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ANNALS OF MICROSCOPY

Vol 8, April 2008

Effects of Methanol, Ethanol and Aqueous Extract of Plantago major on Gram Positive Bacteria, Gram Negative Bacteria and Yeast

Sharifa A.A., Neoh Y.L.., Iswadi M.I., Khairul O., Abdul Halim M., Jamaludin M., Mohamed Azman A.B and H.L. Hing.

Department of Biomedical Sciences, Faculty of Allied Health Sciences, Universiti Kebangsaan Malaysia, 50300 Kuala Lumpur, Malaysia.

ABSTRACT Plantago major which is widely distributed in Malaysia was taken to study the antibacterial effect against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Candida albicans and Candida tropicalis. The whole plant was extracted using methanol, ethanol and aqueous as solvent extractor. The results showed that methanol extracts of Plantago major have antibacterial effects against Gram positive and Gram negative bacteria such as S. aureus with MIC value of 100mg/ ml and E. coli with MIC value of 120mg/ml. The ethanol extract of P. major also showed an antibacterial effect on S. aureus with MIC value of 200mg/ml and on E. coli with MIC value of 140mg/ml. There is no antibacterial effect of methanol and ethanol extracts of P. major on B. subtilis. The aqueous extract of P. major has no antibacterial effect on S. aureus, E. coli and B. subtilis. All these extracts with MIC value between 100­200mg/ml shows no effects on C. albicans and C. Tropicalis at all. INTRODUCTION Plantago major is a perennial plant belonging to Plantaginaceae family. It can grow up to 15 cm high. The leaves grow in rosettes and may ovate to elliptical with parallel venation. The leaves are glabrous and have an entire or irregularly dentate margin. The flowers are small, brownishgreen and long non-ramified spikes. The seeds are quite small with an ovate shape (0.4-0.8x0.8-1.5 mm) and a slightly bitter taste. P.major was introduced to the Nordic countries nearly 4000 years ago (Jonsson, 1983). Phytochemical studies on P. major showed that several flavonoids like plantaginin and homoplantaginin. The flavonoids are antioxidant (Bohm et al. 1998). The methanol, ethanol and aqueous extract contain antibacterial activity against some Gram negative and positive bacteria in addition to a weak anti-mycotic activity. The objective of this paper is to present the antibacterial effect of P. major extracts against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Candida albicans and Candida tropicalis. MATERIALS & METHODS Whole Plantago major plants (stems, leaves and roots) were collected from Cameroon Highland and identified. The fresh specimen was washed, cut into small pieces and dried at room temperature for a week before Soxhlet extracted with methanol, ethanol and aqueous method (Ibrahim et al. 1996). Extracts obtained were tested on S. aureus, B. subtilis, E. coli, C. Albicans, and C. Tropicalis. Sensitivity test was done using disc diffusion with positive effects of extracts samples observed with Scanning Electron Microscopy.

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ANNALS OF MICROSCOPY

Vol 8, April 2008

Figure 1. Untreated S. aureus used as control

Figure 2. S. aureus treated with methanol P. major extract (100mg/ml).

Figure 3. S. aureus treated with ethanol of P. major extract (200mg/ml).

Figure 4. Untreated E. coli used as control

Figure 5. E. coli treated with methanol extract P. major extract (120mg/ml).

Figure 6. E.coli treated with ethanol P. major extract (140mg/ml).

Electron microscopic observation Glass cover slip placed on top of the inhibition zone and the bacterial growth zone was obtained. The cover slip was then fixed in 10% buffered formalin for 2-4 hours. The fixed samples were then washed with phosphate buffers, dehydrated in serial dilution of alcohol up to 70%, left to dried, mounted on stub and gold-coated. The prepared stub was then placed in the LEO VPSEM for observations.

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ANNALS OF MICROSCOPY

Vol 8, April 2008

RESULTS AND DISCUSSIONS The antibacterial effect of various extracts of P. major were seen on S. aureus, and E. coli. After exposure to 200 mg/ml of methanol and ethanol of P. major, the cell wall of S. aureus was crenated and cells were sticky (Figure 2 & 3), almost seen like a shrunken cells as compared to control (Figure 1). With E. coli, methanol and ethanol extracts of P. major showed blebs formation and indentations (Figure 5 & 6) as compare to control (Figure 4). The aqueous extract has no effect on the bacteria tested. This fact is similar to the findings of Hetland et al. (1999). The MIC value of methanol extract was lower than ethanol showing that there was more active antibacterial ingredient in methanol than in ethanol extract. The findings were similar to the findings of Samuelsen et al. (2000) and Hoj et al. (2001). All the three extracts have no effects on the yeast tested. CONCLUSIONS Methanol and ethanol extract of P. major of 100-200mg/ml showed anti-bactericidal activity against both Gram positive and Gram negative bacteria tested. The extract caused the cell wall of Gram positive bacteria to collapse and blebs formations on Gram negative bacteria. All of these extracts have no antibacterial effects on yeast. ACKNOWLEDGEMENTS We would like to acknowledge the support given by Faculty of Allied Health Sciences, Universiti Kebangsaan Malaysia (UKM) and Forest Research Institute of Malaysia (FRIM). REFERENCES Bohm H, Boeing H, Hempel J, Raab B, Kroke A. 1998. Flavonols, flavones ad anthocyanins as native antioxidants of food and their possible role in the prevention of chronic diseases. Zeitschrift fur Ernahrungswissenschaft 37: 147-163 Ibrahim BJ, Kang YH, Sud DY, Han BH 1996. Inhibition effects of Malaysian medicinal plants on the platelet- activating factor (PAF) receptor binding. Natural Product Sciences 2(2): 86-89. Jonsson S. 1983. Blomsterbroken. Markens Urter, Lyng og Traer. Teknologisk Forlag. Oslo Hetland G, Samuelson AB, Lovik M. 1999. Protective effect of Plantago major L. pectin polysaccharide against Streptococcal, pneumoniae infection in mice. Journal of Medicinal Microbiology. 52: 348-357. Samuelson AB. 2000. The traditional uses, chemical constituents and biological activities of Plantago major. L. A review. Journal of Ethnopharmacology 71: 1-21. Hoj Y, Kim AR, Park MH. 2001. The plant origin of herbal medicines and their quality evaluation Sansel Nishibe. Planta Med. 67: 312-316.

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