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In-Vitro Evaluation of the Antimicrobial Activity of Extracts of Bridelia Micrantha on Selected Bacterial Pathogens

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In-Vitro Evaluation of the Antimicrobial Activity of Extracts of Bridelia Micrantha on Selected Bacterial Pathogens Journal of Medicinal Plants Research Vol. 5(20), pp. 5116-5122, 30 September, 2011 Available online at http://www.academicjournals.org/JMPR ISSN 1996-0875 ©2011 Academic Journals Full Length Research Paper In-vitro evaluation of the antimicrobial activity of extracts of Bridelia micrantha on selected bacterial pathogens Adefuye A. O.1, Samie A.2 and Ndip R. N.1,3 * 1Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of Fort Hare, P/Bag X1314, Alice 5700, South Africa. 2Department of Microbiology, University of Venda, Thohoyandou, South Africa. 3Department of Biochemistry and Microbiology, Faculty of Science, University of Buea, Box 63, Buea, Cameroon. Accepted 4 July, 2011 Medicinal plants have been age long remedies for human diseases because they contain components of therapeutic value. In this study, six solvent extracts [dichloromethane, ethyl acetate, acetone, ethanol, methanol (100%), and 40% methanol hydroxide] of the stem bark of Bridelia micrantha and ciprofloxacin were investigated for antimicrobial activity by the agar-well diffusion method against strains of Staphylococcus aureus NCTC 6571 , Shigella sonnei ATCC 29930, Salmonella typhimurium ATCC 13311, Helicobacter pylori ATCC 43526, and Helicobacter pylori 252c. The most active extracts were assayed for MIC 50 using the 96-well microdilution technique and one way ANOVA test was used to determine if there was any statistically significant difference in the MIC 50 of the most active extracts and the control antibiotic (ciprofloxacin). Results obtained indicated that methanol was quantitatively the best solvent for extraction, while ethyl acetate was the least. Zone diameters of inhibition ranged from 0 to 28 mm for the six extracts and 29 to 38 mm for ciprofloxacin. Ethyl acetate and acetone extracts were the most active of all the extracts exhibiting a broad spectrum activity. However, Gram-positive bacteria were more sensitive compared to Gram-negative bacteria. The MIC 50 value ranged from 0.078 to 1.25 mg/ml and 0.078 to 0.625 mg/ml for the acetone and ethyl acetate extracts respectively, with no statistically significant difference in potency (p value = 0.187) when compared to ciprofloxacin. Our findings demonstrate the in-vitro antibacterial activity of the crude extracts of B. micrantha , and therefore provide preliminary scientific evidence to justify the use of the plant in traditional medicine. Key words: Medicinal plant, solvent extract, Bridelia micrantha , MIC 50 , bacteria. INTRODUCTION Throughout life, humans carry a greater number of cells bacteria are pathogenic, thus causing medically important of our indigenous bacteria than of our own human cells, diseases globally. Staphylococcus aureus, Shigella with the skin, respiratory, and gastrointestinal tracts being sonnei, Salmonella enterica serova Tyhimurium , and portals of entry for the introduction of exogenous Helicobacter pylori are medically important pathogens organisms into the body. It is well established that causing life threatening infections such as pneumonia bacterial pathogens can survive in air, soil, and water for and meningitis, nosocomial infections (Lowy, 1998; long periods and their persistence in the environment National Nosocomial Infection Surveillance, 2001), leads to increased risk of infection in human and animal various gastric ailments; shigellosis (Kotloff et al., 1999), hosts (Lemunier et al., 2005). Although the vast majority non-typhoidal salmonellosis (Madigan et al., 2000), of bacteria are harmless or beneficial, quite a few chronic gastritis, peptic ulcer, duodenitis (Ahmed et al., 2007), and an important risk factor for the development of gastric adenocarcinoma and mucosal associated lymphoid tissue (MALT) lymphoma (Mbulaiteye et al., *Corresponding author. E-mail: [email protected] or 2009). These infections have become more difficult to [email protected]. Tel: +27 782696191. Fax: +27 866224759. treat due to the emergence of multidrug resistant strains Adefuye et al. 5117 (MRSA, VRSA, and S. typhimurium DT104) (Akoachere Skirrow’s supplement and incubated microaerobically at 37°C for 5 et al., 2009; Nkwelang et al., 2009) and resistance to days. Confirmed isolates were suspended in 20% glycerol and conventional antibiotics such as ampicillin, amoxicillin, stored at -80°C (Ndip et al., 2008), as working stock. The other organisms were resuscitated in BHI broth and later sub-cultured on chloramphenicol, and trimethoprim - sulfamethoxazole fresh nutrient agar plates 24 h before use (Adeleye et al., 2008). (Chiu et al., 2006; Ndip et al., 2008; Tanih et al., 2010); Pure isolates were then inoculated on fresh nutrient agar slant and as well as to the quinolones and derivatives of kept at 4°C as working stock. fluoroquinolones (nalidixic acid and ciprofloxacin) have emerged (Hakanen et al., 2006). Preparation of plant extracts According to the World Health Organization (WHO), as many as 80% of the world's population depend on The stem bark of B. micrantha was selected based on traditional medicine for their primary healthcare needs ethnobotanical information and identified in collaboration with (Muthu et al., 2006). In sub-Saharan Africa medicinal botanists at the University of Venda, Limpopo Province, South plants have shown great promise in the treatment of Africa where voucher specimens (BPO3) have been deposited. The method described by Ndip et al. (2007) was used with infectious diseases (Ndip et al., 2007). modifications. The harvested plant was air dried for 2 weeks and Bridelia micrantha (Euphorbiaceae) is a semi- ground to fine powder using a blender (ATO MSE mix, 702732, deciduous to deciduous tree up to 20 m tall with a dense England). Analytical grade ethyl acetate, acetone, ethanol, rounded crown and tall, bare stem. It does well in a wide methanol (100%), and (40%) methanol hydroxide (Merck) were variety of climates and it is naturally distributed from the used for extraction. Briefly, dried plant material (2.5 kg) was Sudan in the north to the Eastern Cape in South Africa macerated in five fold excess of the solvent in extraction bottles with the level of the solvent above that of the plant material. The where it is known locally as: bruinstinkhout, mitserie mixture was placed in a shaker (Edison, N.J., USA) at room (Afrikaans), umHlahla-makwaba (Xhosa), temperature (RT) for 48 h and then centrifuged (Model TJ-6 isiHlalamangewibi, umHlahle, umHlalamagwababa, Beckman, USA) at 3000 rpm for 5 min, and later filtered using filter umShonge (Zulu) (Orwa et al., 2009). Virtually all parts of paper of pore size 60 Å. This process was repeated twice for a total the plant are used traditionally to treat different human of three extractions (exhaustive extraction) for each solvent. The ailments including gastritis, salmonellosis, gastro- combined filtrate was concentrated in a rotavapor (BUCHI R461, Switzerland) and the plant extract obtained transferred to labelled enteritis, diarrhea, tapeworms and as an emetic for vials and allowed to stand at RT for 24 h to permit evaporation of poisons (causes vomiting) (Orwa et al., 2009), joint the residual solvent. Stock solutions were prepared by dissolving 3 aches, cough, conjunctivitis, skin problems such as g of the extract in Dimethyl Sulphoxide (DMSO) and the remainder ulcers, boils and rashes, as an antimalarial, for toothache kept in the extract bank. and gum diseases, psychological problems such as neurosis and psychosis (www.blackherbals.com). Determination of solvent extraction strength Extracts of the plant have been reported to be active against E. coli, K . pneumoniae, S. flexneri, and P. To determine the extraction efficiency of different solvents, 100 g of aeruginosa (Samie et al., 2005). It has also been shown fresh plant material was macerated in 300 ml of each extracting solvent, and extraction was done as described previously. The to be a possible principle inhibitor to HIV-1 reverse mass of each solvent extract was measured and presented in a bar transcriptase (Bessong et al., 2005). However, we are not chart, as mass of extract against solvent (Masoko et al., 2008). aware of studies which have investigated B. micrantha for its antimicrobial activity against the selected pathogens Screening of crude extracts for antimicrobial activity which constitute potential health problems in our environment, necessitating the current study. The agar-well diffusion method was used as previously described (Boyanova et al., 2005; Ndip et al., 2007) . In performing this experiment, a total of six solvent extracts were used; the five stated MATERIALS AND METHODS earlier and a dichloromethane extract that already existed in our laboratory. A three day old culture of H. pylori was suspended in Bacterial species sterile normal saline and the density adjusted to equal 0.5 McFarland standard. Bacterial species used in this study include the following reference This was carefully swabbed on fresh BHI agar plates (Oxoid, strains; S. aureus NCTC 6571 , S. tyhimurium ATCC 13311 , S. England) supplemented with 7% laked horse blood (Oxoid, sonnei ATCC 29930, H. pylori ATCC 43526, and H. pylori 252c (a England), and Skirrow’s supplement (Oxoid, England). The plates local drug resistant strain) (Tanih et al., 2010). These strains were were allowed to dry for 3 to 5 min before use. Thereafter, the crude selected based on the frequency of infections and antimicrobial extracts (dichloromethane, ethyl acetate, acetone, ethanol, resistance they exhibit in the developing world (Gangoue-peiboji
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