Alizzia lebbeck (Albizia), Centella asiatica (Gotu Kola), Tinospora cordifolia (Tinospora. Gulancha). The highest activity against the Vibrio harveyi was noticed by ...
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Pelagia Research Library Der Pharmacia Sinica, 2010, 1 (3): 17-22
ISSN: 0976-8688 CODEN (USA): PSHIBD
Antibacterial activity of herbal extract on pathogens isolated from the swollen hind gut of P. Monodon (fabricus) Sankar G, K. Ramamoorthy, K. Sakkaravarthi* and A. Elavarsi, CAS in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai, India ______________________________________________________________________________ ABSTRACT Isolated and identified three species of V. harveyi, V. alginolyticus and V. parahaemolyticus from Swollen Hindgut infected black tiger shrimp seeds were tested against different types of extracts from plants such as Phyllanthus niruri (Stone breaker), Ricinus communis (Castor), Alizzia lebbeck (Albizia), Centella asiatica (Gotu Kola), Tinospora cordifolia (Tinospora Gulancha). The highest activity against the Vibrio harveyi was noticed by Ricinus communis (Castor) in methanol extract (19mm Inhibition zone). The Methanolic extract of R. communis was applied in culture tanks of P. monodon with different dosages which is compared with control tank. The vibrio population decreased (0.64× 104 CFU /ml) at the end of culture in experimental tanks. Hence it is clear that the methanol extract of R. communis played a vital role in the disease resistance of black tiger shrimp P. monodon. Key words: Aquaculture, Herbal extract, P. monodon, Ricinus communis, swollen hind gut, vibrio. ______________________________________________________________________________ INTRODUCTION Aquaculture has become the fastest-growing food-sector on the planet. One of the major constraints faced by shrimp hatcheries is that of mortality caused by disease. Microbial diseases have been reported to be a major limiting factor in production both in wild and cultured shrimp. This has been reported to cause larval mortalities in many hatcheries of the world . Hind Gut syndrome (SHG) was first reported by Lavilla Pitogo et al., in P. monodon post larvae . SHG mainly affected the hindgut and to some extent, the posterior mid gut. PL infected with SHG showed enlargement and distension of the hind-gut folds and its junction with the mid gut. In some cases, swollen mid gut was also noticed. SHG causes gradual mortality in affected post 17 Pelagia Research Library
K. Sakkaravarthi et al Der Pharmacia Sinica, 2010, 1 (3):17-22 ______________________________________________________________________________ larvae, but larvae showed no abnormal swimming behavior. SHG has direct impacts on the hatchery and may also affect grow out systems. The Marine Product Export Development Authority has banned the use Chloramphenicol, Furazolidone, Neomycin, Nalidixicacid, Sulphamet, Nitrofurantoin, Oxytetracycline, Cotrimoxazole and Chloramphenicol were banned in Indian shrimp aquaculture . However, emergence of antibiotic resistant pathogens and ban on the use of antibiotics due to detection of antibiotic residues in shrimp tissues by various importing countries has led to a need for alternatives to antibiotics in hatchery systems . An alternative source for this is using antimicrobial characteristic herbal compounds instead of synthetic antibiotics. Medicinal herbs (MH) have been used as popular medicines to secure health and longevity in land mammals and humans in Asia, especially China, India, Japan, and Korea . There is a developing interest in using MH as a kind of dietary supplement in aquaculture, and in showing the positive effects on growth and the immune response . The abuse of antimicrobial drugs, pesticides, and disinfectants in aquaculture disease prevention and growth promotion has led to the evolution of resistant strains of bacteria and questions of safety . So, this work framed to find out the effective herbal compound which has effective anti bacterial activity against bacteria isolated from Swollen Hind Gut infected shrimp seeds. MATERIALS AND METHODS Collection of Herbal Plants Live and healthy herbal plants such as Phyllanthus niruri (Stone breaker), Ricinus communis (Castor), Alizzia lebbeck (Albizia), Centella asiatica (Gotu Kola), Tinospora cordifolia (Tinospora Gulancha) were collected from different regions of Cuddalore district and were washed twice using freshwater to remove epiphytes and other extraneous matter from the plants. Herbal plants, Alizzia lebbeck and Tinospora cordifolia were obtained from Ayurvedic Research Centre to perform this work. Preparation of crude extract Leaves of the collected plant part were shadow dried, well grinned to make a fine powder. For solvent extraction, sieved powder was soaked with equal part of acetone (1:1), methanol (1:1), hexane (1:1), chloroform (1:1) for 48 hrs . The slurry was then filtered and washed to remove non-soluble fractions. The filtered substance was then centrifuged (20,000gradient for 30 minutes). The extracts were condensed at 350C, until the solvent residue has evaporated. Each 500µg of condensate was impregnated in 5mm diameter sterile paper discs (HIMEDIA, India). Determination of Antibacterial Activity The antibacterial activities were done by disc diffusion method . In vitro antibacterial activity was screened by using Mueller Hinton Agar (MHA) obtained from Himedia (Mumbai). The MHA plates were prepared by pouring 15 ml of MHA media into sterile Petri plates. The plates were allowed to solidify for 5 minutes and 0.1 % inoculums suspension was swabbed uniformly and the inoculums were allowed to dry for 5 minutes. The different plant extracts 500µg of each extract dissolved in appropriate solvent and was applied to sterile paper discs 5mm (Himedia, India). After allowing the solvent to evaporate, the loaded disc was placed on the surface of 18 Pelagia Research Library
K. Sakkaravarthi et al Der Pharmacia Sinica, 2010, 1 (3):17-22 ______________________________________________________________________________ medium and the compound was allowed to diffuse for 5 minutes and the plates were kept for incubation at 37°C for 24 hrs. At the end of incubation, inhibition zones formed around the disc were measured with transparent ruler in millimeter using caliper or scale and recorded. Vibrio population at culture tanks Herbal extracts were added to culture tanks at 10ppm with five days interval in different five tanks with 15 P. monodon seeds and one tank kept as control with out herbal extracts. Fifteen days cultures were carried out. Initial Vibrio load and after 15days Vibrio load has estimated. RESULT AND DISCUSSION The results of antimicrobial activity against the pathogen isolated such as Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio alginolyticus are presented in Figure 1, 2 and 3. The highest activity against the Vibrio harveyi was noticed by Ricinus communis (Castor) in methanol extract (19mm) where as no activity was noticed by Centella asiatica (Gotu Kola) in chloroform extract. The highest activity against Vibrio parahaemolyticus was noticed in Ricinus communis methanol extract (15mm). No activity was noticed in hexane extracts of C. asiatica, A. lebbeck and, P. niruri, chloroform extracts of C. asiatica, A. lebbeck and C. asiatica; acetone extracts of P. niruri. Antimicrobial activity against V. alginolyticus was noticed the highest in methanol extract of Ricinus communis (19mm). No activity was noticed in hexane extracts of C. asiatica, A. lebbeck, T. cordifolia; chloroform extracts of C. asiatica and acetone extracts of T. cordifolia. In water, the Vibrio spp. was found higher in the tanks kept as control (3.99 × 104 CFU /ml) at the end of the culture period. However, among the treated tanks, group IV was showing a reduced count of Vibrio spp. (0.64× 104 CFU /ml). Among the microbes Group II treated showed minimum (1.64 × 104 CFU /ml) followed by Group 111(2.54 × 104 CFU/ml) and Group I (2.51× 104 CFU /ml).(Fig.4)
Fig.1. Antibacterial activity of herbal plant extract against V. harveyi
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K. Sakkaravarthi et al Der Pharmacia Sinica, 2010, 1 (3):17-22 ______________________________________________________________________________
Fig.2. Antibacterial activity of herbal plant extract against V. parahaemolyticus
Fig.3. Antibacterial activity of herbal plant extract against V. alginolyticus Vibrio load in water
5 4 3 2 1 0 Group I
Group III Group IV
Fig.4. Vibrio load in water of P. monodon control and experimental tanks
The present study is aimed to identify the antibacterial activity of herbal extracts against isolated and identified three species of Vibrio such as V. harveyi, V. alginolyticus and V. parahaemolyticus from SHG affected shrimp from a commercial hatchery. Uma et al.,  isolated V. harveyi from SHG affected P. monodon. Similarly, Lightner  isolated V. 20 Pelagia Research Library
K. Sakkaravarthi et al Der Pharmacia Sinica, 2010, 1 (3):17-22 ______________________________________________________________________________ alginolyticus from SHG affected P. monodon. Ruangpan and Kitao  reported in their study that the high abundance of luminescent Vibrio is consistent with occurrence of disease and poor harvest results. Baticados et al.,  reported V. harveyi, a pathogen of P. monodon that causes severe losses. In this study, Antibacterial activity against V. harveyi was found highest in R. communis methanol extract. Correspondingly there was no activity in C. asiatica in chloroform extracts. Samy and Raja  reported that V. parahaemolyticus and V. damsela were resistant to sixteen aqueous plant extracts. In the same way, the current study also shows there were no activity in C. asiatica, A. lebbeck, P. niruri hexane extracts and C. asiatica, A. lebbeck chloroform extracts and C. asiatica P. niruri acetone extracts but the maximum inhibition was found in R. communis methanol extract. Many studies revealed that plant methanol extracts inhibited the growth of tested bacteria more than plant aqueous extracts. Lee Seong Wei et al.,  showed that only aqueous extract of Murraya koenigii leaf can be effective on the growth of Aeromonas hydrophila, Citrobacter freundii, Schewanella putrifacien, V. alginolyticus, V. harveyi and V.vulnificus. Abu Shanab et al.  showed that methanol extracts of the dried ripe berries of Rhus coriaria species inhibited all tested bacteria; however, its aqueous extract showed effect on 2 out of 5tested bacteria. The same way in methanol extracts of R. communis has exposed the maximum inhibition zone against the V. parahaemolyticus and low level of inhibition was found in P. niruri and T. cordifolia. Also highest antibacterial activity against V. alginolyticus was noted in R. communis in methanol extract there is no inhibition activity in C. asiatica, A.lebbeck, T.cordifolia in hexane extracts and C. asiatica chloroform extracts and T. cordifolia acetone extracts. Sree et al.,  studied three menthol extracts of Cynometra iripa was active against all the six pathogens. Where as, Aegiceras corniculatum and Aegialitis cicullsta were active against four of the pathogens. The results revealed that methanol extract of R. communis was having higher antibacterial activity. This may be due to the presence of some inhibitory compounds (alkaloids) or factors present in the plant extracts . Antibacterial activity was found varying with the bioactive compounds of different species and different pathogenic bacteria. Diluting of extracts and crude extracts usually weaken their antimicrobial activity. At lower concentrations, it showed micro biostatic or loss of antimicrobial activity. Low doses may inhibit growth, whereas high doses may arrest the bacterial growth with a few exception, the more concentration or intense the exposure to any germicidal agents, it is likely that target organisms will be digested. CONCLUSION The isolated and identified three species of Vibrio such as V. harveyi, V. alginolyticus and V. parahaemolyticus from SHG affected shrimp was tested against different types of extracts of five different plants. Among these R. communis was showing more activity. The observation made in the present study clearly showed that R. communis extract may be successfully used as a dietary source to enhance the disease resistance.
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K. Sakkaravarthi et al Der Pharmacia Sinica, 2010, 1 (3):17-22 ______________________________________________________________________________ REFERENCES  Jiravanichpaisal P, Miyazaki T, Limsuwan C, J. Aquat. Anim. Health 1994, 6, 27–35.  Lavilla-Pitogo C.R., Paner M.G. and Travina R.D, Diseases in Asian Aquaculture IV, Fish Health Section. Asian Fisheries Society, Manila, 2002, 151-158.  MPEDA, Notification of ban on use of antibiotics/drugs etc. Deputy Director (Aqua.)MPEDA, Ministry of commerce, Govt.of India 700054, 2001.  Karunasagar I., Pai R, and Malathi G.R, Aquaculture, 1994,128; 203-209.  Ji SC, Jeong G.S, SIm Lee SW G, Yoo J.H, Takii K,. Fish Sci. 2007, 73:70–76.  Esiobu N, Armenta L, Ike J, Int. J. Environ. Health Res. 2002, 12, 133–144.  Eloff J.N, J .Ethnopharmacol 1998, 60:1–8.  Murray P.R, Baron E.J, Pfaller M.A, Tenover F.C, and Yolke R.H, Manual of Clinical Microbiology, ASM, Washington, DC. vol. 6, 1995,  Uma A, Saravanabava K, Singaravel R and Koteeswaran A,Tamilnadu journal of Veterinary and Animal Sciences.,4:16-19. 2008.  Lightner D.V, Aquaculture, Elsevier 1988, 8-127.  Ruangpan and Kitao. J. Fish Dis., 1991, 14: 383–388.  Baticados M.C.L, and Padihare J.O, Disease in Asian Aquaculture IV, Fish Health section. Asian Fishery Society, Manila, Philippines. 1992, 531-5 46.  Samy R.P and Patric Raja D, .Acta Botanica Indica 1996, 24:133-114.  Lee Seong Wei, Najiah Musa, Chuah Tse Sengm, Wendy Wee and Noor Azhar Mohd.. African Journal of Biotechnology Vol. 2008, 7 (13), 2275-2278.  Abu-Shanab, B, Adwan G, Abu Safiya D, Adwan K and Abu-Shanab M, J. Islam. Univ. Gaza. 2005, 13 (2): 147-153.  Sree, A Choudhury S, Mukhrjee S.C, Pattnaik P and Bapuji M, Asian Fisheries Science, 2005,18:285-294  Sastry, V.M.V.S. and Rao G.R.K Bot.Mar, 1994, 37:357-360.
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