Protection effect of donkey hide gelatin hydrolysates on UVB-induced photoaging of human skin fibroblasts

Process Biochemistry xxx (xxxx) xxx–xxx

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Protection effect of donkey hide gelatin hydrolysates on UVB-induced photoaging of human skin fibroblasts Jung-Soo Kim, Dongwook Kim, Hee-Jin Kim, Aera Jang

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Department of Animal Products and Food Science, Kangwon National University, Chuncheon, 24341, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Keywords: Donkey hide Peptides Photoaging Procollagen type I MAPK

UVB irradiation is a potent photoaging factor and leads to the formation of skin wrinkles. The objective of this study was to investigate the mechanisms of UVB photoaging inhibition of low molecular weight peptides derived from donkey hide gelatin hydrolysates (LDGH) on human skin fibroblasts. The donkey hide was hydrolyzed using proteases, and the LDGH exhibited higher antioxidant activity than the original hydrolysate and inhibited collagenase and elastase activities. We also found that LDGH increases viability of Hs68 cells after UVB irradiation at 100 mJ/cm2. Moreover, LDGH prevented the decrease in procollagen type I levels caused by exposure to UVB irradiation in Hs68 cells and reduced the up-regulated phosphorylation of p38, ERK, and JNK in the mitogen-activated protein kinase (MAPK) signaling pathway. These findings suggest that LDGH increases synthesis of procollagen type I by decreasing the phosphorylation of MAPK, therefore, LDGH may be useful as an effective anti-photoaging agent.

1. Introduction Skin aging is a complex process that can be divided into intrinsic and extrinsic aging. Intrinsic aging is caused by an innate process, which is highly associated with genetic factors. Extrinsic aging, in contrast, is environmentally derived from external factors such as short wavelength UV light (UVB) and characterized by wrinkling and degradation of skin elasticity [1]. Exposure to UVB irradiation causes skin damage by inducing oxidative stress, which leads to DNA mutations in collagenous tissues that produce the extracellular matrix (ECM) with collagen and elastin [2]. UV-induced skin damage leads to activation of the mitogen-activated protein kinase (MAPK) pathway, which comprises c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 kinase. These MAP kinases activated by UV irradiation increase the activation and expression of matrix metalloproteinases, which lead to degradation of type I collagen in human skin fibroblasts [2]. Thus, inhibition of the MAPK pathway can prevent UVBinduced photoaging. Furthermore, preparation of natural products containing compounds with anti-aging effects, such as antioxidant activity, inhibition of the ECM-degrading enzyme (collagenase and elastase), MAPK, and up-regulation of procollagen type I synthesis, may be beneficial against photoaging. In recent years, diseases induced by UV radiation that cause skin damage became a global concern. In Korea, in particular, it has been reported that the number of people aged 65 or older is continuously

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increasing. Younger and older people, therefore, would be interested in preventing skin aging, thus increasing the market for anti-photoaging products. Recently, many studies have reported that hydrolysates from bioresources can reduce the risk of skin disease [3,4]. Interestingly, some reports have focused on the beneficial effects of low molecular weight peptides in hydrolysates owing to the anti-photoaging and antioxidant effects in the skin [5,6]. Since the absorption capacity of high molecular weight compound derived from animal collagen may have limitation for real-life application to adapt biological activity, the low molecular hydrolysate or peptides could be more efficient. Nimalaratne et al. [7] reported that low molecular weight peptides are more biologically active compared to its original polypeptide or protein. Furthermore, it is have higher chances to be absorbed to the intestinal membranes by resistance to the gastrointestinal digestion. Similarly, Zhuang et al. [8] reported that oral intake collagen hydrolysate (less than 5000 Da) showed higher effect on restoration of antioxidant enzyme activity and protection of collagen synthesis in mice skin from the UV radiation damages than oral intake of original collagen. In addition, Samaranayaka and Li-Chen [9] reported that antioxidant compounds from food sources have been used to prevent aging and UV-induced skin damage as cosmeceuticals. Many antioxidative peptides have been studied from various food proteins likely egg yolk [10] and royal jelly [11]. Especially, donkey hide has been used in the production of gelatin, a valued traditional medicine and food for skin health in China [12] and Korea. In addition, several bioactivities were identified in

Corresponding author. E-mail address: [email protected] (A. Jang).

https://doi.org/10.1016/j.procbio.2018.02.004 Received 12 October 2017; Received in revised form 6 January 2018; Accepted 6 February 2018 1359-5113/ © 2018 Published by Elsevier Ltd.

Please cite this article as: Kim, J.-S., Process Biochemistry (2018), https://doi.org/10.1016/j.procbio.2018.02.004

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solution (0.5 mL) was mixed with an equal volume of 20% trichloroacetic acid (TCA). The mixtures were incubated for 30 min at 25 °C and then centrifuged at 3000g for 15 min. The soluble protein in the 10% TCA supernatant and total protein contents were determined using the bicinchoninic acid (BCA) protein assay kit (Sigma Chemical, USA). Bovine serum albumin (BSA) was used as a standard. DH was calculated as follows:

donkey hide extracts, including antioxidant, neuroprotective, and antidiabetic effects [13,14]. However, there is still a lack of information on the anti-photoaging activities of enzymatic hydrolysates from donkey hide. The present study was undertaken to evaluate the antioxidant and the collagenase and elastase inhibitory effects of low molecular weight peptides derived from donkey hide gelatin, and to determine whether these peptides can attenuate UVB-induced MAPK activation and downregulation of procollagen type I expression in human skin fibroblasts.

DH (%) = [Soluble protein content in 10% TCA/Total protein content in sample] × 100

2. Materials and methods 2.4. Brix and yield

2.1. Materials

Brix of samples was determined using a refractometer (Master-α, ATAGO, Japan). Yield of samples was determined following the method of Gudmundsson and Hafsteinsson [16]. Yield was calculated as follows:

Donkey hide was obtained from a local donkey farm in Icheon, Korea. Foodpro alkaline protease (Danisco Ltd., Co., Copenhagen, Denmark), pancreatin (Bision Co., Gyunggi, Korea), and protease P (Amano Enzyme Inc., Nagoya, Japan) were used. ( ± )-6-Hydroxy2,5,7,8-tetramethyl-chroman-2-carboxylic acid (97%, Trolox), 2,2′azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide (MTT), fluorescein sodium salt, 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH), 2,4,6tripyridyl-s-triazine (TPTZ), collagenase from Clostridium histolyticum, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg, porcine pancreatic elastase, and N-succinyl-(Ala)3-p-nitroanilide were purchased from Sigma Aldrich (St. Louis, MO, USA). Trypsin-EDTA, 1% penicillin/ streptomycin was purchased from Gibco (Carlsbad, CA, USA) and fetal bovine serum (FBS) was obtained from Hyclone (Logan, UT, USA). Dulbecco's phosphate buffered saline, Tween 20, acrylamide, Tris-buffered saline (TBS), and sodium dodecyl sulfate (SDS) were purchased from GenDEPOT (Barker, TX, USA). Dulbecco’s modified Eagle’s medium (DMEM) and Dulbecco's phosphate buffered saline (DPBS) were purchased from Welgene (Daegu, Korea). All other chemicals and reagents used in this study were analytical grade.

Yield (%) = [Dry weight of hydrolysate/Wet weight of raw donkey hide] × 100

2.5. Antioxidant activities 2.5.1. DPPH radical scavenging activity DPPH radical scavenging activity was determined following the method of Blois [17] with slight modifications. Reaction mixture containing 100 μL of methanolic solution with DPPH radicals (0.2 mM) and 100 μL of sample was placed in a 96-well microplate. The mixture was shaken and kept in the dark at 25 °C for 30 min, and then absorbance was read at 517 nm using a UV spectrophotometer (Spectramax M2e, Molecular Devices, USA). A standard curve was established using Trolox solution (0, 10, 30, 50, 70, 100, 110, and 120 μM) and the DPPH values were calculated as μM Trolox equivalents per g of dry matter. 2.5.2. ABTS radical scavenging activity ABTS radical scavenging activity was determined according to Re et al. [18]. Briefly, ABTS radical was produced by reacting a 14 mM ABTS solution with an equal volume of 4.9 mM potassium persulfate in the dark at 25 °C for more than 14 h before use. Next, the ABTS radical solution was diluted in DW and equilibrated at 30 °C to obtain an absorbance of 0.70 ± 0.02 at 735 nm. Sample (50 μL) or Trolox standard solutions (0, 20, 50, 70, 100, 200, 300, and 400 μM in 75 mM PBS) were added to 950 μL of ABTS radical solution and incubated at 30 °C for 30 min in the dark. The absorbance was measured using a UV-spectrophotometer at 735 nm at 30 °C. ABTS radical scavenging activity was calculated using the Trolox standard curve as μM Trolox equivalents per g of dry matter.

2.2. Preparation of donkey gelatin and low molecular weight donkey gelatin hydrolysates Donkey hide was washed with water and preheated in boiling water (100 °C, 20 min) to eliminate impurities. After removing the fat layer, donkey hide was chopped into small pieces (1 × 0.5 × 0.5 cm) and then the extraction of gelatin from donkey hide (Fig. 1) was carried out using an autoclave at high temperature and pressure (121 °C, 1.5 kgf/ cm2). The first extraction was carried out by adding 200 mL of distilled water (DW) to 100 g of donkey hide and soaked for 1 h and then filtering using a mesh sieve with less than 150 μm pore size. The second extraction was carried out by adding 100 mL of DW to the sieved donkey hide for 30 min and then filtering again using a similar sieve. The extracts were mixed and enzymatic hydrolysis of gelatin extracts was then performed using Foodpro alkaline protease (F), pancreatin (P), protease P (PP), or a mixture of proteases (Foodpro alkaline protease, pancreatin, and protease P (F/P/PP)) for 3 h (F3, P3, PP3, F/P/ PP3) or 6 h (F6, P6, PP6, F/P/PP6) at 45 °C, pH 7.0. Each enzyme was added to the gelatin extracts at an enzyme/substrate ratio (E/S) of 0.3:100. The donkey gelatin hydrolysate (DGH) was then heated at 90 °C for 10 min to inactivate the enzymes. To separate low molecular weight peptides from DGH (LDGH, less than 3 kDa), each DGH was passed through a 3 kDa molecular weight cut-off membrane (Ultracel–3 kDa, Millipore, Ireland). DGHs and the corresponding LDGHs were lyophilized and then stored at −20 °C until use.

2.5.3. Oxygen radical absorbance capacity (ORAC) assay ORAC assay was determined according to the method described by Gillespie et al. [19] with slight modifications. Sample, standards, and other reagents were prepared in 75 mM PBS (pH 7.0). The reaction mixtures containing 150 μL of fluorescein (80 nM) and 25 μL of sample or standard were placed in 96-well black microplates and preincubated for 15 min at 37 °C. After mixing with 25 μL of AAPH (150 mM), the fluorescence was recorded every min for 60 min at 37 °C with emission and excitation wavelengths of 485 and 520 nm, respectively, using a fluorescence reader (Spectramax M2e, Molecular Devices, USA). A standard curve was established by measuring Trolox solution (0, 5, 10, 30, 40, 50, 70, and 100 μM) as area under the curve. ORAC values were expressed as μM Trolox equivalents per g of dry matter.

2.3. Degree of hydrolysis 2.5.4. Ferric reducing antioxidant power (FRAP) assay The FRAP assay performed was a slight modification of that described by Benzie and Strain [20]. Briefly, FRAP reagent was freshly

Degree of hydrolysis (DH) was determined following the method of Hoyle and Merritt [15] with slight modifications. Protein hydrolysate 2

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Fig. 1. Separation procedure of low molecular weight peptides from donkey hide gelatin hydrolysates.

Collagenase inhibitory activity = {1-(sample O.D.-blank O.D.)/con O.D.} × 100

prepared by mixing 300 mM acetate buffer, 10 mM TPTZ solution, and 20 mM ferric chloride solution at a ratio of 10:1:1 (v/v) at 37 °C before use. Samples (25 μL) were mixed with FRAP reagent (175 μL) and kept in the dark at 37 °C for 30 min. Absorbance was measured at 590 nm and a standard curve was established using Trolox solution (0, 5, 10, 30, 40, 50, 70, and 100 μM). FRAP value was calculated as μM Trolox equivalents per g of dry matter.

Where sample O.D. and con O.D. represent the optical densities in the presence and absence of sample, respectively, and blank O.D is the optical density in the absence of collagenase.

2.6.2. Elastase inhibitory activity Elastase inhibitory activity was determined using elastase from porcine pancreas and (N-succinyl-(Ala)3-p-nitroanilide). This assay is a modification of the method described by Kraunsoe et al. [22]. Briefly, the elastase was dissolved at 1 unit/mL in 0.2 M Tris-HCl buffer solution (pH 8.0). The 1 mM N-succinyl-(Ala)3-p-nitroanilide was dissolved in 0.2 M Tris-HCl buffer solution (pH 8.0). To determine elastase inhibitory activity, 100 μL of sample in 0.2 M Tris-HCl buffer solution (pH 8.0), 120 μL of 0.2 M Tris-HCl buffer, and 20 μL of 1 mM N-succinyl(Ala)3-p-nitroanilide solution were dispensed into each well of a 96-well plate. The plate was preincubated at 25 °C for 10 min, and then 20 μL of elastase solution was added into each well. After incubation at 25 °C for 20 min, the plate was placed on ice to stop the reaction. Absorbance was measured using a UV spectrophotometer at 405 nm. Ursolic acid (100 μg/mL) was used as a positive control. Elastase inhibitory activity of samples was calculated as follows:

2.6. Collagenase and elastase inhibition activity 2.6.1. Collagenase inhibitory activity Collagenase inhibitory activity was determined using the method of Wuensch and Heidrich [21] with slight modifications. Briefly, collagenase from C. histolyticum was dissolved in 0.1 M Tris-HCl buffer (pH 7.5). To measure collagenase inhibitory activity, 100 μL of sample in 0.1 M Tris-HCl buffer (pH 7.5), 150 μL of collagenase, and 250 μL of 0.1 M Tris-HCl buffer (pH 7.5) containing 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg, 4 mM CaCl2 were mixed and incubated for 20 min at 25 °C in the dark. The reaction was stopped by adding 0.5 mL of 6% citric acid, and then the reaction mixture was separated by adding 1.5 mL of ethyl acetate. Absorbance of the supernatant was determined using a UV spectrophotometer at 320 nm. Collagenase inhibitory activity was determined as follows:

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Table 1 Degree of hydrolysis (%), brix, and yield (%) of donkey hide gelatin hydrolysates. Items

DH Brix Yield

Type

DGH DGH DGH LDGH

Treatment1) F3

F6

P3

P6

PP3

PP6

F/P/PP3

F/P/PP6

88.33 8.6 26.41 4.03

91.34 8.9 26.92 5.20

93.99 9.0 27.75 4.96

94.09 9.0 27.67 5.52

93.26 9.0 27.93 8.37

91.94 9.2 28.39 11.01

86.71 9.0 27.93 8.88

87.03 9.2 27.70 9.73

1) Refer to Fig. 1. DH, degree of hydrolysis; DGH, donkey gelatin hydrolysates; LDGH, low molecular weight peptides from DGH.

Elastase inhibitory O.D.} × 100

activity = {1-(sample

O.D.-blank

136521), p-p38 (sc-17852-R) at a 1:200 dilution, or β-actin (sc-47778) at a 1:3000 dilution (Santa Cruz Biotechnology, CA, USA). After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies either anti-mouse IgG (sc-2005) and antigoat IgG (sc-2004) or anti-rabbit IgG (sc-2020) (Santa Cruz Biotechnology, CA, USA) for 1 ∼ 2 h. The immunoreactive bands were detected using ECL kit (Bio-Rad Laboratories, CA, USA) and AE-9150 Ez-Capture 11 (ATTO, Tokyo, Japan). Signal intensities were determined using CS Analyzer Version 3.00 (ATTO, Tokyo, Japan).

O.D)/con

Where sample O.D. and con O.D. represent the optical densities in the presence and absence of sample, respectively, and blank O.D is the optical density in the absence of elastase. 2.7. Protection of fibroblast from UVB induced photoaging 2.7.1. Human skin fibroblast cell culture Human skin fibroblasts (Hs68, ATCC CRL 1635) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in DMEM supplemented with 1% penicillin/ streptomycin and 10% FBS in 5% CO2 at 37 °C. Cells were cultured until they reached 90% confluence and subcultured following trypsinization. The cells were used for experiments from 5th to 15th passages.

2.8. Statistical analysis Statistical significance of data was determined using SAS software (ver. 9, SAS Institute, USA) using one-way analysis of variance (ANOVA) and the general linear model. Values are expressed as mean and standard error (SEM) according to Tukey’s multiple range test. A value of p < 0.05 was considered to be statistically significant.

2.7.2. UVB irradiation Cells were covered with DPBS prior to UVB irradiation and then subjected to 0.971 mW/cm2 of UVB for 103 s on the surface of the cell to reach 100 mJ/cm2 using UVB lamp (T–15 M, Vilber Lourmat, France). The UVB dosage was measured and calibrated using the UV light meter (YK-35UV, Lutron, Taipei, Taiwan). After UVB irradiation, cells were washed again with fresh DPBS. Subsequently, the cells were incubated in a humidified atmosphere of 5% CO2 at 37 °C for 24 h in serum-free medium containing various concentrations of sample.

3. Results and discussion 3.1. DH, brix, and yield of DGH and LDGH DH, brix, and yield of DGH and LDGH is shown in Table 1. The DH values of all DGH were similar to each other and ranged from 86.71% to 94.09% (p > 0.05). According to Klompong et al. [23], high DH values generally increase solubility of hydrolysates, although the emulsifying capacity may decrease. Because the hydrolysis break down protein structure and results in low molecular weight peptide and change the functionality of protein. Extensive hydrolysis could result in negative effect on the functionality. Sarmadi and Ismail [24] reported that advantage of hydrolysis can be development of hydrophobicity because proteolysis unfolds the protein chains. Also hydrolysis can decrease or increase the hydrophobicity which mostly depends on the nature of the precursor protein and molecular weight peptides generated by enzymes. Since enzymes specificity affects size, amount, composition of free amino acid and peptides, and their amino acid sequence which influences the antioxidant activity of the hydrolysates. In this study, we used Foodpro alkaline protease (endoprotease from Bacillus licheniformis), pancreatin (pancripsin from pig), and protease P (alkaline protease from Aspergillus melleus) which are food grade. Our results show that no difference was found in brix values and yield among DGHs, and values ranged from 8.6% to 9.2% and from 26.41% to 28.39%, respectively. The yield of LGDHs ranged from 4.03% (F3) to 11.01% (PP6) which indicating that the protease P showed higher yield compare to Foodpro alkaline protease and pancreatin. Similarly, Cheung and Chan [25] reported that steelhead skin hydrolysate using protease P indicates the highest yield of low molecular weight hydrolysates (less than 3 kDa) compare to other protease.

2.7.3. Cell viability Cell viability was determined using the MTT assay. Briefly, Hs68 cells were plated at 2 × 105 cells/well in a 48-well plate and treated with various concentrations of sample. After incubation for 24 h, MTT solution (0.5 mg/mL in PBS) was added to each well and incubated for 4 h at 37 °C. The formazan crystals in each well were then dissolved by adding DMSO. Absorbance was read at 540 nm directly in the wells using a UV spectrophotometer (Spectramax M2e, Molecular Devices, USA). 2.7.4. Western blot analysis Cells were harvested and lysed with radio immunoprecipitation assay lysis buffer (150 mM NaCl, 1% deoxicholic acid sodium salt, 0.1% SDS, 1% Triton-X-100, 50 mM Tris-HCl, 2 mM EDTA, pH 7.5; GenDEPOT, Barker, TX, USA) containing 1% protease inhibitor cocktail solution (GenDEPOT, Barker, TX, USA). The lysates were centrifuged at 10,000g for 10 min at 4 °C, and the protein content in the cell was determined using the bicinchoninic acid (BCA) protein assay kit (Sigma Chemical, USA). Cell lysates containing equal amounts of protein (20 μg) were separated by SDS-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (ATTO, Tokyo, Japan). Blots were blocked with 5% (w/v) non-fat milk or 5% (w/v) BSA at 25 °C for 2 h in TBS buffer (150 mM NaCl, 10 mM TrisHCl, pH 7.5) containing 0.05% Tween 20 and then incubated overnight at 4 °C with primary antibody against procollagen type I (sc-8782), JNK (sc-46006), ERK (sc-93), p38 (sc-7972), p-JNK (sc-6254), p-ERK (sc-

3.2. Antioxidant activity of DGH and LDGH Antioxidant activity of peptides from hydrolysates is influenced by the amount of free amino acids and by the sequence and molecular 4

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Table 2 Antioxidant activity of donkey hide gelatin hydrolysates (μM Trolox equivalent/g dry matter). Treatment1)

Items

DPPH ABTS ORAC FRAP

DGH LDGH DGH LDGH DGH LDGH DGH LDGH

F3

F6

P3

P6

PP3

PP6

F/P/PP3

F/P/PP6

3.35 ± 0.26Be 11.86 ± 0.13Aa 42.62 ± 2.35Ba 50.56 ± 3.20Aa 153.51 ± 3.38Be 233.04 ± 2.78Ac 2.54 ± 0.13Ba 3.14 ± 0.17Aa

4.69 ± 0.27Bb 9.42 ± 0.07Ab 40.57 ± 0.44Ba 45.69 ± 1.60Aa 206.45 ± 3.15Bb 223.09 ± 3.40Ad 2.38 ± 0.13Bab 2.64 ± 0.09Ab

4.06 ± 0.47Bcd 11.86 ± 0.27Aa 30.82 ± 2.22Bb 51.59 ± 3.95Aa 211.57 ± 7.65Bab 307.26 ± 2.74Aa 1.91 ± 0.09Bc 2.96 ± 0.11Aab

4.44 ± 0.60Bbc 8.39 ± 0.09Abc 33.64 ± 2.66Bb 47.49 ± 1.60Aa 219.62 ± 2.43Ba 294.55 ± 2.74Ab 1.96 ± 0.14Bc 2.66 ± 0.07Ab

5.49 ± 0.45Ba 6.68 ± 0.08Ae 32.87 ± 0.77Bb 35.18 ± 0.77Ab 167.53 ± 1.80Bd 219.33 ± 2.77Ad 2.37 ± 0.12Bab 2.72 ± 0.13Ab

4.38 ± 0.56Bbc 6.36 ± 0.24Ae 31.07 ± 0.44Bb 34.41 ± 1.78Ab 188.83 ± 1.62Bd 204.88 ± 2.24Ae 2.16 ± 0.14Bbc 2.64 ± 0.10 Ab

4.52 ± 0.20Bbc 7.98 ± 0.23Acd 30.57 ± 0.89Bb 36.20 ± 0.44Ab 168.50 ± 0.44Bc 232.86 ± 5.76Ac 2.51 ± 0.11Ba 3.17 ± 0.23Aa

3.74 ± 0.29Bde 6.92 ± 0.10Ade 31.33 ± 2.66Ab 36.46 ± 3.11Ab 176.82 ± 6.32Bd 217.78 ± 1.59Ad 2.39 ± 0.08Bab 2.82 ± 0.15Aab

Refer to Fig. 1. Each value was expressed as mean ± SD (n = 3).A-B Means within a column with different superscripts differ significantly at p < 0.05.a-e Means within a row with different superscripts differ significantly at p < 0.05. DGH, donkey gelatin hydrolysates; LDGH, low molecular weight peptides from DGH; DPPH, 1,1-diphenyl-2-picrylhydrazyl; ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; ORAC, oxygen radical absorbance capacity; FRAP, ferric reducing antioxidant power

1)

weight of peptides [26]. As shown in Table 2, all LDGHs showed significantly higher DPPH radical scavenging activity, ORAC, and FRAP values than the corresponding DGH (p < 0.05). Especially, LDGH-F3 and LDGH-P3 showed the highest DPPH radical scavenging activity (11.86 and 11.86 μM TE/g of dry matter, respectively) compare to other treatment. In addition, ABTS radical scavenging activity and FRAP value of LDGH-F3 and LDGH-P3 were 50.56 and 51.59 μM TE/g of dry matter and 3.14 and 2.96 μM TE/g of dry matter, respectively compare to those of DGH (p < 0.05). Liu et al. [27] noted that the strong reducing power may be determined according to the increased availability of hydrogen ions (protons and electrons) due to peptide cleavages. Especially, ORAC value of LDGH-P3 (307.26 μM TE/g of dry matter) was higher than that of other treatment (204.88 ∼ 294.55 μM TE/g of dry matter). Compare to other protease, pancreatin (0.3%) was more effective enzyme to produce antioxidant hydrolysates from donkey hide protein. However, hydrolysates from hydrolysis by combination of three enzymes showed no significant antioxidant activities (DPPH, ABTS, ORAC) except FRAP. These results suggest that the antioxidant activity of LDGH is mainly influenced by the enzyme used for hydrolysis and the size of peptides. Some studies have reported similar results. The size of peptides produced by hydrolysis may vary depending on the protease used [24]. Especially, Kim et al. [28] reported that low molecular weight peptides (< 3 kDa) derived from pig skin hydrolysates have higher ORAC values (about 140 μM TE/g of dry matter) than the original hydrolysates (about 30 μM TE/g of dry matter). In addition, Kim et al. [29] reported that low molecular weight peptides (< 3 kDa) from horse bone protein hydrolysates have higher DPPH, ABTS radical scavenging activity, and FRAP values than hydrolysates (> 3 kDa) derived from horse bone protein. Jang et al. [30] also reported that low molecular weight peptides derived from animal protein have higher antioxidant activity than larger molecular weight fractions over 3 kDa. Also, Kawashima et al. [31] reported that low molecular weight peptides such as di- and tri peptides containing aromatic amino acid residues and Tyr, Pro, and His showed strong antioxidant activity. These findings suggest that low molecular weight peptides less than 3 kDa can more easily react with and eliminate free radicals because the disruption of the native protein structure by enzyme hydrolysis resulted in active amino acid as a free radical scavenger. We have found that hydrolysis time is also important for a high antioxidant activity. Especially, LDGH-3 had higher antioxidant activity than LDGH-6. Yang et al. [32] reported that peptides derived from protein hydrolysates resulting from longer enzymatic digestion do not show much more antioxidant activity. Moreover, our results show that the antioxidant activity of LDGH-6 decreased with increasing hydrolysis time. LDGH-3 had the highest antioxidant activity and, therefore, we selected LDGH-F3, P3, PP3, and F/P/PP3 for further studies.

Fig. 2. Collagenase (a) and elastase (b) inhibitory activity of peptides less than 3 kDa from donkey hide gelatin hydrolysates.

3.3. Collagenase and elastase inhibitory activities of LDGH-F3, P3, PP3, and F/P/PP3 The effects of the selected LDGH-F3, P3, PP3, and F/P/PP3 on collagenase and elastase inhibitory activities is shown in Fig. 2. LDGHP3 and LDGH-PP3 showed the highest collagenase inhibitory activity (54.09% and 55.21%, respectively) among LDGHs (p < 0.05; Fig. 2A). Also, LDGH-F3 showed the highest elastase inhibitory activity compared to other LDGHs (p < 0.05), and the value is equivalent to 50 μg/ mL of ursolic acid. Similarly, Kim et al. [5] reported that low molecular weight peptides (< 3 kDa) from horse bone protein hydrolysates inhibit collagenase and elastase activities by about 40% and 28%, respectively. In this study, the collagenase inhibitory activities of all LDGH ranged from 42.31% to 55.21%, and were higher than that reported by Kim et al. [5]. In contrast, the elastase inhibitory activities of all LDGH ranged from 15.10% to 23.68%, and were lower than that reported by Kim et al. [5]. Park et al. [33] reported that porcine placental extracts at 100 μg/mL significantly reduce elastase activity over 25% in a dose 5

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Fig. 3. Effects of LDGH-F3 and LDGH-P3 on Hs68 cell viability.

protect human skin fibroblast cells (Hs68) after exposure to UVB. LDGH-F3 and P3 showed no cytotoxic effect on Hs68 cells at concentrations ranging from 10 to 500 μg/mL (Fig. 3A). To examine the protective effects of LDGH-F3 and LDGH-P3 against UVB-induced cell death, Hs68 cells were treated with different doses (10–500 μg/mL) of LDGH-F3 and LDGH-P3 after UVB irradiation (100 mJ/cm2). As shown in Fig. 3B, the viability of cells treated with LDGH-P3 after UVB irradiation increased significantly with increasing concentration (p < 0.05). In addition, the survival rate of cells treated with LDGH-P3 at 100, 250, and 500 μg/mL after UVB irradiation was higher than LDGH-F3 and UVB irradiated control cells. This protection may result from the higher antioxidant activity of LDGH-P3 as shown in Table 2, which reduces oxidative stress induced by UVB irradiation. Consequently, LDGH-P3 was selected due to its superior antioxidant activity,

dependent manner. Our results suggest that LDGH may play a role as collagenase and elastase inhibitory compound to protect skin from wrinkling. Collagen and elastin are the major structural proteins of ECM in the dermis. Decrease in ECM protein has been known to cause wrinkles and line formation in the skin. Collagen is rapidly degraded by collagenase, and collagenase activity is enhanced by UV irradiation. Elastase activity is also enhanced by UV irradiation, and elastin degradation causes sagging of the skin [34]. Therefore, LDGH-F3 and LDGH-P3, which effectively inhibit collagenase and elastase activities, are ideal candidates for prevention of photoaging.

3.4. Effects of LDGH-F3 and P3 on UVB-exposed Hs68 cell viability LDGH-F3 and LDGH-P3 were selected to determine whether they 6

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3.6. Effects of LDGH-P3 on MAPK expression UVB irradiation causes the production of reactive oxygen species (ROS) and stimulates cell surface growth factor receptors, cytokines and increases the levels of phosphorylated MAPKs, which are comprised of three general classes, p38, ERK, and JNK in human skin fibroblasts. The three classes of MAPKs have been shown to be present in mammalian cells, and phosphorylated MAPKs have been reported to inhibit type I collagen synthesis in human skin fibroblasts [38]. In our study, we investigated whether LDGH-P3 inhibits phosphorylation of p38, ERK, and JNK in UVB-irradiated Hs68 cells. As shown in Fig. 5, UVB irradiation (100 mJ/cm2) of Hs68 cells induced phosphorylation of p38, ERK, and JNK. However, treatment with LDGH-P3 (50–500 μg/mL) significantly reduced activation of ERK (p < 0.05). Activation of p38 and JNK were suppressed by LDGH-P3 only at 100, 250, and 500 μg/mL (p < 0.05). Especially, the levels of pERK and p-JNK after UV irradiation were significantly reduced by LDGH-P3 at 250–500 μg/mL and 100–500 μg/mL, respectively to the levels observed in control cells (p < 0.05). These results show that LDGH-P3 at 250 and 500 μg/mL could reduce the activation of MAPK in irradiated human skin fibroblasts to the levels observed in control cells (p < 0.05). Oxidative stress induced by UVB initiates the MAPK signaling cascade [39]. Free radicals with major species of ROS are react readily with other substances or groups in the body, resulting in cell damage and human disease [40]. Therefore, removal of free radicals and ROS may be one of the most protective defenses of a living body against various diseases. Generally, exposure of human skin fibroblast cells to UVB radiation can generate production of free radical and ROS, which damage cellular components [36]. UV irradiation-induced, photochemically-generated ROS activates cellular responses that mimic those that occur in response to ligand activation of cell surface growth factor such as epidermal growth factor receptor (EGFR) [41]. Downstream from the activated EGFR, the GTP-binding regulatory Ras was activated, and subsequently MAPKs (p38; extracellular signal-regulated kinase, ERK; c-Jun N-terminal kinase, JNK) were activated and phosphorylated [39]. In order to inhibit MAPK-mediated photoaging, many studies have been carried out to evaluate antioxidants to inhibit free radical and ROS by various sources such as chitooligosaccarides [38], pacific cod skin gelatin peptides [42], coffea arabica extract hydrolysates [43]. Ahn et al. [38] reported that chitooligosaccharides derived from chitosan via enzymatic reaction attenuate phosphorylated MAPKs (p-p38, p-ERK, p-JNK) at 50 μg/mL in UVB-irradiated human fibroblast cells and suppress in these activation at 500 μg/mL to the levels of control cell. Lu et al. [44] also reported that peptides from cod skin gelatin hydrolysates inhibited activation of MAPKs at 200 μg/mL to the levels of control cells in UVB-irradiated human fibroblast cells. Anti-oxidation activity is associated with inhibition of MAPK phosphorylation and procollagen type I synthesis in human skin fibroblasts after UVB-induced photodamage [39]. Our results suggest that LDGH-P3 has a great potential as a photoaging inhibitor because it inhibits the phosphorylation of MAPK and the down-regulation of type I collagen by preventing UVB-induced photodamage. Although LDGH-P3 showed antioxidative activity and protected UVB induced photodamage, these results are not always promising the good effect on real life. Because the cell-based study sometimes is not able to estimate the biological activity of LDGH-P3 when applied in the real body. Therefore, further study should be necessary to evaluate the absorption capacity or biological activity of LDGH-P3 in human living body.

Fig. 4. Effect of LDGH-P3 on the expression of procollagen type I in UVB-irradiated Hs68 cells.

collagenase inhibitory activity and cell viability to evaluate if it modulates procollagen type I and MAPKs of human skin fibroblast cells. 3.5. Effects of LDGH-P3 on procollagen type I expression Collagen is a major protein of skin, and it is the main component of skin connective tissue. Skin elasticity is modulated by collagen synthesized mostly from procollagen type I. Among the various type of collagen, type I is the most abundant, comprising between 85% and 90% of total skin collagen. In addition, damage of procollagen type I by UVB results in premature aging of skin [35]. To evaluate the effects of the selected LDGH-P3 on procollagen type I expression in UVB-induced Hs68 cells, the cells were exposed to UVB (100 mJ/cm2) and treated with LDGH-P3 at different doses (10–500 μg/mL) for 24 h. As shown in Fig. 4, UVB irradiation resulted in 1.05% of procollagen type I expression levels relative to control cells. In contrast, after LDGH-P3 treatment (at 500 μg/mL), procollagen type I expression was significantly restored to as high as 11.12% compared to the control group (p < 0.05). Jo et al. [36] reported that microalgal extracts of Tetraselmis suecica (at 31 μg/mL) restore procollagen type I expression levels by 15% in UVB-irradiated human fibroblast cells. Seo et al. [37] also reported that fish scale collagen peptides increase procollagen type I expression levels in UVB-irradiated human fibroblast cells by approximately 3% compared to the control group. These results suggest that LDGH-P3 prevents UVB-induced downregulation of procollagen type I expression by protecting proper regulation of synthesis and degradation against UVB-irradiation. Therefore the LDGH-P3 may have potential for treatment and prevention of photoaging.

4. Conclusions Scientists have mostly focus on plant resources to evaluate natural compounds for enhancing skin health and there is a lack of study that investigate anti-photoaging activity of animal resources. The present 7

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Fig. 5. Effect of LDGH-P3 on the UVB-induced expression of MAP kinases in Hs68 cells.

study shows that the low molecular weight hydrolysates LDGH-P3 derived from donkey hide has antioxidant activity, collagenase and elastase inhibiting activities, and attenuated activation of MAPK by UV irradiation, thereby increasing procollagen type I synthesis. LDGH-P3, therefore, could be used as a potential natural agent for the prevention of photodamage. These results can be used to elucidate the bioactivity of low molecular weight gelatin hydrolysates from animal resource. However, it is recommended that LDGH-P3 to be further tested for its efficacy and bioavailability in human trial.

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