Immunological and Bactericidal Effects of Turmeric (Curcuma longa Linn.) Extract in Pacific White Shrimps (Litopenaeus vannamei Boone)

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1 Ksetsrt J. (Nt. Sci.) 44 : (2010) Immunologicl nd Bctericidl Effects of Turmeric (Curcum long Linn.) Extrct in Pcific White Shrimps (Litopeneus vnnmei Boone) Kittim Vnichkul 1, Nontwith Areechon 1 *, Ngmpong Kongkthip 2, Prpnsk Srispoome 1 nd Niti Chuchird 3 ABSTRACT Pcific white shrimp (Litopeneus vnnmei Boone) hs een one of the min export griculturl products of Thilnd. However, culture of this mrine shrimp hs een retrded continuously y disese prolems tht hve led to decline in production or even the collpse of the frming system in some res of the country. To cope with the disese outreks, some frmers pply ntiiotics, which cn cuse negtive consequences to shrimp products involving drug residues. In this study, turmeric (Curcum long Linn.) extrct contining % (w/w) curcuminoids ws dded to shrimp feed s n immunoenhncement. Shrimp with n verge weight of 12 g were rised with feed contining turmeric extrct t 0, 12.5, 25.0 nd 50.0 mg/kg feed (ppm). The studied prmeters included resistnce ginst pthogenic Virio hrveyi, immune functions nd the totl count of cteri from the shrimp intestines. The results showed significntly (P<0.05) etter resistnce ginst V. hrveyi in shrimps fed with 25 mg/kg feed of turmeric extrct when compred with control. Phenoloxidse ctivity of shrimps fed with 25 nd 50 mg/kg feed of turmeric extrct ws significntly (P<0.05) higher thn the control. All turmeric extrct-treted shrimps showed higher ctericidl ctivity thn the control. However, no significnt (P>0.05) differences were found mongst the vlues of totl hemocyte count nd percent phgocytosis. Totl cteri nd the Virio spp. count from the intestines of shrimps fed with turmeric extrct t ll concentrtions were significntly (P<0.05) lower thn the control. Keywords: Pcific white shrimp, turmeric Curcum long Linn. extrct, immunity INTRODUCTION Mrine shrimp products hve ecome mjor export commodity, s well s mjor source of income for people involved in the shrimp culture industry. Pcific white shrimp (Litopeneus vnnmi Boone) is the min species used for mrine shrimp culture of Thilnd. It is lso the primry peneid shrimp currently eing cultured in Centrl nd South Americ, Chin, Indonesi nd Tiwn (Hsu nd Chen, 2007). As expected in intensive culture systems, disese outreks hve een common in Pcific white shrimp cultures, including prsitic, cteril nd virl infections. Disese outrek is result of environment deteriortion nd stress ssocited 1 Deprtment of Aquculture, Fculty of Fisheries, Ksetsrt University, Bngkok 10900, Thilnd. 2 Deprtment of Chemistry, Fculty of Science, Ksetsrt University, Bngkok 10900, Thilnd. 3 Deprtment of Fishery Biology, Fculty of Fisheries, Ksetsrt University, Bngkok 10900, Thilnd. * Corresponding uthor, e-mil: ffisnw@ku.c.th Received dte : 19/02/10 Accepted dte : 10/05/10

2 Ksetsrt J. (Nt. Sci.) 44(5) 851 with intensive frming nd n increse in the numers of pthogens, such s Virio spp., in the pond wter (Lvill-Pitogo et l., 1998). Antiiotics hve een used in shrimp culture s tretment for cteril disese, which cn cuse residue prolems if good prctice hs not een implemented s prt of the drug regime. In ddition, mesures tht re more stringent hve een exercised y trde counterprts in regrd to contmintion with ntiiotics. Thus, it is impertive for shrimp helth reserchers to investigte lterntive pproches for effective disese prevention nd tretment with miniml negtive consequences to shrimp products. Turmeric (Curcum long Linn.) is ntive plnt of southern Asi nd is cultivted extensively throughout the tropicl prts of the world (Gupt nd Blsurhmnym, 1998). Mny iologicl ctivities hve een ttriuted to the extrcts of C. long nd to its ctive compound (curcumin). These ctivities include ntioxidnt, nti-inflmmtory nd ntiprolifertive properties. Turmeric extrct nd curcumin hve lso een used widely s heptoprotective gent (Mes et l., 2000). Little of the reserch on hers hs investigted mtters of immuno-modultion nd disese resistnce in qutic nimls. Blck tiger shrimp (Peneus monodon Fricius) fed on diet contining turmeric extrcts t 25 mg/kg diet showed resistnce ginst Virio hrveyi nd Virio spp., while n in vitro study showed tht 15 isoltes of Virio spp. were erdicted y extrcts contining 250 mg/l turmeric (Supmtty et l., 2004). Turmeric hs good potentil for ppliction to qutic nimls, ecuse it is nturl product without ny negtive consequences to the hrvest. However, due to limited informtion on its effective nd prcticl ppliction to qutic nimls, study tht is more thorough should e conducted to demonstrte the enefit of this her. The purpose of this study ws to investigte the effect of turmeric (Curcum long Linn.) extrct in feed of the Pcific white shrimp (Litopeneus vnnmei Boone) on its immunity to disese. Bctericidl ctivity of turmeric extrct in the shrimp intestine ws lso studied. MATERIALS AND METHODS Test niml Cliniclly helthy Pcific white shrimp (Litopeneus vnnmei Boone) with weight rnge of g were otined from commercil frm in Thilnd nd cclimted in cement tnks with dimensions m contining g/kg chlorinted se wter for two weeks efore commencing the experiment. The tnks were covered with lck plstic to mintin the wter temperture, nd the se wter ws chnged regulrly to mintin optimum wter conditions throughout the tril. There were four tretments (one control group nd three concentrtions of turmeric extrct t 12.5, 25 nd 50 mg/kg (ppm) feed) replicted in triplicte (three tnks per tretment). Ech tnk ws stocked with 15 shrimps. Turmeric extrct nd nlysis Turmeric (rhizome) ws chopped into smll pieces, dried nd ground finely nd then mcerted in 95% ethyl lcohol for 10 h. Filtrtion nd evportion were used to otin crude extrct of turmeric. The mount of ctive content in the turmeric extrct (curcuminoids) ws nlyzed y high performnce liquid chromtogrphy (HPLC) using the method descried y Kongkthip nd Kongkthip (2005). The turmeric extrct ws kept in refrigertor t 4 C efore use. The turmeric extrct ws dissolved in 95% ethyl lcohol efore eing mixed with feed ccording to the experimentl concentrtions nd then ir-dried efore coting with squid oil.

3 852 Ksetsrt J. (Nt. Sci.) 44(5) Disese resistnce of Pcific white shrimp ginst viriosis Virio hrveyi ws isolted from disesed Pcific white shrimp (Litopeneus vnnmei Boone) nd identified, s descried y Buchnn nd Gions (1974). A suspension of V. hrveyi ws prepred in sterile 1.5% NCl nd diluted to the predetermined concentrtion. After two weeks of the feeding tril, 10 shrimps from ech tretment nd the control were injected intrmusculrly on sixth segment with 0.1 ml of cteril suspension contining CFU/ ml of V. hrveyi. Ded shrimps were removed dily nd the numer recorded for 7 d. Bcteri were isolted from the heptopncres to confirm the cuse of mortlity. Mortlity rtes were sttisticlly compred. Immune functions Ten shrimps from the control nd tretment groups were smpled for immune nlysis in the fourth week of the feeding tril. Totl hemocyte count Hemolymph ws collected from the ventrl sinus of ech shrimp. A syringe (3 ml) contining 1 ml of nticogulnt (K % L- cystein) with needle (26 G) ws used to drw 0.5 ml of shrimp lood. The totl hemocyte count ws recorded using hemcytometer nd clculted s the numer of lood cells (totl hemocytes/mm 3 ). Phenoloxidse ctiviy (modified from Supmtty et l. (2000)) Hemolymph ws collected from ech shrimp y plstic syringe (3mL) nd the hemocytes were seprted nd wshed three times with shrimp sline. Hemocyte lyste (HLS) ws otined y suspending the prepred hemocytes in ccodylte uffer ph 7.4, sonicted t 30 mplitudes for 5 sec, followed y centrifugtion t 10,000 rpm t 4 C for 20 min. Phenoloxidse ctivity ws mesured from the HLS y dding 200 µl of 0.1% trypsin in ccodylte uffer in 200 µl HLS, followed y 200 µl of L-dihydroxyphenyllnine (L-DOPA). Enzyme ctivity ws mesured s the sornce of dopchrome t the 490 nm wvelength. The protein concentrtion in the HLS ws determined y Lowry s method (Lowry et l., 1951). The phenoloxidse ctivity ws expressed s IU/min/ mg protein (1 unit of phenoloxidse = OD 490 / min/mg protein). Phgocytic ctivity The phgocytic ctivity ws mesured s descried y Supmtty et l. (2000). Shrimp hemolymph (200 µl) ws smered on glss slide nd incuted t room temperture for 1 h. The non-dherent cells were removed nd wshed three times with shrimp sline. Bker s yest ws dded to the hemolymph lyer nd incuted for 2 h t room temperture nd then wshed three times with shrimp sline, ir dried nd stined with Wright-Giems solution. The phgocytosis percentge ws enumerted from the numer of phgocytizing cell in 100 hemocytes. Bctericidl ctivity Shrimp serum ws diluted with 2.6% NCl y twofold method in multiwell-plte. A suspension of V. hrveyi ws dded into the diluted serum nd incuted t room temperture for 3 h efore enumerting the numer of cteri y spred plte technique on thiosulfte citrte ile sucrouse gr (TCBS). The results were recorded s the lowest dilution tht killed 50% of the V. hrveyi compred to the control. Totl cteril count from shrimp intestine After four weeks of the feeding tril, shrimp intestines were removed, homogenized nd tenfold diluted with sterile 1.5% NCl. A spred plte technique ws used to determine the totl cteril count nd totl Virio spp. count on plte count gr (PCA) nd TCBS, respectively. Sttisticl nlysis Sttisticl nlysis ws performed using

4 Ksetsrt J. (Nt. Sci.) 44(5) 853 nlysis of vrince (ANOVA) nd Duncn s new multiple rnge test t P = 0.05 (Duncn, 1995). RESULTS Turmeric extrct nlysis Anlysis of turmeric extrct y HPLC identified % (w/w) of curcuminoids tht contined three ctive ingedients, nmely curcumin, desmethoxycurcumin nd isdesmethoxycurcumin, nd voltile oil. Disese resistnce ginst V. hrveyi After chllenging with V. hrveyi for 7 d, there were significnt (P<0.05) differences in the mortlity rtes. Shrimps fed with 25 mg/kg turmeric extrct hd the lowest mortlity rte of ± %, while the control hd the highest mortlity t %. However, the significnt difference ws found only etween the 25 mg/kg turmeric group nd the control (Figure 1). Immune functions Totl hemocyte count The totl hemocyte counts of the control nd tretment groups (concentrtions of turmeric t 12.5, 25, 50 mg/kg) were ± , ± , ± nd ± cells/ml, respectively, which were not significntly (P> 0.05) different (Figure 2) % Mortlity Concentrtion Control 12.5 ppm 25 ppm 50 ppm Figure 1 Mortlity rte of Litopeneus vnnmei rised with feed contining different concentrtions of turmeric extrct nd chllenged with Virio hrveyi. Totl Hemocyte (x 10 6 cells/ml) Control 12.5 ppm 25 ppm 50 ppm Concentrtion Figure 2 Totl hemocytes count of Litopeneus vnnmei rised with feed contining different concentrtions of turmeric extrct.

5 854 Ksetsrt J. (Nt. Sci.) 44(5) Phenoloxidse ctivity Shrimps tht were fed with 25 nd 50 mg/kg turmeric extrct hd significntly (P<0.05) higher ctivity of phenoloxidse thn the control nd the shrimps tht were fed with 12.5 mg/kg turmeric extrct, with vlues of ± , ± , ± nd ± unit/min/mg protein, respectively (Figure 3). Phgocytic ctivity There were high percentges of phgocytosis in the shrimps fed with 12.5 nd 25 mg/kg of turmeric extrct t ± nd ± %, respectively, ut these vlues were not significntly (P> 0.05) different from the control, which hd vlue of ± 9.986%. Shrimps tht were fed with turmeric extrct t 50 mg/kg hd the lowest percentge of phgocytosis, which ws not significntly (P> 0.05) different from the control, ut ws significntly (P<0.05) different from the other turmeric-treted groups (Figure 4) (unit/min/mg Protien) Concentrtion Control 12.5 ppm 25 ppm 50 ppm Figure 3 Phenoloxidse ctivity of Litopeneus vnnmei rised with feed contining different concentrtions of turmeric extrct % phgocytosis Control 12.5 ppm 25 ppm 50 ppm Concentrtion Figure 4 Percent phgocytosis of Litopeneus vnnmei rised with feed contining different concentrtions of turmeric extrct.

6 Ksetsrt J. (Nt. Sci.) 44(5) 855 Bctericidl ctivity Bctericidl ctivity (Tle 1) t serum dilution of 1:256-1:512 ws found in shrimps fed with 25 mg/kg turmeric extrct, which ws the highest level when compred with other tretments. Shrimps fed with 12.5 nd 50 mg/kg hd the sme ctericidl ctivity levels (1:128-1:256), while the control hd the lowest ctericidl ctivity (1:64-1:128). Totl cteril count from shrimp intestine The totl cteril count of the control ws ± CFU/gm, which ws significntly (P< 0.05) higher thn in the turmerictreted groups. The totl Virio spp. count of the control ws ± CFU/gm, which ws significntly (P< 0.05) higher thn in the turmeric-treted groups (Tle 2). DISCUSSION Turmeric extrct In this study, extrct from turmeric using 95% ethyl lcohol contined % (w/w) curcuminoids tht consisted of curcumin, desmethoxycurcumin nd isdesmethoxycurcumin mixed with voltile oil. According to Supmtty et l. (2005), extrction of turmeric with ethyl lcohol yielded higher level of ctive ingredient thn from other solvents. Supmtty et l. (2004) found 21.57% curcuminoids from turmeric extrcted y lcohol, which ws lower thn in the current study. The source of the turmeric plnt nd the extrction process re key fctors tht determine the curcuminoid content. The mount of curcuminoids indicted the degree of ctericidl ctivity nd immunostimulnt effects in the tested nimls. Disese resistnce ginst V. hrveyi Resistnce ginst experimentl infection with V. hrveyi ws clerly elevted in shrimps tht were fed with turmeric extrct. All turmeric-treted groups hd less mortlity thn the control, even though the differences were significnt only etween the 25 mg/kg turmeric group nd the control. This finding ws similr to previous report on resistnce to viriosis in lck tiger shrimps fed with turmeric extrct (Vnichkul et l., 2007). Supmtty et l. (2005) compred Tle 1 Bctericidl ctivity of Litopeneus vnnmei rised with feed contining different concentrtions of turmeric extrct. Tretment Bctericidl ctivity Control 1:64-1:128 Turmeric extrct 12.5 mg/kg 1:128-1:256 Turmeric extrct 25 mg/kg 1:256-1:512 Turmeric extrct 50 mg/kg 1:128-1:256 Tle 2 Totl cteri nd Virio spp. counts (men±stndrd devition) from intestines of Litopeneus vnnmei rised with feed contining different concentrtions of turmeric extrct ( 10 9 CFU/g). Tretment Bcteri Control 12.5 mg/kg 25 mg/kg 50 mg/kg Totl count ± ± ± ± Totl Virio spp. Count 10.67± ± ± ±2.07 Row vlues with different superscript letters re significntly different t (P<0.05).

7 856 Ksetsrt J. (Nt. Sci.) 44(5) the virl nd cteril inhiition ctivities of three hers: turmeric (Curcum long), Androgrphis pnicult nd Clincnthus mutns. They found y in vitro study tht extrcts of ll the hers could inhiit, s well s erdicte, the shrimp pthogenic cteri, Virio spp. nd white spot virus, for which the turmeric extrct showed the highest efficcy. By in vivo study, Supmtty et l. (2004) prepred feed contining different concentrtions of turmeric tht ws then fed to lck tiger shrimps (Peneus monodon Fricius) for two weeks. They found tht 5 nd 25 mg/kg turmeric extrct could enhnce the survivl rte of lck tiger shrimps infected experimentlly with Virio hrveyi. In ddition, voltile oil in the turmeric extrct hs een reported to show ctericidl effects (Lutomoski et l., 1974; Bhvnishnkr nd Murthy, 1986). There hve een very limited studies on the effects of turmeric on qutic nimls. Dey nd Chndr (1995) reported the production of disese-resistnt fry of Indin mjor crp (Ctl ctl) y spwn tretment with turmeric, neem leves nd grlic powder. Immune functions The study on the effects of turmeric extrct on Pcific white shrimp immunity showed positive effect with three of the four prmeters studied. Totl hemocyte count ws not ffected y the her extrct. The ctericidl ctivity, prmeter tht showed the ility of shrimp hemolymph to reduce certin mounts of cteri, of ll turmeric-treted shrimps hd higher rnge thn in the control. This ctivity is elieved to e n importnt defense mechnism ginst cteril systemic infection in shrimps. Phenoloxidse nd phgocytic ctivity ws lso significntly different etween the treted nd the control group. Interestingly, the most effective concentrtion of turmeric extrct relted to these prmeters ws 25 mg/kg. There hve een very few studies on turmeric with regrd to enhncing immunity in invertertes. Supmtty et l. (2004 ) reported the negtive effects of feed supplemented with 50 nd 200 mg/kg of turmeric extrct (with 21.57% curcuminoids) on the immune functions of lck tiger shrimp (P. monodon Fricius). After 8 weeks of the feeding tril, shrimps tht were fed with turmeric extrct hd lower totl hemocyte counts nd phenoloxidse ctivity thn the control. These findings might hve een cused y poor feed consumption nd the rte of feed uptke of the treted shrimps. Totl cteril count from shrimp intestine The current study indicted clerly the ctericidl ctivity of turmeric extrct in the intestines of Pcific white shrimp, in which the totl cteril counts nd totl Virio counts of ll turmeric-treted groups were significntly lower thn for the control. This result ws different from Supmtty et l. (2004), s they did not find ny significnt chnges in the cteril count in the heptopncres nd intestines of lck tiger shrimps fed with turmeric extrct t 50 nd 200 mg/kg, which might hve een relted to the low curcuminoids content in the extrct nd poor feed consumption y the experimentl shrimps. The mechnism of the immunostimulnt effect of hers in nimls is not clerly understood. The immunostimulnt effect cn e chieved y four mechnisms: ctivtion of phgocytosis, stimultion of the firolsts, incresing respirtory ctivites nd incresed moility of leucocytes (Guri-Fkim, 2006). Extrcts from roots nd eril prts from vrious species of hers hve een ssessed for their phgocytic potentil in nimls nd ll ethnolic root extrcts incresed phgocytosis y in vitro study (Guri-Fkim, 2006). It is lso useful to understnd how niml cells rect to hers nd their extrcts. Gupt nd Blsurhmnym (1998) reported n in vitro nlysis of the effect of turmeric on endothelil cells from the humn umilicl vein. They found tht cells cultured in medi with turmeric showed prolifertive response.

8 Ksetsrt J. (Nt. Sci.) 44(5) 857 CONCLUSION The results from the current study showed tht the ppliction of turmeric extrct t 25 mg/kg feed for two weeks, nd one month feeding of Litopeneus vnnmei Boone showed positive effects on: resistnce to viriosis; immune functions; nd ctericidl ctivity in shrimp intestines. Thus, the extrct of this her cn e used s n effective immunoenhncement tht cn e pplied for the prevention of viriosis in mrine shrimp. The extrct of this her lso showed n effective ctericidl effect in shrimp intestines. Cution will e required in the extrction process nd in determining the most effective mount of the ctive components in the turmeric extrct. ACKNOWLEDGEMENTS This work ws supported y the Ksetsrt University Reserch nd Development Institute (KURDI). The uthors re grteful to the Nturl Products nd Orgnic Synthesis Reserch Unit (NPOS), Deprtment of Chemistry, Fculty of Science, Ksetsrt University for support with the HPLC nlysis. LITERATURE CITED Bhvnishnkr, T.N. nd S. Murthy Curcumin-induced ltertion in the glucose metolism of Escherichi coil. J. Gen. Appl. Microiol. 32(4): Buchnn, R.E. nd N.E. Gions The Shorten Bergey s Mnul of Determintion Bcteriology. 8 th ed. The Willims & Wilkins Co. Bltimore. Dey, R.K. nd S. Chndr Preliminry studies to rise disese resistnt seed (fry) of Indin mjor crp, Ctl ctl (Hm.) through herl tretment of spwn. Fish. Chimes Duncn, D.W Multiple-rnge nd multiple F-test. Biometrics. 11: Guri-Fkim, A Medicinl plnts: Trditions of yesterdy nd drugs of tomorrow. Mol. Aspects Med. 27: Gupt, R.K. nd L. Blsurhmnym The turmeric effect. World Ptent Informtion 20: Hsu, Si-Wei nd J.C. Chen The immune response of white shrimp Peneus vnnmei nd its susceptiility to Virio lginolyticus under sulfide stress. Aquculture 271: Kongkthip, N. nd B. Kongkthip Extrction of Her. Nturl Products nd Orgnic Synthesis Reserch Unit (NPOS), Deprtment of Chemistry, Fculty of Science, Ksetsrt University, Bngkok. Lvill-Pitogo, C.R., E.M. Leno nd M.G. Pner Mortlities of pond-cultured juvenile shrimp, Peneus monodon, ssocited with dominnce of luminescent viriosis in the rering environment. Aquculture 164: Lowry, O.H., N.J. Roserough, A.L. Frr nd R.J. Rndell Protein mesurement with the folin phenol regent. J. Biol. Chem. 193: Lutomoski, J., B. Kezi nd W. Desk Effect of lcohol extrct nd ctive ingredient from Curcum long on cteri nd fungi. Plnt Med. 26(1): Mes, M.D., M.D. Rmirez-Fortos, C.M. Aguiler nd A. Gill Nutritionl nd phrmcologicl effect of Curcum long L. extrcts. Recent Res. Dev. Nutr. 3: 157. Supmtty, K., U. Ekpnithnpong, Y. Itmi nd J. Ksornchndr The immune system in lck tiger shrimp, Peneus monodon Fricius: I. Techniques on immunologicl ssessment nd lood component in lck tiger shrimp, Peneus monodon Fricius. Songklnkrin J. Sci. Technol. 22 (Suppl.): Supmtty, K., N. Suntornchreonnon, M.

9 858 Ksetsrt J. (Nt. Sci.) 44(5) Boonyrtplin nd J. Rungsri Effect of three Thi medicinl plnts on growth performnce, immune functions nd disese resistnce in lck tiger shrimp (Peneus monodon Fricius), pp In Proceedings of the JSPS-NRCT Interntionl Symposium, Mngement of Food Sfety in Aquculture nd HACCP Dec Ksetsrt University, Thilnd. Supmtty, K., K. Suntornchreonnon, M. Boonyrtplin, J. Rungsri, T. Tttnon nd T. Klowkling Effects of Thi medicl plnts on pthogenic cteril, growth performnce, helth condition nd disese resistnce in lck tiger shrimp (Peneus monodon Fricius). Songklnkrin J. Sci. Technol. 27 (Suppl. 1): Vnichkul, K., N. Areechon nd N. Kongkthip Appliction of turmeric (Curcum long Linn.) extrct in lck tiger shrimp (Peneus monodon Fricius) culture, pp In Proceedings of 45 th Ksetsrt University Annul Conference, Self-sufficient griculture : Projects initited y His Mjesty the King, 30 Jn- 2 Fe Ksetsrt University, Bngkok.