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Guru Prasad Srinivasan, Caroline Regina Delma, Anandajothi Elamaran, and Somasundaram Thirugnanasambandan Somasundaram, Cytotoxic effect ofacetone extract of Curucma longa in Brine Shrimp, Artemia salina (L), Int.J.Curr.Biotechnol., 2014, 2(9):1-6.

Cytotoxic effect of acetone extract of Curucma longa in Brine Shrimp, Artemia salina (L)

Guru Prasad Srinivasan*1,2, Caroline Regina Delma1, Anandajothi Elamaran1, and SomasundaramThirugnanasambandan Somasundaram1

1Faculty of Marine Sciences, Center of Advanced Study in Marine Biology, Annamalai University, Parangipettai, TN,India.2Dept. Of Microbiology, Sri Sankara Arts and Science college, Enathur, Kanchipuram.

A R T I C L E I N F O A B S T R A C T

Article History:Received 10 September 2014Received in revised form 15 September 2014Accepted 20 September 2014Available online 23 September 2014

Key words:Curcuma longa, cytotoxicity, brine shrimp,Artemia salina, acetone, FT-IR.

Penaeid shrimps compleletly rely on the innate immune system to combat microbial infec-tions. White spot syndrome virus (WSSV) is a dreadly virus that causes severe infection incultured shrimps causing mass mortality. In recent years, focus on shrimp vaccination/im-mune stimulation has shifted from traditional antigenic stimulation to stimulation of theinnate immune response, localization and characterization of WSSV proteins that interact andsuppress the anti-viral response of the shrimps. In addition, prophylactic procedures(immunomodulators) were followed to enhance viral protection of the host system. Medici-nal herbs and their products have taken lead role in antiviral research for the past few decades.In this study, polyphenol extract from the traditional medicinal herb, Curcuma longa wasused to evaluate its cytotoxic effect in the brine shrimps. The curcuma longa extract (CLE)from the rhizome was prepared with acetone and this was validated with commercial standardCurcumin in the FT-IR. The FT-IR results revealed to have the major groups contributing tothe presence of curcumin in the extract. The brine shrimp lethality bioassay (BSLB) resultshowed the LC50 value to be 47.97 µg/ml. This value is found to be a safer dose whencompared to the earlier reports. Hence, the acetone extract of CLE is much suitable forimmunization studies in the penaeid shrimps.

*Corresponding author.Email address: harinigpra@gmail.comMobile: 919976355585

IntroductionPenaeid shrimps are the major marine species which areeconomically important and cultured worldwide. In India,in the last decade, notable increase in shrimp productionwas observed. It has increased from 35,500 tonnes in1990-91 to about 2,16,500 tonnes in 2011-12 with an exportvalue of US $ 3.5 billion. Although the production hasincreased, the annual loss due to diseases met by thenation was estimated and valued at Rs.1022.1 Crores(approximately US $ 165 millions) (Kalaimani, 2013). Inthe Indian scenario, the epidemic diseases are WSSVinfection, LSS, combination of WSSV and LSS, white gutand slow growth syndrome. Of this, WSSV is the primepathogen that cause high mortality and huge economicloss (Flegel, 2008). Despite the fact that many strategieslike immunization with inactivated pathogens, DNAvaccines, siRNA have been used to protect shrimpsagainst WSSV, there is no effective antiviral therapy tocurtail the outbreaks and prevalence of WSSV (Jha et al.,2006, Lu et al., 2009, Tharntada et al., 2009, Witteveldt etal., 2004, 2006, Zhao et al., 2009). Antibiotics, which areused in culture ponds can alleviate bacterial and viralinfections, but overdose could lead to bacterial resistanceand development of new diseases (Rodgers and Furones,2009). Lacking an adaptive immune system, shrimps rely

ISSN: 2321 - 8371

completely on the cellular innate immunity to combatinvading pathogens (Bachère et al. , 2004).Immunomodulators have been proven to be effective andenhance the immune protection of the animals. Non-specific immune response induced usingimmunostimulants will be apt to battle diseases incrustaceans. Use of immunostimulants has increasedbecause of the frequent outbreaks of bacterial, viral,parasitic and fungal infections in culture ponds,hatcheries and aquaculture stations (Anderson, 1992).Immunostimulants in field as supplementary diets havebeen reported to reduce the risk of several pathogenicinfections. Several studies have reiterated the use ofpolyphenols as immunomodulators in shrimps againstbacterial and viral infections (Immanuel et al., 2004;Praseetha, 2005; Direkbusarakom, 2004; Micol et al., 2005).It has been hypothesized that these bioactive compoundsantagonize viral replication in the host cells and enhancethe host’s innate immune response (Citarasu, 2010).Medicinal herbs and their products have taken lead rolein antiviral research for the past few decades. Extracts ofdifferent medicinal herbs showed results againstInfectious Haematopoietic Necrosis Virus (IHNV),Infectious Pancreatic Necrosis Virus (IPNV),Oncorhynchus masou virus (OMV) and Yellow head virus(YHV) in fishes and penaeid shrimp (Direkbusarakom etal., 1993, 1998 a and 1998 b).

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Turmeric (Curcuma longa) a very common kitchen spice,has been used in ayurveda since ancient times. Themolecular components present in this medicinal planthave multifaceted clinical applications. Even powderedrhizome is used to treat cuts, wounds, bruises and inflamedjoints (Surh, 2002). Although, many studies are availableon the potential use of curcumin against several humanpathological conditions, reports on the application ofcurcumin to treat shrimp diseases are very scarce.Supamattaya et al., (2005) have evaluated the in vitroefficacy of Curcuma longa extract against 15 differentisolates of Vibrio spp. The toxicity of any botanical extractneeds to be addressed prior to its application in a livingsystem. This paper discusses the preparation,characterization of crude C. longa extract and itsnanoemulsion and their relative toxicity in brine shrimps.

Materials and methods:Preparation of Curcuma longa extract (CLE):The dried rhizome of Curcuma longa was crushed tofine powder and the polyphenols was extracted withacetone and filtered. The clear filtrate was thenevaporated to dryness under vacuum in a rotaryevaporator (IKA, Germany).

Fourier Transform Infrared spectroscopy:The FT-IR analysis was carried out to identify thefunctional groups present in the Curcuma longa extract(CLE). The CLE was constituted in methanol and IRspectra was recorded with a FTIR spectrophotometer(Shimadzu, Japan). Curcumin standard was obtained fromSigma Aldrich, USA.

Brine shrimp lethality bioassay (BSLB):Brine shrimp Artemia salina (L) also known as seamonkeys, are marine invertebrates about 1 mm in sizeand are being used widely to assess the toxicity of plantmetabolites. The mature brine shrimp nauplii were usedas the test organisms. The Curcuma longa extract (CLE)was dissolved in 10% DMSO and the cytotoxic activitywas evaluated using Brine shrimp lethality bioassaymethod where 7 graded doses (15.625, 31.25, 62.5, 125,250, 500 and1000 µg/mL) were used (Figure 1). DMSOwas used as the negative control. The larvae wereincubated undisturbed for 24 hours. The animal numberswere counted after 24 h. Larvae were considered dead ifthey did not exhibit any internal or external movementduring several seconds of observation. They were notfed during the experiment. The dead nauplii in thetreatment groups were compared with the control groupto ensure that the mortality could be attributed only toCLE and not starvation.

ResultsFourier Transform Infrared spectroscopy:The functional groups present in CLE were identifiedbased on their vibrational frequencies by FTIR analysisusing curcumin (Sigma Aldrich, USA) as the referencecompound (Figure 2). The IR spectra of the CLE revealedbands at 690.51 cm-1 ,which could be attributed to thebending vibrations of C-H groups. Absorption bands at1029 cm-1

and 1114.86 cm-1 indicated the presence of C-O-

H and C-O-C groups respectively. Stretching vibrationsof -CH3 and C=C gave characteristic bands at 1456.26 cm-

1 and 1664.57 cm-1

respectively. Peaks at 2046.47 cm-1 and

2353.16 cm-1 can be ascribed to CO groups. Stretching

vibrations corresponding to O-H groups in carboxylicacid gave characteristic peaks at 2522.89 cm-1

and 2594.26cm-1. Peaks at 2833.43 cm-1

and 2943.37 cm-1 were observed

which indicated the presence of C-H groups. Absorptionbands at 3342.64 cm-1

correspond to the stretching

vibrations of O-H groups in phenols.

Brine shrimp lethality bioassay:The BSLB was performed to evaluate the cytotoxic effectof the CLE. Figure 3 illustrates the survival rate of brineshrimp nauplii in response to CLE treatment. The survivalrate was found to be 72.22 ± 1.202, 62.22 ± 1.764, 41.11 ±0.882, 33.33 ± 1.155, 7.78 ± 1.453 at 15.625, 31.25, 62.5, 125and 250 µg/ml respectively. The mortality rate wasobserved to be 100 % in 500 and 1000 µg/mLconcentrations. The LC50 value was calculated and foundto be 47.97 µg/ml.

DiscussionImmunization using different strategies to combatpathogenic infections in the field of aquaculture has beenthe topic of debate for the scientific community. Previousreports on the DNA vaccine, siRNA, etc., were found tobe good at laboratory scale but the feasibility in the aquafarming sector in developing nations has yet to be proven.Curcuma longa, a natural immunomodulator was usedin this study to evaluate its cytotoxic effect. Many reportson the extracts of C.longa and its derivatives have beenattributed with diverse bioactivities which includeantioxidant (Menon and Sudheer, 2007), anti-inflammatory (Surh et al., 2001), antiproliferative (Ono etal., 2013) antiprotozoal (Nagajyothi et al., 2012),antimicrobial (Ronita De et al., 2009), anti-allergic (Kurupand Barrios 2008), antiulcer (Tuorkey and Karolin, 2009),antidyspeptic (Deitelhoftt et al., 2002) and antidepresant(Yu et al., 2002).

Curcumin extract has also been found to alleviatemicrobial diseases in aquatic animals by modulating theirimmune system (Misra 2004; Misra et al., 2006; Sahu etal., 2007, Behera et al., 2011). Desai et al., (2002) havepatented a composition containing extracts fromtraditional medicinal plants including Curuma longa forthe management of viral and bacterial diseases in aquaticorganisms. In concordance, several reports havedemonstrated the immunomodulatory activities ofcurcumin extract in shrimps (Malar and Charles, 2013;Chandrakala et al., 2013; Lawhavinit et al., 2011).Although the antimicrobial activities of C. longa extractin penaeid shrimps have been reported by severalresearchers, studies on their activity against WSSV arevery scanty. Balasubramanian et al., (2007) screened 20species of Indian traditional medicinal plants includingC. longa against WSSV infection in marine shrimp andfreshwater crab. Their study reported that C. longa failedto inactivate WSSV. Similarly, Supamattaya et al., (2005)have reported that C. longa extract didnot showpromising activity in WSSV challenging experimentsdespite showing profound anti-vibrio activity in penaeidshrimps. Keeping this in view, the present study wasattempted to evaluate the immunomodulatory effect ofCLE against WSSV infection in penaeid shrimps.Curcumin was extracted from the rhizome of Curcumalonga using acetone. Acetone is an ideal solvent to extractcurcumin owing to its low boiling point and less toxicityas compared to other organic solvents like ethanol andmethanol (Popuri and Pagala, 2013). Revathy et al., (2011)have also demonstrated that acetone exhibited maximumyield of curcuminoids.

FTIR analysis gives an overview of the functional groupspresent in a compound. The FT-IR spectra of CLErevealed peaks corresponding to that of the standardcurcumin. The results corroborated with the FT-IRspectra of curcumin obtained by Kolev et al., (2005).Presence of vibrational peaks corresponding to phenolic

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Figure 1: Brine shrimp lethality bioassay. Various concentrations of the CLE (15.625, 31.25, 62.5, 125, 250, 500 and1000µg/mL) in 10% DMSO was added in triplicates to the microplate wells containing Artemia nauplii. Each well contained20 nauplii. 10% DMSO was used as control.

Figure 2: FT-IR spectra of standard Curcumin (A) and CLE (B). Stretching vibrations at 690.51 cm-1, 1029 cm-1 and

1114.86 corresponds to C-H, C-O-H and C-O-C groups respectively. Peaks at 3342.64 cm-1, 2522.89 cm-1 and 2594.26

correspond to the stretching vibrations of O-H groups in phenols and carboxylic acid respectively.

Figure 3: Cytotoxic effect of CLE. The figure illustrates the survival rate of Artemia nauplii in in response to CLEtreatment.

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-OH, aromatic C=C, C-O-C and enol C-O groups confirmsthat CLE contains curcumin as the major component(Shrivastava et al., 2013; Bich et al., 2009; Coates, 2000).Any compound (or) drug from natural source must beevaluated for its toxicity before it is proceeded fortranslational studies. The BSLB is widely being used todetermine the cytotoxic effect of heavy metals, pesticides,natural plant extracts (Price, 1974; Meyer, 1982). The brineshrimp (A. salina) is a lower invertebrate of the marineecosystem. The brine shrimp cytotoxicity test is anefficient, rapid and inexpensive test which requires verysmall amount of sample and the assay correlates wellwith the cytotoxicity (Sutha devaraj et al., 2013). Dimethylsulfoxide (DMSO) was reported to be a safer solvent ascompared to other organic solvent (Wu, 2014). Hence,the CLE was suspended in 10% DMSO and tested forcytotoxicity. The brine shrimp assay results showed thetoxicity level increased with with increasingconcentrations. The LC50 value obtained in this studywas 47.97 µg/ml. Previous studies have reported a lowerLC50 value with ethanol extract of C. longa as comparedto the present study (Khattak et al., 2005; R.Akter et al.,2012). This indicates that the acetone extract of C. longais found to be relatively less toxic than the ethanolicextract as hypothesized by Popuri and Pagala (2013).Taken together, these observations suggest that theacetone extract can further be used for immunizationstudies with penaeid shrimps. The acetone extract ofCurucma longa with the LC50 will be a saferimmunomodulator with low or no side effects.Acknowledgements: The authors thank Department ofBiotechnology, Government of India for the financialsupport (BT/AAQ/Indo-Norway/183196/2007).

ReferencesAkter R, Mohammed A. Satter., Khan M.S., Rahman M.S.,and Ahmed N.U., 2012. Cytotoxic effect of five medicinalplants extracts using Brine shrimp (Artemia salina) test.Bangladesh. J. Sci. Ind. Res. 47(1):133-136.

Anderson D.P., 1992. Immunostimulants, adjuvants, andvaccine carriers in fish: application to aquaculture. Ann.Rev. Fish. Dis. 281-307.

Ashok Kumar Popuri and Bangaraiah Pagala. 2013.Extraction of Curcumin from Turmeric Roots InternationalJournal of Innovative Research & Studies, May: Vol 2,(5):289-299.

Bachère E., Gueguen Y., Gonzalez M., De Lorgeril J.,Garnier J. and Romestand B., 2004. Insights into the anti-microbial defense of marine invertebrates: the penaeidshrimps and the oyster Crassostrea gigas. Immunol.Review 198: 149–168. doi: 10.1111/j.0105-2896.2004.00115.x

Balasubramanian G., Sarathi M., Rajesh Kumar S., SahulHameed A.S., 2007. Screening the antiviral activity ofIndian medicinal plants against white spot syndromevirus in shrimp. Aquaculture. 263:15–19. doi: 10.1016/j.aquaculture.2006.09.037

Behera T., Swain P., Sahoo S.K., Mohapatra D., Das B.K.,2011. Immunostimulatory effects of curcumin in fish,Labeo rohita (H.) Indian Journal of Natural Products andResources Vol. 2(2), June, pp. 184-188

Bich V.T., Thuy N.T., Binh N.T., Huong N.T.M., Yen P.N.D.,Luong T.T., 2009. in: D.T. Cat, A. Pucci, K. Wandelt (Eds.),Physics and Engineering of New Materials, Springer

Publications, Berlin, pp. 271–278.

Chandrakala N., Sasikala P., Sankar Ganesh P., PrabakaranM., Sampath Kumar G., 2013. Preliminary screening ofCurcuma longa (Linn) extract against Penaeus monodon(Fab) pathogenic Vibrio sp. Indian Journal of AppliedMicrobiology ; January-June, 16 (1): 21-29.

Deitelhoftt P., Peterowicz O., and Muller B., 2002. Anti-dyspeptic properties of turmeric root extract (TRE).Phytomedicine .7: 92.

Direkbusarakom S., 2004. Application of medicinal herbsto aquaculture in Asia. Walailak Journal of Science andTechnology 1 (1): 7–14.

Direkbusarakom S., Ezura Y., Yoshimizu M., HerunsaleeA., 1998a. Efficacy of Thai traditional herb extracts againstfish and shrimp pathogenic bacteria. Fish Pathology 33(4):437–441.

Direkbusarakom S., Herunsalee A., Boonyaratpalin S.,Danayadol Y., Aekpanithanpong U., 1993. Effect ofPhyllanthus spp against yellow head baculovirusinfection in black tiger shrimp, Penaeus monodon. In:shariff, M., Arthur, J.R., Subasinghe, R.P. (Eds.), Diseasein Asian Aquaculture II. Fish Health Section. AsianFisheries Society, Manila, pp. 81–88.

Direkbusarakom S., Ruangpan L., Ezura Y., Yoshimizu M.,1998b. Protective Efficacy of Clinacanthus nutans onyellow-head disease in black tiger shrimp (Penaeusmonodon). Fish Pathology 33(4): 401–404.

Flegel T.W., Lightner D.V., Lo C.F., Owens L., 2008. Shrimpdisease control: past, present and future. In: Diseases inAsian aquaculture (Bondad-Reantaso MG, Mohan CV,Crumlish M, Subasinghe RP, eds). Fish Health. AsianFisheries Society, Manila, Philippines VI: 355-78.

Immanuel G., Vincy Bai V.C., Palavesam A., Peter MarianM., 2004. Effect of butanolic extracts from terrestrialherbs and seaweeds on the survival, growth andpathogen (Vibrio parahaemolyticus) load on shrimpPenaeus indicus juveniles. Aquaculture. 236:53-65.doi:10.1016/j.aquaculture.2003. 11.033

Jha R.K., Xu Z.R., Shen J., Bai S. J., Sun J.Y., Li W.F., 2006.The efficacy of recombinant vaccines against white spotsyndrome virus in Procambarus clarkii. Immunol. Lett.105:68–76.

John Coates., 2000. Interpretation of Infrared Spectra, APractical Approach. Encyclopedia of AnalyticalChemistry R.A. Meyers (Ed.). John Wiley & Sons Ltd

Kalaimani N., Ravisankar T., Chakravarthy N., Raja S.,Santiago T.C. and Ponniah A.G., 2013. Economic Lossesdue to Disease Incidences in Shrimp Farms of India.Fishery Technology. 50 : 80 - 86

Khattak S., Rehman S., Ullah Shah H., Ahmad W., andAhmad M., 2005. Biological effects of indigenousmedicinal plants Curcuma longa and Alpinia galangal.Fitoterapia, 76: 254-257.

Kolev T.M., Velcheva E.A., Stamboliyska B.A., SpitellerM., 2005. DFT and experimental studies of the structureand vibrational spectra of curcumin. Int. J. Quantum.Chem. 102(6):1069–1079.

Kurup V.P., Barrios C.S., 2008. Immunomodulatory effects

5 Int.J.Curr.Biotechnol. Volume 2; Issue 9; September, 2014

of curcumin in allergy. Mol. Nutr. Food. Res. Sep;52(9):1031-9. doi: 10.1002/mnfr.200700293.

Lu Y., Liu J., Jin L., Li X., Zhen Y., Xue H., et al., 2009.Passive immunization of crayfish (Procambius clarkii)with chicken egg yolk immunoglobulin (IgY) againstWhite Spot Syndrome Virus (WSSV). Appl. Biochem.Biotechnol. 159: 750–8.

Menon V.P., Sudheer A.R., 2007. Antioxidant and anti-inflammatory properties of curcumin. Adv. Exp. Med. Biol.595:105-25.

Meyer B.N., Ferrigni N.R., Putnam J.E., Jacobson L.B.,Nichols D.E., McLaughlin J.L., 1982. Brine shrimp: aconvenient general bioassay for active plant constituents.Planta. Medica. 45(5): 31-34.

Micol V., Caturla N., Pe ìrez-Fons L., Ma ìs V., Pe ìrez L.,Estepa A., 2005. The olive leaf extract exhibits antiviralactivity against viral haemorrhagic septicaemiarhabdovirus (VHSV). Antiviral. Res. 66(2–3):129–136

Misra C.K., 2004. Comparative study on the effect ofdifferent immunostimulants on the immune system of L.rohita (Hamilton, 1822). PhD thesis, CIFE (InlandAquaculture) pp. 231.

Misra C.K., Das B.K., Mukherjee S.C., Pattnaik P., 2006.Effect of multiple injections of â-glucan on non-specificimmune response and disease resistance in Labeo rohitafingerlings. Fish. Shellfish. Immunol. 20: 305–319.

Nagajyothi F., Zhao D., Weiss L.M., Tanowitz H.B., 2012.Curcumin treatment provides protection againstTrypanosoma cruzi infection. Parasitol. Res. Jun: 110(6):2491-99. doi: 10.1007/s00436-011-2790-9.

Ong-ard Lawhavinit., Pronchai Sincharoenpokai.,Patcharee Sunthornandh., 2011. Effects of ethanol tumeric(Curcuma longa Linn.) extract against shrimp pathogenicVibrio spp. and on growth performance and immunestatus of White shrimp (Litopenaeus vannamei).Kasetsart. J. (Nat.Sci.) 45:70– 77.

Ono M., Higuchi T., Takeshima M., Chen C., Nakano S.,2013. Antiproliferative and apoptosis-inducing activityof curcumin against human gallbladder adenocarcinomacells. Anticancer. Res. May; 33(5):1861-66.

Praseetha., 2005. Enrichment of brine shrimp Artemiafranciscana with commercial probiotics and herbalextracts and their resistance against shrimp pathogenVibrio sp. (Vibrio parahaemolyticus and Vibrio damsela),M.Phil, Dissertation, Manonmaiam SundaranarUniversity, India

Price K.S., Waggy G.T., Conway R.A., 1974. Brine shrimpbioassay and seawater BOD of petrochemicals. J. Water.Pollut. Control. Fed: 46:63 77.

Revathy S., Elumalai S., Merina Benny and Benny Antony.2011. Isolation, purification and identification ofCurcuminoids from Turmeric (Curcuma longa L.) byColumn Chromatography Journal of ExperimentalSciences. 2(7): 21-25.

Rodgers C.J., Furones M.D., 2009. Antimicrobial agentsin aquaculture: practice, needs and issues. CIHEAM.OPTIONS Meìditerraneìennes 86:41-59. Series A:Mediterranean Seminars. The use of veterinary drugsand vaccines in Medi- terranean aquaculture.

Ronita De., Parag Kundu., Snehasikta Swarnakar.,Ramamurthy T., Abhijit Chowdhury G., Balakrish Nair.,Asish K., Mukhopadhyay., 2009. Antimicrobial activityof Curcumin against Helicobacter pylori isolates fromIndia and during infections in Mice. Antimicrob. AgentsChemother. April; 53(4):1592-1597. doi: 10.1128/AAC.01242-08.

Sahu S., Das B.K., Mishra B.K., Pradhan J., Sarangi N.,2007. Effect of Allium sativum on the immunity of Labeorohita infected with Aeromonas hydrophila. Journal ofApplied Ichthyology 23: 80-86.

Shrivastava A., Sharma J., Jain S., Aggrawal K.L., 2013.Incompatibility studies by high performance thin-layerchromatography: In case of curcumin. Asian. J. Pharm.7:103-9

Supamattaya K., Suntornchareonnon N., BoonyaratpalinM., Ruangsri J.,Tattanon T., Klowkliang T., 2005. Effectsof Thai medicinal plants on pathogenic bacterial, growthperformance, health condition and disease resistance inblack tiger shrimp (Penaeus monodon Fabricius)Songklanakarin J. Sci. Technol. 27 (1): 55-70

Surh Y. J., 2002. Anti-tumor promoting potential ofselected spice in- gredients with antioxidative and anti-inflammatory activities: a short review. Food. Chem.Toxicol. 40: 1091–1097.

Surh Y. J., Chun K.S., Cha H.H., Han S.S., Keum Y.S., ParkK.K. and Lee S.S., 2001. Molecular mechanism underlyingchemopreventive activities of anti-inflammatoryphytochemicals: down regulation of COX-2 and iNOSthrough suppression of NF-êB activation. Mutat. Res.480–481: 243–268.

Sutha Devaraj., Sabariah Ismail., Surash Ramanathan.,Mun FeiYam., 2013. In vivo toxicological investigationsof standardized ethanolic extract of Curcumaxanthorrhiza Roxb. rhizome. J. Nat. Prod. Plant. Resour.,3 (1):67-73.

Tharntada S., Ponprateep S., Somboonwiwat K., Liu H.,Söderhäll I., Söderhäll K., et al., 2009. Role ofantilipopolysaccharide factor from the black tiger shrimp,Penaeus monodon, in protection from white spotsyndrome virus infection. J. Gen. Virol. 90:1491–98.

Thavasimuthu Citarasu., 2010. Herbal biomedicines: anew opportunity for aquaculture industry Aquacult. Int.18:403–414. doi: 10.1007/s10499-009-9253-7.

Tuorkey M., Karolin K., 2009. Anti-ulcer activity ofcurcumin on experimental gastric ulcer in rats and its effecton oxidative stress/antioxidant, IL-6 and enzymeactivities. Biomed. Environ. Sci. Dec; 22(6):488-95. doi:10.1016/S0895-3988(10)60006-2.

Ulhas Manohar Desai., Chittur ThelakkatAchuthankutty., Rayadurga Anantha Sreepada., 2001.Composition for treating white spot syndrome virus(WSSV) infected tiger shrimp penaeus monodon and aprocess for preparation thereof. Patent number: 6440466,February 21, 2001.

Vidhya malar H.L., Maria charles P., 2013. Effect of turmericCurcuma longa (Linn.) extract on immunity andresistance to Vibrio harveyi in Black Tiger shrimpPenaeus monodon. International Journal of Research inZoology. 3(2): 21-26.

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Witteveldt J., Cifuentes C.C., Vlak J.M., Van Hulten M.C.,2004. Protection of Penaeus monodon against white spotsyndrome virus by oral vaccination. J. Virol. 78:2057–61.

Witteveldt J., Vlak J.M., Van Hulten M.C., 2006. Increasedtolerance of Litopenaeus vannamei to white spotsyndrome virus (WSSV) infection after oral applicationof the viral envelope protein VP28. Dis. Aquat. Organ.70:167–70.

Wu C., 2014. An important player in brine shrimp lethalitybioassay: The solvent. J. Adv. Pharm. Technol. Res. 5:578.

Yu Z.F., Kong L.D., and Chen Y., 2002. Antidepressantactivity of aqueous extracts of Curcuma longa in mice. J.Ethnophamacol.83: 161- 165.

Zhao Z.Y., Yin Z.X., Xu X.P., Weng S.P., Rao X.Y., DaiZ.X., et al., 2009. A novel C-type lectin from the shrimpLitopenaeus vannamei possesses anti-white spotsyndrome virus activity. J. Virol. 83:347–56.