Chiang Mai J. Sci. 2018; 45(1) 249

Chiang Mai J. Sci. 2018; 45(1) : 249-262http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Antioxidant, Anti-tyrosinase and ImmunomodulatoryActivities of the Enzymatic Boiled Venus ClamHydrolysateChanutchamon Sutthiwanjampa and Sang Moo Kim*

Department of Marine Food Science and Technology, Gangneung-Wonju National University, Gangneung,

Gangwon-do 25457, Korea.

*Author for correspondence; e-mail: smkim@gwnu.ac.kr

Received: 7 April 2016

Accepted: 8 July 2016

ABSTRACT

The antioxidant, anti-tyrosinase and immunomodulatory activities of the boiledVenus clam hydrolysate (BVH) produced by Alcalase was determined. The amino acidcomposition of BVH was rich in Gly, Glu, Asp, Thr, Ala, Ser, Pro, Val and Leu. The IC

50

values of BVH on antioxidant activity were 201.11 mg/mL for DPPH scavenging activity,424.61 mg/mL for hydrogen peroxide radical scavenging activity and 66.78 mg/mLfor hydroxyl radical scavenging activity. The BVH exhibited strong anti-tyrosinase activity,with the IC

50 value of 458.54 mg/mL. Furthermore, the BVH (0.5-5 mg/mL) stimulated the

production of nitric oxide, as well as pro- and anti-inflammatory cytokines by stimulatingRAW 264.7 cells through the activation of PI3K/Akt and NF-kB pathways. Therefore, theBVH might be utilized as a food or cosmeceutical ingredient because of its strong antioxidant,anti-tyrosinase and immunomodulatory activities.

Keywords: antioxidant, anti-tyrosinase, clam, enzymatic hydrolysate, immunomodulatory activity

1. INTRODUCTION

Recently, biofunctional activities such asanti-aging, whitening, anti- imflammatory andimmunomodulatory of various compoundshave attracted more attention. Free radicalsand other reactive oxygen species (ROS),which are normally formed during theoxidative metabolic process, causes manyhealth disorders, such as hypertension,cardiovascular, cancer, diabetes mellitus,neurodegenerative, and inflammatory diseasesas well as aging [1]. Tyrosinase is a keyenzyme in the melanin biosynthetic pathway.

Hence, tyrosinase inhibitors are of importantinterest and attention in food industry due toenzymatic browning in fruit and vegetables,as well as in cosmetic industry due toskin-whitening effect [2]. The immune systemis essential and important to prevent andcontrol infection and neoplasia. Enzymatichydrolysis of dietary proteins gives a rapidand reproducible method for the productionof considerable bioactive peptides respondedto the immune system. These peptide arelikely to become potential health beneficial

250 Chiang Mai J. Sci. 2018; 45(1)

food ingredients or nutraceutical preparations[3].

Although several biofunctional activitiesof the enzymatic protein hydrolysate such asantioxidant, antihypertensive, antithrombotic,antibacterial and immunomodulatoryactivity have been reported [4], theimmunomodulatory activity of marineshellfish hydrolysates has not yet beenreport so far. In our previous study [5],the enzymatic boiled Venus clam hydrolysatehad strong hyaluronidase and elastaseinhibitory activities. In this study, its furtherbiofunctional activities such as antioxidant,anti-tyrosinase and immunomodulatoryactivities were determined.

2. MATERIALS AND MEHODS

2.1 Raw Material and ReagentsFresh Venus clams (Gomphina melanaegis)

were purchased from a local market(Gangneung, Korea) and transported to thelaboratory in an ice box within one h priorto use. Alcalase 2.4 L was purchasedfrom Novozyme (Bagsvaerd, Denmark). 1,1-Diphenyl-2-pycryl-hydrazyl (DPPH),ferrous sulfate, hydrogen peroxide, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS), pyrogallol, peroxidase, L-ascorbicacid, mushroom tyrosinase, and L-3,4′-dihydroxyphenylalanine (L-DOPA) werepurchased from Sigma Chemical Co.(St. Louis, MO, USA). Macrophage cell lineRAW 264.7 was purchased from Korean CellLine Bank (Seoul, Korea). Roswell ParkMemorial Institute medium (RPMI-1640),fetal bovine serum (FBS) and penicillin werepurchased from Lonza (Walkersville, MD,USA). TRIzol reagent and Superscript IIIRT were purchased from Invitrogen(Carlsbad, CA, USA). GoTaq Flexi DNApolymerase was purchased from Promega

(Madison, WI, USA). PCR primers werepurchased from Bioneer (Daejeon, Korea).Anti-Akt, anti-phospho-inhibitor of Akt,anti-nuclear factor I-κB-α, anti-phospho-inhibitor of nuclear factor I-κB-α ,and anti-actin were purchased fromSanta Cruz Biotechnology (Santa Cruz, CA,USA). Horseradish peroxidase-conjugatedanti-rabbit or anti-mouse secondaryantibodies were purchased from StressgenBiotechnologies (Stressgen, CA, USA).Enhanced chemiluminescence (ECL) solutionwas purchased from Thermo Scientific(Waltham, MA, USA). All of the otherreagents that were used were of analyticalgrade.

2.2 Preparation of Sample and EnzymaticHydrolysis of Boiled Venus Clams UsingAlcalase

The boiled Venus clam hydrolysate (BVH)was prepared according to the method ofour previous study [5]. The Venus clamswere washed using tap water and were thenboiled at 100 °C for 20 min. The boiledVenus clams were washed again usingdistilled water and were exuviated usinga scalpel. The exuviated clams werehomogenized, lyophilized (Type FD-1000;Eyela, Tokyo, Japan), and then stored at-40 °C prior to use.

2.3 Analysis of Amino Acid CompositionTwenty five g of sample was hydrolyzed

in 5 mL of 6 N HCl at 110 °C for 24 h.The hydrolysate was dried and dissolved in1 mL of distilled water. Then the samplesolution was filtered using a 0.2 mm celluloseacetate syringe filter unit (Toyo Roshi KaishaLtd., Tokyo, Japan). The resulting solutionwas analyzed using an amino acid analyzer(Hitachi, Tokyo, Japan).

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2.4 Determination of AntioxidantActivity2.4.1 DPPH radical scavenging activity

DPPH radical scavenging activitywas determined according to the slightlymodified method of Blois (1958) [6].Two hundred mL of sample was mixed with50 mL of 0.15 mM DPPH solution inethanol. After stood for 30 min in the darkat room temperature, the absorbance at515 nm was determined a microplate reader(EL-800; Biotek, Vermont, USA).

2.4.2 Superoxide anion radicalscavenging activity

Superoxide radical scavenging activitywas determined according to the modifiedmethod of Marklund and Marklund (1974)[7]. Three mL of 50 mM Tris-HCl buffer(pH 8.5) and 0.2 mL of 7.2 mM pyrogallolwere mixed with 0.2 mL of sample.The mixture was incubated at 25 °C for10 min and then 1 mL of 1 N HCl was addedto terminate the reaction. The absorbanceat 420 nm was measured.

2.4.3 Hydrogen peroxide radicalscavenging activity

Hydrogen peroxide radical scavengingactivity was determined according tothe method of Muller (1985) [8]. Onehundred mL of 0.1 M phosphate buffer(pH 5.0) and 100 mL of sample solution weremixed. Twenty L of 2 mM peroxide wasadded to the mixture and was then incubatedat 37 °C for 5 min. Each 30 mL of 1.25 mM2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1 U/mL ofperoxidase were added to the mixture,which was then incubated at 37 °C for10 min. The absorbance at 405 nm wasmeasured.

2.4.4 Hydroxyl radical scavenging activityHydroxyl radical scavenging activity

was determined according to the slightlymodified method of Sudha et al. (2011) [9].One mL of the sample was mixed with1 mL of 1.5 mM FeSO

4, 0.7 mL of 6 mM

hydrogen peroxide and 0.3 mL of 20 mMsodium salicylate. After incubated at 37 °Cfor 1 h, the absorbance of the salicylatecomplex at 562 nm was measured.

Antioxidant activity was calculated by thefollowing equation;

Scavenging activity (%) = {[(A - B) - (C - D)]/(A - B)}×100

where A was the control; B was thecontrol blank; C was the sample; D was thesample blank.

2.5 Determination of Anti-tyrosinaseActivity

Anti-tyrosinase activity was determinedaccording to the modified method ofChang et al. (2007) [10]. One hundred mLof the sample was mixed with 880 mL of2 mM L-3,4′-dihydroxyphenylalanine(L-DOPA) dissolved in 50 mM phosphatebuffer (pH 6.8) and was then incubated at 25°C for 20 min. Afterwards, 20 mL oftyrosinase (1000 U/mL in phosphate buffer,pH 6.8) was added to initiate the reaction.The mixture was incubated at 25 °C for10 min. The increase in absorbance at492 nm due to the formation of dopachromewas monitored using a spectrophotometer(V-300; JASCO, Tokyo, Japan). Test samplesreplaced by distilled water was used for thecontrol, while the enzyme solution wasreplaced by buffer solution was used for theblank. Anti-tyrosinase activity was calculatedby the following equation;

252 Chiang Mai J. Sci. 2018; 45(1)

Anti-tyrosinase activity (%) = {[(A - B) - (C -D)]/(A - B)} ×100

where A was the control; B was thecontrol blank; C was the sample; D was thesample blank.

2.6 Immunomodulatory Activity2.6.1 Cell line and culture

The macrophage cell line RAW 264.7was grown in a culture flask usingRPMI-1640 medium containing L-glutaminesupplemented with 10% fetal bovine serum(FBS) and 100/U mL penicillin in 5% CO

2

at 37 °C. After incubated for 3 days, thecells were removed from the culture flaskby scraping and centrifuging at 1,800 g for3 min. The medium was removed andthe cells were then resuspended in freshRPMI-1640 medium. Cell numbers werecounted with a haemocytometer usingTrypan blue exclusion staining and microscopy(Motic AE31; Motic China Group Co. Ltd.,Xiamen, China).

2.6.2 Macrophage proliferation assayOne hundred mL of BVH at different

concentrations (0.5, 1, 5, 10, 50, 100 and 250mg/mL) was added to 100 mL of RAW264.7 cells (1×106 cells/well) in RPMI-1640medium containing 10% FBS plated in a96-well microplate. After incubated at 37 °Cfor 24 hours in a humidified atmospherecontaining 5% CO

2, 20 mL of the WST-1

solution was added to the well, and thenfurther incubated at 37 °C for 1-2 hours.The optical density at 450 nm was measuredusing a microplate reader (EL-800; BioTekInstruments, Winooski, VT, USA). Theabsorbance (A) was translated intomacrophage proliferation ratio (%) = At/Ac×100, where At and Ac were theabsorbance of the test group and the controlgroup, respectively.

2.6.3 Nitrite assayThe immunomodulatory activity of the

protein hydrolysate was determined on thebasis of nitric oxide (NO) production inmacrophage culture supernatants. The nitriteconcentration was measured using theGriess reaction [11]. One hundred mL ofRAW 264.7 cells (1×105 cells/well) was platedin a 96-well microplate and was incubatedat 37 °C for 24 h in a 5% CO

2 incubator.

The cultured cells were treated with 100 mLof BVH solutions at different concentrations(0.5, 1 and 5 mg/mL) or lipopolysaccharidesolution (LPS, 1 mg/mL), the positive control.After incubated at 37 °C for 24 h, 100 mLof the cultured cell supernatant was mixedwith an equal volume of Griss reagent[1% (w/v) sulphanilamide and 0.1% (w/v)N-(1-naphtyl)ethylenediamine hydrochloridein 2.5% (v/v) phosphoric acid] and incubatedat room temperature for 10 min. Theabsorbance at 540 nm was measured using amicroplate reader. The NO production ofthe macrophage cells was calculated using astandard curve obtained using NaNO2

(1-200mM in culture medium).

2.6.4 Prostaglandin E2 assay

For measuring the prostaglandin E2

(PGE2) concentration, one mL of RAW

264.7 cells (1×105 cells/well) in RPMI-1640medium containing 10% FBS and BVH atdifferent concentrations (0.5 to 5 mg/mL) orlipopolysaccharide solution (LPS, 1 mg/mL),the positive control, in a 24-well microplatewas incubated at 37 °C for 72 h in a 5%CO

2 incubator. The PGE

2 concentration of

the supernatants was measured according tothe manual of the examination agent box(R&D System Inc., Minneapolis, MN, USA).

2.6.5 Reverse transcription-polymerasechain reaction (RT-PCR)

The total RNA of RAW 264.7 cells

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treated with LPS and the samples wereextracted using TRIzol reagent accordingto manufacturer’s protocol and were kept at-80 °C until use. The concentration of RNAwas determined using a spectrophotometerbefore constructing cDNA with an oligo-(dT)

20 primer and Superscript III RT.

The PCR amplification was carried out usingGoTaq Flexi DNA polymerase and specificprimers. Reverse transcriptase amplificationwas performed using an initial denaturationat 94 °C for 3 min; 30 cycles of denaturation(94 °C for 30 s), annealing (56 °C for 40 s),extension (72 °C for 1 min) and final extension

step at 72 °C for 10 min. The products ofRT-PCR were run in 1% agarose gelelectrophoresis stained with ethidiumbromide at 100 voltages for 20 min. The gelwas visualized under UV transillumination,analyzed using an image analysis software(Kodak Digital Science, Kennesaw, GA, USA),and was evaluated using relative intensity ofβ-actin, the reference standard. The forwardand reverse primers for inducible NO synthase(iNOS), cycloxygenase (COX)-2, tumornecrosis factor-α (TNF-α), interleukin (IL)1-β, IL-6, IL-10, IL-12 and β-actin wereused in this study (Table 1).

Table 1. Sequence of primer pairs used in reverse transcription-polymerase chain reaction.

COX-2, cyclooxygenase-2; IL, interleukin; iNOS, inducible nitric oxide synthase; TNF-α, tumornecrosis factor-α.

Gene, directioniNOS

ForwardReverse

COX-2ForwardReverse

IL-1βForwardReverse

IL-6ForwardReverse

IL-10ForwardReverse

IL-12ForwardReverse

TNF-αForwardReverse

β-actinForwardReverse

Primer sequence

5′-CCCTTCCGAAGTTTCTGGCAGCAGC-3′5′-GGCTGTCAGAGCCT CGTGGCTTTGG-3′

5′-CCCCCACAGTCAAAGACACT-3′5′-GAGTCCATGTTCCAGGAGGA-3′

5′-ATGGCAACTATTCCTGAACTCAACT-3′5′-CAGGACAGGTATAGATTCTTTCCTTT-3′

5′-TTCC TCTCTGCAAGAGACT-3′5′-TGTATCTCTCTGAAGGACT-3′

5′-TACCTGGTAGAAGTGATGCC-3′5′-CATCATGTATGCTTCTATGC-3′

5′-CCACAAAGGAGGCGAGACTC-3′5′-CTCTACGAGGAACGCACCTT-3′

5′-ATGAGCACAGAAAGCATGATC-3′5′-TACAGGCTTGTCACTCGAATT-3′

5′-TGGAATCCTGTGGCATCCATGAAAC-3′5′-TAAAACGCAGCTCAGTAACAGTCCG-3′

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2.6.6 Western blot analysisRAW 264.7 cells were lysed in

phosphatase inhibitor solution. Then 35 mgof protein from the cell lysate was applied to10% sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). After electrophoresis, theproteins were transferred onto polyvinylidenefluoride (PVDF) membranes and themembranes were blocked using 5% skimmilk in tris-buffered saline solution containing0.1% Tween-20. The membranes were thenimmunoblotted using primary antibodies,Anti-Akt, anti-phospho-inhibitor of Akt,anti-nuclear factor I-κB, anti-phospho-inhibitor of nuclear factor I-κB and anti-β-actin, and were then incubated withhorseradish peroxidase-conjugated anti-rabbitor anti-mouse secondary antibodies ina shaking incubator at 60×g at roomtemperature for 4.5 h. The blotswere developed using an enhancedchemiluminescence (ECL) solution.

2.7 Statistical AnalysisEach experiment was performed at least

in triplicate, and data were expressed as themean values±standard deviation (SD).Statistical analysis of the data was carried outby one way analysis of variance (ANOVA)and Duncan’s New Multiple Range Testusing the SPSS software package (Version 12.0;SPSS Inc., Chicago, IL, USA), with a p valueof < 0.05 set as the level of significance.

3. RESULTS AND DISCUSSION

Based on our previous study [5],the fraction with molecular weight of 26.1kDa was obtained from the enzymatic boiledVenus clam hydrolysate prepared usingAlcalase at optimal hydrolysis condition(E/S, 2.02%; time, 4.11 h; W/S, 69.74 mL/g), followed by the fractionation using aS-200 HR size exclusion chromatography.The boiled Venus clam hydrolysate exhibited

hyaluronidase and elastase inhibitory activitywith specific activity of 141.15 and 81.36%⋅mL/mg, respectively. Therefore, furtherbiofunctional activities including antioxidant,anti-tyrosinase and immunomodulatoryactivities of boiled Venus clams hydrolysatewere determined in this study.

3.1 Amino Acid CompositionThe amino acid composition of BVH

is shown in Table 2. The BVH containedmost of amino acid responsible forantioxidant activity (Val, Leu, Tyr, Phe, Lys,His, Gly, Ala and Arg) [12]. For proteinhydrolysates and peptide, an increase inhydrophobicity would increase theirsolubility in lipid resulting in increasingtheir antioxidative [13]. In this study, thetotal hydrophobic amino acids (Gly, Ala, Val,Ile, Leu, Phe, and Pro) concentration ofBVH was 47.9%. Therefore, the BVH mightpossess antioxidant activity due to its highamount of hydrophobic amino acid.

Table 2. Amino acid compositions of theboiled Venus clam hydrolysate (BVH).

Amino acid

AspThrSerGluGlyAlaValIle

LeuTyrPheLysHisArg

HyproPro

Total

Composition

(nM)0.930.790.731.171.280.730.660.330.480.050.190.360.040.300.380.6813.28

(%)10.208.688.0112.8414.108.037.243.665.310.502.083.980.463.274.187.47100

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3.2 Antioxidant Activity3.2.1 DPPH radical scavenging activity

The DPPH radical scavenging activity ofBVH exhibited a concentration-dependentmanner (Figure 1A). The IC

50 value of BVH

against DPPH was 201.11 mg/mL, whichwas higher than <10 mg/mL of L-ascorbicacid, a positive control. Hence, BVH hadsignificantly weaker DPPH radical scavengingactivity than L-ascorbic acid. However,The IC

50 value of BVH against DPPH

radical (201.11 g/mL) was much lower than2.62 mg/mL of blue mussel (Mytilus edulis)protein hydrolysate [14]. Therefore, the BVHmight be a good antioxidant material becauseof its strong DPPH radical scavengingactivity. The changes in the size, level andcomposition of free amino acids or smallpeptides usually affects antioxidant activity.The low-MW protein hydrolysate fractionshad higher ABTS radical scavenging activityand Fe2+-chelating ability, while high-MWprotein hydrolysate fractions had strongerDPPH radical scavenging activity and reducingpower [1]. Normally, the strong DPPH orother radical scavenging activities of proteinhydrolysates or peptides are associatedwith hydrophobic amino acids or overallhydrophobicity [15]. On the other hand, theaccumulation of shorter peptides and aminoacids could make the peptide fractionsmore hydrophilic. The increased polarity ofthe low-MW protein hydrolysate fractionsresults in more difficult to react with thehydrophobic DPPH radicals [16]. Therefore,the strong DPPH scavenging activity ofBVH might be due to its high compositionof hydrophobic acids (47.9%) and highmolecular weight (26.1 kDa) [5].

3.2.2 Superoxide anion radical scavengingactivity

Superoxide anion radical (O-•) is a highlytoxic species, and can promote oxidative

reactions due to its ability to reduce transitionmetals, release protein-bound metals andform perhydroxyl radicals, which initiatelipid oxidation [17]. Therefore, the scavengingof this radical is important to reduce theoxidative reaction. The superoxide radicalscavenging activity of BVH increased in adose-dependent manner (Figure 1B). At 500mg/mL, the superoxide radical scavengingactivity of BVH was 13.56%, which waslower than 66.80% of L-ascorbic acid, apositive control. However, the BVH exhibitedhigher superoxide radical scavenging activitythan those of black soybean peptides (1 and9% of Fra-I and Fra-II, respectively) [17].

3.2.3 Hydrogen peroxide radicalscavenging activity

It is crucial for the cells to removehydrogen peroxide as an antioxidantdefense because hydrogen peroxide candamage several cellular componentstogether with ROS by generating hydroxylradicals and singlet oxygen through Fentonreaction [18]. The IC50

value of BVH againsthydrogen peroxide was 424.61 mg/mL,which was higher than 27.24 mg/mL ofL-ascorbic acid (Figure 1C). However, theIC

50 value of BVH was much lower than

those of Arca Subcrenata hydrolysates byAlcalase (19.09 mg/mL) and papain (23.52mg/mL) [19]. Therefore, the BVH can beutilized as an antioxidant ingredient to quenchthe hydrogen peroxide radical.

3.2.4 Hydroxyl radical scavenging activityRemoval of hydroxyl radical is probably

one of the most effective defenses of a livingbody against various diseases since hydroxylradical easily reacts with biomolecules, suchas amino acids, proteins, and DNA resultingin cell damage and human disease [20].The hydroxyl radical scavenging activity ofBVH increased as its concentration increased

2

256 Chiang Mai J. Sci. 2018; 45(1)

(Figure 1D). The IC50

value of BVH againsthydroxyl radical was 66.78 mg/mL. At10 mg/mL, the BVH exhibited hydroxylradical scavenging activity of 32.74%, whichwas lower than 64.51% of L-ascorbic acid.However, The IC

50 value of BVH (66.78

mg/mL) was much lower than that of blacksoy bean peptides (Fra-I, 10.56 mg/mL;Fra-II, 7.49 mg/mL; Fra-III, 1.74 mg/mL)[17]. Therefore, the BVH might be utilizedas a potent hydroxyl radical scavenger.

The biofunctional activities of enzymaticprotein hydrolysates are dependent on manyfactors, such as (1) the type of enzyme used,(2) the degree of hydrolysis, and (3) the aminoacid composition, sequence and configurationof peptide [1]. Several amino acids such asGly, Ala, Cys, Val, Met, Tyr, Leu, Phe, Trp,Lys, His and Arg were generally accepted asan antioxidant [12, 21]. Kim et al. reportedthat the protein hydrolysates or peptides

containing Tyr, Pro, Met, Cys, Gly, Ala andHis exhibited strong antioxidant activity [22].The BVH contained 52.44% of amino acidresponsible for antioxidant activity (Table 2).Li et al. reported that hydrophobicity ofprotein hydrolysate or peptides are related tostrong antioxidant activity [15]. In addition,Zhu et al. also reported that the low molecularweight protein hydrolysate showed morehydrophilic and results in more difficult to

Figure 1. Antioxidant activities of the boiled Venus clam hydrolysate (BVH) at differentconcentrations, DPPH scavenging activity (A), Superoxide radical scavenging activity (B),Hydrogen peroxide radical scavenging activity (C) and Hydroxyl radical scavenging activity(D).

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react with the hydrophobic free radicals [16].Therefore, the BVH might be superior toother protein hydrolysate sources to be anantioxidant agent due to its high compositionof amino acid response to antioxidant(52.44%), high composition of hydrophobicacids (47.9%) and high molecular weight(26.1 kDa) [5].

3.3 Anti-tyrosinase ActivityThe anti-tyrosinase activity of BVH is

shown in Figure 2. The tyrosinase inhibitoryactivity of BVH increased as its concentrationsincreased (10-250 mg/mL) (Figure 2).The BVH exhibited significantly weakeranti-tyrosinase activity than kojic acid, apositive control, because the IC50

value ofBVH (458.54 μg/mL) was much higherthan 22.30 μg/mL of kojic acid. However,the anti-tyrosinase activity of BVH wasmore than 50% at 500 μg/mL, which was10 times stronger than 50% at 5 mg/mL ofjelly fish (Rhopilema esculentum) umbrella collagenhydrolysates [23]. In regard to the actionmechanism of tyrosinase inhibitors, blockingoxidative pathway and binding the enzymeactivity sites may be mainly involved toinhibit the activity of tyrosinase [23]. Schurinket al. [24] reported that peptides containingat least one Arg residue, often in combinationwith a Phe residue, are always strongtyrosinase-binding peptide, as well aspeptides containing Val, Ala and/orLeu residues are strong tyrosinase-inhibitingpeptide. BVH contains 25.93% of tyrosinase-inhibiting amino acids. Theses amino acidsmight lead BVH to act as a tyrosinase-bindinginhibitor, which resulted in tyrosinaseinhibition. Therefore, the BVH might be anatural tyrosinase inhibitor and utilized as afunctional ingredient in cosmetic or foodindustries.

3.4 Immunomodulatory ActivityThe BVH did not affect the viability

of RAW 264.7 cells at concentrations up to250 mg/mL (data not shown). Thus, theinhibitory effect of BVH on the RAW264.7 cells was deemed not to be attributableto cytotoxicity. The NO release from RAW264.7 cells by BVH at the concentrations of0.5, 1 and 5 mg/mL is shown in Figure 3A.The amount of NO release from RAW264.7 cells increased in a dose-dependentmanner. At 5 mg/mL of BVH, the amountof NO released was 32.67 mM, which wascomparable to the amount of NO producedby LPS at 1 mg/mL, the positive control.Therefore, the BVH could be a strongstimulant for the NO production of RAW264.7 cells. Hence, the BVH might stimulatethe mRNA expression of NO synthase byincreasing an inducible nitric oxide synthase(iNOS). Thus, an agarose gel analysis of theRT-PCR products using primers for iNOSmRNA was used to determine the mRNAexpression of iNOS in RAW 264.7 cellsby the BVH. The obvious and clear bandswere observed in the agarose gel by BVH(Figure 3B and C). This result indicated

Figure 2. Tyrosinase inhibitory activities ofthe boiled Venus clam hydrolysate (BVH) atdifferent concentrations.

258 Chiang Mai J. Sci. 2018; 45(1)

that the increase in the NO release mightbe due to the increased expression ofiNOS as the result of the activation ofRAW 264.7 cells in the presence of BVH.Therefore, the BVH with molecular mass of

26.1 kDa could strongly stimulate RAW 264.7cells, which was similar to results of theprotein smilaxin isolated from Smilax glabrarhizomes (30 kDa) [25].

Figure 3. Amount of nitric oxide (NO) released (A), mRNA expression of the gene foriNOS and graphic analysis of the polymerase chain reaction product for RNA levels of iNOS(B) of the RAW 264.7 cells stimulated with the boiled Venus clam hydrolysate for 18 h atdifferent concentrations. The mean values in the same column with different alphabets aresignificantly different (p < 0.05).

The BVH at 5 mg/mL also induced theproduction of prostaglandin E

2 (PGE

2),

a pro-inflammatory cytokine, 3.2 ng/mLfrom RAW 264.7 cells, which was comparableto that of the positive control, LPS(1 mg/mL) (Figure 4A). This result wassimilar to the trend of NO release. TheBVH also enhanced the mRNA expressionof prostaglandin syntheses by increasing

a cyclooxygenase-2 (COX-2) concentrationin RAW 264.7 cells (Figure 4B and C).Therefore, the increase in the amount ofPGE

2 produced by BVH might be due to

the increase in the expression of COX-2,which was up-regulated in the activatedRAW 264.7 cells and thus promoted therelease of PGE

2.

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Furthermore, the BVH stimulated theinduction of mRNA expressions of othercytokines including tumor necrosis factor-α(TNF-α), IL-1β, IL-6, IL-10 and IL-12 atthe concentrations of 0.5, 1 and 5 mg/mL(Figure 5), which was similar to the trend ofNO and PGE

2 released. It is known that

the balance between pro-inflammatory andanti-inflammatory cytokines is crucial to thehost’s response to infection. In this study,the BVH increased the mRNA expression byincreasing not only the concentration ofpro-inflammatory (PGE

2, TNF-α, IL-1β

and IL-6), but also anti-inflammatory (IL-10and IL-12) cytokines (Figure 5E and F,respectively). Hence, the released IL-10 andIL-12 might suppress the excessive of

macrophage activation avoiding thepotential aggravation at the recoveringsites. Karnjanapratum et al. (2012) reportedthat sulfated polysaccharide from Capsosiphonfulvescens could enhance the production ofboth pro-inflammatory and anti-inflammatorycytokines from RAW 264.7 cells, whichimplied its considerable immunostimulationwithout potential occurrence of severeinflammation [26]. Therefore, the BVHmight be an immunostimulating proteinhydrolysate, which could activate themacrophages through the pro-inflammatorycytokines production, while suppressingtheir over-activation by releasing anti-inflammatory cytokines.

Figure 4. Amount of prostaglandin E2 (PGE

2) production (A), mRNA expression of the

gene for COX-2 and graphic analysis of the polymerase chain reaction product for RNAlevels of COX-2 (B) of the RAW 264.7 cells stimulated with the boiled Venus clam hydrolysatefor 18 h at different concentrations. The mean values in the same column with differentalphabets are significantly different (p < 0.05).

260 Chiang Mai J. Sci. 2018; 45(1)

Figure 5. mRNA expression of TNF-α, IL-1β, IL-6, IL-10 and IL-12 (A), and graphicanalysis of the polymerase chain reaction product for RNA levels of TNF-α (B), IL-1β (C),IL-6 (D), IL-10 (E) and IL-12 (F) of the RAW 264.7 cells stimulated with the boiled Venusclam hydrolysate at different concentrations after incubated for 18 h. The mean values in thesame column with different alphabets are significantly different (p < 0.05).

To investigate how the BVH inducedthe release of pro-inflammatory andanti-inflammatory mediator in RAW 264.7cells, further experiment was carried out.Phosphatidylinositol 3-kinase (PI3K) is alipid-modifying enzyme which catalyzesthe phosphorylation of phosphoinositides.These lipid products act as secondarymessengers to activate downstream proteinkinases including AktDprotein kinase B [27].It is known that the phosphorylation andactivation of Akt plays an important rolein TNF-a production [28]. In this study,the BVH stimulated the TNF-α expressionin RAW 264.7 cells. The production of NOby iNOS increased when macrophageswere activated. The production of TNF-αand iNOS is mediated through the PI3K/Aktsignaling pathway. The Akt, which is activatedby PI3K, is involved in modulating theactivation of MAPKs, which in turn stimulatesthe production of various cytokines, includingTNF-α and NO, in macrophages [29].Therefore, Akt is known to play important

functions in the immune system. Thephosphorylation of Akt induced by BVH isshown in Figure 6. The phosphorylation ofAkt increased as the concentration of BVHincreased (0.5-5 g/mL). Based on this result,Akt was phophorylated by the treatment ofBVH, and the translocation of nuclearfactor-kB (NF-κB) into the nucleus in RAW264.7 cells might be subsequently induced.

Figure 6. The effect of the boiled Venus clamhydrolysate at different concentrations on Ik-B degradation and Akt phosphorylation inmurine macrophages cells.

Chiang Mai J. Sci. 2018; 45(1) 261

NF-kB, an important transcription factorin inflammation, immunity and cellproliferation, is a critical activator in theexpression of iNOS, COX-2 and variouscytokines in macrophages [30]. NF-κB islocated in the cytosol of quiescent cells.Upon activation, it is translocated into thenucleus to initiate the transcription oftarget immune response genes, such as iNOSand TNF-α [29]. The degradation andphosphorylation of I-kB is necessary torelease NF-kB from the cytoplasmic NF-kB/I-kB complex to allow its subsequenttranslocation to the cell nucleus [31]. Thefurther experiment was conducted toinvestigate whether the BVH could stimulatethe phosphorylation of I-kB and activationof NF-kB pathway. The expression of I-kBa,a degradation of I-kB, and phosphorylationof I-kB induced by BVH are shown inFigure 6. The BVH induced the degradationof I-kB and stimulated the phosphorylationof I-kB. Therefore, it was concluded thatthe BVH promoted the degradation andphosphorylation of I-kB, and the NF-kBpathway might be activated resulting in thestimulation of cytokines in RAW 264.7 cell.This result was corresponded to the result ofOzes et al. (1999), in which Akt promotedNF-kB activation by directly phosphorylatingI-kB-a in response to TNF-a. Therefore, theBVH stimulated immunomodulation ofRAW 264.7 cells via PI3KDAkt signalingpathway, an upstream stimulator of NF-kBin RAW 264.7 cells [32].

4. CONCLUSION

The boiled Venus calm proteinhydrolysate manufactured using Alcalaseexhibited strong antioxidant and anti-tyrosinase activities. In addition, the boiledVenus clam hydrolysate resulted in theconsiderable production of NO as well as

pro- and anti-inflammatory cytokines bystimulating RAW 264.7 cells throughthe activation of PI3K/Akt and NF-kBpathways. Hence, the boiled Venus calmprotein hydrolysate could be a strongimmunostimulator. The in vitro assaysperformed in this study revealed that theboiled Venus clam hydrolysate might bedeveloped as a food or cosmeceuticalingredient.

ACKNOWLEDGEMENTS

We authors thank Prof. Seong Soo Jooin the Department of Marine MolecularBiotechnology, Gangneung-Wonju NationalUniversity for helping western blot analysis.This research was supported by the KoreaSea Grant Program (Gang Won Sea Grant)funded by the Ministry of Oceans andFisheries in Korea.

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