)joebxj1vcmjtijoh$psqpsbujpo …downloads.hindawi.com/journals/jir/2016/4086591.pdfresearch article...

Research ArticleDietary Administration of Banana (Musa acuminata) PeelFlour Affects the Growth Antioxidant Status CytokineResponses and Disease Susceptibility of Rohu Labeo rohita

Sib Sankar Giri1 Jin Woo Jun1 Venkatachalam Sukumaran2 and Se Chang Park1

1Laboratory of Aquatic Biomedicine College of Veterinary Medicine and Research Institute for Veterinary ScienceSeoul National University Seoul 151742 Republic of Korea2Department of Biotechnology Periyar Maniammai University Thanjavur Tamil Nadu 613403 India

Correspondence should be addressed to Venkatachalam Sukumaran drvsukumargmailcom andSe Chang Park parksecsnuackr

Received 24 November 2015 Revised 4 April 2016 Accepted 14 April 2016

Academic Editor Ghislain Opdenakker

Copyright copy 2016 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

To explore the feasibility ofMusa acuminata (banana) peels as a feed additive effects of banana peel flour (BPF) on the growth andimmune functions of Labeo rohita were evaluated Diets containing five different concentrations of BPF (0 [basal diet] 1 [B1]3 [B3] 5 [B5] and 7 [B7]) were fed to the fish (average weight 153 g) for 60 daysThe final weight gain and specific growth ratewere higher (119875 lt 005) in the B5 group The most significant improvements in immune parameters such as lysozyme alternativecomplement pathway leukocyte phagocytic superoxide dismutase and catalase activities were observed in the B5 group Howeverthe B5 group exhibited the lowest malondialdehyde activity IgM and glutathione peroxidise activities were significantly elevatedin the treatment groups except in B1 after only 30 days of feeding Of the examined cytokine-related genes IL-1120573 TNF-120572 andHSP70 were upregulated in the head kidney and hepatopancreas and expressions were generally higher in the B3 and B5 groupsMoreover B5 group challenged withAeromonas hydrophila 60 days after feeding exhibited the highest survival rate (70119875 lt 005)These results suggest that dietary BPF at 5 could promote growth performance and strengthen immunity in L rohita

1 Introduction

Global aquaculture production is dominated by freshwaterfish species (564) especially carp species (719 242 mil-lion tons in 2010) [1] Intensive rearing of aquaculture fishspecies generates environmental stress to fish which canincrease susceptibility to various pathogens such as virusesbacteria fungi and parasites [2] this has led to a huge eco-nomic loss The most common and frequently encounteredbacterial pathogen in freshwater aquaculture is Aeromonashydrophila which causes severe damage to carps [3] Treat-ment of infections by using antibiotics and chemotherapeu-tics at the farm level is either prohibited or infeasible becauseit may result in an increase in drug-resistant pathogens envi-ronmental hazards and food safety concerns [4] Thereforedisease management approaches in aquaculture should be

based on preventive measures rather than disease control ortreatment as they are likely to be more cost-effective Cur-rently use of ldquoimmunostimulantsrdquo is gaining interest becauseof their potential in overcoming the limitations of vaccinesand probiotics [5]

Fish respond to infectious agents via both specific andnonspecific mechanisms but they rely primarily on nonspe-cific immune responses [6] Therefore boosting fish innateimmunity may be the most promising approach for diseaseprevention Plant extracts are known to promote growthstimulate appetite enhance tonicity and immunostimulationfacilitate maturation of cultured species and possess stressreduction sexual stimulation and antipathogenic propertiesin fish [7] Products of several herbal plants for exampleAzadirachta indica [5] Psidium guajava [8] Rheum officinale[9] Withania somnifera [10] Rehmannia glutinosa [2] Ficus

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2016 Article ID 4086591 11 pageshttpdxdoiorg10115520164086591

2 Journal of Immunology Research

carica polysaccharide [11] emodin [12 13] and Achyranthesaspera [14] have been reported to enhance fish immunityMoreover some products of herbal plants have been reportedto alter the expression of cytokine- and immune-related genesin fish [2 8 11 14] They play vital role in host innateimmunity and are indispensable for the use and activation ofmacrophages neutrophils and lymphocytes at infection sitesfor pathogen elimination [15]

Banana (Musa spp) is vital for food security in manytropical and subtropical countries and they are the mostpopular fruit in industrialized countries [16] Banana is thesecond leading fruit produced after citrus contributing toapproximately 17 of the worldrsquos total fruit production it iscultivated in over 130 countries [17] Banana peel which con-stitutes up to 35 of the ripe fruit is a household and indus-trial food waste discarded in large quantities [18] It is rich indietary fibre proteins essential amino acids vitamins poly-unsaturated fatty acids and potassium [19] Soluble fibresare well known to lower serum cholesterol and help reducethe risk of colon cancer [20] Bioactive compounds likeflavonoids tannins phlobatannins alkaloids glycosidesanthocyanins and terpenoids were found in banana peelsand these compounds have been reported to exert variousbiological and pharmacological effects (antibacterial anti-hypertensive antidiabetic and anti-inflammatory activities)[21] Further antioxidant compounds (eg prodelphinidinspolyphenols catecholamines and carotenoids) [22] and highamount of micronutrients [23] were found in the peels ofgenusMusaThe presence of various bioactive compounds inbanana peels suggests that the peels possess variousmedicinalproperties and may be useful as immunostimulant [24]

The effect of oral immunostimulants on the immuneresponse in aquatic animals is strongly associated with thedose and duration of application In the present study weaimed to investigate the effects of dietary supplementationof banana peels on the growth antioxidant status immuneparameters and cytokine gene expression of L rohita and itssusceptibility toA hydrophila infectionWe also explored thepotential of banana peel as a feed additive in aquaculture

2 Materials and Methods

21 Diet Preparation Bananas (Musa acuminata) were pur-chased from the local market (Thanjavur Tamil Nadu) witha skin colour index of 6 (all yellow) [27] The bananas wererinsed thoroughly in tap water followed by double distilledwater and acetone to remove any contaminants They werethen separated into pulps and peels The peels were cut intopieces of about 5 cm2 by using a kitchen knife and air dried inthe oven at 50∘C for 24 h Then the pieces were ground intoa fine powder by using the laboratory grinder The obtainedflour called BPF was stored at minus20∘C until use

The basal diet (Table 1) was prepared using a previouslydescribed method [8] Proximate analysis of the basal diet[28] revealed a composition of 331 proteins 89 lipidsand 12 ash The basal diet was used as the control diet Thebasal diet was supplemented with BPF at five concentrations0 (control diet) 1 (B1) 3 (B3) 5 (B5) and 7 (B7) Allingredients were blended thoroughly into a mixture and then

Table 1 Formulation and chemical composition of basal diet (g kgminus1dry matter)

Ingredients Concentrations (g kgminus1)Ground nut oil cake 390Rice bran 340Soybean meal 170Fish meal 78Vegitable oil 20Mineral and vitamin mixturea 2Proximate analysis (g kgminus1)Crude protein 331Crude lipid 89Ash 120

aEvery 250 g of mineral-vitamin mixture (Supplevite-M Sarabhai ZydusAnimal Health Ltd India) provides vitamin A 500000 IU vitamin D3100000 IU vitamin B2 02 g vitamin E 75 units vitamin K 01 g calciumpantothenate 025 g nicotinamide 01 g vitamin B12 06mg choline chlo-ride 15 g calcium 75 g manganese 275 g iodine 01 g iron 075 g zinc15 g copper 02 g and cobalt 0045 g

pelletized air-dried ground and sieved into an appropriatepellet sizeThe pellets were then stored at minus20∘C until furtheruse

22 Experimental Design Labeo rohita fingerlings (meanbodyweight 153 g) obtained from local fish farm (ThanjavurTamil Nadu India) were acclimatised to laboratory condi-tions in 500 L plastic tanks at 26 plusmn 2∘C for 2 weeks and fedthe basal diet Approximately 20 of the water in all tankswas exchanged daily and 100 of the water was exchangedevery week Basic physiochemical parameters of the waterwere measured every week [29] Oxygen and ammoniaconcentrations were 61ndash73120583gmLminus1 and 003ndash006120583gmLminus1respectively and pH ranged from 7 to 8 throughout the studyperiod

The fish were randomly divided into five experimentalgroups with three replicates in each group Tank capacitywas 200 L and each tank contained 25 fish (ie per group25 times 3 = 75 fish) The fish were fed one of the five diets for60 days and three times (at 0800 1300 and 1800 hours)at 3ndash5 of body weight daily The amount of feed consumedwas determined by daily recovery of excess feed which wasthen adjusted every 15 days by batch weighing after 24 h ofstarvation

23 Growth Performance Ten fish were randomly selectedfrom each tank (ie 10 times 3 = 30 fish per group) at day 0 day30 and day 60 after experimental feeding and batch-weighedto estimate growth performance Growth performance andsurvival rate of the fish were calculated using the followingformulae weight gain (WG gfish) =119882

119905minus1198820 specific growth

rate (SGR) = 100 times [(ln119882119905minus ln119882

0)119905] feed conversion ratio

(FCR) = FI(119882119905minus1198820) where119882

119905and119882

0were final and initial

weights of the fish respectively 119905 is the duration of feeding(in days) and FI is feed intake

Journal of Immunology Research 3

24 Sample Collection At the end of 30 days and 60 daysof experimental feeding five fish from each tank (ie 5times 3 = 15 fish per group) were collected to study immuneand antioxidant parameters Blood samples were collected bycaudal venipuncture with a 1 mL syringe after anesthetizingthe fish with diluted MS222 (Sigma-Aldrich USA) Theblood samples were transferred into Eppendorf tubes Aftercollection blood was centrifuged at 2000timesg for 10min at4∘C The obtained blood leucocytes and plasma were storedat minus20∘C for further analysis

241 Immune Parameters

(1) Serum Lysozyme Activity Lysozyme activity wasmeasuredaccording to the method described by Ellis [30] Brieflythe lysozyme substrate was 02mgmLminus1 of freeze-driedMicrococcus lysodeikticus (Sigma USA) suspension in 005MPhosphate Buffer Solution (PBS) pH 62 Serum (100 120583L) wasadded to 19mL of the bacterial suspension and the reductionin absorbance at 530 nm was measured after 05min and45min at room temperature Results were expressed in unitsof lysozyme activity mLminus1 serum One unit of lysozymeactivity was defined as the amount of enzyme that producesa decrease in absorbance of 0001minminus1mLminus1 serum

(2) Alternative Complement Pathway (ACP) Activity AssayACP activity was determined and calculated using themethod of Yano [31] by using rabbit red blood cells (RaRBC)Briefly the RaRBC were adjusted to 2 times 108 cells mLminus1 in001M ethylene glycol tetraacetic acidndashmagnesiumndashgelatinveronal buffer (EGTAndashMgndashGVB) The 100 lysis value wasobtained by lysing of 100mL of RaRBC with 34mL of dis-tilled water andmeasuring the optical density of haemolysateat 414 nm against distilled water The test serum was dilutedand different volumes ranging from 01 to 025mLweremadeup to 025mL total volume before being allowed to react with01mL of RaRBC in test tubes After incubation at 20∘C for90min with occasional shaking 315mL of a saline solutionwas added to each tube and centrifuged at 1600timesg for 10minat 4∘C The optical density of the supernatant was measuredat 414 nm using a spectrophotometer (Beckman InstrumentsInc California USA) The volume of serum that produces50 haemolysis (ACH

50) was determined and the number of

ACH50UmLminus1 was calculated for each group

(3) Leucocyte-Phagocytic Assay Staphylococcus aureus wascultured in an agar culture medium for 24 h at 37∘C Thecell number was adjusted to 5 times 106 CFU mLminus1 Phago-cytic activity (PA) of the blood leukocytes was determinedaccording to the method described by Cai et al [32] Brieflyblood sample was collected into heparinized centrifuge tubeand shaken well Same volume of bacterial suspension wasadded to the tube The tubes were then kept at 28∘C in awater bath for 30min and shaken every 10min Followingincubation solution was centrifuged at 1500timesg for 5min andthe precipitate was used further The upper layer of the pre-cipitate was used to make blood slides Slides were air driedfixed with methanol for 3min and then stained with Swissstaining solution for 5min The number of phagocytotic and

unphagocytotic leukocytes was counted under the micro-scope PA was expressed as phagocytic rate (PR)

PR () = [100

times (phagocytic leucocytes) (total leucocytes)minus1] (1)

(4) Immunoglobulin M (IgM) Assay Plasma total IgM levelsweremeasured according to themethod described by Sharmaet al [10] Briefly blood sample was centrifuged for 5min at1000timesg to get clear cells 01mL of plasma was placed intoa serum vial and added 01mL of 12 polyethylene glycol(PEG) that had been suspended in deionised water and incu-bated at room temperature for 2 h under constant mixingFollowing centrifugation at 5000timesg for 10min supernatantwas collected and protein concentration was determinedProtein reading from supernatant was the amount of proteintaken out by absorption to PEG Total immunoglobulin wasexpressed as unit mg mLminus1 Total immunoglobulin = totalprotein in individual sample plasma minus total protein taken outby absorption to polyethylene glycol

242 Antioxidant Parameters

(1) SuperoxideDismutase (SOD) andMalondialdehyde (MDA)Assay SOD activity was determined using an enzymaticassay method with a reagent kit (Randox Crumlin UK) asdescribed by Zhang et al [33] MDA content was measuredusing barbituric acid reaction chronometry [34]

(2) Measurement of Catalase (CAT) Activity and GlutathionePeroxidise (GPx) Activity CAT activity was measured by therate of decrease in H

2O2absorbance at 240 nm by using

a commercially available kit (Sigma-Aldrich USA) GPxactivitywasmeasured using a commercial kit (Ransel RS-504Randox) according to the manufacturerrsquos instructions Spe-cific activity was expressed as GPx unit mgminus1 of total protein

25 Expressions of Immune-Related Genes in the Head Kidneyand Hepatopancreas Thehead kidney and hepatopancreaseswere dissected from nine fish per group at the end of thefeeding trial Total RNA was extracted from the tissues(kidney and hepatopancreas) by using TRIZOL reagent(Invitrogen USA) according to the manufacturerrsquos instruc-tions RNA concentration and purity were analysed usinga spectrophotometer (Thermo Scientific USA) and qualitywas checked using 1 agarose gel containing 05120583gmLminus1ethidium bromide RNAwas reverse-transcribed to cDNA byusing the SuperScript cDNA synthesis kit (Life Technolo-gies) according to themanufacturerrsquos instructions Real-timePCR analysis of IL-1120573 IL-10 iNOS TNF-120572 TGF-120573 HSP70NF-120581B and a housekeeping gene (120573-actin) was performedusing CFX96 Real-Time PCR (Bio-Rad Laboratories Inc)according to standard protocols with primer sequences andthermocycling conditions as indicated in Table 2 To verifythe accuracy of each amplicon melt curve analysis was per-formed after amplification All samples were run in parallelwith the housekeeping gene to normalize cDNA loadingGene expression results were analysed using the 2minusΔΔCT


Table 2 Real-time primer sequences and thermocycling conditions

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

IL-1120573ACCCCACAAAACATCGGCCAACC 95∘C 30 s 40 cycles of 95∘C 5 s 615∘C 30 s

and 72∘C 30 s [25]TCTTCTCCATTTCCACCCTCTC

IL-10CGCAGTGCAGAAGAGTCGAC 95∘C 30 s 40 cycles of 95∘C 5 s 615∘C 30 s

and 72∘C 30 s GU256643CCCGCTTGAGATCCTGAAATAT

TNF-120572CTCAACAAGTCTCAGAACAATCAGG 95∘C 30 s 40 cycles of 95∘C 5 s 615∘C 30 s

and 72∘C 30 s [25]TCCTGGTTCCTTCTCCAATCTAGCT

iNOSGGAGGTACGTCTGCGAGGAGGCT 95∘C 30 s 40 cycles of 95∘C 5 s 611∘C 30 s

and 72∘C 30 s [8]CCAGCGCTGCAAACCTATCATCCA

TGF-120573ACGCTTTATTCCCAACCAAA 95∘C 30 s 40 cycles of 95∘C 5 s 615∘C 30 s

and 72∘C 30 s AF13694GAAATCCTTGCTCTGCCTCA

NF-120581BTATTCAGTGCGTGAAGAAG 95∘C 30 s 40 cycles of 95∘C 5 s 615∘C 30 s

and 72∘C 30 s LN590704TATTAAAGGGGTTGTTCTGT

HSP70GGCAGAAAGTTTGATGACCCA 95∘C 30 s 40 cycles of 95∘C 5 s 615∘C 30 s

and 72∘C 30 s [26]GCAATCTCCTTCATATTCACC

120573-actinAGACCACCTTCAACTCCATCATG 95∘C 30 s 40 cycles of 95∘C 5 s 615∘C 30 s

and 72∘C 30 s [25]TCCGATCCAGACAGAGTATTTACGC

method after verifying that the primers were amplified withan efficiency of approximately 100 [26] whereΔΔCT = [(CTgene of interest minus CT120573-actin) treatment group minus (CT gene ofinterest minusCT120573-actin) control group]The CT is defined as thePCR cycle at which the fluorescent signal of the reporter dyecrosses an arbitrarily placed threshold

26 Challenge Test Seven-day lethal dose 50 (LD50) for

A hydrophila MTCC-1739 was 107 CFUmL as determinedearlier in our laboratory [8] and this strain was reisolatedfrom experimentally infected fish in our laboratory At thetermination of the feeding trial 10 fish from each tank (ie 3times 10 = 30 fish per group) were selected for the challenge testThe fish (119899 = 30) were injected intraperitoneally with 100 120583Lof phosphate-buffered saline (PBS) containing 1 times 107 live Ahydrophila For negative control 10 fish were injected withPBS only The challenged fish were kept under observationfor 14 days and fed the basal diet Mortality of the fish in eachtank was observed over 14 days

27 Statistical Analysis One-way analysis of variance(ANOVA) was used to analyse the data Multiple com-parisons were performed using Tukeyrsquos test to analyse thedifferences between treatments All statistical analyses wereperformed using the OriginPro software (version 8 Origin-Lab Corporation Northampton USA) The level of signifi-cance was set at 119875 lt 005 and the results were expressed asmean plusmn SEM

3 Results

31 Growth Performance Effects of BPF on the growthperformance of L rohita are shown in Table 3 After 30 daysof feeding significantly higher WG and SGR were observed

in the B5 group than in the control However 30 days ofBPF administration had no significant effects on FCR At theend of the trial WG and SGR were higher in the treatmentgroups and the highest (119875 lt 005) WG (8361 plusmn 152 g) andSGR (286 plusmn 0023 g) values were observed in the B5 group(compared to the control) FCR was significantly lower in theB5 group (439 plusmn 002) than in the control

32 Immune Parameters Results of LA andACP activities areshown in Table 4 Serum lysozyme activity was significantlyhigher in the B5 group during the whole period of the trialthan in the control ACP activity was significantly higherin the treatment groups except B1 at any point of timeand it was the highest in the B5 group (Table 4) A slightdecline in LA and ACP activities was observed at a higherBPF concentration (ie B7) during the trial period

Effects of dietary administration of BPF on PA and IgMof L rohita are shown in Table 5 After 30 days of feedingsignificantly higher PAwas observed in the treatment groupsexcept B1 than in the control However PA was significantlyhigher in the treatment groups at the end of the feeding trialand the highest activity was recorded in the B5 group (4870plusmn168) IgM level was significantly higher in the treatmentgroups except B1 during the 30 days of feeding However 60days of feeding with BPF did not have a significant effect onIgM levels although a slight increase in IgM level (comparedto the control) was recorded in the B1 group SurprisinglyIgM levels were lower in the other treatment groups than inthe control and the lowest level (119875 lt 005) was observed inthe B7 group

33 Antioxidant Parameters SOD activity was significantlyhigher in the B5 andB7 groups than in the control at any point


Table 3 Effects of banana peel flour (BPF) on the growth performance of Labeo rohita

Parameters Control B1 B3 B5 B7Initial weight (g) 1537 plusmn 041 1562 plusmn 024 1518 plusmn 068 1521 plusmn 027 156 plusmn 0460ndash30 days of feeding

WG (g) 3806 plusmn 044a 3865 plusmn 028ab 3924 plusmn 057ab 4097 plusmn 039b 3963 plusmn 048ab

SGR 294 plusmn 0017a 295 plusmn 0041a 308 plusmn 0032b 314 plusmn 0024cb 303 plusmn 0015ab

FCR 584 plusmn 021 583 plusmn 018 581 plusmn 029 578 plusmn 036 581 plusmn 024Survival () 100 100 100 100 100

0ndash60 days of feedingFinal weight gain (g) 7419 plusmn 166a 7706 plusmn 109ab 8142 plusmn 122b 8361 plusmn 152b 8007 plusmn 118b

SGR 263 plusmn 0047a 266 plusmn 0053ab 278 plusmn 0031b 286 plusmn 0023b 272 plusmn 0029ba

FCR 461 plusmn 003a 454 plusmn 001a 448 plusmn 005ba 436 plusmn 002b 452 plusmn 003ba

Survival () 100 9866 100 100 9733Note WG = weight gain SGR = specific growth rate FCR = feed conversion ratioValues in the same column with different superscript letters are significantly different (P lt 005) Values are presented as mean plusmn SEM (119899 = 30 fish in eachgroup)

Table 4 Lysozyme (LA) and alternative complement pathway(ACP) activities observed on different sampling days after feedingLabeo rohita with banana peel flour (BPF) supplemented diets

DietImmune response

LA (unit mLminus1) ACP (ACH50

b unit mLminus1)30 days 60 days 30 days 60 days

Control 693 plusmn 173a 712 plusmn 164a 1031 plusmn 218a 1114 plusmn 178a

B1 715 plusmn 23ab 746 plusmn 191a 1079 plusmn 273a 1191 plusmn 281a

B3 761 plusmn 264ab 821 plusmn 11b 1184 plusmn 311b 1317 plusmn 306c

B5 812 plusmn 251b 869 plusmn 196b 1367 plusmn 153c 1431 plusmn 221c

B7 793 plusmn 152ba 806 plusmn 087ba 1276 plusmn 201cb 1294 plusmn 182cb

Values are expressed as mean plusmn SEM (n = 15) Mean values in the samecolumn with different superscript letters vary significantly (P lt 005)

Table 5 Phagocytic activity (PA) and immunoglobulin (IgM)activities observed on different sampling days after feeding Labeorohita with banana peel flour (BPF) supplemented diets

DietImmune response

PA () IgM (unit mgmLminus1)30 days 60 days 30 days 60 days


B1 3620 plusmn 21ab 3976 plusmn 127b 540 plusmn 016a 591 plusmn 019a

B3 3766 plusmn 264b 4406 plusmn 239b 716 plusmn 026b 536 plusmn 031ab

B5 4133 plusmn 251c 4870 plusmn 168c 843 plusmn 032c 508 plusmn 021ab

B7 4220 plusmn 152c 4516 plusmn 137b 733 plusmn 014b 434 plusmn 017b


of time (Table 6) The highest SOD activity was observed inthe B5 group at any point of time

After 30 days of feeding significantly lower MDA activ-ities were observed in the B3 B5 and B7 groups than in thecontrol (Table 6) However at end of the trial significantlylower MDA activities were recorded in the B5 and B7 groups

Table 6 Superoxide dismutase (SOD) and malondialdehyde(MDA) activities observed on different sampling days after feedingLabeo rohita with banana peel flour (BPF) supplemented diets

DietImmune response

SOD activity (unit mLminus1) MDA (nmol mLminus1)30 days 60 days 30 days 60 days


B1 4383 plusmn 088a 473 plusmn 062ad 883 plusmn 043ab 761 plusmn 043ab

B3 4736 plusmn 065ab 4993 plusmn 091bd 771 plusmn 026bc 712 plusmn 032ab

B5 4928 plusmn 095b 525 plusmn 078b 723 plusmn 031c 666 plusmn 024b

B7 492 plusmn 119b 487 plusmn 053cd 756 plusmn 019cb 732 plusmn 044b


The lowest MDA activity was recorded in the B5 group after60 days of feeding

CAT activity was significantly higher in the B5 and B7groups than in the control (Table 7) and the highest CATactivity was recorded in the B5 group

GPx activities were significantly higher in the B5 and B7groups than in the control after 30 days of feeding and thehighest GPx activity was recorded in the B7 group (Table 7)

34 Gene Expression Analysis Expression profiles of immune-related genes were examined in the head kidney and hep-atopancreas of the fish (119899 = 9) at the end of the feedingtrial Transcription levels of TNF-120572 IL-1120573 and HSP70 wereupregulated in the organs of fish fed the experimental diets(Figure 1) whereas expressions of IL-10 iNOS NF-120581B andTGF-120573 were downregulated (Figure 2)

The B5 group exhibited the highest (119875 lt 005) TNF-120572mRNA expression in the head kidney and hepatopancreastissues (Figure 1(a)) IL-1120573mRNA expression was the highest(119875 lt 005) in the head kidneys of the B5 group followedby B3 whereas IL-1120573 expression was the lowest in the hep-atopancreas (Figure 1(b))HSP70 expressionwas significantlyhigher in the head kidneys of the B3 and B7 groups and


Table 7 Catalase (CAT) activity and glutathione peroxidase (GPx) level measured on different sampling days after feeding Labeo rohitawithbanana peel flour (BPF) supplemented diets

DietImmune response

CAT activity (unit mLminus1) GPx (unit per mgminus1 of protein)

30 days 60 days 30 days 60 days


B1 1417 plusmn 081ab 1483 plusmn 062ab 1683 plusmn 057ab 1731 plusmn 054a

B3 1463 plusmn 052ac 1652 plusmn 046bc 196 plusmn 064bc 1706 plusmn 081a

B5 1641 plusmn 067bc 178 plusmn 083c 2104 plusmn 072c 1654 plusmn 047a

B7 1582 plusmn 079c 152 plusmn 061ca 2136 plusmn 092c 1572 plusmn 091a

Values are expressed as mean plusmn SEM (n = 15) Mean values in the same column with different superscript letters vary significantly (P lt 005)

0123456789

10

Head kidney Hepatopancreas

TNF-120572

expr

essio

n re

lativ

e to 120573

-act

in

CB1B3

B5B7

lowast

lowast

lowastlowast

(a)


CB1B3

B5B7

0

05

1

15

2

25

3

IL-1120573

expr

essio

n re

lativ

e to 120573

-act

inlowast

lowast

lowast

lowast

(b)


CB1B3

B5B7

0

05

1

15

2

25

HSP

70 ex

pres

sion

relat

ive t

o 120573

-act

in lowast

lowast

(c)

Figure 1 RelativemRNA expressions of upregulated genes (TNF-120572 IL-1120573 andHSP70) in the head kidney and hepatopancreas of Labeo rohitafed banana peel flour (BPF) supplemented diets A significant difference is denoted by an asterisk (119875 lt 005) Each bar represents mean plusmnSEM (119899 = 9)



CB1B3

B5B7

0

05

1

15

2

25IL

-10

expr

essio

n re

lativ

e to 120573

-act

in

lowast

lowast

lowast

lowast lowast

(a)


CB1B3

B5B7

002040608

112141618

2

iNO

S ex

pres

sion

relat

ive t

o 120573

-act

in

lowastlowast

(b)


CB1B3

B5B7

002040608

112141618

2

NF120581

B ex

pres

sion

relat

ive t

o 120573

-act

in

lowastlowast

lowastlowastlowast

(c)


CB1B3

B5B7

0

05

1

15

2

25

TGF-120573

expr

essio

n re

lativ

e to 120573

-act

in

lowast

(d)

Figure 2 Relative mRNA expressions of IL-10 iNOS NF-120581B and TGF-120573 in the head kidney and hepatopancreas of Labeo rohita fed bananapeel flour (BPF) supplemented diets A significant difference is denoted by an asterisk (119875 lt 005) Each bar represents mean plusmn SEM (119899 = 9)

the highest HSP70 expression was observed in the B5 group(Figure 1(c)) However dietary treatments had no significanteffect on HSP70 expression in the hepatopancreas

Effects of BPF on IL-10 iNOS NF-120581B and TGF120573 expres-sions are shown in Figure 2 IL-10 expression was downreg-ulated in the B3 and B5 groups and the lowest level wasobserved in the B5 group (Figure 2(a)) Expression of iNOSwas downregulated (119875 lt 005) in the B5 and B7 groups(Figure 2(b)) In addition a slight increase in iNOS expres-sion was observed in the hepatopancreas of the B3 group(Figure 2(b)) NF-120581B expression was downregulated in boththe B5 and B7 groups (Figure 2(c)) TGF-120573 expression wassignificantly lower in only the head kidney of the B5 group(Figure 2(d))

35 Challenge Test Dietary supplementation of BPF fora longer period enhanced the resistance of L rohita to

A hydrophila infection The highest postchallenge survival(70) was recorded in the B5 group whereas the lowestpostchallenge survival rate (20) was observed in the fishfed the basal diet The B3 group exhibited a survival rate of566 followed by the B7 (40) and B1 (2666) groupsNo mortality was observed in the group injected with PBSalone Typical symptoms of haemorrhagic septicaemia wereobserved inmoribund or dead fish Colonies ofA hydrophilawere isolated from dead fish

4 Discussion

In the present study dietary supplementation with 5 BPF(ie B5) for 60 days significantly increased WG and SGR inL rohita Recently we demonstrated that dietary administra-tion of emodin (at 30mg kgminus1 of diet) [13] or guava leaves(at 05) [8] for 60 days significantly increased the growth


performance of L rohita Nonprotein energy sources such ascarbohydrates and lipids in the diets have the ability to sparedietary proteins by reducing the catabolism of proteins forenergy which can improve retention and ultimately growthin animals [35] Further significant reduction in FCR in theB5 group suggested that the fish utilised dietary nutrientsmore efficiently when feed was supplemented with BPFHowever owing to the complexity of the components used inherbal preparations the direct cause of growth stimulation isunclear

A complex system of numerous types of antioxidants(eg catalase glutathione SOD and various peroxidases)is present in aquatic animals [9] SOD GPx and CAT areimportant biochemical parameters and the first line of antiox-idant enzymatic defence Therefore measurement of theseantioxidant parameters may provide a hint of the antioxi-dant status in fish and these parameters can serve as bio-markers for oxidative stress [33] In the present study sup-plementation of 5 BPF (ie B5) for 60 days resulted inthe highest SOD and CAT activities whereas MDA activitywas lowered considerably (119875 lt 005) In addition supple-mentation of BPF significantly enhanced GPx for up to 30days and thereafter increment was not significant Similarresults were observed in L rohita [13] Megalobrama termi-nalis [33] Ctenopharyngodon idella [36] and Megalobramaamblycephala [12] after dietary administration with herbalimmunostimulants Our results revealed that BPF at anappropriate concentration could stimulate the secretion ofantioxidant enzymes as well as antioxidants which can effi-ciently eliminate excess free radicals and regulate the balanceof free radical in the body resulting in improved antioxi-dant ability [33] Further lower MDA content observed inthe present study also strengthened the higher antioxidantpotential of BPF because MDA has strong biotoxicity andcan damage cell structure and function [37] Bioactive com-pounds such as phenolic compounds in banana peel maybe responsible for the antioxidant activity [22] Antioxidantpotential of banana peels has also been reported in previousstudies [22 24]

As the first line of defence various peptides such aslysozyme antibodies and complement factors inhibit theadhesion and colonization of microorganisms leading to theprevention of infection and disease [35] Phagocytosis hasalso been recognised as an important cellular process in thenonspecific immune system of fish [38] In the present studydietary supplementation with 3 (B3) or 5 BPF (B5) for60 days augmented (119875 lt 005) serum LA ACP and PAin fish with the highest degree of activity exhibited by theB5 group Several recent studies have reported that dietarysupplementation of plant products enhanced these immuneparameters in carp [2 5 8 10 13] which is consistent withour results However higher levels of BPF administration fora longer period had no significant effect on these immuneparameters This is most likely because higher doses ofimmunostimulants administered over longer periods oftenlead to immunosuppression but the exact underlying reasonis still unclear

The predominant antibody type in fish IgM is used toidentify and neutralize foreign objects such as bacteria and

viruses [39] In the present study supplementation of 3 to7 BPF improved (119875 lt 005) IgM levels in fish up to 30days and thereafter the levels gradually decreased Similartrend of IgM levels has also been reported in previous studies[8 10 13] In line with earlier reports it can be suggestedthat stimulation of IgM level is a temporary phenomenonattributable to immunostimulants

Our study also provides evidence that dietary supplemen-tation with BPF could efficiently regulate the expressions ofcertain cytokine-related genes in L rohita IL-120573 and TNF-120572 expression levels are generally considered to be indicatorsof inflammatory response [40] and they can regulate theproduction of other cytokines [2] Coexpression of IL-1120573 andTNF-120572 is not unusual because they share similar roles in theinitiation of immune response [41] We found that dietaryadministration of 5 BPF (B5) significantly upregulated theexpressions of IL-1120573 and TNF-120572 in both examined tissuesRecently we demonstrated that administration of 03 or05 guava leaves for 60 days significantly augmented theexpressions of IL-1120573 and TNF-120572 in the head kidney intestineand hepatopancreas of L rohita [8] Dietary administrationof R glutinosa augmented the expressions of IL-1120573 and TNF-120572 genes in the common carp Cyprinus carpio [2] Howeverabundance of the two genes differed in the same organ of fishthat were fed various levels of BPF

iNOS and nitric oxide (NO) are recognised immuno-regulatory factors in the defence against various pathogensin fish [8] Recently we observed that dietary administrationof guava leaves significantly downregulated the expressionsof iNOS and NF-120581B in L rohita [8] In the present study weobserved that dietary administration of BPF downregulatedthe expressions of NF-120581B in the examined tissues whereasiNOS expression was significantly downregulated in only thehead kidney in the B5 group In contrast R glutinosa rootpowder enhanced iNOS gene expression in the head kidneyofC carpio [2]Themechanismbywhich BPF downregulatediNOS or NF-120581B expression is not yet known Howeverinhibition of NO activity by water extracts of banana peelshas been reported in the literature [42]

HSPs are conserved proteins induced by heat and severalnoxious stimuli including thermal shock heavy metalsviruses oxygen free radicals and pathological stress [43]HSP70 has a number of functions including maintenanceof cellular homeostasis and general protective effects on allliving organisms after stress [44] Liu et al [9] reportedan increase in HSP70 mRNA expression in Macrobrachiumrosenbergii after dietary administration of anthraquinoneextract for 6 or 8 weeks However HSP70 expression inthe blood of grass carp was remarkably downregulated afteradministration of Ficus carica polysaccharide [11] In thisstudy expression of HSP70 was significantly elevated in thehead kidney of the B3 and B5 groups but BPF had nosignificant effect onHSP70 expression in the hepatopancreasDietary emodin and high doses of vitamin C increasedHSP70 mRNA expression in Wuchang bream under hightemperature stress which is consistent with the results of thepresent study [12] However further studies are necessaryto elucidate the mechanism by which BPF affects HSP70expression in L rohita


IL-10 and TGF-120573 are regulatory cytokines with multi-functional roles in the immune system IL-10 limits the mag-nitude of immune responses to foreign pathogens [45] In thisstudy IL-10 expression was downregulated in the examinedtissues of L rohita fed a B3 or B5 diet We observed a counterrelationship between the expressions of TNF-120572 and IL-10These results correspond with those of previous studies [2 814] Swain et al [46] examined themechanism of IL-10 induc-tion by blocking NF-120581B signalling with BAY11-7082 in kidneycell culture of Catla catla Blocking of NF-120581B suppressed IL-10 induction by lipopolysaccharides suggesting that IL-10was induced through the NF-120581B signalling pathway in Catlacatla Normally TGF-120573 inhibits B-andT-cell propagation anddifferentiation antagonises proinflammatory cytokines (IL-1120573 TNF-120572 and IFN-120574) and blocks expressions of IL-1120573 andIL-12 receptors [36] In the present study TGF-120573 expressionwas significantly downregulated in the head kidney of the B5groups alone which is consistent with the results of previousstudies [2 8] Active constituents of banana peels may beresponsible for observed anti-inflammatory properties Forexample vitamin E which is present in banana peel is knownto have potent anti-inflammatory activities [47] Pectin isan eco-friendly and biodegradable polysaccharide present inbanana peel and it has been used as an antimicrobial andanti-inflammatory agent [48] Anti-inflammatory activitiesof banana peel extracts have also been reported in previousstudies [42 49]

After challenging with A hydrophila all BPF-fed groupsexhibited higher survival than the control and the highestsurvival (70) was observed in the B5 group Our resultsindicated that BPF assisted in the control of microbialpathogens as well as infections Higher survival in B5 fedgroup may be due to the augmentation of immune param-eters (LA PA ACP SOD and CAT) decreased level ofMDA and stimulation of immune-related genes Recentlyseveral studies have shown that fish fed with herbal dietsexhibited higher resistance to pathogen infections [2 810 13 14 33] Active components present in banana peelhave higher antibacterial activities against gram-positive andgram-negative pathogens [48 49] Moreover hydroxyapatitenanoparticles derived from banana peel pectin had strongantibacterial activity against S aureus and Escherichia coli[48]

5 Conclusions

Present study reveals that dietary administration of 5 BPF(B5) for 60 days has the potential to modulate growth param-eters antioxidant status immune responses and expressionof cytokine genes in L rohita In addition 5 BPF upreg-ulated HSP70 expression and enhanced the postchallengesurvival rate of L rohita Therefore banana peel can be usedas a feed additive in aquaculture to improve fish growthand disease resistance Therefore this inexpensive and easilyavailable natural immunostimulant may represent a viablealternative to prophylactic use of chemicals in freshwateraquaculture However further studies should be conductedto better understand themechanisms underlying dietary BPFin fish

Additional Points

Venkatachalam Sukumaran present address is Departmentof Zoology Kundavai Nachiyar Government Arts Collegefor Women (Autonomous) Thanjavur Tamil Nadu 613007India

Competing Interests

The authors declare no conflict of interests

Acknowledgments

Authors gratefully acknowledge the research grant receivedfrom National Research Foundation of Korea Ministry ofEducation (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 Maiti M Shetty M Shekar I Karunasagar and I Karun-asagar ldquoEvaluation of two outer membrane proteins Aha1 andOmpWofAeromonas hydrophila as vaccine candidate for com-mon carprdquo Veterinary Immunology and Immunopathology vol149 no 3-4 pp 298ndash301 2012

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

[5] 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

[6] C Uribe H Folch R Enriquez and G Moran ldquoInnate andadaptive immunity in teleost fish a reviewrdquo Veterinarni Medic-ina vol 56 no 10 pp 486ndash503 2011

[7] M Reverter N Bontemps D Lecchini B Banaigs and P SasalldquoUse of plant extracts in fish aquaculture as an alternative tochemotherapy current status and future perspectivesrdquo Aqua-culture vol 433 pp 50ndash61 2014

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

[9] B Liu X Ge Y He et al ldquoEffects of anthraquinones extractedfromRheum officinaleBail on the growth non-specific immuneresponse of Macrobrachium rosenbergiirdquo Aquaculture vol 310no 1-2 pp 13ndash19 2010

[10] 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 resistanceto Aeromonas hydrophila in Labeo rohita (Hamilton) finger-lingsrdquo Fish and Shellfish Immunology vol 29 no 3 pp 508ndash5122010


[11] 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 and Shellfish Immunology vol42 no 1 pp 132ndash137 2015

[12] J H Ming J Xie P Xu X P Ge W B Liu and J Y Ye ldquoEffectsof emodin and vitamin C on growth performance biochemicalparameters and two HSP70s mRNA expression of Wuchangbream (Megalobrama amblycephala Yih) under high tempera-ture stressrdquo Fish and Shellfish Immunology vol 32 no 5 pp651ndash661 2012

[13] S S Giri S Jai Suda V Sukumaran and S C Park ldquoDietaryemodin affects the growth performance immune responsesand disease resistance of Labeo rohita against Aeromonashydrophilardquo Aquaculture International vol 24 no 1 pp 85ndash992016

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

[15] C Svanborg G Godaly and M Hedlund ldquoCytokine responsesduringmucosal infections role in disease pathogenesis and hostdefencerdquo Current Opinion in Microbiology vol 2 no 1 pp 99ndash105 1999

[16] A DrsquoHont F Denoeud J-M Aury et al ldquoThe banana (Musaacuminata) genome and the evolution of monocotyledonousplantsrdquo Nature vol 488 no 7410 pp 213ndash217 2012

[17] FAO FAOSTAT Food and Agriculture Organization GenevaSwitzerland 2013

[18] T H Emaga S N Ronkart C Robert B Wathelet and MPaquot ldquoCharacterisation of pectins extracted from bananapeels (Musa AAA) under different conditions using an exper-imental designrdquo Food Chemistry vol 108 no 2 pp 463ndash4712008

[19] T H Emaga R H Andrianaivo B Wathelet J T Tchango andM Paquot ldquoEffects of the stage of maturation and varieties onthe chemical composition of banana and plantain peelsrdquo FoodChemistry vol 103 no 2 pp 590ndash600 2007

[20] J L Kelsey ldquoA review of research on effect of fibre intake onmanrdquo American Journal of Clinical Nutrition vol 31 pp 142ndash159 1978

[21] A Pereira andM Maraschin ldquoBanana (Musa spp) from peel topulp ethnopharmacology source of bioactive compounds andits relevance for human healthrdquo Journal of Ethnopharmacologyvol 160 pp 149ndash163 2015

[22] L P G Rebello A M Ramos P B Pertuzatti M T Barcia NCastillo-Munoz and I Hermosın-Gutierrez ldquoFlour of banana(Musa AAA) peel as a source of antioxidant phenolic com-poundsrdquo Food Research International vol 55 pp 397ndash403 2014

[23] S Sundaram S Anjum P Dwivedi and G K Rai ldquoAntioxidantactivity and protective effect of banana peel against oxidativehemolysis of human erythrocyte at different stages of ripeningrdquoApplied Biochemistry and Biotechnology vol 164 no 7 pp 1192ndash1206 2011

[24] W Rattanavichai and W Cheng ldquoDietary supplement ofbanana (Musa acuminata) peels hot-water extract to enhancethe growth anti-hypothermal stress immunity and diseaseresistance of the giant freshwater prawnMacrobrachium rosen-bergiirdquo Fish and Shellfish Immunology vol 43 no 2 pp 415ndash426 2015

[25] S S Giri S S Sen and V Sukumaran ldquoRole of HSP70 in cyto-plasm protection against thermal stress in rohu Labeo rohitardquoFish and Shellfish Immunology vol 41 no 2 pp 294ndash299 2014

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using realtime quantitative PCR and the 2minus998779998779CTmethodrdquoMethods vol 25 pp 402ndash408 2001

[27] M Soltani R Alimardani andM Omid ldquoPrediction of bananaquality during ripening stage using capacitance sensing systemrdquoAustralian Journal of Crop Science vol 4 no 6 pp 443ndash4472010

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

[29] 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

[30] A E Ellis ldquoLysozyme assayrdquo in Techniques in Fish ImmunologyJ S Stolen T C Fletcher D P Anderson B S Robertson andW B Van Muiswinkel Eds pp 101ndash103 SOS Publications FairHaven NJ USA 1990

[31] T Yano ldquoAssays of hemolytic complement activityrdquo in Tech-niques in Fish Immunology J S Stolen T C Fletcher D PAnderson S L Kaattari andA E Rowley Eds pp 131ndash141 SOSPublications Fair Haven NJ USA 1992

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

[33] C-N Zhang X-F Li W-N Xu et al ldquoCombined effects ofdietary fructooligosaccharide and Bacillus licheniformis oninnate immunity antioxidant capability and disease resistanceof triangular bream (Megalobrama terminalis)rdquo Fish and Shell-fish Immunology vol 35 no 5 pp 1380ndash1386 2013

[34] H H Draper E J Squires H Mahmoodi J Wu S Agarwaland M Hadley ldquoA comparative evaluation of thiobarbituricacid methods for the determination of malondialdehyde inbiological materialsrdquo Free Radical Biology and Medicine vol 15no 4 pp 353ndash363 1993

[35] M A Esteban ldquoAn overview of the immunological defenses infish skinrdquo ISRN Immunology vol 2012 Article ID 853470 29pages 2012

[36] L Weifen Z Xiaoping S Wenhui et al ldquoEffects of Bacilluspreparations on immunity and antioxidant activities in grasscarp (Ctenopharyngodon idellus)rdquo Fish Physiology and Biochem-istry vol 38 no 6 pp 1585ndash1592 2012

[37] B A Freeman and J D Crapo ldquoBiology of disease free radicalsand tissue injuryrdquo Laboratory Investigation vol 47 no 5 pp412ndash426 1982

[38] B Magnadottir ldquoInnate immunity of fish (Overview)rdquo Fish andShellfish Immunology vol 20 no 2 pp 137ndash151 2006

[39] I Fridovich ldquoSuperoxide radical and superoxide dismutasesrdquoAnnual Review of Biochemistry vol 64 pp 97ndash112 1995

[40] C J Secombes T Wang S Hong et al ldquoCytokines and innateimmunity of fishrdquo Developmental and Comparative Immunol-ogy vol 25 no 8-9 pp 713ndash723 2001

[41] B Gorgoglione T Wang C J Secombes and J W HollandldquoImmune gene expression profiling of Proliferative Kidney Dis-ease in rainbow troutOncorhynchusmykiss reveals a dominanceof anti-inflammatory antibody and T helper cell-like activitiesrdquoVeterinary Research vol 44 no 1 article 55 2013


[42] P Phuaklee S Ruangnoo and A Itharat ldquoAnti-inflammatoryand antioxidant activities of extracts from Musa sapientumpeelrdquo Journal of the Medical Association of Thailand vol 95 pp142ndash146 2012

[43] E D I VegaM R Hall BMDegnan andK JWilson ldquoShort-term hyperthermic treatment of Penaeus monodon increasesexpression of heat shock protein 70 (HSP70) and reducesreplication of gill associated virus (GAV)rdquoAquaculture vol 253no 1ndash4 pp 82ndash90 2006

[44] N Basu A E Todgham P A Ackerman et al ldquoHeat shockprotein genes and their functional significance in fishrdquo Genevol 295 no 2 pp 173ndash183 2002

[45] 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

[46] B Swain M Samanta M Basu et al ldquoMolecular characteri-zation inductive expression and mechanism of interleukin-10gene induction in the Indian major carp catla (Catla catla)rdquoAquaculture Research vol 43 no 6 pp 897ndash907 2012

[47] A Scalbert I T Johnson andM Saltmarsh ldquoPolyphenols anti-oxidants and beyondrdquo The American Journal of Clinical Nutri-tion vol 81 no 1 pp 215ndash217 2005

[48] D Gopi K Kanimozhi N Bhuvaneshwari J Indira and LKavitha ldquoNovel banana peel pectin mediated green route forthe synthesis of hydroxyapatite nanoparticles and their spectralcharacterizationrdquo Spectrochimica Acta Part A Molecular andBiomolecular Spectroscopy vol 118 pp 589ndash597 2014

[49] P Jain M H Bhuiyan K R Hossain and S C Bachar ldquoAnti-bacterial and antioxidant activities of local seeded bananafruitsrdquo African Journal of Pharmacy and Pharmacology vol 5no 11 pp 1398ndash1403 2011

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


carica polysaccharide [11] emodin [12 13] and Achyranthesaspera [14] have been reported to enhance fish immunityMoreover some products of herbal plants have been reportedto alter the expression of cytokine- and immune-related genesin fish [2 8 11 14] They play vital role in host innateimmunity and are indispensable for the use and activation ofmacrophages neutrophils and lymphocytes at infection sitesfor pathogen elimination [15]

Banana (Musa spp) is vital for food security in manytropical and subtropical countries and they are the mostpopular fruit in industrialized countries [16] Banana is thesecond leading fruit produced after citrus contributing toapproximately 17 of the worldrsquos total fruit production it iscultivated in over 130 countries [17] Banana peel which con-stitutes up to 35 of the ripe fruit is a household and indus-trial food waste discarded in large quantities [18] It is rich indietary fibre proteins essential amino acids vitamins poly-unsaturated fatty acids and potassium [19] Soluble fibresare well known to lower serum cholesterol and help reducethe risk of colon cancer [20] Bioactive compounds likeflavonoids tannins phlobatannins alkaloids glycosidesanthocyanins and terpenoids were found in banana peelsand these compounds have been reported to exert variousbiological and pharmacological effects (antibacterial anti-hypertensive antidiabetic and anti-inflammatory activities)[21] Further antioxidant compounds (eg prodelphinidinspolyphenols catecholamines and carotenoids) [22] and highamount of micronutrients [23] were found in the peels ofgenusMusaThe presence of various bioactive compounds inbanana peels suggests that the peels possess variousmedicinalproperties and may be useful as immunostimulant [24]

The effect of oral immunostimulants on the immuneresponse in aquatic animals is strongly associated with thedose and duration of application In the present study weaimed to investigate the effects of dietary supplementationof banana peels on the growth antioxidant status immuneparameters and cytokine gene expression of L rohita and itssusceptibility toA hydrophila infectionWe also explored thepotential of banana peel as a feed additive in aquaculture

2 Materials and Methods

21 Diet Preparation Bananas (Musa acuminata) were pur-chased from the local market (Thanjavur Tamil Nadu) witha skin colour index of 6 (all yellow) [27] The bananas wererinsed thoroughly in tap water followed by double distilledwater and acetone to remove any contaminants They werethen separated into pulps and peels The peels were cut intopieces of about 5 cm2 by using a kitchen knife and air dried inthe oven at 50∘C for 24 h Then the pieces were ground intoa fine powder by using the laboratory grinder The obtainedflour called BPF was stored at minus20∘C until use

The basal diet (Table 1) was prepared using a previouslydescribed method [8] Proximate analysis of the basal diet[28] revealed a composition of 331 proteins 89 lipidsand 12 ash The basal diet was used as the control diet Thebasal diet was supplemented with BPF at five concentrations0 (control diet) 1 (B1) 3 (B3) 5 (B5) and 7 (B7) Allingredients were blended thoroughly into a mixture and then

Table 1 Formulation and chemical composition of basal diet (g kgminus1dry matter)

Ingredients Concentrations (g kgminus1)Ground nut oil cake 390Rice bran 340Soybean meal 170Fish meal 78Vegitable oil 20Mineral and vitamin mixturea 2Proximate analysis (g kgminus1)Crude protein 331Crude lipid 89Ash 120

aEvery 250 g of mineral-vitamin mixture (Supplevite-M Sarabhai ZydusAnimal Health Ltd India) provides vitamin A 500000 IU vitamin D3100000 IU vitamin B2 02 g vitamin E 75 units vitamin K 01 g calciumpantothenate 025 g nicotinamide 01 g vitamin B12 06mg choline chlo-ride 15 g calcium 75 g manganese 275 g iodine 01 g iron 075 g zinc15 g copper 02 g and cobalt 0045 g

pelletized air-dried ground and sieved into an appropriatepellet sizeThe pellets were then stored at minus20∘C until furtheruse

22 Experimental Design Labeo rohita fingerlings (meanbodyweight 153 g) obtained from local fish farm (ThanjavurTamil Nadu India) were acclimatised to laboratory condi-tions in 500 L plastic tanks at 26 plusmn 2∘C for 2 weeks and fedthe basal diet Approximately 20 of the water in all tankswas exchanged daily and 100 of the water was exchangedevery week Basic physiochemical parameters of the waterwere measured every week [29] Oxygen and ammoniaconcentrations were 61ndash73120583gmLminus1 and 003ndash006120583gmLminus1respectively and pH ranged from 7 to 8 throughout the studyperiod

The fish were randomly divided into five experimentalgroups with three replicates in each group Tank capacitywas 200 L and each tank contained 25 fish (ie per group25 times 3 = 75 fish) The fish were fed one of the five diets for60 days and three times (at 0800 1300 and 1800 hours)at 3ndash5 of body weight daily The amount of feed consumedwas determined by daily recovery of excess feed which wasthen adjusted every 15 days by batch weighing after 24 h ofstarvation

23 Growth Performance Ten fish were randomly selectedfrom each tank (ie 10 times 3 = 30 fish per group) at day 0 day30 and day 60 after experimental feeding and batch-weighedto estimate growth performance Growth performance andsurvival rate of the fish were calculated using the followingformulae weight gain (WG gfish) =119882

119905minus1198820 specific growth

rate (SGR) = 100 times [(ln119882119905minus ln119882

0)119905] feed conversion ratio

(FCR) = FI(119882119905minus1198820) where119882

119905and119882

0were final and initial

weights of the fish respectively 119905 is the duration of feeding(in days) and FI is feed intake










PR () = [100
































3 Results

















DietImmune response










DietImmune response











DietImmune response















DietImmune response









0123456789

10


TNF-120572

expr

essio

n re

lativ

e to 120573

-act

in

CB1B3

B5B7

lowast

lowast

lowastlowast

(a)


CB1B3

B5B7

0

05

1

15

2

25

3

IL-1120573

expr

essio

n re

lativ

e to 120573

-act

inlowast

lowast

lowast

lowast

(b)


CB1B3

B5B7

0

05

1

15

2

25

HSP

70 ex

pres

sion

relat

ive t

o 120573

-act

in lowast

lowast

(c)




CB1B3

B5B7

0

05

1

15

2

25IL

-10

expr

essio

n re

lativ

e to 120573

-act

in

lowast

lowast

lowast

lowast lowast

(a)


CB1B3

B5B7

002040608

112141618

2

iNO

S ex

pres

sion

relat

ive t

o 120573

-act

in

lowastlowast

(b)


CB1B3

B5B7

002040608

112141618

2

NF120581

B ex

pres

sion

relat

ive t

o 120573

-act

in

lowastlowast

lowastlowastlowast

(c)


CB1B3

B5B7

0

05

1

15

2

25

TGF-120573

expr

essio

n re

lativ

e to 120573

-act

in

lowast

(d)






4 Discussion














5 Conclusions


Additional Points


Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























PR () = [100
































3 Results

















DietImmune response










DietImmune response











DietImmune response















DietImmune response









0123456789

10


TNF-120572

expr

essio

n re

lativ

e to 120573

-act

in

CB1B3

B5B7

lowast

lowast

lowastlowast

(a)


CB1B3

B5B7

0

05

1

15

2

25

3

IL-1120573

expr

essio

n re

lativ

e to 120573

-act

inlowast

lowast

lowast

lowast

(b)


CB1B3

B5B7

0

05

1

15

2

25

HSP

70 ex

pres

sion

relat

ive t

o 120573

-act

in lowast

lowast

(c)




CB1B3

B5B7

0

05

1

15

2

25IL

-10

expr

essio

n re

lativ

e to 120573

-act

in

lowast

lowast

lowast

lowast lowast

(a)


CB1B3

B5B7

002040608

112141618

2

iNO

S ex

pres

sion

relat

ive t

o 120573

-act

in

lowastlowast

(b)


CB1B3

B5B7

002040608

112141618

2

NF120581

B ex

pres

sion

relat

ive t

o 120573

-act

in

lowastlowast

lowastlowastlowast

(c)


CB1B3

B5B7

0

05

1

15

2

25

TGF-120573

expr

essio

n re

lativ

e to 120573

-act

in

lowast

(d)






4 Discussion














5 Conclusions


Additional Points


Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of







































3 Results

















DietImmune response










DietImmune response











DietImmune response















DietImmune response









0123456789

10


TNF-120572

expr

essio

n re

lativ

e to 120573

-act

in

CB1B3

B5B7

lowast

lowast

lowastlowast

(a)


CB1B3

B5B7

0

05

1

15

2

25

3

IL-1120573

expr

essio

n re

lativ

e to 120573

-act

inlowast

lowast

lowast

lowast

(b)


CB1B3

B5B7

0

05

1

15

2

25

HSP

70 ex

pres

sion

relat

ive t

o 120573

-act

in lowast

lowast

(c)




CB1B3

B5B7

0

05

1

15

2

25IL

-10

expr

essio

n re

lativ

e to 120573

-act

in

lowast

lowast

lowast

lowast lowast

(a)


CB1B3

B5B7

002040608

112141618

2

iNO

S ex

pres

sion

relat

ive t

o 120573

-act

in

lowastlowast

(b)


CB1B3

B5B7

002040608

112141618

2

NF120581

B ex

pres

sion

relat

ive t

o 120573

-act

in

lowastlowast

lowastlowastlowast

(c)


CB1B3

B5B7

0

05

1

15

2

25

TGF-120573

expr

essio

n re

lativ

e to 120573

-act

in

lowast

(d)






4 Discussion














5 Conclusions


Additional Points


Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























DietImmune response










DietImmune response











DietImmune response















DietImmune response









0123456789

10


TNF-120572

expr

essio

n re

lativ

e to 120573

-act

in

CB1B3

B5B7

lowast

lowast

lowastlowast

(a)


CB1B3

B5B7

0

05

1

15

2

25

3

IL-1120573

expr

essio

n re

lativ

e to 120573

-act

inlowast

lowast

lowast

lowast

(b)


CB1B3

B5B7

0

05

1

15

2

25

HSP

70 ex

pres

sion

relat

ive t

o 120573

-act

in lowast

lowast

(c)




CB1B3

B5B7

0

05

1

15

2

25IL

-10

expr

essio

n re

lativ

e to 120573

-act

in

lowast

lowast

lowast

lowast lowast

(a)


CB1B3

B5B7

002040608

112141618

2

iNO

S ex

pres

sion

relat

ive t

o 120573

-act

in

lowastlowast

(b)


CB1B3

B5B7

002040608

112141618

2

NF120581

B ex

pres

sion

relat

ive t

o 120573

-act

in

lowastlowast

lowastlowastlowast

(c)


CB1B3

B5B7

0

05

1

15

2

25

TGF-120573

expr

essio

n re

lativ

e to 120573

-act

in

lowast

(d)






4 Discussion














5 Conclusions


Additional Points


Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















DietImmune response









0123456789

10


TNF-120572

expr

essio

n re

lativ

e to 120573

-act

in

CB1B3

B5B7

lowast

lowast

lowastlowast

(a)


CB1B3

B5B7

0

05

1

15

2

25

3

IL-1120573

expr

essio

n re

lativ

e to 120573

-act

inlowast

lowast

lowast

lowast

(b)


CB1B3

B5B7

0

05

1

15

2

25

HSP

70 ex

pres

sion

relat

ive t

o 120573

-act

in lowast

lowast

(c)




CB1B3

B5B7

0

05

1

15

2

25IL

-10

expr

essio

n re

lativ

e to 120573

-act

in

lowast

lowast

lowast

lowast lowast

(a)


CB1B3

B5B7

002040608

112141618

2

iNO

S ex

pres

sion

relat

ive t

o 120573

-act

in

lowastlowast

(b)


CB1B3

B5B7

002040608

112141618

2

NF120581

B ex

pres

sion

relat

ive t

o 120573

-act

in

lowastlowast

lowastlowastlowast

(c)


CB1B3

B5B7

0

05

1

15

2

25

TGF-120573

expr

essio

n re

lativ

e to 120573

-act

in

lowast

(d)






4 Discussion














5 Conclusions


Additional Points


Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















CB1B3

B5B7

0

05

1

15

2

25IL

-10

expr

essio

n re

lativ

e to 120573

-act

in

lowast

lowast

lowast

lowast lowast

(a)


CB1B3

B5B7

002040608

112141618

2

iNO

S ex

pres

sion

relat

ive t

o 120573

-act

in

lowastlowast

(b)


CB1B3

B5B7

002040608

112141618

2

NF120581

B ex

pres

sion

relat

ive t

o 120573

-act

in

lowastlowast

lowastlowastlowast

(c)


CB1B3

B5B7

0

05

1

15

2

25

TGF-120573

expr

essio

n re

lativ

e to 120573

-act

in

lowast

(d)






4 Discussion














5 Conclusions


Additional Points


Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























5 Conclusions


Additional Points


Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















)joebxj1vcmjtijoh$psqpsbujpo …downloads.hindawi.com/journals/jir/2016/4086591.pdfresearch article...

Documents