Ancient Science of Life  Vol : XXII(4) April, May, June 2003 3DJHV EMERGENCE OF MESUA FERREA LINN. LEAF EXTRACT AS A POTENT BACTERICIDE Rupa Mazumder1, Sujata G. Dastidar2, S.P. Basu3, Avijit Mazumder1 and Sanjay Kumar1 1 Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi – 835 215. 2 Division of Microbiology, Department of Pharmaceutical Technology, Jadavpur University, Kolkata – 700 032, West Bengal 3 Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj, Jharpokaria – 757 087, Orissa. Received : 16-01-2003 Accepted: 26-02-2003 ABSTRACT : The methanolic extract of leaves of Mesua ferrea Linn. were tested for its antibacterial potentiality against 103 various strains of bacteria including Staphylococcus aureus, Bacillus spps. Klebsiella spps., Streptococus pneumoniae, Sarcina lutea, Lactobacilus arabinosus, Escherichia coli, shigellae, salmonellae, Proteus spps., Pseudomonas spps. and the vibrios. Significant antibacterial effects were produced by the extract against Staphylococcus aureus, Bacillus sppa., lactobacilli, Escherichia coli, shigellae and salmonellae and the results were compared with standard antibiotic ciprofloxacin. Further the extract was proved to be bacterial in its action. INTRODUCTION Mesua ferrea Linn. ( Family : Clusiaceae : Guttiferae) commonly known as ‘ Naagkesar’ (Bengali, Hi ndi a nd Punjabi), ‘ Naagchampa’ (Gujarat, Kon. a nd M ar) and ‘ Naagakeshara’ (Sanskrit) is a well kn own medium – sized or large evergreen tree with its various parts having tr emendous use in t he India n traditional syste m of m edicine for the treatment of vari ous di seases. The barks are used as astr ingent an d i n combinat ion with ginger as a sudori fic. The leaves and flowers are used in snake bite a nd s corpion strings , flower buds are used in dysentery, flowers are used as astringent, stomachic and expectorant, unripe fruits have sudorific effects, seed oil is used externally for cuta neous affections as an embrocation in rheumatism (1,2). Antibacterial and ant ifungal activiti es of t he flowers of the f lowers of Mesua ferrea Linn. Have already been reported (3). The present study wa s underta ken to inve stigate t he antibacterial activity of Mesua ferrea Linn. the leaf extract of MATERIALS AND METHODS Plant Material The leav es of Mesua ferrea Lin n ( Family : Clusiaceae : Guttiferae) wer e coll ected fro m Assam, India. In November, 2000. The plant part was authenti cated by Centra l Nati onal Herbarium, Botanical Survey of I ndia, Botanical Garde n, Howr ah – 711 103, We st Bengal, India [CNH/I – I (54)/2001 – Tech.II]. Preparation of the plant extract The wa shed and drie d mat ured lea ves a fter collection were coarsely powdered 9114.1 gm and extra cted with met hanol i n a soxhlet at below 60 0C. The extract was evaporated to dryness at low temperature under vacuum in a vacuum dessicator. The yield of the methanol Pages 160 - 165 1 extract with respect to dry powdered material was calculated to be 24.72% w/w. compared with those obt ained wi th s tandard antibiotic Ciprofloxacin. Preparation of samples Dimethyl sulfoxide (DMSO) was us ed as the solvent to dissolve the dry powered extract for the antibacterial tests. Ciplofloxacin solutions were prepared by using sterile distilled water and were used as st andard for the comparison of the antibacterial potency of the leaf extract. Determination of mode of antibacterial action of the extract Chemicals All c hemicals an d s olvents us ed in th is experiment wer e of AR gra de and obtai ned from BDH (Poole, UK). Microorganisms One hundr ed thr ee strains of bacter ia belonging to 12 di fferent g enera w ere te sted in this st udy. S. aure us AM 8/ 98, E.coil AM 8/98,P. mirabilis AM 8/98, Kl ebsiella pneumoniae RM 8/98 and Ps eudomonas spp. Were collected from S.C.B. Medical College, Cuttack, Ori ssa, India; E. coi l VC Sona wave 3:37C, S. typhi ATCC 6539, S. aureus NCTC 7447 AND S. pne umoniae NCTC 7465 were collected from I nstitute of Microbial Technology, Cha ndigarh, India. W e had collected B.subti lis CD/ 99/1, Lactobacillus arabinosus CD/99/1, E.coil CD/99/1, B.cereus var myc oides, S. a ureus ATCC 29737 and Sarcina Iut ea CD/99/1 fr om Ce ntral Drugs Laboratory, Kolkata, India. All the remaining strains w ere procured f rom the D ivision of Microbiology, Depart ment of Pharamaceutical Technol ogy, J adavpur University, Kolkata, India. In vitro tests for antibacterial efficacy of the extract. The mi nimum i nhibitory c oncentrations (MIC) of the extract agai nst the various tested strains wer e det ermined by agar dilution technique ( 4). The anti bacterial pot entiality of the extract was assa yed by disc di ffusion method (5,6) and the results so obtained were A hi ghly se nsitive ba cterial strai n, S. aure us ML 161, to the extrac t w as grow n in sterile nutrient brot h medi um overni ght, 2 ml fr om which wer e added to 4 ml of steril e nutrie nt broth and incubated for 2 hr at 37 0C, so that the culture attained logarithmic pha se of growth. After 2 hr incubation t he extract was added at a higher concentration than its MIC value for that particular strain. The number of colony for ming unit (CFU/ ml) was determined by Miles and Mishra’s method (7) at an int erval of 2 hr upt o 6 hr a nd then after 18 hr starting from zero hour. RESULTS AND DISCUSSION The results of the determination of MIC of the extract agai nst 103 var ious tested bacter ial strains are r ecorded i n Tabl e. 1. This shows that the extract was mo stly active against the Staphylococcus aureus, Bacillus sppa., Lactobacillus arabinosus, Escherichia coli, Shigellae a nd Prote us s pps., but moder ately active against the Klebsiell ae spp., Streptococcus pneumoniae, Sarcina lutea, Salmonellae typhimurium, Pseudomonas spps. and the vibrios. The comparative results of t he anti bacterial assay of the extract and Ci profloxacin are depicted in Table 2. The M IC of th e ex tract against the most sensitive strain S. aureus ML 161 was found to be 50µg/ ml. At the logarit hmic growth phase of t he culture, when CFU count of the strain wa s 9.8 x 10 6 CFU/ ml, 100µg/ ml, of the extr act was added. Subseque ntly, the CFU count of the cu lture was found to decrease a fter 2,4 and 6 hr and it ul timately Pages 160 - 165 2 reduced t o z ero at t he e nd of 18 hr. Thus it can be concl uded that t he m ethanolic extract of the leaf of M. ferrae (Linn) is bactericidal in its action. (Table 3 and Fig.1). In summary, the metha nolic extract of the leaves of Mesua ferrea Linn appe ars to ha ve potent bacterial eff ect against both gram positive and gra m nega tive str ains, mostl y against the organisms causing dysentery. An attempt to identify a nd isolat e the chemical component(s) which i s responsible for t his activity is being carried out. ACKNOWLEDGMENTS The aut hors are grate ful to the authority of Jadavpur Univer sity, Kolkata – 700 032, West Bengal, India, for supplying of bacterial strains in this wo rk. T he taxonom ic identification of th e plant speci men by Central National Herbarium, Botanical Survey of India , Shi bpur, Howra h, We st Bengal, India, is also grateful acknowledged. REFERENCES 1. Satyavati, G. v. et. al., Medicinal Plants of India, Vol.2, ICMR, New Delhi., 242, (1987). 2. Chatterjee, Asima and Pakrashi, Satyesh Chandra., “The Treatise of Indian Medicinal P lants”, Vol.2, Publi cation and I nformation Dire ctorate, CSI R, Dr. K.S. Krishnan Marg, New Delhi – 110 012., pp.163-164, (1992). 3. Ray, P .G. and Majum dar, S.K ., “A ntimicrobial activity of som e Indian Plants”, E conomic – Botany 30, 4, 317 – 320, (1976). 4. Mazumder, R., Mendiratta, T., Mondal, S.C. and Mazumder, A., “Antimicrobial potency of the leaf-stalk extract of Curcuma longa (Linn)”, Anc. Sci, of Life XX, 92-96, (2000). 5. Mariam, T. G ., Murthy, P.N ., Ranganathan, P., Hymet e, A a nd Da ka, K., “ Antimicrobial screening of Ru mex abyssiricus and Rumex nervosus”, The East ern Pharmacist 36, 33, 131 – 133, (1993). 6. Pelczar, M.J., Chan, E.C. S. and Krie g, N.R., “Mi crobiology – Concepts and Application”, Int. edn., McGraw Hill, New York, pp 578 – 581, (1993). 7. Miles, A. A. and Mis hra, S. S., “ The estimation of the bacterial power of bloo”, J. Hyg Ca mb 38, 732, (1938). Pages 160 - 165 3 Table 1. Bacterial inhibitory spectrum of the methanolic extract of leaves of Mesua ferrea Linn. Bacteria Staphylococcus aureus No. of tested 40 - No. of bacteria inhibited by the extract of (µg/ml) 5 10 25 50 100 200 - - 10 09 10 - 03 >200 11 - Bacillus spp. 03 - --- Klebsiella spp. 02 - --- Streptococcus 01 - --- - 01 - Sarcina lutea 01 - --- - 01 - Lactobacillus arabinosus 01 - --- 01 - Escherichia coli 06 - --- 02 03 01 Shigella spp. 12 - --- 03 06 03 Salmonella spp. 04 - --- - 03 01 Proteus mirabilis 01 - --- Pseudomonas spp. 02 - --- Vibrio cholerae 30 - 01 - 01 pneumoniae Total Strains - - 01 - - - 01 - 03 07 01 07 13 103 Table 2. A comparative account of the assay results (in terms of diameters of zones of inhibition) of methanolic extract of Mesua ferrea Linn. leaf and Ciproflxacin. Bacteria Extract (µg/ml) Ciprofloxacin (µg/ml) Staphylococcus aureus ATCC 29737 7.00 14.83 17.66 Bacillus cereus var mycoides 7.50 11.00 15.83 17.83 12.00 14.66 16.83 8.50 15.00 16.66 11.50 17.00 21.00 Lactobacillus arabinosua CD/99/1 8.00 Escherichia coli ROW 7/12 7.00 Shigella dysenteriae 6 8.50 Pages 160 - 165 10.50 4 Table 3. Mode of antibacterial activity of the methanolic extract Mesua ferrea Linn. leaf and S. aureus ML161. Time (hours) CFU count /ml for the extract Zero 9.8 X 106 2 9.6 X 104 4 8.9 X 102 6 9.8 X 102 18 0 Fig . 1 : Graphical representation of the mode of antibacterial activity of the methanol extract of Mesua ferrea Linn. leaf extract against S. aureus ML 161 Pages 160 - 165 View publication stats 5