Sundaram Ravikumar et al. / Journal of Pharmacy Research 2011,4(6),1854-1856 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Bioactive potential of a against bacterial fish pathogens Sundaram Ravikumar1*, Ramar Vinoth2, Gopi Palani Selvan1 1School of Marine Sciences Department of Oceanography and Costal Area Studies, Alagappa University, Thondi Campus- 623409, Tamil Nadu, India; 2Center of Advance studies in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai –608 502, Tamil Nadu, India Received on: 11-02-2011; Revised on: 16-03-2011; Accepted on:21-04-2011 ABSTRACT The antibacterial activity of ethanolic extracts of leaf and root seagrass Syringodium isoetifolium was evaluated by disc diffusion method. Among the parts seagrass root extracts exhibited antibacterial activity against chosen fish pathogens. The disc diffusion assay of root extract showed maximum inhibition against Aeromonas hydrophila (14 mm) and also it’s showed minimum inhibitory concentration and minimum bactericidal concentration value at 600µg.ml-1. Preliminary phytochemical analysis of the plant parts revealed that, the presence of active compounds such as carbohydrates, flavanoids, alkaloids, tannin and saponin glycosides. The results conclude that, the Syringodium isoetifolium might be the potential sources of antibacterial compounds for the treatment of fish diseases.

Keywords: Disc diffusion assay, fish pathogens, Minimum Bactericidal Concentration, Minimum Inhibitory Concentration, Seagrass, Syringodium isoetifolium. INTRODUCTION Aquaculture has become an important economic activity in many countries. pounds[7]. The major pharmaceutically valuable phytochemical compounds In large-scale production facilities, where aquatic animals are exposed to stressful investigated in the present study were alkaloids, catachin, steroids, terpenoid, conditions, problems related to diseases and deterioration of environmental total sugar, flavanoids, protein, tannin, phenolic compound, anthroquinone conditions often occur resulted serious economic losses. Prevention and control and saponin. of diseases have led to a substantial increase in the use of veterinary medicines [1]. Bacterial diseases are responsible for heavy mortalities in both culture and Disc diffusion assay: wild fishes throughout the world. Aeromonas, Pseudomonas and Edwardsiella Filter paper disc method was used for testing of the plant extract against 5 tarda are the major bacterial fish pathogens which are widely distributed in bacterial fish pathogens. Whatman No. 1 filter paper disc (6mm diameter) was aquatic ecosystems[2,3]. In coastal regions, fish have been suffered from impregnated with different concentration of crude plant extracts (200, 400, Vibriosis, a bacterial disease causing losses in the fish production [4]. Control 600, 800 and 1000 µg disc-1) and placed on Muller Hinton Agar (HIMEDIA, of fish disease is currently based almost entirely on chemotherapy [5]. Decreased Mumbai) plate which was previously swabbed with bacterial fish pathogens viz., efficacy and resistance of pathogens to antibiotics has necessitated the Aeromonas hydrophila, Bacillus subtilis, Vibrio harveyii, Vibrio parahaeomo development of new alternatives [6]. In this study, seagrass of Syringodium lyticus and Serratia sp. Ethanol without the extracts is maintain as control. All isoiethifolium leaf and root extracts were tested against five bacterial fish the plates were incubated at 37ºC under static conditions. After 24 hrs, the pathogens. zone of inhibition appearing around the discs were measured and recorded in millimeter diameter. Triplicate samples were maintained for each bacterial MATERIALS AND METHODS strain.

Sample collection: The fresh leaves and roots of Syringodium isoetifolium were collected along Minimum Inhibitory Concentration (MIC): Palk Strait region of Thondi coast (Latitude 9°44’N and Longitude 79°10’E). The MIC is defined as the lowest concentration of the test compounds, which All the collected samples were washed thrice with tape water, twice with inhibits bacterial growth. MIC determination 0.5 ml of varied concentration -1 distilled water to remove the adhering salts and other associated animals and of extracts (200, 400, 600, 800 and 1000 µg. ml ) were mixed with 0.5 ml of then shade dried for 10 days. nutrient broth and 50 µl of bacterial inoculum. Whole setup in duplicate was incubated at 37°C for 24hrs. After the incubation period, turbidity was ob- Extraction of bioactive compounds: served. The MIC was calculated by the concentration in which inhibition of The dried seagrass leaf and root samples of Syringodium isoetifolioum were bacterial growth was maximum. soaked in ethanol and water mixture (3:1) for one week for the extraction of bioactive compounds. The colored solvent was filtered by using Whatman Minimum Bactericidal Concentration (MBC): filter paper No.1 and further distillation was carried out by using rotary flask To avoid the possibility of mis-interpretations due to the turbidity of insoluble evaporator (SUPERFIT, INDIA). The condensed extract was then kept in compounds, the MBC is determined by sub-culturing the above (MIC) serial petriplates for evaporation. Then collected seagrass extract were weighed and dilutions in nutrient agar plates and incubating at 37°C for 24 hrs. MBC was stored in sterilized plastic tubes in refrigerator (4°C) for further assay. The recorded as the lowest concentration that prevents the growth of bacterial percentage of extraction from each sample was calculated using the following colony on this solid media. formula: RESULTS Percentage of extraction (%)= Weight of the extract (g) / Weight of the plant The in vitro antibacterial activity of seagrass leaf and root extracts that were material (g) ×100 screened against chosen fish pathogens reveals that, the root extracts showed maximum sensitivity against all fish pathogens. The ethanolic root extracts Phytochemical analysis: of seagrass Syringodium isoetifolium showed considerable inhibitory effect (6 The herbal product was screened for the qualitative analysis of natural com- – 14 mm) against both gram positive and gram negative bacterial pathogens. The highest zone of inhibition was observed in root extract against Aeromonas hydrophila (14 mm) at a concentration of 1000 µg. ml-1 and minimum inhibi- tion was observed against Aeromonas hydrophila, Vibrio parhaemolyticus and *Corresponding author. Serratia sp. (6 mm) at various concentration (400, 600 and 1000 µg. ml-1 Sundaram Ravikumar respectively). Whereas, leaf extract showed inhibitory activity against two pathogens viz., Aeromonas hydrophila (maximum 9 mm at 1000 µg. ml-1) and School of Marine Sciences Bacillus subtilis (6 mm at 1000 µg. ml-1). Surprisingly no inhibition was ob- Department of Oceanography and Costal Area served against Vibrio harveyi, Vibrio paraheamolyticus and Serratia sp. by the Studies,Alagappa University, leaf extracts (Table.1). Thondi Campus- 623409, Tamil Nadu, India

Journal of Pharmacy Research Vol.4.Issue 6. June 2011 1854-1856 Sundaram Ravikumar et al. / Journal of Pharmacy Research 2011,4(6),1854-1856 Table 1. Antibacterial activity of different concentration of ethanolic negative bacteria including Aeromonas hydrophila, Vibrio harveyi, Vibrio crude extracts of seagrass leaf and root against chosen fish pathogens. parahaemolyticus and Serratia sp. Alam et al., [26] reported the antimicro- bial activity of ethanolic extract of Xylocarpus granatum and found to ex- Name of the fish pathogens Zone of inhibition (mm) hibit good antibacterial activity at 400 µg.disc-1dose level against most of the 200(µg) 400(µg) 600(µg) 800(µg) 1000 (µg) gram positive and gram negative bacteria. The sensitivity of Syringodium Seagrass leaf extract isoietifolium extract to fish pathogens might be due to the presence of phy- Aeromonas hydrophila - - - 6±0.8 9±1.2 tochemical constituents Viz., Carbohydrate, flavonoids, alkaloids and tannins. Bacillus subtilis - - - - 6±1 Engel et al., [27], reported that, the extracts from the seagrass Thalassia Vibrio harveyi - - - - - Vibrio parahaeomolyticus - - - - - testudinum inhibits the growth of S. aggregatum due to the presence of Serratia sp. - - - - - flavones glycoside, thalassiolin. Okeke et al, [ 28], reported that, the phy- Seagrass root extract tochemical compounds viz., glycoside, saponin, tannin, flavonoids, terpenoides, Aeromonas hydrophila - 6±0.9 7±0.8 11±0.65 14±0.78 Bacillus subtilis - - 7±1.2 10±0.6 12±1.75 alkaloids have antimicrobial activity. Fennel et al.,[29] reported that, the Vibrio harveyi - - - - 7±0.8 antibacterial activity exhibited by the marine plant parts could be due to the Vibrio parahaeomolyticus - - - - 6±1.2 presence of phytochemical like alkaloids, tannin, flavonoids and sugars present Serratia sp. - - 6±1.45 8±0.8 12±1.8 in the plant extracts. “±” - Standard deviation, “-”- indicates no zone of inhibition. The marine are to have reported are very heterogeneous mixtures of Table 2. Minimum Inhibitory concentration (MIC) and Minimum Bac- single substances which may act in a synergistic or antagonistic manner. Mix- tericidal concentration (MBC) of Syringodium isoetifolium Leaf and root tures of active constituents showed a broad spectrum of biological and phar- macological activity [30-32]. Arwa et al.,[33], reported that, the presence of extracts against fish pathogens sugar somehow facilitated the growth of the microorganisms and hence an- tagonizing the antibacterial activity of active compounds in the extracts. Name of the Pathogen Leaf extract Root extract MIC (µg) MBC (µg) MIC (µg) MBC (µg) Flavonoids are containing one carbonyl group complexes with extracellular and soluble protein and with bacterial cell wall [34], thus exhibits antibacterial Aeromonas hydrophila 1000 1000 600 600 activity through these complexes. Tannin [35] form irreversible complexes Bacillus subtilis 1000 1000 800 800 with proline rich proteins, resulting in the inhibition of cell protein synthesis Vibrio harveyi 1000 1000 1000 1000 of bacteria [36]. Generally the gram positive bacteria are believed to be more Vibrio paraheomolyticus 1000 1000 1000 1000 susceptible having only an outer peptidoglycan layer which is not effective Serratia sp. 1000 1000 600 600 permeability barrier, where as the gram negative bacteria possesses an outer phospholipidic membrane carrying the structural lipopolysaccharide com- Table 3. Phytochemical constituents of Seagrass Syringodium isoetifolium pound. This makes the cell wall impermeable to drug constituents. In spite of (leaf and root) the barriers the phytochemical constituents were effective in controlling the growth of these pathogenic strains. It is also concluded from present study Parameters Leaf Root that, the S.isoetifolium proved as excellent source for novel antibacterial Steroids - - compounds for the management of fish bacterial diseases. Investigations are Terpenoids - - in progress to identify the active antibacterial compounds from the seagrass Carbohydrates - + Flavanoids - + root extract by bioassay guided fractionation. Proteins - - Alkaloids + + ACKNOWLEDGEMENTS: Tannin + + The authors are grateful to the authorities of Alagappa University for provid- Phenolic compound - - Anthroquinone for glycosides - - ing facilities and also the Indian Council of Medical Research, New Delhi for Catachin - - financial assistance. Saponin glycosides + + “+”- presence, “–”- absence REFERENCE The Minimum inhibitory concentration (MIC) and Minimum bactericidal 1. 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Journal of Pharmacy Research Vol.4.Issue 6. June 2011 1854-1856