OriginalArticle Journal of Indian System of Medicine Antimicrobial activity of Symphorema polyandrum Wight. Seeds 1 Sarang Lakhmale , Rabinarayan Acharya 2 1 Scholar,Dept.ofDravyaguna(saranglakhmale@gmail.com), 2 AssociateProfessor,Dept.ofDravyaguna,InstituteforPostgraduateTeachingandResearchin Ayurveda,GujaratAyurvedUniversity,Jamnagar,Gujarat 361008. JISM1327NReceivedforpublication:June16,2013;Accepted:June14,2014 How to cite the article: Sarang Lakhmale & Rabinarayan Acharya, Antimicrobial activity of Symphorema polyandrum, J-ISM, V2 N2, Apr-June 2014, pp.66-70 Abstract Symphorema polyandrum Wight. seeds are reported for its use in the management of snake bite, scorpion stings and associated skin ailments. The alcoholic extracts of its seed was screened for in vitro antimicrobial activity following agar diffusion method and compared with Gentamycin, Cefpodoxime, Streptomycin as standard antibacterial and Amphotericin B, Fluconazole and Clotrimazole as standard antifungal agents. For antibacterial assay B. subtilis, S. aureus, S. epidermidis, E. coli, K. pneumonia , S. typhi and for antifungal activity on A. flavus and C. albicans as test organisms in different concentrations (25μg/ml, 50μg/ml, 100μg/ml and 250μg/ml), were evaluated following standard procedures. It was observed that the seed extract is effective against all six bacteria and two fungal strains, when compared to standard drugs. The zone of inhibition for antibacterial activity of the test drug, against 6 strains of bacterias was found between 11 to 15 mm and 12 to 17 mm for two fungal strains. Keywords:Antimicrobial activity, Symphorema polyandrum, Introduction Materials and methods Symphorema polyandrum Wight. belonging to family Verbenaceae, commonly known by tribal people as 'Badichang or Mahasindhu' grows in different parts of Odisha, Chatisgargh and Madhay Pradesh. Different parts of S. polyandrum Wight., has been reported for various ethnopharmacological uses viz. snake bite, scorpion stings, cat bite, mad dog bite and mosquito bite [1,2,3]. Microbes are having important role in spreading various local or systemic infections [4]. Ayurveda advocates the management of various infectious diseases with herbal remedies. The antibiotic resistance has become a global concern [5]. Recent literature survey shows that its seeds though reported for its ethno medicinal claim for management of different aliments but have not been evaluated for its antimicrobial activities. Hence the present study was designed to assess the antimicrobial activity of its seed. Collection of plant material 66 S. polyandrum Wight. was identified by studying its morphological characters with the help of various floras [6,7,8]. A voucher specimen (herbarium) of the sample has been preserved in the institute Pharmacognosy laboratory (vide no. 6059.) Its mature seeds were in the month of May June 2012; shade dried and was coarsely powdered to mesh 60# and kept in airtight glass jar bottle for future use. Preparation of extract: 1g of S. polyandrum seed powder was extracted with methanol by sonicating it for 10 min and then keeping it overnight. Next day after filtration, methanol evaporated, then by taking weight of residue, 4 different concentrations 25 μg/ml, 50 μg/ml, 100μg/ml, 250μg/ml of the sample, were prepared. These are used for determination of Journal of Indian System of Medicine Vol.2-Number 2, April-June, 2014 Sarang Lakhmale & Rabinarayan Acharya, Antimicrobial activity of Symphorema polyandrum, pp.66-70 antimicrobial activity and coded as SP. Determination of microbial load for plant material Microbial load of the test sample was done by total viable aerobic count method [9,10]. To 500 mg, accurately weighed sample, 1-2 drops of Tween80 and a homogeneous suspension was prepared by slowly adding 5 ml of sterile buffered sodium chloride peptone (SBSCP) solution of pH -1 7.0. This suspension was diluted 10 onwards as required in sterile dilution blanks (SBSCP). One ml each from these aliquots was added to sterile melted and cooled top agar (Soyabean casein digest agar, for fungal count Potato dextrose agar medium used) tubes. These tubes were poured to sterile petridish and allowed to solidify. These plates were incubated 0 at 30-35 C for 48 hours. The numbers of colonies were counted and the results were expressed in Cfu/g. Cfu / g = Number of average colonies Dilution X Volume plated Antimicrobial activity of plant materials Culture conditions: The antimicrobial efficacy of these plant materials was tested on 6 different strains, 3 Gram positive bacteria namely Bacillus subtilis (NCIM 2063) Staphylococcus aureus (NCIM 2079) & Staphylococcus epidermidis (NCIM 2439); 3 Gram negative bacteria namely Escherichia coli (NCIM 2065), Klebsiella pneumoniae (NCIM 2719) and Salmonella typhi (NCIM 2501) as well as 2 fungal strains namely Aspergillus flavus (NCIM 1028) and Candida albicans (NCIM 3471). All cultures were obtained from National Chemical Laboratory, Pune. 24 hours old cultures of all these organisms were inoculated in sterile broths and incubated till 0.5 Mcfarland standard turbidity obtained, and then used for assay. The antimicrobial activity of methanol extracts of SP seed was studied in different concentrations (25 μg/ml, 50 μg/ml, 100μg/ml, 250 μg/ml) against six pathogenic bacteria and two fungal strains. 0.3 ml of different extracts as well as Streptomycin, Fluconazole and Clotrimazole standards were added in the Agar well for the assay; while Gentamycin, Cepodoxime and Amphotericin B standard antibiotics discs were used [11]. Antimicrobial assay Sterile soybean casein digest agar (25 ml per plate) used for antibacterial activity and sterile sabouraud agar (25ml per plate) used for antifungal activity. 20 ml sterile medium was poured aseptically in sterile plates and allowed to solidify. Then 0.5 ml of culture was inoculated in 5 ml sterile, melted, cooled medium and poured them on solidified agar plates aseptically. After solidification of medium, wells were made with the help of cup borer and 0.3 ml of sample was inoculated. Three Standard drug solutions were added in the well and other standard antibiotic discs were directly placed on agar surface and pressed with the help of sterile forceps aseptically. For diffusion purpose, plates were placed in refrigerator for 20-25 mins. Then 0 plates were incubated at 37 C for 24 hrs except sabouraud agar plates and plates containing K. 0 pneumoniae organism, they were incubated at 30 C for 24-48 hrs. After incubation, zone of inhibition was measured with Himedia antibiotic zone scale- c [12]. Pathogen study Same extracts were used as for antimicrobial activity assay, these extracts were transferred to specialized mediums given below and incubated at their optimum temperature for growth, then after incubation plates were observed and results were concluded [13] . Selective differential mediums according to pathogens:     Pseudomonas aeruginosa - Citrimide agar Salmonella typhi - TSI agar slant, XLD agar Escherichia coli- EMB agar Staphylococcus aureus - Mannitol salt agar Result and discussion: Microbial load: The observations of the microbial load of S. polyandrum seed showed that the tasted samples, when collected from their natural sources, are either free or within prescribed limit of the microbes [14]. When the samples were tested for bacterial contents, Journal of Indian System of Medicine Vol.2-Number 2, April-June, 2014 67 Sarang Lakhmale & Rabinarayan Acharya, Antimicrobial activity of Symphorema polyandrum, pp.66-70 all the samples were found free of common pathogens and bacterial and fungal count was under permissible limit (Table 1). Antimicrobial activity: References: [1] R. C. Misra; Therapeutic uses of some seeds among the tribals of Gandhamardarn hill range, Orissa; Indian Journal Of Traditional Knowledge vol.3 No. 1 January 2004:105115; 114p. The antimicrobial activity of methanol extracts of SP seed was studied in different concentrations (25μg/ml, 50μg/ml, 100μg/ml, 250μg/ml) against six pathogenic bacterial strains (Table 2) (three Gram positive B.subtilis NCIM 2063, S.aureus NCIM 2079 & S. epidermidis NCIM 2439; three Gram negative (E. coli NCIM 2065, K. pneumoniae NCIM 2719 and S. typhi NCIM 2501) and two fungal strains (Table 3) (S. flavus NCIM 1028 and C.albicans NCIM 3471). Antibacterial and antifungal potential of extracts were assessed in terms of zone of inhibition (ZOI). The result showed that the extracts of all samples were found to be effective against all the microbes tested. The antibacterial and antifungal activity of the SP increased linearly with increase in concentration of extracts (μg/ml). As compared with standard drugs, the results revealed that in the extracts for all six bacterial activity, were around equally sensitive and for fungal activity C. albicans showed good result as compare to S. flavus. The growth inhibition zone measured ranged from 11-15 mm for all the sensitive bacteria, and ranged [2] Sarita Das, S. K .Dash and S. N. Padhy; Ethno-medicinal information from Orissa state, India, A Review; J. Hum. Ecol., 14(3): 165-227 (2003); 219p. from12-19 mm for fungal strains (Table 2 and 3). The inhibitory effect of SP showed in mm at 25, 50, 100, 250μg/ml were against B.subtilis 11, 12, 13, 14.5; S.aureus 11, 11.5, 13, 14.5; S. Epidermidis 11, 11.5, 12, 14.5; E. coli 0, 11.5, 12, 13.5; K. pneumoniae 11, 12, 13, 14; S. typhi 11, 12, 12.5, 14.5; and two fungal strains S. flavus 12, 12.5, 14, 16 and C.albicans 14, 15, 16, 17 respectively. [8] Haines HH, The Botany of Bihar and Orissa, part II-IV. Dehradun (India); Bishen Singh Mahendrapal Singh, 1988. 703p. Conclusion: The zone of inhibition for antibacterial activity of the test drug, against 6 bacterial strains was found between 11 to 15 mm and 12 to 17 mm for two fungal strains. However, further studies would be necessary at different concentration and by different extraction media to increase the efficacy of the test drug. 68 [3] Sarang P Lakhmale, Rabinarayan Acharya, Nikita Yewatkar 'Ethnomedicinal claims on antivenom activity of certain fruit and seed drugs - a review' Ayurpharm Int J Ayur Alli Sci., Vol.1, No.1 (2012) Pages 21 - 29 ISSN No. 22784772. [4] Understanding Microbes in Sickness and in Health, U.S. department of health and human services, National Institutes of Health,National Institute of Allergy and Infectious Diseases, NIH Publication No. 09-4914, September 2009, www.niaid.nih.gov. [5] Zinn C. S, Rosdahl V. T et al. An international multicenter study of antimicrobial consumption and resistance in Staphylococcus aureus isolates from 15 hospitals in 14 countries. Microb Drug Resist; 2004. (10); 169-176p. [6] Saxena H. O, Brahman M. The Flora of Orissa; volume III, Orissa Forest Development Corporation Ltd. Bhubaneswar 751001, December 1995; 1396p [7] Hooker J. D. The flora of British India, Vol. IV. Dehradun, India, (London); Bishen Singh Mahendra Pal Singh, 1997. 560p. [9] Anonymous. Indian Pharmacopoeia. Delhi: Government of India, Ministry of Health and Family WelfareController of Publications, 1996; 1(1) 37-43p. [10] Anonymous. Quality control methods for herbal materials, W.H.O Monograph for limitation of microbes, WHO Press, World Health Organization, 20-Avenue Appia, 1211 Geneva 27; Switzerland, 1998. 75p. [11]Anonymous. Indian Pharmacopoeia. Delhi: Government of India, Ministry of Health and Family WelfareController of Publications, 1996; 1(1) 37-39p. [12] Dorman H. J. D, Deans S. G. Antimicrobial agents from plants. Antimicrobial activity of plant volatile oils, Journal of Applied Microbiology; Feb-2000.88(2), 308316p. [13] Anonymous. Indian Pharmacopoeia. Delhi: Government of India, Ministry of Health and Family WelfareController of Publications, 1996. 1(1); 43-49p. [14] Anonymous. The Ayurvedic Pharmacopoeia of India. Part-II, Volume-II, First edition, Ministry of Health and Family Welfare, Government of India, Department of Indian Systems of Medicine & Homoepathy, 2008; 2(2):199p. Journal of Indian System of Medicine Vol.2-Number 2, April-June, 2014 Sarang Lakhmale & Rabinarayan Acharya, Antimicrobial activity of Symphorema polyandrum, pp.66-70 PLATE - 1 Photographs of antimicrobial study of S. polyandrum Wight.seeds Fig. a: Showing zone of inhibition by standard against B. subtilis Fig. b: Showing zone of inhibition by SP against B. subtilis Fig. c: Showing zone of inhibition by standard against S. aureus Fig. d: Showing zone of inhibition by SP against S. aureus Fig. e: Showing zone of inhibition by standard against S. epidermidis Fig. f: Showing zone of inhibition by SP against S. epidermidis Fig. g: Showing zone of inhibition by standard against E. coli Fig. h: Showing zone of inhibition by SP against E. coli SP - Symphorema polyandrum Wight. seed Journal of Indian System of Medicine Vol.2-Number 2, April-June, 2014 69 Sarang Lakhmale & Rabinarayan Acharya, Antimicrobial activity of Symphorema polyandrum, pp.66-70 Plate 2 Photographs of antimicrobial and antifungal study of S. polyandrum Wight. seeds Fig. a: Showing zone of inhibition by standard against K. pneumoniae Fig. b: Showing zone of inhibition by SP against K. pneumoniae Fig. c: Showing zone of inhibition by standard against S. typhi Fig. d: Showing zone of inhibition by SP against S. typhi Fig. e: Showing zone of inhibition by standard against A. flavus Fig. f: Showing zone of inhibition by SP against A. flavus Fig. g: Showing zone of inhibition by standard against C. albicans Fig. h: Showing zone of inhibition by SP against C. albicans 70 Journal of Indian System of Medicine Vol.2-Number 2, April-June, 2014