)JOEBXJ1VCMJTIJOH$PSQPSBUJPO …downloads.hindawi.com/journals/jir/2015/256510.pdf · lysozyme,...

Research ArticleChlorophytum borivilianum Polysaccharide FractionProvokes the Immune Function and Disease Resistance ofLabeo rohita against Aeromonas hydrophila

Sib Sankar Giri1 Shib Sankar Sen2 Cheng Chi1 Hyoun Joong Kim1 Saekil Yun1

Se Chang Park1 and V Sukumaran3

1Laboratory of Aquatic Biomedicine College of Veterinary Medicine and Research Institute for Veterinary ScienceSeoul National University Seoul 151742 Republic of Korea2School of Life Sciences Jawaharlal Nehru University New Delhi 110067 India3Department of Biotechnology Periyar Maniammai University Thanjavur Tamil Nadu 613403 India

Correspondence should be addressed to Se Chang Park parksecsnuackr and V Sukumaran drvsukumargmailcom

Received 7 May 2015 Revised 16 September 2015 Accepted 29 October 2015

Academic Editor Clelia M Riera

Copyright copy 2015 Sib Sankar Giri et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The present study aimed to investigate the effects of Chlorophytum borivilianum polysaccharide (CBP) as a dietary supplementadministered at varying concentrations with feed (basal diet) on various cytokine-related responses in Labeo rohita fingerlingsImmune parameters and immune-related gene expressions were measured at 3rd 4th and 5th week after feeding The resultsrevealed that dietary administration of CBP at 02 and 04 for 4 weeks significantly upregulated serum lysozyme and phagocyticactivity Complement C3 and respiratory burst activity (RBA) were significantly higher after 4 weeks of CBP feeding The immunerelated genes IL-8 IL-1120573 TNF-120572 and iNOS were downregulated (119875 lt 005) in groups with 02 and 04 CBP supplemented dietsat week 4 Expression of anti-inflammatory cytokines (IL-10 and TGF-120573) was also downregulated (119875 lt 05) after 4 weeks of feedingwith 02 to 08 CBP However five weeks of CBP administration had no significant effect on immune gene expression exceptTNF-120572 and IL-8 Fish fed with 04 CBP for 4 weeks showed maximum resistance against Aeromonas hydrophila (733 survival)compared to control From these results we recommend that CBP administration at 04 for 4 weeks could effectively improveimmune response and disease resistance in L rohita

1 Introduction

Indian aquaculture production consists mainly (sim70) ofthree major carps Labeo rohita Catla catla and Cirrhi-nus mrigala [1] Intense culture practices generate potentialenvironmental stressors that lead to high susceptibility ofcaptive fish species to various diseases including viralbacterial fungal and parasitic infections Moreover com-mercial aquaculture has been hampered by economic lossas a result of infectious diseases [2] Typically antibioticsvaccines and chemotherapeutics are used for disease controlin aquaculture However treatment with antibiotics leads tothe development of drug resistant pathogens environmentalhazards and food safety problems [3] Further single vaccineapplication is only effective against one type of pathogen

and the vaccination of juvenile fish is labor intensive andexpensive [4]Therefore it is imperative to develop natural oreco-friendly therapeutics to ensure the sustainability of aqua-culture Since fish depend primarily on innate immune func-tion rather than on specific immunity immunostimulantsplay a major role in disease resistance by enhancing innateimmunity [5] Additionally several physiological parameterssuch as respiratory burst activity (RBA) nitric oxide synthaselysozyme bactericidal activity and antibody response serveas good immunological indicators in fish

Cytokines protein mediators produced by immune cellsare responsible for host innate defense mechanisms Sev-eral cytokine and immune-related genes have been identi-fied and characterized in fish [2 6ndash8] Interleukins (ILs)tumor necrosis factors (TNFs) transforming growth factor

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2015 Article ID 256510 10 pageshttpdxdoiorg1011552015256510

2 Journal of Immunology Research

(TGF) chemokines and interferons (IFNs) are types ofcytokines which require cytokine receptors These cytokinesare reported to have proinflammatory anti-inflammatoryand pathogen-killing activities [9] Hence understandingdifferential cytokine expression can enable the developmentof immunostimulants for aquaculture [10] Further obtainedinformation has the added advantage of identifying potentialindirect immunological markers for aquatic species

Several studies have investigated the enhancement ofgrowth immunity and disease resistance in fish throughdietary administration of immunostimulants Stimulationof fish immunity through dietary immunostimulants is ofhigh interest for commercial aquaculture [8] Several herbalpreparations have been reported to enhance the immuneresponses in fish [2 4 7 11ndash15] For example Rehmanniaglutinosa root products enhanced the growth and immuneparameters ofCyprinus carpio [2] Seed ofAchyranthes asperaenhanced the immune responses inCatla catla [7]The root ofWithania somnifera improved the disease resistance of Labeorohita against Aeromonas hydrophila [13] Recently we foundthatPsidium guajava leaves as feed additive could increase thegrowth performance disease resistance and cytokine geneexpression of Labeo rohita [14] Herbal therapeutic productsare less expensive and have greater accuracy compared withchemotherapeutic agents providing a potential solution forthe problems facing aquaculture today [15]

Chlorophytum borivilianum L (Liliaceae) is a traditionalrare medicinal herb in India and considered ldquowhite goldrdquoor ldquodivya aushadrdquo in Indian systems of medicine Its roottubers are widely used for various therapeutic applica-tions The traditional uses of C borivilianum tubers againstdiseases such as diabetes mellitus dysuria diarrhea anddysentery are well documented in the literature [16 17]Several studies have reported that C borivilianum possessesvarious pharmacological properties such as antimicrobial[18] anti-inflammatory [19] antioxidant [20 21] antistress[22] antidiabetic [23] immunomodulatory [16 24] antiulcer[25] and anticancer effects [26] Major phytochemical con-stituents isolated from the roots of C borivilianum includesteroidal saponins fructans and fructooligosaccharides phe-nolic compounds acetylated mannans and proteins [16 1821 22] Polysaccharide fractions derived from the root tubersof C borivilianum have been shown to exhibit modulation ofhuman natural killer (NK) cell activity humoral response tosheep red blood cells (SRBCs) and enhanced immunoglob-ulin G levels in rats [16] Moreover polysaccharides fromnatural resources are a class of macromolecules that canefficiently affect the immune system and therefore havemuchimportance in basic and applied research [6] Hence wehypothesize that polysaccharide fractions of C borivilianummay boost the immune response of teleosts However veryfew studies have investigated the medicinal properties of Cborivilianum

To date no investigation has been conducted to exploitthe potential efficacy of C borivilianum as a feed additive inmodulating fish growth and immune response The presentstudywas designed to investigate the effects of dietary supple-mentation of a polysaccharide extracted fromC borivilianumroot on the growth performance immune parameters and

expression of several immune-related genes in L rohitaFurther the efficacy of this polysaccharide in the diseaseresistance of L rohitawas also investigated through challengestudy

2 Materials and Methods

21 Diet Preparation Dried roots of Chlorophytum boriv-ilianum were collected from a safed musli farm Indore(Madhya Pradesh) India The roots were dried in sunlightand coarsely powdered for extraction The powdered rootswere subjected to hot water extraction following the methoddescribed by Thakur et al [16] The purified polysaccharidefraction was lyophilized and stored at minus20∘C for further use

A basal diet comprising 39 groundnut oil cake 34rice bran 20 soybean meal 5 fish meal and 2 mineraland vitaminmixture (every 250 g ofmineral-vitaminmixtureprovided vitamin A 500000 IU vitamin D3 100000 IUvitamin B2 02 g vitamin E 75 units vitamin K 01 gcalcium pantothenate 025 g nicotinamide 01 g vitaminB12 06mg choline chloride 15 g calcium 75 g manganese275 g iodine 01 g iron 075 g zinc 15 g copper 02 gand cobalt 0045 g) was prepared [28] Proximate analysisof basal diet performed according to AOAC method [29]revealed 373 protein 86 lipid and 121 ash The basaldiet was considered as control diet The C borivilianum rootpolysaccharide (CBP)was supplemented into basal diet at fivelevels 0 (basal diet) 01 (D1) 02 (D2) 04 (D3) and08 (D4) All the ingredients were blended thoroughly in amixture pelletized air-dried ground and sieved into properpellet size All feeds were stored at 4∘C until use

22 Cytotoxicity of CBP Rat macrophage cells (ATCC CRL-2192) were routinely cultured in DMEM (Dulbeccorsquos Mod-ified Eagle Medium) supplemented with 10 horse serum(HS) penicillin G (500 UmL) streptomycin (5000 120583gmL)and 35mgmL of D-glucose at 37∘C and 5 CO

2in a

humidified incubator (Sanyo Japan) To maintain exponen-tial growth cells were seeded at 1 times 105 cellsmL and pas-saged every 5-6 days Chlorambucil (Sigma-Aldrich USA) atconcentration 600120583gmL was used as positive control [16]Cell viability was assessed using ATPlite kit (Perkin ElmerUSA) CBP was assayed at various concentrations starting at10mgmL Test was performed in triplicate For this 50120583Lof CBP sample was added to each well along with 50 120583L ofcell suspensionThe cells and samples were incubated at 37∘Cfor 24 h in a humidified (5 CO

2) incubator (Sanyo Japan)

Measurement of cell proliferationwas performedusingMicroBeta 1450 plate reader (Perkin Elmer USA) following ATPlitekit instructions

23 Test Fish and Experimental Protocol Labeo rohita fin-gerlings (mean bodyweight 103 plusmn 007 g) were acclimatisedto laboratory conditions for 2 weeks in 500-litre plasticquarantine tanks at 26 plusmn 2∘C Fish were fed with basal dietduring the acclimatisation period About 20 of the waterin all tanks was exchanged daily and 100 of the water wasexchanged once a week Basic physiochemical parameters

Journal of Immunology Research 3

of the water were measured every week [30] The O2and

ammonia concentrations were ranged from 61 to 73mg Lminus1and 003 to 006 ppm respectively and pH was ranged from70 to 80 throughout the study period

Fish were randomly stocked in fifteen tanks with astocking density of 30 fish per tankwith triplicates per dietarytreatment Capacity of each tank was 200 litres All the fishwere fed with one of the five diets (basal diet D1 D2 D3 andD4) for 5 weeks at the rate 2ndash4 of body weight and fed twicea day (900 and 1700) Any uneaten feed leftwas removed afterone hour of feeding anddriedweighed andfinally subtractedfrom the total amount of supplied diets to calculate the actualfeed intake The water quality was checked regularly

24 Growth Performance Ten fish were randomly collectedfrom each tank at end of 3rd 4th and 5th week of feedingfor batch weighing Growth performances and survival rateof fish were calculated using the following formula

Weight gain (WG gfish) = 119882119905minus1198820

Specific growth rate (SGR)

= 100 times [(ln119882119905minus ln119882

0)

119905]

Feed conversion ratio (FCR) = FI(119882119905minus1198820)

Survival () = 100 times ( final no fishinitial no of fish

)

(1)

where 119882119905and 119882

0were final and initial weight of fish

respectively ldquo119905rdquo is the duration of feeding (in days) FI is feedintake

25 Immune Parameters Five fish were randomly collectedfrom each tank at end of 3rd 4th and 5th week of feeding tomeasure the immune parametersThus a total of 15 fish (3 times 5= 15) were collected in each group for immunological assaysBlood samples were collected by caudal venipuncture using a1 mL syringe after anesthetizing the fish with diluted MS222(Sigma-Aldrich USA) The blood samples were transferredinto microcentrifuge tubes After collection blood was cen-trifuged at 3500 g at 4∘C for 10min and obtained serum wasstored at minus20∘C

251 Serum Lysozyme Activity Lysozyme activity (LA) wasmeasured according to the method described by Ellis[31] One unit of lysozyme activity was defined as theamount of enzyme producing a decrease in absorbance of0001minminus1mLminus1 serum

252 Complement C3 Assay The serum complement C3level was assayed using a C3 kit (Biocompare CA USA)C3 level analysis included measurement of the increase inturbidity after immunity response of C3 and its increasedantibody [32] Results of C3 activity were expressed asC3mgmLminus1

253 Phagocytic Assay Aeromonas hydrophila MTCC-1739was cultured in tryptic soy agar medium (Sigma-Aldrich)for 24 h at 37∘C The cell number was adjusted to 5 times106 CFUmL The phagocytic activity of the blood leukocytewas determined according to themethod ofCai et al [33]Thephagocytic activity () was calculated as using the formula

[100 times (phagocytic leucocytes) (total leucocytes)minus1] (2)

254 Respiratory Burst Activity (RBA) The RBA of phago-cytes was measured using the nitroblue tetrazolium (NBTSigma-Aldrich) assay following themethod of Secombes [34]with previously described modifications [35] Color devel-opment was measured at 630 nm with a spectrophotometer(Bio-Rad India)

26 Expressions of Immune-Related Genes Head kidney wascollected from 15 fish per group frozen in liquid nitrogen andstored at minus80∘C Total RNA was extracted from head kidneyusing TRIZOL (Invitrogen USA) according to the manu-facturerrsquos instructions RNA concentration and purity werequantified using a spectrophotometer (NanoDrop 2000cThermo scientific) and the quality was checked using a1 agarose gel containing 05120583gmLminus1 ethidium bromideRNA was reverse-transcribed to cDNA using a SuperScriptcDNA synthesis kit (Life Technologies USA) following themanufacturerrsquos instructions Real-time PCR analysis of IL-8IL-1120573 IL-10 iNOS TNF-120572 and TGF-120573 and a housekeepinggene (120573-actin) was carried out with CFX96 Real-Time PCR(Bio-Rad Laboratories Inc) following standard protocolswith the primer sequences and thermocycling conditionsindicated in Table 1 To verify the accuracy of each ampliconmelt curve analysis was performed after amplification Allsamples were run in parallel with the housekeeping gene inorder to normalize cDNA loading Gene expression resultswere analyzed using the 2minusΔΔCT method after verifying theprimers amplified with an efficiency of approximately 100[36] and data for all treatment groups were compared tothose of the control groups

27 Challenge Test The seven-day lethal dose 50 (LD50) for

A hydrophila MTCC-1739 was 107 CFUmL as determinedearlier in our laboratory [28] At the end of the feedingtrial ten fish from each tank (3 times 10 = 30 fish per group)were picked out for challenge test The fish were injected ipwith 100 120583L of phosphate buffer saline (PBS) containing 1 times107 live A hydrophila The challenged fish were kept underobservation for 2 weeks and fed a basal diet The mortality offish in each tank was observed over the course of 14 days

28 Statistical Analysis One way analysis of variance(ANOVA) was used to analyze the data Multiplecomparisons were performed with Tukeyrsquos test to analyzethe differences between treatments All statistical analyseswere performed using the OriginPro software (version 8OriginLab Corporation Northampton USA) The level ofsignificance was set at 119875 lt 005 and the results are expressedas mean plusmn SEM


Table 1 Real-time primer sequences and thermocycling conditions

Target gene Primer sequence (51015840 to 31015840) Thermocycling conditions Referenceaccession number

IL-1120573 ATCTTGGAGAATGTGATCGAAGAGGATACGTTTTTGATCCTCAAGTGTGAAG

95∘C 30 s 40 cycles of 95∘C 5 s 615∘C30 s and 72∘C 30 s AM932525

IL-10 AAGGAGGCCAGTGGCTCTGTCCTGAAGAAGAGGCTCTGT

95∘C 30 s 40 cycles of 95∘C 5 s 611∘C30 s and 72∘C 30 s AB010701

iNOS GGAGGTACGTCTGCGAGGAGGCTCCAGCGCTGCAAACCTATCATCCA


TNF120572 CCAGGCTTTCACTTCACGGCCATAGGAATCGGAGTAG

95∘C 30 s 40 cycles of 95∘C 5 s 611∘C30 s and 72∘C 30 s FN543477

TGF-120573 ACGCTTTATTCCCAACCAAAGAAATCCTTGCTCTGCCTCA

95∘C 30 s 40 cycles of 95∘C 5 s 605∘C30 s and 72∘C 30 s AF136947

IL-8 GGGTGTAGATCCACGCTGTAGGGTGCAGTAGGGTCCA

95∘C 30 s 40 cycles of 95∘C 5 s 605∘C30 s and 72∘C 30 s Self-design

120573-actin AGACCACCTTCAACTCCATCATGTCCGATCCAGACAGAGTATTTACGC

95∘C 30 s 40 cycles of 95∘C 5 s 605∘C30 s and 72∘C 30 s [27]

3 Results

31 Cytotoxicity Evaluation Evaluation of cytotoxicity anddetermination of IC

50was carried out on mouse macrophage

cell line The IC50

values for chlorambucil and CBPwere 3262 120583gmL and 89117 120583gmL respectively This highIC50value of CBP confirmed its nontoxic nature

32 Growth Performance Dietary administration of CBP tocaptive carp increased growth parameters such as percentweight gain (PWG) and specific growth rate (SGR) althoughthe increment was not significant at any point in time (datanot shown) However a significant reduction in FCR valuewas observed after 4 weeks of feeding with 04 CBP (groupD3) No mortality was recorded in any group during theexperimental feeding

33 Immunological Parameters Results of the immunologi-cal parameters are shown in Figure 1 Serum lysozyme activity(Figure 1(a)) showed maximum peak at 4 weeks of feedingregime Fish fed 02ndash08 CBP supplemented diets for 4weeks had significantly higher lysozyme activity and thehighest was in the group (D3) fed 04 CBP CBP had nosignificant effect of lysozyme activity at 5 weeks of feedingexcept in the group fed 02 CBP

In the case of complement C3 activity a significantincrement was observed in dietary supplementation of 02ndash08 CBP for 4 weeks and the highest C3 activity wasrecorded in the group fed 04 CBP (Figure 1(b)) Howeverdietary CBP administration for 5 weeks had no significanteffect on C3 activity except in the group fed 02 CBP

CBP administration for 3 weeks had no significant effecton phagocytic activity (PA) (Figure 1(c)) and respiratorybursts activity (RBA) (Figure 1(d)) in rohu However CBPadministration at 02ndash08 (D2ndashD4) for 4 weeks enhancedPA and RBA compared to the control (119875 lt 005) and highestactivitieswere observed in the group (D3) fed 04CBPAfter

5weeks ofCBP feeding significantly higher PA andRBAwereobserved in groups D2 and D1 respectively

34 Expression of Immune Related Genes The expressionprofiles of six immune-related genes in the head kidney offish were investigated at the end of 3 4 and 5 weeks ofCBP administration The expression levels of four proin-flammatory cytokines (IL-8 IL-1120573 iNOS and TNF-120572) wereupregulated after 4 weeks of feeding (Figure 2) whereas twoanti-inflammatory cytokines (IL-10 and TGF-120573) were down-regulated after four weeks of feeding with CBP (Figure 3)

The IL-1120573 expression was upregulated at 04 and 08CBP administration for 3 weeks (119875 lt 005) while after 4weeks of CBP supplementation higher expression (119875 lt 005)was observed in groups administered 02 and 04 CBP(Figure 2(a)) TNF-120572 expression was higher (119875 lt 005) inthe group fed 08CBP after 3 weeks (Figure 2(b)) Howevermaximum (119875 lt 005) TNF-120572 expression was achieved ingroups administered 02 and 04CBP groups after 4weeksof feeding Administration of 02ndash08 CBP for 4 weeksprovoked (119875 lt 005) IL-8 expression in the head kidney ofrohu and this trend continued until the end of the trial inthe groups administered 02 and 04 CBP (Figure 2(c))Dietary administration of CBP upregulated iNOS expression(119875 lt 005) at 4 weeks of feeding compared to the control(Figure 2(d)) and the highest expression levels were in thegroup (D3) administered 04 CBP After 5 weeks onlythe group administered 01 CBP exhibited a higher iNOSexpression level In general prolonged supplementation ofCBP attenuated the expected levels of IL-1120573 TNF-120572 andiNOS expression

Effects of CBP on IL-10 and TGF-120573mRNA expression areshown in Figure 3 Dietary administration of CBP attenuatedthe expected rate of expression of IL-10 and significant atten-uation was observed at 4 weeks of 02ndash08 CBP administra-tion (Figure 3(a)) However 5 weeks of CBP supplementationhad no significant effect on IL-10 and TGF-120573 transcription Asignificant attenuation of TGF-120573 expression was observed in


05

101520253035404550

3 4 5Assay schedule (weeks)

CD1D2

D3D4

Lyso

zym

e act

ivity

(U m

Lminus1)

lowast

lowast lowastlowast

lowast

(a)

0

02

04

06

08


lowast

lowast lowast

lowast lowast

Com

plem

ent C

3(m

g m

Lminus1)

CD1D2

D3D4

(b)

0

5

10

15

20

25

30

35

40

3 4 5

PA (

)

Assay schedule (weeks)

lowast

lowast lowastlowast

lowast

CD1D2

D3D4

(c)

0

02

04


RBA

(OD630)

lowastlowast

CD1D2

D3D4

(d)

Figure 1 Serum lysozyme activity complement C3 phagocytic activity (PA) and respiratory burst activity (RBA) of Labeo rohita (119899 = 15) atthe end of weeks 3 4 and 5 of feeding with a graded level of CBP Bars represent the mean plusmn SEM and asterisks represent levels of significantdifferences compared to the control (119875 lt 005) Note C (basal diet) D1 (basal diet + 01 CBP) D2 (basal diet + 02 CBP) D3 (basal diet +03 CBP) D4 (basal diet + 04 CBP)

the group fed 04 and 08 CBP at 3 weeks of feeding but4 weeks of CBP administration attenuated (119875 lt 005) TGF-120573expression (Figure 3(b)) in all experimental groups

35 Challenge Test Results of the challenge study revealedthat fish fed 04CBP (groupD3) exhibited highest postchal-lenge survival (733) followed by 02 CBP (group D2) fedgroup (60 survival) and 08 CBP (group D4) supplemen-tation (533 survival) (Figure 4) Fish on the control dietwithout CBP exhibited the lowest postchallenge survival rate(266)

4 Discussion

Chlorophytum borivilianum is renowned for its ability toboost immune function [24] In the present study we report

the effects of C borivilianum polysaccharide (CBP) onimmune response and disease resistance of rohu (L rohita)We found that CBP had no significant effect on growthperformance but administration of 04 CBP resulted inlower (119875 lt 005) FCR The significant reduction in FCRsuggests that the fish utilize nutrients more efficiently whentheir diet is supplemented with CBP

Lysozyme present in the mucus plasma and other bodyfluids of most fish species plays an important role in medi-ating protection against microbial invasion [37] Our studyshowed that fish fed with 02ndash08 CBP supplemented dietsfor 4 weeks had significantly higher serum lysozyme activityOur results are in agreement with earlier investigations whichreport the increment of serum lysozyme activity throughdietary administration of Ficus carica polysaccharide [8] andAstragalus polysaccharides [6] in grass carp and common


0

05

1

15

2

25


IL-1120573

expr

essio

n re

lativ

e to120573

-act

in lowastlowast

lowastlowast

CD1D2

D3D4

(a)

0

05

1

15

2

25

3


TNF-120572

expr

essio

n re

lativ

e to120573

-act

in

lowast

lowastlowast

lowast

CD1D2

D3D4

(b)

0

05

1

15

2

25


IL-8

expr

essio

n re

lativ

e to120573

-act

in

lowastlowastlowast

lowastlowast

CD1D2

D3D4

(c)

0

05

1

15

2

25


iNO

S ex

pres

sion

relat

ive t

o 120573

-act

inlowast

lowast

lowastlowast

CD1D2

D3D4

(d)

Figure 2 The relative mRNA expression of IL8 IL-1120573 iNOS and TNF-120572 in the head kidney of Labeo rohita at the end of weeks 3 4 and 5of feeding with graded level of CBP Bars represent the mean plusmn SEM and asterisks represent levels of significant differences compared to thecontrol (119875 lt 005) Note C (basal diet) D1 (basal diet + 01 CBP) D2 (basal diet + 02 CBP) D3 (basal diet + 03 CBP) D4 (basal diet +04 CBP)

carp respectively We observed that higher levels (04ndash08) of CBP supplementation for 5 weeks had no significanteffect on LA in the present study This is likely becausehigher doses of immunostimulants for longer durations leadto immunosuppression [11]

The complement system is an important component ofthe innate immune response and plays a critical role inalerting as well as in clearing potential pathogens in the host[38] In the present study CBP supplementation significantlystimulated the complement C3 level up to 4 weeks andthereafter it declined Our results are in agreement with theresults of Yang et al [8] Complement activation is usuallybeneficial to fish but constant activation could cause sideeffects and immune suppression to the host [39] Henceenhancement of serum complement C3 in CBP fed groupsup to 4 weeks after administration may be beneficial to fish

Phagocytosis is responsible for early activation of theinflammatory response before antibody production [40]Phagocytosis-associated respiratory burst activity (RBA) isconsidered to be a vital indicator of nonspecific defensein fish [41] Respiratory bursts are produced by phagocytesin order to attack invasive pathogens during phagocytosisand it is being used to evaluate the defense ability againstpathogens [41] In this study enhanced PA and RBA (119875 lt005) were observed in groups D2 D3 and D4 (administered02ndash08 CBP) after 4 weeks and highest was in D3 (ie04 CBP) group Similarly dietary administration of guavaleaves enhanced (119875 lt 005) the phagocytic activity of bloodleucocytes in L rohita [14] In Cyprinus carpio and Larim-ichthys crocea RBA of phagocytic cells has been significantlyenhanced after feeding with a mixture of two herbal extracts[41] Dietary A aspera seeds enhanced RBA in carp Catla


0

05

1

15

2

25


IL-10

expr

essio

n re

lativ

e to120573

-act

in

CD1D2

D3D4

lowastlowast

lowast

(a)

002040608

112141618

2


TGF-120573

expr

essio

n re

lativ

e to120573

-act

in

lowastlowastlowast

lowast

CD1D2

D3D4

(b)

Figure 3 The relative mRNA expression of IL-10 and TGF-120573 in the head kidney of Labeo rohita at the end of weeks 3 4 and 5 afteradministration of varying concentrations CBP Bars represent themean plusmn SEM (119899 = 15) and asterisks represent levels of significant differencescompared to the control (119875 lt 005) Note C (basal diet) D1 (basal diet + 01 CBP) D2 (basal diet + 02 CBP) D3 (basal diet + 03 CBP)D4 (basal diet + 04 CBP)

266

100

366

60733

53

0

20

40

60

80

100

120

Control NC D1 D2 D3 D4

Postc

halle

nge s

urvi

val (

)

CBP supplementation level

lowast

lowast

lowast

lowast

lowast

Figure 4 Effect of dietary CBP administration on the survival of Labeo rohita (119899 = 30) after challenge with A hydrophila Postchallengesurvival (percent) in each dietary group is indicated at the top of each column Bars represent the mean plusmn SEM and asterisks represent levelsof significant differences compared to the control (119875 lt 005) Note NC (negative control) C (basal diet) D1 (basal diet + 01 CBP) D2(basal diet + 02 CBP) D3 (basal diet + 03 CBP) D4 (basal diet + 04 CBP)

catla [7]The increased activities of serum lysozyme comple-ment C3 PA and RBA in the present study reveal that dietarysupplementation of CBP has a significant role in enhancingthe immune response in rohu However increased levels ofthis dietary supplementation over longer periods decreasedlysozyme and complement C3 RBA and phagocytic activ-ities suggesting that higher levels of CBP supplementationmay depress fish immunity This is most likely becausehigher doses of immunostimulants administered over longerperiods often lead to immunosuppression [14]

Beside the enhancement of immune responses in rohuour study also provides evidence that CBP can effectivelyregulate the expressions of certain cytokine-related genessuch as Il-8 IL-1120573 TNF-120572 iNOS IL-10 and TGF-120573 To thebest our knowledge this is the first report about the effect

of CBP on cytokine gene expressions in fish Therefore thepresent results of the gene expression study are comparedwith earlier studies reported about the effect of herbalpreparations on cytokine gene expressions in fish

IL-1120573 is a proinflammatory cytokine that affects almostevery cell type in concert with another proinflammatorycytokine TNF [42] Both IL-1120573 and TNF-120572 are cytokines thatinduce an inflammatory response IL-1120573 plays an importantrole in host response to microbial invasion tissue injury andimmunological reactions while TNF-120572 is crucial for diversecellular responses like cell proliferation differentiation andinduction of other cytokines [43] In the present study fishgroups fed with 02 and 04 CBP supplemented dietsshowed significantly higher IL-8 IL-1120573 and TNF-120572 expres-sion than the control however their expression abundance


exhibited differences in the same organ of fish fed differentlevels of CBP Many studies have demonstrated that herbalimmunostimulants can induce proinflammatory cytokinesGrass carp fed with F carica polysaccharide for three weeksexhibited strong upregulation of IL-1120573 and TNF-120572 expression[8] The oral administration of Spirulina platensis augmentedthe expression of IL-1120573 and TNF-120572 genes in common carp[44]

Production of iNOS is a well-known immunoregulatoryfactor in fish challenged with various pathogens [45] Wefound that dietary administration of CBP for 4-week changes(119875 lt 005) iNOS transcription in rohu Transcription ofiNOS was upregulated in the head kidney of common carpfollowing Rehmannia glutinosa supplementation for 80 days[2] Recently we demonstrated that dietary administration ofguava leaves at 05ndash15 attenuated the expected expressionof iNOS in head kidney intestine andhepatopancreas of rohu[14] which is in contrast to the result of the present studyHowever the upregulation of IL-8 IL-1120573 and iNOS geneexpression in the present study may increase production ofreactive-nitrogen intermediates that can damage pathogens[46]

IL-10 is a multifunctional cytokine with immunosup-pressive and cytokine synthesis inhibitory functions [47]The prime function of IL-10 is to counteract proinflamma-tory cytokines such as IL-1120573 and TNF-120572 to prevent tissuedamage [48] Another cytokine TGF-120573 generally inhibitsB and T cell proliferation and differentiation antagonizesproinflammatory cytokines such as IL-1120573 TNF-120572 and IFN-120574and suppresses the expression of IL-1120573 and IL-2 receptors Italso directly targets effector T cells and Treg cells to ensureself-tolerance [49] In the present study IL-10 and TGF-120573expression was downregulated in the head kidney of rohufed 02minus08 CBP for 4 weeks Similarly IL-10 expressionwas downregulated in the kidney of Catla catla fed a 10A aspera supplemented diet [7] Common carp fed a dietsupplemented with R glutinosa had lower IL-10 and TGF-120573 expression levels in the kidney spleen and intestine [2]Oral administration of spirulina in common carp inducesa decrease in IL-10 gene expression which is similar to thedecrease that we observed [44] Contrary to our resultsZhang et al [50] reported that bath delivery of immunos-timulants can induce higher expression of TGF-120573 in rainbowtrout fry In a recent study we found that rohu fed dietsupplemented with guava leaves exhibited downregulation inIL-10 and TGF-120573 gene transcription [14] Therefore we sug-gest that downregulation of the anti-inflammatory cytokinesIL-10 and TGF-120573 may favor the enhanced expression ofproinflammatory cytokines in CBP-fed fish

After challenge with A hydrophila the highest postchal-lenge survival rate (733) was exhibited by the fish groupfed a 04 CBP supplemented diet while the second-highest survival (633) was exhibited by the group supple-mented with 02 CBP The enhanced immune parameters(egLA ACP PA andRBA) stimulation in proinflammatorycytokines and declines in anti-inflammatory cytokines inrohu administered 04 CBP might be associated withimproved resistance against A hydrophila and the resultinghigher postchallenge survival percentages Further the group

administered 08 CBP did not exhibit the highest postchal-lenge survival and this may be associated with the results ofimmune responses and immune gene expression RecentlyWang et al [2] reported that dietary administration of Rglutinosa enhanced the postchallenge survival of commoncarp against A hydrophila infection Oral administration ofazadirachtin increased the resistance of gold fish Carassiusauratus against A hydrophila infection [15] Grass carp fedwith F carcica polysaccharide had remarkably higher resis-tance against Flavobacterium columnare [8] However theeffect of C borivilianum polysaccharide oral administrationshould be investigated further to elucidate the molecularmechanism by which disease resistance is conferred

5 Conclusions

This study has provided the first evidence for how C borivil-ianum provokes immune responses in fish The administra-tion of CBP influences both innate and humoral immunity inrohu and provokes the expression of immune related genesAmong the different dietary level of CBP administration at04 (D3) for 4 weeks efficiently upregulated (119875 lt 005)the immune parameters and expressions of proinflammatorycytokines (IL-8 IL-1120573 TNF-120572 and iNOS) anddownregulatedthe expressions anti-inflammatory cytokines (TGF-120573 andIL-10 gene) As CBP administration at 04 was shown toprovide extraordinary protection in fish against pathogenchallenge CBP at 04 for four weeks can be used as a feedadditive in carp breeding to improve immunity and diseaseresistance However further studies must be carried out toexplore the active compounds in CBP and their detailedmechanisms in stimulating immunity

Conflict of Interests

Authors declare that there is no conflict of interests regardingthe publication of this paper

Authorsrsquo Contribution

Sib Sankar Giri and Shib Sankar Sen have equal contribution

Acknowledgments

Sib Sankar Giri is a recipient of BK21 PLUS postdoctoralfellowship Shib Sankar Sen acknowledges DSKPDF UGCGoI Se Chang Park gratefully acknowledges the researchgrant received from National Research Foundation of KoreaMinistry of Education (NRF-2014R1A2A1A11050093)

References

[1] FAOThe State of World Fisheries and Aquaculture FAO RomeItaly 2012

[2] J-L Wang X-L Meng R-H Lu et al ldquoEffects of Rehmanniaglutinosa on growth performance immunological parametersand disease resistance to Aeromonas hydrophila in commoncarp (Cyprinus carpio L)rdquo Aquaculture vol 435 pp 293ndash3002015


[3] B Austin and D A Austin Bacterial Fish Pathogens Diseasesof Farmed and Wild Fish Springer-Praxis Chichester UK 4thedition 2007

[4] F S Zanuzzo E C Urbinati M L Rise J R Hall G W Nashand A K Gamperl ldquoAeromonas salmonicida induced immunegene expression in aloe vera fed steelhead trout Oncorhynchusmykiss (Walbaum)rdquo Aquaculture vol 435 pp 1ndash9 2015

[5] M Sakai ldquoCurrent research status of fish immunostimulantsrdquoAquaculture vol 172 no 1-2 pp 63ndash92 1999

[6] C Yuan X Pan Y Gong et al ldquoEffects of Astragalus polysac-charides (APS) on the expression of immune response genesin head kidney gill and spleen of the common carp Cyprinuscarpio Lrdquo International Immunopharmacology vol 8 no 1 pp51ndash58 2008

[7] R Chakrabarti P K Srivastava N Verma and J SharmaldquoEffect of seeds ofAchyranthes aspera on the immune responsesand expression of some immune-related genes in carp Catlacatlardquo Fish amp Shellfish Immunology vol 41 no 1 pp 64ndash692014

[8] X Yang J L Guo J Y Ye Y X Zhang and W Wang ldquoTheeffects of Ficus carica polysaccharide on immune responseand expression of some immune-related genes in grass carpCtenopharyngodon idellardquo Fish amp Shellfish Immunology vol 42no 1 pp 132ndash137 2015

[9] G Biswas H Korenaga H Takayama T Kono H Shimokawaand M Sakai ldquoCytokine responses in the common carp Cypri-nus carpioL treatedwith bakerrsquos yeast extractrdquoAquaculture vol356-357 pp 169ndash175 2012

[10] P K Sahoo and M Sakai ldquoImmunostimulants in aqua-culturerdquo in Recent Advances in Aquaculture Research GKoumoundouros Ed pp 204ndash229 Transworld Research Net-work Trivandrum India 2010

[11] G Yin L Ardo K D Thompson A Adams Z Jeney andG Jeney ldquoChinese herbs (Astragalus radix and Ganodermalucidum) enhance immune response of carp Cyprinus carpioand protection againstAeromonas hydrophilardquoFish and ShellfishImmunology vol 26 no 1 pp 140ndash145 2009

[12] J Xie B Liu Q Zhou et al ldquoEffects of anthraquinone extractfrom rhubarb Rheum officinale Bail on the crowding stressresponse and growth of common carpCyprinus carpio var JianrdquoAquaculture vol 281 no 1ndash4 pp 5ndash11 2008

[13] A Sharma A D Deo S T Riteshkumar T I Chanu and ADas ldquoEffect of Withania somnifera (L Dunal) root as a feedadditive on immunological parameters and disease resistance toAeromonas hydrophila in Labeo rohita (Hamilton) fingerlingsrdquoFish amp Shellfish Immunology vol 29 no 3 pp 508ndash512 2010

[14] S S Giri S S Sen C Chi et al ldquoEffect of guava leaves onthe growth performance and cytokine gene expression of Labeorohita and its susceptibility to Aeromonas hydrophila infectionrdquoFish amp Shellfish Immunology vol 46 no 2 pp 217ndash224 2015

[15] S Kumar R P Raman P K Pandey S Mohanty A Kumarand K Kumar ldquoEffect of orally administered azadirachtin onnon-specific immune parameters of goldfish Carassius auratus(Linn 1758) and resistance against Aeromonas hydrophilardquo Fishand Shellfish Immunology vol 34 no 2 pp 564ndash573 2013

[16] M Thakur P Connellan M A Deseo C Morris and V KDixit ldquoImmunomodulatory polysaccharide from Chlorophy-tum borivilianum rootsrdquo Evidence-Based Complementary andAlternative Medicine vol 2011 Article ID 598521 7 pages 2011

[17] Z Khanam O Singh R Singh and I U H Bhat ldquoSafed musli(Chlorophytumborivilianum) a review of its botany ethnophar-macology and phytochemistryrdquo Journal of Ethnopharmacologyvol 150 no 2 pp 421ndash441 2013

[18] D S Ghorpade and P V Thakare ldquoPhytochemical screeningand antimicrobial activity of Chlorophytum species leaves ofMelghat regionrdquo International Journal of Pharmacognosy andPhytochemical Research vol 6 no 1 pp 141ndash145 2014

[19] S LDeore and S S Khadabadi ldquoAntiinflammatory and antioxi-dant activity ofChlorophytum borivilianum root extractsrdquoAsianJournal of Chemistry vol 20 no 2 pp 983ndash986 2008

[20] S R Ahmad P Kishan and K Abul ldquoIn vitro antioxidant prop-erties of Chlorophytum borivilianum (Santapau amp Fernandez)rdquoWorld Journal Pharmacy and Pharmaceutical Sciences vol 3 pp937ndash947 2014

[21] M F Ashraf M Abd Aziz J Stanslas I Ismail and M AbdulKadir ldquoAssessment of antioxidant and cytotoxicity activities ofsaponin and crude extracts of Chlorophytum borivilianumrdquoTheScientific World Journal vol 2013 Article ID 216894 7 pages2013

[22] S L Deore and S S Khadabadi ldquoScreening of antistress proper-ties of Chlorophytum borivilianum tuberrdquo Pharmacologyonlinevol 1 pp 320ndash328 2009

[23] M Mujeeb S A Khan M Ali A Mall and A AhmadldquoAntidiabetic activity of the aqueous extract of ChlorophytumborivilianumL in streptozotocin induced-hyperglycemic ratsmdasha preliminary studyrdquo Journal of Pharmacy Research vol 2 no 1pp 51ndash53 2009

[24] M Thakur S Bhargava and V K Dixit ldquoImmunomodulatoryactivity of Chlorophytum borivilianum Sant Frdquo Evidence-BasedComplementary and Alternative Medicine vol 4 no 4 pp 419ndash423 2007

[25] S K Panda D Das and N K Tripathy ldquoStudies on anti-ulceractivity of root tubers of Chlorophytum borivilianum santapauand fernandesrdquo International Journal of Pharmaceutical SciencesReview and Research vol 9 no 2 pp 65ndash68 2011

[26] S L Deore and S S Khadabadi ldquoAntiproliferative activity ofsaponin fractions of Chlorophytum borivilianumrdquo Pharmacog-nosy Journal vol 2 no 16 pp 29ndash33 2010

[27] M Samanta M Basu B Swain P Panda and P JayasankarldquoMolecular cloning and characterization of toll-like receptor 3and inductive expression analysis of type I IFN Mx and pro-inflammatory cytokines in the Indian carp rohu (Labeo rohita)rdquoMolecular Biology Reports vol 40 no 1 pp 225ndash235 2013

[28] S S Giri S S Sen andV Sukumaran ldquoEffects of dietary supple-mentation of potential probiotic Pseudomonas aeruginosaVSG-2 on the innate immunity and disease resistance of tropicalfreshwater fish Labeo rohitardquo Fish and Shellfish Immunologyvol 32 no 6 pp 1135ndash1140 2012

[29] AOAC Official Methods of Analyses Association of OfficialAnalytical Chemists Washington DC USA 16th edition 1997

[30] APHA AWWA and WEF Standard Methods for the Examina-tion ofWater andWasteWater American Public Health Associ-ation AmericanWaterWorks AssociationWater EnvironmentAssociation Washington DC USA 20th edition 1998

[31] A E Ellis ldquoLysozyme assayrdquo in Techniques in Fish ImmunologyJ S Stolen T C Fletcher D P Anderson B S Robertsonand W B Van Muiswinkel Eds pp 101ndash103 SOS PublicationsHaven NJ USA 1990

[32] LThomas Clinical Laboratory Diagnostics TH-Books Verlags-gesellschaft Frankfurt Germany 1st edition 1998


[33] W-Q Cai S-F Li and J-Y Ma ldquoDiseases resistance ofNile tilapia (Oreochromis niloticus) blue tilapia (Oreochromisaureus) and their hybrid (female Nile tilapiaxmale blue tilapia)to Aeromonas sobriardquo Aquaculture vol 229 no 1ndash4 pp 79ndash872004

[34] C J Secombes ldquoIsolation of salmonid macrophages and anal-ysis of their killing activityrdquo in Techniques in Fish ImmunologyJ S Stolen T C Fletcher D P Anderson B S Robertson andW B VanMuiswinkel Eds pp 137ndash154 SOS Publications FairHaven NJ USA 1990

[35] X Geng X-H Dong B-P Tan et al ldquoEffects of dietaryprobiotic on the growth performance non-specific immunityand disease resistance of cobia Rachycentron canadumrdquo Aqua-culture Nutrition vol 18 no 1 pp 46ndash55 2012

[36] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the2minus998779998779CT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[37] R Harikrishnan C Balasundaram and M-S Heo ldquoEffectof probiotics enriched diet on Paralichthys olivaceus infectedwith lymphocystis disease virus (LCDV)rdquo Fish amp ShellfishImmunology vol 29 no 5 pp 868ndash874 2010

[38] H Boshra J Li and J O Sunyer ldquoRecent advances on thecomplement systemof teleost fishrdquoFishampShellfish Immunologyvol 20 no 2 pp 239ndash262 2006

[39] P Taylor M Botto and MWalport ldquoThe complement systemrdquoCurrent Biology vol 8 no 8 pp R259ndashR261 1998

[40] Y-Z Sun H-L Yang R-L Ma and W-Y Lin ldquoProbioticapplications of two dominant gut Bacillus strains with antag-onistic activity improved the growth performance and immuneresponses of grouper Epinephelus coioidesrdquo Fish amp ShellfishImmunology vol 29 no 5 pp 803ndash809 2010

[41] J Jian and Z Wu ldquoEffects of traditional Chinese medicine onnonspecific immunity and disease resistance of large yellowcroaker Pseudosciaena crocea (Richardson)rdquo Aquaculture vol218 no 1ndash4 pp 1ndash9 2003

[42] M O Huising R J M Stet H F J Savelkoul and BM L Verburg-Van Kemenade ldquoThe molecular evolution ofthe interleukin-1 family of cytokines IL-18 in teleost fishrdquoDevelopmental and Comparative Immunology vol 28 no 5 pp395ndash413 2004

[43] L Wei B Sun M Chang Y Liu and P Nie ldquoEffects ofcyanobacterial toxinmicrocystin-LR on the transcription levelsof immune-related genes in grass carp Ctenopharyngodonidellardquo Environmental Biology of Fishes vol 85 no 3 pp 231ndash238 2009

[44] H Watanuki K Ota A C M A R Tassakka T Kato and MSakai ldquoImmunostimulant effects of dietary Spirulina platensison carpCyprinus carpiordquoAquaculture vol 258 no 1ndash4 pp 157ndash163 2006

[45] J P J Saeij R J M Stet A Groeneveld L B M Verburg-Van Kemenade W B Van Muiswinkel and G F WiegertjesldquoMolecular and functional characterization of a fish inducible-type nitric oxide synthaserdquo Immunogenetics vol 51 no 4-5 pp339ndash346 2000

[46] J P J Saeij W B Van Muiswinkel A Groeneveld andG F Wiegertjes ldquoImmune modulation by fish kinetoplastidparasites a role for nitric oxiderdquo Parasitology vol 124 no 1 pp77ndash86 2002

[47] R Savan D Igawa and M Sakai ldquoCloning characterizationand expression analysis of interleukin-10 from the commoncarp Cyprinus carpio Lrdquo European Journal of Biochemistry vol270 no 23 pp 4647ndash4654 2003

[48] K W Moore R L de Waal Malefyt R L Coffman andA OrsquoGarra ldquoInterleukin-10 and the interleukin-10 receptorrdquoAnnual Review of Immunology vol 19 pp 683ndash765 2001

[49] M O Li and R A Flavell ldquoContextual regulation of inflamma-tion a duet by transforming growth factor-120573 and interleukin-10rdquo Immunity vol 28 no 4 pp 468ndash476 2008

[50] Z Zhang T Swain J Boslashgwald R A Dalmo and J KumarildquoBath immunostimulation of rainbow trout (Oncorhynchusmykiss) fry induces enhancement of inflammatory cytokinetranscripts while repeated bath induce no changesrdquo Fish ampShellfish Immunology vol 26 no 5 pp 677ndash684 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


(TGF) chemokines and interferons (IFNs) are types ofcytokines which require cytokine receptors These cytokinesare reported to have proinflammatory anti-inflammatoryand pathogen-killing activities [9] Hence understandingdifferential cytokine expression can enable the developmentof immunostimulants for aquaculture [10] Further obtainedinformation has the added advantage of identifying potentialindirect immunological markers for aquatic species

Several studies have investigated the enhancement ofgrowth immunity and disease resistance in fish throughdietary administration of immunostimulants Stimulationof fish immunity through dietary immunostimulants is ofhigh interest for commercial aquaculture [8] Several herbalpreparations have been reported to enhance the immuneresponses in fish [2 4 7 11ndash15] For example Rehmanniaglutinosa root products enhanced the growth and immuneparameters ofCyprinus carpio [2] Seed ofAchyranthes asperaenhanced the immune responses inCatla catla [7]The root ofWithania somnifera improved the disease resistance of Labeorohita against Aeromonas hydrophila [13] Recently we foundthatPsidium guajava leaves as feed additive could increase thegrowth performance disease resistance and cytokine geneexpression of Labeo rohita [14] Herbal therapeutic productsare less expensive and have greater accuracy compared withchemotherapeutic agents providing a potential solution forthe problems facing aquaculture today [15]

Chlorophytum borivilianum L (Liliaceae) is a traditionalrare medicinal herb in India and considered ldquowhite goldrdquoor ldquodivya aushadrdquo in Indian systems of medicine Its roottubers are widely used for various therapeutic applica-tions The traditional uses of C borivilianum tubers againstdiseases such as diabetes mellitus dysuria diarrhea anddysentery are well documented in the literature [16 17]Several studies have reported that C borivilianum possessesvarious pharmacological properties such as antimicrobial[18] anti-inflammatory [19] antioxidant [20 21] antistress[22] antidiabetic [23] immunomodulatory [16 24] antiulcer[25] and anticancer effects [26] Major phytochemical con-stituents isolated from the roots of C borivilianum includesteroidal saponins fructans and fructooligosaccharides phe-nolic compounds acetylated mannans and proteins [16 1821 22] Polysaccharide fractions derived from the root tubersof C borivilianum have been shown to exhibit modulation ofhuman natural killer (NK) cell activity humoral response tosheep red blood cells (SRBCs) and enhanced immunoglob-ulin G levels in rats [16] Moreover polysaccharides fromnatural resources are a class of macromolecules that canefficiently affect the immune system and therefore havemuchimportance in basic and applied research [6] Hence wehypothesize that polysaccharide fractions of C borivilianummay boost the immune response of teleosts However veryfew studies have investigated the medicinal properties of Cborivilianum

To date no investigation has been conducted to exploitthe potential efficacy of C borivilianum as a feed additive inmodulating fish growth and immune response The presentstudywas designed to investigate the effects of dietary supple-mentation of a polysaccharide extracted fromC borivilianumroot on the growth performance immune parameters and

expression of several immune-related genes in L rohitaFurther the efficacy of this polysaccharide in the diseaseresistance of L rohitawas also investigated through challengestudy

2 Materials and Methods

21 Diet Preparation Dried roots of Chlorophytum boriv-ilianum were collected from a safed musli farm Indore(Madhya Pradesh) India The roots were dried in sunlightand coarsely powdered for extraction The powdered rootswere subjected to hot water extraction following the methoddescribed by Thakur et al [16] The purified polysaccharidefraction was lyophilized and stored at minus20∘C for further use

A basal diet comprising 39 groundnut oil cake 34rice bran 20 soybean meal 5 fish meal and 2 mineraland vitaminmixture (every 250 g ofmineral-vitaminmixtureprovided vitamin A 500000 IU vitamin D3 100000 IUvitamin B2 02 g vitamin E 75 units vitamin K 01 gcalcium pantothenate 025 g nicotinamide 01 g vitaminB12 06mg choline chloride 15 g calcium 75 g manganese275 g iodine 01 g iron 075 g zinc 15 g copper 02 gand cobalt 0045 g) was prepared [28] Proximate analysisof basal diet performed according to AOAC method [29]revealed 373 protein 86 lipid and 121 ash The basaldiet was considered as control diet The C borivilianum rootpolysaccharide (CBP)was supplemented into basal diet at fivelevels 0 (basal diet) 01 (D1) 02 (D2) 04 (D3) and08 (D4) All the ingredients were blended thoroughly in amixture pelletized air-dried ground and sieved into properpellet size All feeds were stored at 4∘C until use

22 Cytotoxicity of CBP Rat macrophage cells (ATCC CRL-2192) were routinely cultured in DMEM (Dulbeccorsquos Mod-ified Eagle Medium) supplemented with 10 horse serum(HS) penicillin G (500 UmL) streptomycin (5000 120583gmL)and 35mgmL of D-glucose at 37∘C and 5 CO

2in a

humidified incubator (Sanyo Japan) To maintain exponen-tial growth cells were seeded at 1 times 105 cellsmL and pas-saged every 5-6 days Chlorambucil (Sigma-Aldrich USA) atconcentration 600120583gmL was used as positive control [16]Cell viability was assessed using ATPlite kit (Perkin ElmerUSA) CBP was assayed at various concentrations starting at10mgmL Test was performed in triplicate For this 50120583Lof CBP sample was added to each well along with 50 120583L ofcell suspensionThe cells and samples were incubated at 37∘Cfor 24 h in a humidified (5 CO

2) incubator (Sanyo Japan)

Measurement of cell proliferationwas performedusingMicroBeta 1450 plate reader (Perkin Elmer USA) following ATPlitekit instructions

23 Test Fish and Experimental Protocol Labeo rohita fin-gerlings (mean bodyweight 103 plusmn 007 g) were acclimatisedto laboratory conditions for 2 weeks in 500-litre plasticquarantine tanks at 26 plusmn 2∘C Fish were fed with basal dietduring the acclimatisation period About 20 of the waterin all tanks was exchanged daily and 100 of the water wasexchanged once a week Basic physiochemical parameters








= 100 times [(ln119882119905minus ln119882

0)

119905]



)

(1)

where 119882119905and 119882






























3 Results



cell line The IC50











05

101520253035404550


CD1D2

D3D4

Lyso

zym

e act

ivity

(U m

Lminus1)

lowast

lowast lowastlowast

lowast

(a)

0

02

04

06

08


lowast

lowast lowast

lowast lowast

Com

plem

ent C

3(m

g m

Lminus1)

CD1D2

D3D4

(b)

0

5

10

15

20

25

30

35

40

3 4 5

PA (

)


lowast

lowast lowastlowast

lowast

CD1D2

D3D4

(c)

0

02

04


RBA

(OD630)

lowastlowast

CD1D2

D3D4

(d)




4 Discussion





0

05

1

15

2

25


IL-1120573

expr

essio

n re

lativ

e to120573

-act

in lowastlowast

lowastlowast

CD1D2

D3D4

(a)

0

05

1

15

2

25

3


TNF-120572

expr

essio

n re

lativ

e to120573

-act

in

lowast

lowastlowast

lowast

CD1D2

D3D4

(b)

0

05

1

15

2

25


IL-8

expr

essio

n re

lativ

e to120573

-act

in

lowastlowastlowast

lowastlowast

CD1D2

D3D4

(c)

0

05

1

15

2

25


iNO

S ex

pres

sion

relat

ive t

o 120573

-act

inlowast

lowast

lowastlowast

CD1D2

D3D4

(d)






0

05

1

15

2

25


IL-10

expr

essio

n re

lativ

e to120573

-act

in

CD1D2

D3D4

lowastlowast

lowast

(a)

002040608

112141618

2


TGF-120573

expr

essio

n re

lativ

e to120573

-act

in

lowastlowastlowast

lowast

CD1D2

D3D4

(b)


266

100

366

60733

53

0

20

40

60

80

100

120


Postc

halle

nge s

urvi

val (

)


lowast

lowast

lowast

lowast

lowast












5 Conclusions






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























= 100 times [(ln119882119905minus ln119882

0)

119905]



)

(1)

where 119882119905and 119882






























3 Results



cell line The IC50











05

101520253035404550


CD1D2

D3D4

Lyso

zym

e act

ivity

(U m

Lminus1)

lowast

lowast lowastlowast

lowast

(a)

0

02

04

06

08


lowast

lowast lowast

lowast lowast

Com

plem

ent C

3(m

g m

Lminus1)

CD1D2

D3D4

(b)

0

5

10

15

20

25

30

35

40

3 4 5

PA (

)


lowast

lowast lowastlowast

lowast

CD1D2

D3D4

(c)

0

02

04


RBA

(OD630)

lowastlowast

CD1D2

D3D4

(d)




4 Discussion





0

05

1

15

2

25


IL-1120573

expr

essio

n re

lativ

e to120573

-act

in lowastlowast

lowastlowast

CD1D2

D3D4

(a)

0

05

1

15

2

25

3


TNF-120572

expr

essio

n re

lativ

e to120573

-act

in

lowast

lowastlowast

lowast

CD1D2

D3D4

(b)

0

05

1

15

2

25


IL-8

expr

essio

n re

lativ

e to120573

-act

in

lowastlowastlowast

lowastlowast

CD1D2

D3D4

(c)

0

05

1

15

2

25


iNO

S ex

pres

sion

relat

ive t

o 120573

-act

inlowast

lowast

lowastlowast

CD1D2

D3D4

(d)






0

05

1

15

2

25


IL-10

expr

essio

n re

lativ

e to120573

-act

in

CD1D2

D3D4

lowastlowast

lowast

(a)

002040608

112141618

2


TGF-120573

expr

essio

n re

lativ

e to120573

-act

in

lowastlowastlowast

lowast

CD1D2

D3D4

(b)


266

100

366

60733

53

0

20

40

60

80

100

120


Postc

halle

nge s

urvi

val (

)


lowast

lowast

lowast

lowast

lowast












5 Conclusions






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































3 Results



cell line The IC50











05

101520253035404550


CD1D2

D3D4

Lyso

zym

e act

ivity

(U m

Lminus1)

lowast

lowast lowastlowast

lowast

(a)

0

02

04

06

08


lowast

lowast lowast

lowast lowast

Com

plem

ent C

3(m

g m

Lminus1)

CD1D2

D3D4

(b)

0

5

10

15

20

25

30

35

40

3 4 5

PA (

)


lowast

lowast lowastlowast

lowast

CD1D2

D3D4

(c)

0

02

04


RBA

(OD630)

lowastlowast

CD1D2

D3D4

(d)




4 Discussion





0

05

1

15

2

25


IL-1120573

expr

essio

n re

lativ

e to120573

-act

in lowastlowast

lowastlowast

CD1D2

D3D4

(a)

0

05

1

15

2

25

3


TNF-120572

expr

essio

n re

lativ

e to120573

-act

in

lowast

lowastlowast

lowast

CD1D2

D3D4

(b)

0

05

1

15

2

25


IL-8

expr

essio

n re

lativ

e to120573

-act

in

lowastlowastlowast

lowastlowast

CD1D2

D3D4

(c)

0

05

1

15

2

25


iNO

S ex

pres

sion

relat

ive t

o 120573

-act

inlowast

lowast

lowastlowast

CD1D2

D3D4

(d)






0

05

1

15

2

25


IL-10

expr

essio

n re

lativ

e to120573

-act

in

CD1D2

D3D4

lowastlowast

lowast

(a)

002040608

112141618

2


TGF-120573

expr

essio

n re

lativ

e to120573

-act

in

lowastlowastlowast

lowast

CD1D2

D3D4

(b)


266

100

366

60733

53

0

20

40

60

80

100

120


Postc

halle

nge s

urvi

val (

)


lowast

lowast

lowast

lowast

lowast












5 Conclusions






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















05

101520253035404550


CD1D2

D3D4

Lyso

zym

e act

ivity

(U m

Lminus1)

lowast

lowast lowastlowast

lowast

(a)

0

02

04

06

08


lowast

lowast lowast

lowast lowast

Com

plem

ent C

3(m

g m

Lminus1)

CD1D2

D3D4

(b)

0

5

10

15

20

25

30

35

40

3 4 5

PA (

)


lowast

lowast lowastlowast

lowast

CD1D2

D3D4

(c)

0

02

04


RBA

(OD630)

lowastlowast

CD1D2

D3D4

(d)




4 Discussion





0

05

1

15

2

25


IL-1120573

expr

essio

n re

lativ

e to120573

-act

in lowastlowast

lowastlowast

CD1D2

D3D4

(a)

0

05

1

15

2

25

3


TNF-120572

expr

essio

n re

lativ

e to120573

-act

in

lowast

lowastlowast

lowast

CD1D2

D3D4

(b)

0

05

1

15

2

25


IL-8

expr

essio

n re

lativ

e to120573

-act

in

lowastlowastlowast

lowastlowast

CD1D2

D3D4

(c)

0

05

1

15

2

25


iNO

S ex

pres

sion

relat

ive t

o 120573

-act

inlowast

lowast

lowastlowast

CD1D2

D3D4

(d)






0

05

1

15

2

25


IL-10

expr

essio

n re

lativ

e to120573

-act

in

CD1D2

D3D4

lowastlowast

lowast

(a)

002040608

112141618

2


TGF-120573

expr

essio

n re

lativ

e to120573

-act

in

lowastlowastlowast

lowast

CD1D2

D3D4

(b)


266

100

366

60733

53

0

20

40

60

80

100

120


Postc

halle

nge s

urvi

val (

)


lowast

lowast

lowast

lowast

lowast












5 Conclusions






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

05

1

15

2

25


IL-1120573

expr

essio

n re

lativ

e to120573

-act

in lowastlowast

lowastlowast

CD1D2

D3D4

(a)

0

05

1

15

2

25

3


TNF-120572

expr

essio

n re

lativ

e to120573

-act

in

lowast

lowastlowast

lowast

CD1D2

D3D4

(b)

0

05

1

15

2

25


IL-8

expr

essio

n re

lativ

e to120573

-act

in

lowastlowastlowast

lowastlowast

CD1D2

D3D4

(c)

0

05

1

15

2

25


iNO

S ex

pres

sion

relat

ive t

o 120573

-act

inlowast

lowast

lowastlowast

CD1D2

D3D4

(d)






0

05

1

15

2

25


IL-10

expr

essio

n re

lativ

e to120573

-act

in

CD1D2

D3D4

lowastlowast

lowast

(a)

002040608

112141618

2


TGF-120573

expr

essio

n re

lativ

e to120573

-act

in

lowastlowastlowast

lowast

CD1D2

D3D4

(b)


266

100

366

60733

53

0

20

40

60

80

100

120


Postc

halle

nge s

urvi

val (

)


lowast

lowast

lowast

lowast

lowast












5 Conclusions






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

05

1

15

2

25


IL-10

expr

essio

n re

lativ

e to120573

-act

in

CD1D2

D3D4

lowastlowast

lowast

(a)

002040608

112141618

2


TGF-120573

expr

essio

n re

lativ

e to120573

-act

in

lowastlowastlowast

lowast

CD1D2

D3D4

(b)


266

100

366

60733

53

0

20

40

60

80

100

120


Postc

halle

nge s

urvi

val (

)


lowast

lowast

lowast

lowast

lowast












5 Conclusions






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















5 Conclusions






Acknowledgments


References


























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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