European Journal of Biological Sciences 4 (3): 63-67, 2012 ISSN 2079-2085 © IDOSI Publications, 2012 DOI: 10.5829/idosi.ejbs.2012.4.3.6455

Phytochemicals of the and its Antibacterial and Insecticidal Activities

Aswathi Elizabeth Mani, Velammal Aiyamperumal and Jamila Patterson

Suganthi Devadason Marine Research Institute, Tuticorin, India

Abstract: Qualitative test of phytochemicals from methanol extracts of sea grass Syringodium isoetifolium showed positive activity with phytoconstituents such as saponins, resins, proteins, carbohydrates, glycosides, acidic compounds, reducing sugars, cardiac glycosides, phenols and alkaloids but showed negative activity to tannins, sterols, terpenoids, steroids, catachols and flavanoids. Antibacterial activities of methanol and acetone solvent extracts of sea grass against 17 human pathogens and 5 fish pathogens showed best activity in acetone extract also the same extract at 20 mg/ml exhibited good insecticidal activity.

Key words: Syringodium Isoetifolium Antibacterial Activity Insecticidal Activity Phytochemical Analysis

INTRODUCTION the need of the hour is to search for antibiotics with unique potential for which the pathogens may not have Sea grasses are flowering belonging to developed resistance [9] and so the search for new [1] restricted to shallow marine waters antibiotics is a continuous process. Insects, weeds and with sufficient sunlight penetration. It also occurs in phytopathogenic microorganisms cause great damage to lagoons and protected areas on muddy or sandy agriculture. Insects and mites produce damage and loss of substrate. Sea grass beds are among the most productive crops in both quality and quantity by innumerable ways. ecosystems in the world [2] and serve as nurseries for In search to discover new prototype insecticides, natural many species of fish and shellfish [3]. products are used as models, following a design based on They play an important role in stabilizing the biochemical principles or employing quantitative structure sediment by preventing sediments re suspension, as well activity relationship [10]. Number of families are as trapping particles and recycling nutrients [4]. Sea known to produce alkaloids, phenols and oils which have grasses are well documented for the presence of potent been used as insect control since long [11]. Many workers diverse secondary metabolites [5]. It has been realized documented that the sea grass in particular Halophila that many of these metabolites are being biologically and Posidonia posses novel metabolites [12, 13]. Sea active and of biomedical importance and could be used grasses are a rich source of secondary metabolites, as potential drugs. The roots of Enhalus acoroides is particularly phenolic sulfate, flavone sulfate, nonsulfate used as remedy against stings of different kinds of rays phenol, fatty acids and sterols [14, 15]. The present and scorpion. Halophila sp. is a strong medicine against study was undertaken to investigate the qualitative test malaria and skin diseases and is found to be very effective for phytoconstituents of the sea grass Syringodium in early stages of leprosy [6]. Aquaculture has been the isoetifolium and its antibacterial activity against 17 human world’s fastest growing food production system for pathogens and 5 fish pathogens and insecticidal activity the past decade [7, 8]. It is also being viewed as the only against rice weevil Sitophilus oryzae. alternative to augment fish production due to over exploitation of natural fishery resources. However, MATERIALS AND METHODS disease outbreaks in aquaculture facilities are recognized as an important limiting factor to production and trade. Fresh S. isoetifolium was collected from Tuticorin, There is a growing concern about the use and particularly Gulf of Mannar southeast coast of India. The samples the abuse of antimicrobial drugs not only in human were brought to the laboratory in plastic bags and washed medicine and agriculture but also in aquaculture. Hence with seawater twice and then with distilled water to

Corresponding Author: Aswathi Elizabeth Mani, Suganthi Devadason Marine Research Institute, Tuticorin, India. Tel: +91-4612336488. 63 Europ. J. Biol. Sci., 4 (3): 63-67, 2012

remove potential contaminants and epiphytes. The leaves oryzae were introduced into each petri plates and were separated and dried in the shade. The dried leaves sufficient amount of food was provided to the test were powdered using an electric blender and were stored organisms so that the view that death due to starvation in the refrigerator for further use. About 10 g of the could be ruled out. Numbers of dead insects were counted powdered sea grass was subjected to extraction with 100 after 24 hours and the percentage of mortality was noted. ml of methanol and acetone solvents using Soxhlet extractor for 6 hours at 50°C. The extract was filtered Phytochemical Analysis: The phytochemicals such as through Whatman No. 1 filter paper [16]. The filtrate was tannins, saponins, phenols, resins, proteins, acidic evaporated and dried at 55-60°C [17] and kept in the compounds, reducing sugar, steroids, terpenoids, cardiac refrigerator till use [18]. The concentrates were glycosides, sterols catachols, alkaloids, carbohydrates, reconstituted with their respective extracts. flavanoids and glycosides analysis were carried out by Seventeen human pathogens such as Vibrio the standard method [20] using concentrated crude cholerae, Bacillus cereus, Proteus vulgaris, Escherichia extracts of methanol and acetone. coli, Shigella dysenteriae, Salmonella paratyphi, Shigella sp, Streptococcus mutans, Staphylococcus RESULTS aureus, Pseudomonas fluorescens, Klebsiella pneumoniae, Streptococcus pyogenes, Shigella sonnei, The qualitative test of phytochemicals from methanol Micrococcus luteus, Salmonella typhi, Bacillus subtilis, and acetone extracts of seagrass Syringodium Enterococcus faecalis were obtained from the Christian isoetifolium is presented in Table 1. The methanol extract Medical College Hospital, Vellore. Five fish pathogens; of Syringodium isoetifolium showed the presence of like Vibrio harveyi, Vibrio sclintis, Vibrio protein, resin, glycosides, reducing sugar, saponins, parahaemolyticus, Vibrio alginolyticus, Vibrio acidic compounds, Phenols, cardiac glycosides and anguillarum were obtained from Fisheries Department, alkaloids. The phytoconstituents such as carbohydrates Tuticorin. All the bacterial pathogens were grown on and phenols were moderately present. Steroid, Tannins, nutrient agar and maintained at 4°C. Sterols, Terpenoids, Catachols and flavanoids were The extracts for the analysis of phytoconstituents absent. In the case of acetone extract reducing sugar, were prepared by solvent extraction method. The saponins, acidic compounds were present. insecticidal activity of the extracts of the sea grass was Phytoconstituents such as proteins and alkaloids were tested using contact bioassay method [19] using rice moderately present where as resin; steroids, tannins, weevil Sitophilus oryzae. glycosides, carbohydrates, sterols, terpenoids, phenols, cardiac glycosides, catachols and flavanoids were absent. Antibacterial Activity: Antibacterial activity of the The antibacterial activity of S. isoetifolium extracts extracts of Syringodium isoetifolium was analyzed using against 17 human pathogens is represented in Table 2. well diffusion technique. The wells with 5mm diameter The acetone extracts had good activities against 9 human were punched with a sterile cork borer [16] on to the pathogens compared to the methanolic extract. The zone Muller Hinton agar plates that was previously inoculated of inhibition exhibited by B. cerus, B. subtilis and with the bacterial cultures. The wells were filled with 50 µl Shigella sp. was 7mm with the acetone extract compared of both solvent extracts. Plates were kept in the to the methanol extracts with the zone of inhibition of 2 refrigerator for 2 hours and then incubated at 37°C for 24 mm, 4 mm and 2 mm for the same species of bacteria. hours. Wells containing the solvent alone were used as This was followed by S.sonnei and S.dysentriae with the negative control. Antibacterial activities were evaluated zone of inhibition of 6 mm.The methanol extracts for both by measuring the inhibition zone in millimeters. these organisms showed a zone of inhibition of 2 mm and 3 mm respectively. This was followed by P. fluorescens Insecticidal Activity: 1g of the dried extract was dissolved with 5mm zone of inhibition with the acetone extract. in 1ml of methanol and acetone and from that 2, 3, 5, 10, The methanol extracts of P. fluorescens had a zone of 20, 40 µl were poured in separate petri plates and allowed inhibition of 2mm. P. vulgaris had a zone of inhibition of to evaporate overnight to get the same concentration in 4 mm which showed moderate activity compared to the mg/ml of the extract. Control with solvents alone were methanol extract of this organism. Low activity was seen taken in separate petri plates and evaporated in S. partyphi and S. aureus with a zone of inhibition of 3 overnight.Ten healthy adults of rice weevil Sitophilus mm in the acetone extract whereas very low activity was

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Table 1: Phytochemicals of the methanol and acetone extracts of seagrass Syringodium isoetifolium 7 Phytoconstituents Methanol Extract Acetone Extract 6 Protein Present Moderately present 5 Resin Present Absent 4 Steroid Absent Absent 3 Tannins Absent Absent 2 Glycosides Present Absent Reducing Sugar Present Present 1 Ani bacterial effects levels Carbohydrates Moderately Present Absent 0 Methanol extract Acetone extract Saponins Present Present Acidic compounds Present Present Solvents Sterols Absent Absent V.harveyi, V. Anguillarum V. Sclintis Terpenoids Absent Absent V. parahaemolyticus V. Al ginol yticus Phenols Moderately present Absent Fig. 1: Anti bacterial effect against fish pathogens Cardiac Glycosides Present Absent Catachols Absent Absent 10 Alkaloids Present Moderately present 9 Flavanoids Absent Absent 8 7 6 Table 2: Antibacterial activity of acetone and methanol extracts of S. 5 isoetifolium against 17 human pathogens 4 Acetone extract Methanol extract Negative control 3 Human pathogens ------Zone of Inhibition in mm------2 S.paratyphi 6 2 - 1 Anti insectidal activity levels activity insectidal Anti P.vulgaris 3 2 - 0 S.aureus 4 1 - Acetone Methanol B.cerus 3 1 - Concentration of solvent extracts S.dysentriae 7 2 - 2mg 4mg 5mg 10mg 20mg 40mg P. fluorescens 6 3 - Shigella sp. 52 -Fig. 2: Anti Insecticidal activity of seagrass extracts B.subtilis 7 2 - V.chlorae 7 4 - antibacterial activities against fish pathogens than E.coli 1 - - methanol extract. Out of 5 bacterial fish pathogens 3 K.pneumoniae - - - exhibited antibacterial activity. The acetone extract S.mutants - - - of,V. harveyi, V. parahaemolyticus and V. anguillarum S.pyogens 1 - - showed good activities with a zone of inhibition of 7 mm, M.luteus - - - 6 mm and 5 mm respectively when compared to the S.typhi - - - ethanol extracts which showed a zone of inhibition of 3 E. faecalis - - - mm, 3 mm and 2 mm and it revealed poor activities. No antibacterial activity was seen in V. sclintis and V. seen in methanol extract of only 1mm zone of inhibition. alginolyticus in acetone and methanol extracts. No zone of inhibition was seen in pathogens V. cholorae The insecticidal activity of both the extracts of E. coli, K. pneumoniae, S. mutants, S. pyogens, M. luteus, Syringodium isoetifolium is shown in Fig. 2. Both acetone S. typhi, E. faecalis in the methanol extracts. Trace and methanol extracts of Syringodium isoetifolium were activity was seen in S.mutans, V.chlorae in acetone found to cause mortality of rice weevil. The acetone extracts. extract showed 100% mortality at the concentrations of 20 The antibacterial activity of ethanol and methanol and 40mg/ml compared to the methanol extracts which extracts of S. isoetifolium against 5 fish pathogens is showed only 70% mortality at 20 and 40 mg/ml after 24 represented in Fig. 1. The acetone extract showed best hours of exposure.

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DISCUSSION CONCLUSION

The qualitative phytochemical analysis showed most Our study showed that the crude extract of of the phytoconsitutents with methanol extraction Syringodium isoetifolium showed appreciable compared to acetone extract. The presence of sugar antibacterial activity against Gram positive and Gram somehow facilitated the growth of microorganisms and negative human and fish pathogens. Further study is hence antagonizing the antibacterial activity of active underway to elucidate the structure of compound and compounds of the extracts [21]. The seagrass Thalasia the mechanism of inhibition of pathogens. However, the testudium inhibits the growth of S. aggregatum due to the abundant availability of sea grass along the Indian presence of flavones and glycosides [22]. coastline opens up a new avenue for the entry of Bacterial infection causes high rate of mortality in pharmaceutical industries in developing new drugs for human population and aquaculture organisms [23]. human and aquaculture remedies. The revolutionized therapy of infectious diseases by the use of antimicrobial drugs has certain limitations due to ACKNOWLEDGEMENT changing patterns of resistance in pathogens and side effects they produced. These limitations demand for Authors are thankful to the Director of SDMRI for improved pharmacokinetic properties, which necessitate providing all facilities to carry out this work. continued research for new antimicrobial compounds for the development of drug [24]. Hence the interest in marine REFERENCES organisms as a potential and promising source of pharmaceutical agents has increased during recent years 1. Den Hartog, C., 1970. The of the [25]. Several species of seagrass produce antimicrobial World. Amsterdam. North Holland Publication. Co., compounds that may act to reduce or control microbial pp: 275. growth. In our study we report that acetone was the best 2. Duarte, C.M. and C.L. Chiscano, 1999. Seagrass solvent compared to methanol in inhibiting human and biomass and production: Area Assessment. Aquatic fish pathogens. Our results coincide with earlier report of Botany, 65: 159-174. acetone solvent extract for extracting antimicrobial 3. Levine, S.N., D.T. Rudnick J.R. Kelly, compounds from British marine algae [26]. Acetone was R.D. Morton, L.A. Buttel and K.A. Carr, 1990. the best solvent for extracting the effective antimicrobial Pollutant dynamics as influenced by sea grass materials from Sargassum myricystum, Turbinaria beds: experiments with tributyltin in Thalassia conoides, Hypnea musiformis, Gracilaria edulis and microcosms. Marine Environmental Research, Halimedia gracilis [27]. Our present findings is 30: 297-322. consistent with some earlier reports which concluded that 4. Duarte, C.M., 2002. The future of seagrass meadows. the active fractions of acetone and hexane roots extracts Environmental Conservation, 29(2): 192-206. of Cymodocea serrulata could be effectively used as an 5. Puglisi, M.P., S. Engel., P.R Jensen and W. Fenical alternative bioactive agents to rectify the problem of 2007. Antimicrobial activities of extracts from unknown diseases disseminated from the poultry borne Indo-Pacific marine plants against marine pathogens food products [28]. The variation of antibacterial activity and saprophytes. Mar. Biol., 150: 531-554. of the extracts might be due to distribution of 6. De la Torre-Castro, M. and P. Rönnbäck, 2004. antimicrobial substances, which varied from species to Links between humans and seagrasses-an example species [29]. The drug resistance may be due to pre- from tropical East . Ocean Coast existing factor in the microorganisms, or it may be due to Manag., 47: 361-87. some acquired factors [30]. Previously it was reported that 7. Muir, J.F., 1995. Aquaculture development trends: the acetone and hexane leaf extracts were found to show Perspective for food security, contribution to the strong antimicrobial and anti-inflammatory activities [31]. international conference on the sustainable Our findings well collaborates with the results of contribution of fisheries to food security. Organized insecticidal activity of a gorgonid associated winged by the Government of Japan in collaboration with oyster, Pteria chinensis [32] and they reported acetone FAO, Kyoto, Japan. 4 - 6th December 1999. Rome, solvent was good for insecticidal activity when compared FAO, KC/F1/95/TECH/ 4:133. to other such as ethyl acetate and dichloromethane 8. Tacon, A.G., 1997.Contribution to fish supplies, FAO solvents. fish circular 886, Review, 1: 17-21.

66 Europ. J. Biol. Sci., 4 (3): 63-67, 2012

9. Patil, R.G., S. Jeyasekaran. A. Shanmugan and R. Jeya 21. Arwa, P.S., J.C. Onyango and R. Nyunja, 2008. Shakila, 2001. Control of bacterial pathogens, Phytochemical compounds and antimicrobial activity associated with fish disease, by antagonistic marine of Rhoicissus plant (Rhoicissus revilli) (Planch). actinomycetes isolated from marine sediments. Indian Plant Sci. Res., 1(3): 68-73. Journal of Marine Science, 30(4): 264 - 267. 22. Engel, S., M.P. Puglisi and P.R. Jensen, 2006. 10. Duke, S.O., J.J Menn and J.R. Plimmer, 1993. Antimicrobial activity of extracts from trophical Challenges of pest control with enhanced atlantic marine plants against marine pathogens and toxicological and environmental safety, an overview. saprophytes. Mar. Biol., 149: 991-1002. In: Pest control with enhanced environmental safety. 23. Kandhasamy, M. and K.D. Arunachalam, 2008. ACS Symposium Series, pp: 52. Evaluation of in vitro antibacterial property of 11. Nivsarkar, M., B. Cherian and H. Padh, 2001. seaweeds of southeast coast of India. Afr. J. Alpha-terthienyl: A plant derived new generation Biotechnol., 7(12): 1958-1961. insecticide. Current Science, 81(6): 667- 672. 24. Al- Haj, N.A., N.I. Mashan and M.N. Shamudin, 2009. 12. Mac Millan, J.R. 2001. Aquaculture and Antibiotic Antibacterial activity in marine algae Eucheuma resistance: A negligible public risk. World denticulatum against Staphylococcus aureus and Aquaculture, 49: 51-68. Streptococcus pyogenes. Res. J. Biol. Sci., 13. Dumay, O., J. Costa, J.M. Desjobert and G. Peergent, 4(4): 519 - 524. 2004. Variations in the concentration of 25. Kim, I.H. and J.H. Lee, 2008. Antimicrobial activities phenolic compounds in seagrass, Posidonia oceanic against methicillin resistant Staphylococcus aureus under conditions of competition. Phytochemistry, 65 from macroalgae. J. Ind. Eng. Chem., 14: 568-572. (24):3211 - 3220. 26. Hornsey, I.S. and D. Hide, 1974. The production of 14. McMillan, C., 1983. Sulfated flavonoids and leaf antimicrobial substances by British marine algae. morphology of the Halophila ovalis-H. minor Antibiotic producing marine algae. British Phycology complex (hydrocharitaceae) in the Pacific Islands and Journal, 9: 353-361. . Aquatic Botany, 16: 337-347. 27. Kolanjinathan and G.D Stella, 2009. Antibacterial 15. Gillan, F.T., R.W. Hogg and E.A. Drew, 1984. activity of marine macroalgae against human The sterol and fatty acid compositions of seven pathogens. Recent Res. Sci. Technol., 1(1): 20-22. tropical sea grasses from North Queensland, 28. Ravikumar, S., M. Syed Ali, P. Anandh, M. Australia. Phytochemistry, 23: 2817-2821. Ajmalkhan and M. Dhinakaraj, 2011. Antibacterial 16. Adomi, P.O., 2006. Antibacterial activity of aqueous activity of Cymodocea serrulata root extract against and ethanol extracts of the stem bark of Alstonia chosen poultry pathogens. Indian Journal of Science boonei and Morinda lucida. Scientific Research and and Technology, 4(2): 98-100. Essay, 1(2): 50-53. 29. Lustigman, B. and C. Brown, 1991. Antibiotic 17. Lima-Filho, J.M., F.F Ana, M.F. Sissi and production by marine algae isolated from Newark, M.M. Vania, 2002. Antibacterial activity of extracts of New Jersey Coast. Bulletin of Environmental six macroalgae from the northeastern Brazilian coast. Contamination and Toxicology, 49: 329-335. Braz. J. Microbiol., 33: 4. 30. Sparks, A.K., 1981. Bacterial diseases of invertebrates 18. Ergene, A., P. Guler, S. Tans, S. Miric, E. Hamzaoglu other than insects, In: Davidson E.W. [Ed] and A. Duran, 2006. Antibacterial and antifungal Pathogenesis of Invertebrate Microbial Diseases, activity of Heracleum sphondylium.African J. of Allanhheld, Oosmuro, Totowa, New Jersy, pp: 323. Biotech., 5: 1087-1089. 31. Kunle, O., J. Okogun, E. Egamana. E. Emojevwe and 19. Brossalis, A.M., G.E. Ferraro, V.S. Martino, M. Shock, 2003. Antimicrobial activity of carious R. Pinzone, J.D. Coussiom and J.C. Alveearez, 1999. extracts and carvacrol from Lippia multiflora leaf Argentine plants as potential source of insecticidal extract. Phytomed. 10: 59-61. compounds. Ethnopharacology. 67: 219-223. 32. Chellaram, C., G.K. Mary Elizabeth and P. Jamila, 2005. 20. Sofowora, A., 1931. Medicinal Plans and Traditional Insecticidal and herbicidal activity of a gorgonid Medicine in Africa. Spectrum Book. Ibadan. associated winged oyster, Pteria chinensis. SDMRI Research Publication. 9:178 -181.

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