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 (pbp2Ëˆ) 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
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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.
1
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