Antioxidant, immunomodulatory and antiproliferative ...

Antioxidant, immunomodulatory and antiproliferative effects

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of gelatin hydrolysate from unicorn leatherjacket skin

Supatra Karnjanapratum1, Yvonne C. O’Callaghan2, Soottawat Benjakul1 and Nora O’Brien2,*

1

Department of Food Technology, Faculty of Agro-Industry, Prince of Songkla University. Hat Yai, Songkhla 90112, Thailand 2

School of Food and Nutritional Sciences, University College Cork, Cork, Ireland

*To whom correspondence should be addressed. Tel: +353 21 4902884 Fax: +353 21 4270244, E-mail: [email protected] (N.M. O’Brien)

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.7504 This article is protected by copyright. All rights reserved.

Abstract BLACKGROUND:

The

in

vitro

cellular

bioactivities

including,

antioxidant,

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immunomodulatory and antiproliferative effects of a gelatin hydrolysate (GH) prepared from unicorn leatherjacket skin, using partially purified glycyl endopeptidase, were investigated in order to optimise the use of fish skin waste products as functional food ingredients. RESULTS: GH under the tested concentrations (750-1500 µg mL-1) protected against H2O2induced DNA damage in U937 cells. GH also protected against the H2O2-induced reduction in cellular antioxidant enzyme activities, superoxide dismutase (SOD) and catalase (CAT), in HepG2 cells. GH demonstrated immunomodulatory potential by reducing pro-inflammatory cytokine (interleukin-6 (IL-6) and IL-1β) production and nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Cell proliferation in human colon cancer (Caco-2) cells was significantly reduced in dose-dependent manner following incubation with GH. CONCLUSION: These results indicate that GH has several bioactivities which support its potential as a promising functional food ingredient with various health benefits.

Keywords: Gelatin hydrolysate, Unicorn leatherjacket skin, Antioxidant activity, Immunomodulatory effect, Antiproliferative activity

This article is protected by copyright. All rights reserved.

INTRODUCTION The enzymatic hydrolysis of proteins has become an established method for

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producing peptides with potentially enhanced bioactivity.1 Numerous studies have shown that marine hydrolysates, prepared using various enzymes are novel sources of bioactive peptides. Several enzymes such as digestive, microbial and plant proteases, including trypsin, pepsin, collagenase, protamex, alcalase, papain, etc., have been used to obtain hydrolysates.2-4 Hydrolysates of several skin gelatins derived from tuna (Thunnus spp.), hoki (Johnius belengerii), unicorn leatherjacket (Aluterus monoceros) and jumbo flying squid (Rhopilema esculentum) have been shown to possess antioxidant activity.2,5,6 Bioactive peptides with antioxidant activity have also exhibited immunomodulatory and anticancer potential.

7,8

A

protein hydrolysate derived from a salmon by-product and prepared by peptic hydrolysis significantly inhibited intracellular reactive oxygen species generation, lipid peroxidation, and enhanced the level of glutathione in Chang liver cells.9 Additionally, this hydrolyste possessed anti-inflammatory activity by inhibiting nitric oxide (NO) production and proinflammatory cytokines including tumor necrosis factor-α (TNF- α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in RAW264.7 macrophage cells.9 Flying squid skin gelatin hydrolysates prepared using different commercial proteases demonstrated anti-proliferative effects in human breast carcinoma (MCF-7) and glioma (U87) cell lines, along with 2,2′azinobis(3-thylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity and ferric ion reducing power.3 Unicorn leatherjacket (Aluterus monoceros) is a widespread fish species, known from the western Indian Ocean, the eastern Pacific, and the both sides of the Atlantic.10 Unicorn leatherjacket has been used for fish fillet production in Thailand and other countries in SouthEast Asia. As a consequence, a large amount of skin has been produced as a by-product, which can be further used as a good source for production of marine bioactive peptides. In a


previous study, we found that an endogenous protease in unicorn leatherjacket skin could be exploited via an autolysis-assisted process, in combination with thermal and enzymatic

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hydrolysis, to produce an antioxidant gelatin hydrolysate.6 We partially purified glycyl endopeptidase (GE) from papaya latex which showed potential to produce an antioxidant gelatin hydrolysate from fish skin gelatine.4,11 Moreover, the gelatin hydrolysate prepared using GE in combination with the autolysis-assisted process acted as a multifunctional additive which retarded the deterioration of frozen fish mince by acting as both an antifreeze and antioxidant agent.12 To elucidate its potential for use as functional food ingredients the present study investigated the antioxidant activity, immunomodulatory and anticancer effects of a gelatin hydrolysate from unicorn leatherjacket skin in various cell culture model systems.


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MATERIALS AND METHODS Chemicals Dulbecco's modified Eagle's medium (DMEM), Hanks balanced salt solution (HBSS) and non-essential amino acids were purchased from Sigma-Aldrich Chemical Co. (Dublin, Ireland). Human histiocytic lymphoma cells (U937 cells), mouse leukaemic macrophages (RAW264.7 cells), human hepatoma cells (HepG2 cells) and human colon carcinoma cells (Caco-2 cells) were purchased from the European Collection of Animal Cell Cultures (Salisbury, UK). Fetal bovine serum (FBS) was purchased from Invitrogen (Paisley, Scotland). Costar cell culture plastics were supplied by Fisher Scientific (Dublin, Ireland). All other cell culture chemicals and reagents were from Sigma Chemical Co. All solvents used were of HPLC grade.

Preparation of gelatin hydrolysate from unicorn leatherjacket skin Preparation of fish skins The skins of unicorn leatherjacket (Aluterus monoceros) were obtained from a dock, Songkhla, Thailand. Three different lots of skins were collected. For each lot, skins were pooled and used as a composite sample. The skins were washed with iced tap water (0-2°C) and cut into small pieces (0.5×0.5 cm2). The pretreated skins were then prepared by removing non-collagenous proteins using the method of Kaewruang et al.13 Fish skins (0.5×0.5 cm2) were soaked in 0.05 mol L-1 NaOH with a skin/alkaline solution ratio of 1:10 (w/v). Following pretreatment, skins were washed with tap water until neutral or faintly basic pH of wash water, the autolysis was then


conducted using pretreated skin following the method of Karnjanapratum and Benjakul.4 The resulting autolysed skin was used as substrate for preparation of gelatin hydrolysate.

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Preparation of partially purified glycyl endopeptidase (GE) from papaya (Carica papaya) latex Fresh papaya latex was collected from green papaya fruit cultivated in Songkhla, Thailand. The latex was then transferred to a beaker and stored below 10 °C and used within 3 h. The crude extract was prepared using the method of Kittiphattanabawon et al.14 The glycyl endopeptidase was fractionated from crude extract using the method of Karnjanapratum and Benjakul.7 Aqueous two phase system (ATPS) with 10% PEG 6000 and 10% ammonium sulphate (NH4)2SO4 was used for fractionation of glycyl endopeptidase. The obtained GE was stored at -40 °C until use. Production of gelatin hydrolysates Autolysed skins solutions (3%, w/v) were mixed with GE (8%, based on solid matter) and incubated at 40 °C for 60 min.4 After enzyme inactivation by heating at 90 °C for 15 min, the resulting gelatin hydrolysate was centrifuged at 9,000×g at 4 °C for 20 min. The supernatant was collected and lyophilised. The gelatin hydrolysate powders were referred to as ‘GH’. The powders were placed in polyethylene bag and stored at -40 °C until use. The storage time was not longer than 2 months.

Cell culture U937, HepG2 and RAW264.7 cells were grown in RPMI-1640 supplemented with 10% FBS. Cells were cultured in the absence of antibiotics. The cells were grown at 37 °C in a 5% (v/v) CO2 atmosphere in a humidified incubator. Reduced serum media (25 mL L-1 FBS) was used for all experiments. Cell proliferation


U937, HepG2 and RAW264.7 cells (2×104 cells/mL) was supplemented with 0-40 mg mL-1, 0-1000 µg mL-1 and 0-1500 µg mL-1 gelatine hydrolysate (GH), respectively, in 96-

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well flat-bottom plates with a final volume of 200 µL at 37 ºC for 24 h. Following incubation, cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (MTT I proliferation kit, Roche Diagnostics; Burgess Hill, West Sussex, UK) according to the manufactures instructions. Absorbance was read at 570 nm using a microplate reader (Thermo Scientific Varioskan® Flash Multimode Reader, Fisher Scientific UK Ltd., Leicestershire, UK). The IC50 value (the concentration of sample that induced 50% decrease in viable cells) for each sample was calculated using the data obtained from the MTT assay and Prism software (version 4.0, GraphPad Inc., San Diego, CA, USA).

Determination of DNA damage (Comet assay) U937 cells (1×105 cells/mL) were treated with GH (2.5, 5.0 and 7.5 mg/mL) for 24 h in a 24-well plate with a final volume of 1 mL media, containing reduced FBS (25 mL L-1 ) at 37 °C. Following incubation, cells was treated with 40 and 60 μmol L-1 H2O2 for 30 min. Oxidative DNA damage in the U937 cells was assessed using the Comet assay as described by McCarthy et al.15 Briefly, slides were prepared by coating with 10 g L-1 normal gelling agarose (NGA). Cells (30 µL) were then mixed with 10 g L-1 low melting point (LMP) agarose, placed on a microscope slides, covered with a coverslip and the mini-gels were allowed to solidify on ice. Slides were then placed in cold lysis solution (2.5 mol L-1 NaCl, 100 mmol L-1 EDTA, 10 mmol L-1 tri(hydroxymethyl)-aminomethane, fresh 10 mL L-1 Triton® X-100 and 100 mL L-1 dimethyl sulfoxide) for 1.5 h at 4 ºC. Slides were aligned in a horizontal gel electrophoresis tank (Horizon® 20·25, GIBCO BRL Life Technologies, Gaithersburg, MD, USA) which was filled with fresh electrophoresis solution (1 mmol L-1 EDTA, 300 mmol L-1 NaOH; pH 13). Slides were allowed to sit in this buffer for 30 min.


Electrophoresis was conducted at 20V, 300 mA for 25 min at 4 ºC. After electrophoresis, the slides were neutralized using 0.4 mol L-1 Tris for 5 min (x3) and rinsed with distilled water.

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Slides were stained with ethidium bromide (20 mg L-1) for 5 min and rinsed with distilled water. The Komet 5.5 image analysis software (Kinetic Imaging, Liverpool, UK) was used to score 50 cells for each slide using a fluorescence microscope (Optiphot-2, Nikon, Iowa, USA). DNA damage was expressed as percentage tail DNA.

Antioxidant enzyme activity assays: Superoxide dismutase (SOD) and catalase (CAT) activities HepG2 cells (2 × 105 cells mL-1, 5 mL) were incubated with gelatin hydrolysate (125 and 250 µg mL-1) for 24 h at 37 °C. Following incubation, cells were exposed to 2 mM H2O2 for 2 h. Cells were harvested, sonicated and centrifuged (15000 rpm, 30 min) at 4 °C and the supernatant was collected for the determination of antioxidant enzyme activity. The activity of total cellular superoxide dismutase (SOD) was determined using the method of Misra and Fridovich.16 The supernatant was diluted in 0.05 M potassium phosphate buffer (pH 7) and xanthine, xanthine oxidase, and cytochrome c were added. The xanthine oxidase system generates superoxide anion which reduces cytochrome c, and this reaction is inhibited by SOD. The reduction in cytochrome c was used to determine the activity of SOD present in the samples from a standard curve. Samples were read at 550 nm at 20 min intervals for at least five readings. Catalase (CAT) activity was determined using a modification of the method of Baudhuin et al.17, where any remaining H2O2 was determined as a yellow ‘peroxy titanium sulfate’. One unit of catalase activity was defined as the amount of catalase required to decompose 1µmol H2O2 per min at pH 7.5 and 25 °C. The absorbance was measured at 465 nm.


SOD and CAT activity were determined relative to the protein content as SOD and CAT units mg-1 proteinin cell homogenate, respectively. The protein content of the samples

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was quantified by the bicinchoninic acid (BCA) protein assay as previously described.18 Data were expressed as a percentage of untreated, control cells.

Cytokine production (Immunomodulatory activity) RAW264.7 cells, at a density of 2×105 cells mL-1, were seeded in 96-well plates in the presence of lipopolysaccharide (LPS, 0.5 mg L-1) and treated with test samples (750, 1000 and 1500 mg mL-1) for 24 h at 37 ºC. Production of the cytokines IL-6 and IL-1β was determined using ELISA kits (eBioscience mouse IL-6 and IL-1β ELISA Ready-SET-Go kits, Insight Biotechnology, Wembley, U.K.). Absorbance was read at 450 nm using a microplate reader. Data were expressed as a percentage of the LPS-stimulated RAW264.7 cell control.

Nitric oxide (NO) secretion NO secretion was determined using the Griess reagent. RAW264.7 cells were plated at a density of 1×105 cells mL-1 in a 96-well plate and incubated for 48 h. NO production was induced with LPS (2 μg mL-1) and cells were co-treated with different concentration of gelatin hydrolysate (600-1500 μg mL-1) for 24 h. The cultured supernatant (50 μL) was plated into a 96 well plate and 50 μL of Griess reagent (1:1 of 1% sulphanilamide in 5% phosphoric acid and 0.1% N-1-naphtyl-ethylenediamine dichloride in water) was added. Sodium nitrite was used to generate a standard curve. The plate was incubated at room temperature for 10 min and the absorbance was measured at 540 nm using a Thermo Scientific Varioskan Flash microplate reader.


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Antiproliferative effect Caco-2 cells (2×104 cells mL-1) were cultured in DMEM supplemented with 100 mL L-1 FBS and 10 mL L-1 non-essential amino acids in 96-well flat-bottom plates. The different concentrations of gelatin hydrolysates (0-1.0 mg mL-1) were then added with a final volume of 200 µL at 37 ºC for 24 h. Following incubation, cell viability was assessed using the MTT assay and the IC50 value was calculated as mentioned above.

Statistical analysis A completely randomized design (CRD) was used for the statistical analysis of physical and chemical analysis. All data was subjected to analysis of variance (ANOVA) and mean comparisons was carried out using Duncan’s multiple range test. 19 Statistical analysis was performed using the statistical Package for Social Sciences (SPSS for windows: SPSS Inc., Chicago, IL, USA). The results with P0.05) reduced to control levels at the highest concentration of GH used (7.5 mg mL-1). A similar trend was observed in cells exposed to 60 µmol L-1 H2O2. Few studies have previously investigated the DNA protective effects of fish protein hydrolysates. Yarnpakdee et al.22 reported that an antioxidant protein hydrolysate prepared from Nile tilapia protein isolate effectively inhibited H2O2 and peroxyl radical induced plasmid DNA (pUC18) damage. The protective effect of tuna liver hydrolysates was related to their scavenging of H2O2, hydroxyl radical and chelating activity toward Fe2+. Such activities led to the inhibition of the Fenton reaction, and therefore, protected the supercoiled pBR322 plasmid DNA from oxidant-induced strand breaks.23 In our previous study, we reported that the antioxidant gelatin hydrolysate (GH) prepared using glycyl endopeptidase from unicorn leatherjacket skin possessed an ability to donate electrons and scavenge free radicals4; which is likely to be the mechanism by which GH protects against oxidant-induced DNA damage in U937 cells as reported in the present study.

Effect of gelatin hydrolysate (GH) on induction of antioxidant enzyme activities in HepG2 cells The ability of GH to protect against a H2O2-induced depletion of the cellular antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) was investigated in HepG2 cells (Figure 3). The levels of SOD and CAT activity were significantly decreased (P0.05). This result suggested that GH

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was probably able to enhance the antioxidant enzyme activities in non-stimulated HepG2 cells. However, GH demonstrated an ability to prevent the decrease in SOD and CAT enzyme activity induced by H2O2, especially at the highest concentration used (250 µg mL-1) with approximately 17% and 20% increase, respectively (P