolates were further identified as Methicillin resistant staphylococcus aureus by disc diffusion method as recommended by the Clinical Laboratory Standards Institute (CLSI), using standard antibiotic discs containing oxacillin (5 μg/ml), vancomycin (30 μg/ml), cephalexin (30 μg/ml), levofloxacin (5 μg/ml), ciprofloxacin (5 μg/ml), tetracycline (30 μg/ml), cotrimoxazole (25 μg/ml), gentamicin (30 μg/ml), clindamycin (2 μg/ml) and rifampicin (5 μg/ml). Methicillin resistant staphylococcus aureus confirmation was done using Oxoid® DR0900 penicillin binding protein (pbp2ˈ) latex agglutination test kits. Pulverised Moringa oleifera root bark was defatted with n-hexane to
yield hexane fraction (HEF). The dried marc was extracted with methanol using Soxhlet extractor to obtain crude methanol extract (ME). Methanol extract was adsorbed on Silical gel (60-200 mesh) and eluted in succession to obtain dichloromethane fraction (DMF), ethyl acetate fraction (EAF) and methanol fraction (MEF). Qualitative phytochemical analyses of the extracts were carried out using standard procedures. The antimicrobial activities of ME, HEF, DMF, EAF and MEF were evaluated on the MRSA, the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were recorded and compared with the standard disc antimicrobial test results. The extract fractions were analysed using gas chromatographic-mass spectrometry (GC-MS) for their bioactive compounds. The preliminary acute toxicity and sub-acute toxicity of ME and HEF were evaluated. Statistical analysis was done with ANOVA followed by Duncan post Hoc test using SPSS v 17 software. Characterised clinical isolates yielded 58 S. aureus strains. Antibiotic susceptibility tests indicated varied percentages of MRSA that were resistant to various antibiotics thus: oxacillin (62.1 ± 3.2%), vancomycin (60.4 ± 3.8%), cephalexin (55.2 ± 1.2%), levofloxacin (56.9 ± 2.2%), ciprofloxacin (56.9 ± 0.9%), tetracycline (65.5 ± 2.3%), cotrimoxazole (68.9 ± 0.8%), gentamicin (67.2 ± 1.3%), clindamycin (62.1 ± 3.3%) and rifampicin (62.1 ± 4.1%). Latex agglutination test confirmed 39 strains of the clinical isolates to be MRSA. The S. aureus isolates resistant to all the antibiotics including vancomycin at 30 μg/ml were sensitive to the extract and all its fractions: ME: MIC (3.0 ± 0.1 to 5.0 ± 0.5 mg/ml) and MBC (3.0 ± 0.1 to 6.0 ± 0.5 mg/ml); EAF: MIC (5.0 ± 1.1 to 8.0 ± 0.5 mg/ml) and MBC (5.0 ± 0.5 to 8.0 ± 0.5 mg/ml); DMF MIC (8.0 ± 1.1 to 10 ± 0.5 mg/ml) and MBC (8.0 ± 0.5 to 10 ± 0.5 mg/ml);
HEF: MIC (7.0 ± 0.5 to 8 ± 1.1 mg/ml) and MBC (7.0 ± 0.5 to 9 ± 0.5 mg/ml), MEF: MIC (9.0 ± 1.1 to 10.0 ± 0.5 mg/ml) and MBC (9.0 ± 0.5 to 10.0 ± 0.5 mg/ml). Phytochemical analysis of the extracts showed the presence of alkaloids, glycosides, steroids, terpenoids, flavonoids, saponins, tannins, resins, reducing sugars, proteins, fats and oil and carbohydrates. GC-MS analysis revealed over 100 distinct compounds, some of which are stigmasterol
(C29H48O), eugenol (C10H12O2), oxime (C3H7NO ) and ergosta-4, 22-dien-3-one (C28H44O). The oral acute toxicity test showed the LD50 of ME as 3663.96 mg/kg and HEF as 1934.15mg/kg, with no significant change (P > 0.05) in the hematological, serum biochemical parameters and weight of the rats.

CHAPTER ONE
1.0 Introduction

In the last few decades there has been an exponential growth in the field of herbal medicine. It is getting popularized in developing and developed countries owing to its natural origin and lesser side effects [1]. Herbal drugs constitute a major share of all the officially recognized systems of health in India viz. Ayurveda, Yoga, Unani, Siddha, Homeopathy and Naturopathy, except Allopathy. More than 70% of India’s 1.1 billion population still use these
non-allopathic systems of medicine [2]. In many developing countries, a large proportion of the population relies on traditional practitioners and their armamentarium of medicinal plants in order to meet health care needs. Although modern medicines may exist side-by-side with such traditional practice, herbal medicines have often maintained their popularity for historical and cultural reasons. Such products have become more widely available commercially, especially in developed countries. Use of herbal medicines in developed countries has expanded sharply in the latter half of the twentieth century. In India, herbal drugs are an integral part of the Indian system of medicine (Ayurveda), which is an ancient and mainstream system [3]. The evaluation of various plant products according to their traditional uses and medicinal value based on their therapeutic efficacy leads to the discovery of newer and recent drugs for treatingvarious ailments. This fact forms the basis for the development of new drugs from various plant sources. One of such plants of medicinal value is Moringa olifera, belonging to the family Moringaceae, commonly known as ‘sahajan’ in Hindi, Horse radish in English. It is a small, fast, growing, evergreen, or deciduous tree that usually grows up to 10 or 12 m in height. It is distributed among Sub Himalayan Tracts, Assam, Bengal and Peninsular India [4]. Various properties are attributed to it like antispasmodic, diuretic, expectorant and abortifacient [5].

PROJECT TOPIC- ANTIMICROBIAL ACTIVITY OF METHANOL EXTRACT AND FRACTIONS OF MORINGA OLEIFERA LAM. ROOT BARK ON CLINICAL ISOLATES OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS

 

1.1 History of Moringa oleifera

It is a small or medium-sized tree, attractive enough to be a focal point in the tropics and sub-tropics owing to its creamy – white, sweetly scented flowers and light –green, tripinnately compound foliage [1-3]. It is a native to India, occurring wild in the sub- Himalayan regions of Northern India and cultivated throughout the country. It is commonly known as Sajina, sajna (Bengali); horseradish tree, drumstick tree (English); Sahinjan, mungna (Hindi); murinna, muringa, tishnagandha (Malyalam); sevaga, segata (Marathi); Sohanjana (Punjabi); Sobhanjana, sigru, murungi, dvishiguru (Sanskrit) and Sehjan(Urdu) in varied Indian languages and regions [4,5]. It also thrives well in Pakistan, Bangladesh, Sri Lanka, tropical Africa, Arabia, Philippines, Cambodia and Central, North and South America [6-10]. Described as “one of the most amazing trees God has created”, almost every part of drumstick viz. bark, root, fruit, flowers, leaves, seed and gum is a rich repository of proteins, vitamins and minerals including potassium, calcium, phosphorus, iron, folic acid as well as β carotene. Leaves can be eaten fresh, cooked or stored as dry powder for many months without refrigeration, without loss of nutritional value. Almost all the parts of this plant have been used for various ailments in the indigenous medicine of South Asia [11, 12]. The named
varieties of moringa include Jaffna or Yazhpanam, grown in various parts of South India, (producing 60-90 cm long pods), Chavakacheri murungai, (producing pods 90-120 cm long), Chemmurungai (with red tipped fruits), Kadumurungai, Palmurungai, Puna murungai (with thick pulp and bitter taste), Kodikkal Murungai etc. [13,14]. The Horticultural College & Research Institute of Tamil Nadu Agricultural University has released two improved annual moringa varieties (PKM1, PKM2) within a span of 10 years, for commercial cultivation [15, 16]. The folklore claims and ancient literature report
moringa to be an abortifacient antidote, antirheumatic, bactericide, diuretic, ecbolic, emetic, expectorant, purgative, rubefacient, stimulant, tonic, vermifuge and vesicant [17-20].

PROJECT TOPIC- ANTIMICROBIAL ACTIVITY OF METHANOL EXTRACT AND FRACTIONS OF MORINGA OLEIFERA LAM. ROOT BARK ON CLINICAL ISOLATES OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS

 

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