Preparation, purification and identification of cadmium-induced osteoporosis-protective peptides from chicken sternal cartilage

Journal of Functional Foods 51 (2018) 130–141

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Preparation, purification and identification of cadmium-induced osteoporosis-protective peptides from chicken sternal cartilage

T

Xiaoling Lina, Liu Yanga, Min Wanga, Ting Zhangc, Ming Liangc, Erdong Yuana,d, ⁎ Jiaoyan Rena,b,d, a

School of Food Sciences and Engineering, South China University of Technology, Guangzhou 510641, China Sino-Singapore International Joint Research Institute, Guangzhou Knowledge City 510000, China c Infinitus (China) Company Ltd., Guangzhou 510665, Guangdong, China d Overseas Expertise Introduction Center for Discipline Innovation of Food Nutrition and Human Health (111Center), Guangzhou 510641, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Chicken sternal cartilage peptides (CSCP) Purification Identification Cadmium-induced osteoporosis Antioxidant activity Protective effect

Five novel peptides (GGAP, QIGPA, QLGPA, MPKYA and QGPAN) were identified from the purified sub-fraction (F1-c) of chicken sternal cartilage hydrolysate produced using alcalase (E/S 0.50%, pH 7.0, 55 °C, 9 h). The protective effect of the peptides was evaluated based on a cadmium-induced osteoporosis model (48 hr-LD50 of 3.2 μM) by analyzing the cell viability, mitochondrial membrane potential (ΔΨm) and apoptosis rate. Except for MPKYA and GGAP, the other three identified peptides which contained the same GPA amino acid sequence showed favorable reversal effects with cell viability ranging from 62.93 to 90.96%, a significant increase of ΔΨm reaching more than 61.28% and a percentage of early apoptotic cells lower than 25.48% (p < 0.05). This research suggests that peptides derived from chicken sternal cartilage that counteract Cd-induced osteoporosis have application potential as functional foods for the prevention of osteoporosis.

1. Introduction Osteoporosis is a progressive bone-weakening disease that generally occurs with aging (Ganguly et al., 2017). It is characterized by low bone mineral density and microarchitectural deterioration of bone tissues, which usually leads to the development of brittle bones susceptible to fracturing (Consensus, 2001; Mithal & Kaur, 2012). Fractures can be serious, and contribute to the substantial morbidity and mortality. (Khosla et al., 2017). The treatment of osteoporosis is costly and therefore imposes a heavy economic burden on patients, their families as well as society at large (Fischer et al., 2017; Mohd-Tahir & Li, 2017). In addition to immutable factors such as age and gender, osteoporosis is also affected the modifiable risks such as lifestyle (lack of calcium and vitamin D intake, insufficient physical activity, smoking, alcohol consumption and glucocorticoid administration) and environmental factors (Berarducci, 2004). An outbreak of itai-itai disease in Japan had garnered great attention in the 1940′s (Inaba et al., 2005). In recent years, a myriad of cross-sectional and prospective studies of different populations, mainly from China (Lv et al., 2017), the United States (Wu, Magnus, & Hentz, 2010) and Norway (Dahl et al., 2014)s have provided evidence for a correlation between cadmium exposure and low bone mineral density, as well as an increased risk of osteoporosis. The ⁎

epidemiological research also indicated that even low chronic exposure to cadmium, as is still taking place in industrialized countries, can contribute to the development of osteoporosis and bone fracture (Mezynska & Brzóska, 2018). Therefore, cadmium-induced osteoporosis could not be ignored. Cadmium (Cd), a heavy metal as well as a widespread environmental contaminant, has gained public attention due to the increased discarding of electronic waste such as electronic components and batteries of cell phones and computers (Guo et al., 2010). In addition, the ingestion of contaminated food (rice, wheat, leafy greens, potatoes and carrots) or water, active or passive inhalation of tobacco smoke, and occupational use are also considered as risk factors for humans (Järup & Åkesson, 2009). The divalent Cadmium cation (Cd2+), as the major toxic form of Cd, is closely related disorders of skeleton. It has been demonstrated that increasing exposure to Cd was consistent with the loss of the bone mineral (Horiguchi et al., 2010). Several mechanisms that aim to explain the phenomenon of Cd-induced osteoporosis have been proposed. Firstly, Cd2+ was found to affect the proliferation, differentiation and apoptosis of the osteoblasts (Mohajeri, Rezaee, & Sahebkar, 2017). In this case, it was shown to change the mitochondrial transmembrane potential (ΔΨm) which contributed to apoptosis (W. Liu et al., 2014). Furthermore, Cd depletes the important intracellular

Corresponding author at: School of Food Science and Engineering, South China University of Technology, Wushan Rd. 381, Guangzhou 510641, China. E-mail address: [email protected] (J. Ren).

https://doi.org/10.1016/j.jff.2018.09.036 Received 8 June 2018; Received in revised form 5 September 2018; Accepted 7 September 2018 1756-4646/ © 2018 Published by Elsevier Ltd.

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Biochem Co., Ltd. (Shanghai, China). Mouse pre-osteoblast cell line MC3T3-E1 (sub-clone 14), which originated from the bone and calvaria of the newborn C57BL/6 mice and is considered as a good model for the study of in vitro osteoporosis, was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells below passage 10 were used for this study. Trypsin, neutral protease, alcalase, papain and flavourzyme were purchased from Guangxi Nanning Pangbo Biological Engineering Co. Ltd. (Nangning, China). The reagents 2,2-diphenyl-1picrylhydrazyl (DPPH), 2.2′-azo-bis-2-amidinopropane dihydrochloride (AAPH), cadmium acetate, thiazolyl blue tetrazolium bromide (MTT) and 4,6-Diamidino-2-phenylindole dihydrochloride (DAPI) were obtained from Sigma-Aldrich (Europe). Phosphate-buffered saline (PBS), alpha-minimum essential medium (ɑ-MEM), and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies Corp. (Grand Island, NY, USA). Cell Counting Kit-8 (CCK-8) was from Beyotime Institute of Biotechnology (Haimen, China). The mitochondrial membrane potential (ΔΨ) assay kit with JC-1 was from Yeasen Biotechnology Co., Ltd (Shanghai, China). The Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Dojindo Laboratories (Tokyo, Japan). The other chemical reagents used in the present study were of analytical pure grade and were obtained from local commercial sources.

antioxidant glutathione and binds to the sulfhydryl groups of proteins, leading to the excessive generation of reactive oxygen species (ROS) (Brzóska, Rogalska, & Kupraszewicz, 2011; Mohajeri et al., 2017). Furthermore, it was found to indirectly affect the metabolism of vitamin D and minerals (Brzóska & Moniuszko-Jakoniuk, 2005). Based on these complex mechanisms, several antioxidant natural products such as curcumin (Mohajeri et al., 2017), grape and apple juices (Ruiz et al., 2018) have been investigated for their protective effect against Cd-induced damage. Because of the complex composition of most of the compounds, the actual mechanisms that help protect against Cd-induced osteoporosis are not fully understood. There is an urgent need to identify efficient prophylactic-therapeutic factors against Cd-induced osteoporosis. To date, numerous bioactive food factors have been investigated for their favorable protective effects on osteoblasts, including the administration of calcium, vitamin D3, flavone and collagen (Eaton-Evans, 1994). Among these, type-I collagen, which is widely distributed in connective tissues (ligaments and cartilage) was found to play a significant role in the maturation and mineralization of osteoblasts (Barnes et al., 2017). Chicken sternal cartilage has become a potential source of collagen as an abundant by-product of the chicken breast procession industry. However, collagen cannot be efficiently digested and absorbed by the human body due to its large molecular weight (Sontakke, Jung, Piao, & Chung, 2016) and triple-helix structure (Ratanavaraporn, Damrongsakkul, Sanchavanakit, Banaprasert, & Kanokpanont, 2017). Conversely, small peptides such as buffalo casein-derived bioactive peptides (Reddi, Shanmugam, Kapila, & Kapila, 2016; Reddi, Shanmugam, Tanedjeu, Kapila, & Kapila, 2018) and whey-derived peptides (Pandey, Kapila, & Kapila, 2018) have been proved to promote the proliferation and differentiation of the osteoblasts. In order to improve the availability of chicken sternal collagen, basic methods were applied to convert the collagen into small molecules. Enzymatic hydrolyzation is a favorable choice to release encoded bioactive peptides with various functions. To date, only few studies have investigated the optimal hydrolysis conditions for the industrial production of chicken sternal peptides. In addition, valorization of these chicken-breast processing wastes as high-value peptides can not only reduce environmental pollution but also increase the economic value in the food industry (Henchion, McCarthy, & O’Callaghan, 2016). In vitro experiments have confirmed that collagen peptides can stimulate the proliferation and differentiation of MC3T3-E1 cells (Liu et al., 2014), and even alter the expression of bone-related genes in osteoblasts (Daneault, Prawitt, Fabien Soulé, Coxam, & Wittrant, 2017). However, all these cell models were set up without Cd induction, and thus cannot well mimic Cd-induced osteoporosis. It is still not known whether the chicken sternal cartilage peptides are beneficial for the protection against Cd-induced osteoporosis. Furthermore, the mechanisms mediating the reversal effect of the peptides with known sequences should also be investigated. In the present study, the hydrolysis conditions were evaluated for the preparation of small molecular weight peptides with relatively high antioxidant activity from chicken sternal cartilage. The optimal hydrolysates were successively purified by chromatography on DEAE-52 cellulose and a Sephadex G-15 column, and the target fraction was analyzed by UPLC-ESI-MS/MS for identification. The protective effect of the identified peptides in the Cd-induced cell model of osteoporosis was evaluated using the CCK-8 assay, Flow cytometric mitochondrial membrane potential assay and annexin V-FITC/PI staining.

2.2. Preparation of chicken sternal cartilage peptides (CSCP) Prior to hydrolyzing, chicken sternal cartilage (CSC) was dried at 40 °C followed by smashing and filtration through a 40 mesh, yielding a CSC powder. In the process of SCS powder, the five parameters type of enzyme (trypsin, neutral protease, alcalase, papain, flavourzyme), enzyme to substrate ratio (E/S: 0.25, 0.50 and 0.75% w/w), pH (6.0, 6.5 and 7.0), temperature (50, 55 and 60 °C) and time (2, 4 and 9 h), were optimized in single-factor experiments. The pH of the mixture was maintained constant during the hydrolysis by frequent addition of 30% NaOH. At the end of the reaction, the solution was heated in a boiling water bath for 10 min to inactivate the enzymes and then centrifuged at 8000 rpm for 30 min. The supernatant was lyophilized to obtain chicken sternal cartilage peptides (CSCP) for subsequent analysis. Protein content was determined using the Kjeldahl method ab HYP308 Digest Stove with a KDN-103F Automatic Nitrogen Determinator (Shanghai Xianjian Instrument Co. Ltd., Shanghai, China). The molecular weight distribution CSCP was analyzed using an Agilent 1200 HPLC system (Agilent, USA) equipped with a TSK G2000 SWXL column (TOSOH Co., Japan) according to the protocol of Su et al. (Su, Ren, Zhao, & Sun, 2013). The degree of hydrolysis (DH, %) was determined using the o-phthalaldehyde (OPA) method. (Kokkaew, Thawornchinsombut, & Park, 2016; Nielsen, Petersen, & Dambmann, 2001). The DPPH radical scavenging activities of the different CSCP were estimated as described by Gu et al. (Gu et al., 2012) The ORAC Assay was performed in black 96-well microplates according to published methods (Dávalos, Miguel, Bartolome, & Lopez-Fandino, 2004; Ou, Hampsch-Woodill, & Prior, 2001).

2.3. Purification of chicken sternal cartilage peptides (CSCP) 2.3.1. Anion-exchange chromatography Chicken sternal cartilage which hydrolyzed for 9 h was used for further purification. A CSCP solution (2 mL, 50 mg/mL) was separated in a glass column (1.6 × 30 cm) which was filled with DEAE-52 cellulose (Guangzhou Qiyun Ltd., Guangzhou, China) pre-equilibrated using distilled water, and stepwise eluted at a flow rate of 0.5 mL/min with 150 mL distilled water, 0.01, 0.05, 0.1 and 0.2 M NaCl solution. Each eluted fraction (5 mL) was collected and monitored at 214 nm. Five fractions (F1, F2, F3, F4 and F5) were pooled and lyophilized, and their ORAC antioxidant activities were measured.

2. Materials and methods 2.1. Materials Chicken sternal cartilage was collected from the supermarket (Guangzhou, China). The targeted peptides were synthesized by GL 131

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2.6. Evaluation of the protective effects of identified peptides in the cell model of Cd-induced osteoporosis

2.3.2. Size exclusion chromatography The F1 fraction (2 mL, 30 mg/mL) was further purified on a Sephadex G-15 column (1.6 × 60 cm), and pre-equilibrated using distilled water. The fraction was eluted with distilled water at a flow rate of 0.5 mL/min. Each eluted fraction (5 mL) was collected and peptides were detected at 214 nm. Finally, three sub-fractions (F1-a, F1-b and F1-c) were collected, lyophilized, and their ORAC antioxidant activities were measured.

2.6.1. MTT-based assay of peptide toxicity MC3T3-E1 cells were seeded into 96-well plates at a density of 5 × 103/well. After 24 h, the cells were treated with complete medium containing various concentrations (0.1 and 0.5 mM) of the identified peptides, and incubated for 48 h. Then, the MTT assay was conducted following the same procedure as described in Section 2.5. 2.6.2. CCK-8-based assay for the effects of peptide pre-treatment MC3T3-E1 cells were seeded into 96-well microplates (3 × 103 cells/well). After 24 h, the cells were pre-treated with the identified peptides (0.1 and 0.5 mM) for 48 h. Then, the treated cells were incubated with 3.2 μM Cd(Ac)2 for 48 h. The cell viability was assessed using the CCK-8 assay, according to the manufacturer’s protocol. Briefly, 10 μL of CCK-8 reagent was added to the cells, followed by incubation for 2 h at 37 °C, after which the absorbance at 450 nm was measured using a microplate reader (Biotech, Inc., USA). All the results were expressed as the absorbance minus that of the control group.

2.4. Identification of chicken sternal cartilage peptides (CSCP) by ESI-MS/ MS The fraction with the highest ORAC value after Sephadex G-15 separation was subjected to ESI-MS/MS analysis. An aliquot comprising 5 μL of the peptide solution (1 mg/mL) was first separated on an ultraperformance liquid chromatography system (Agilent 1290, Agilent Technologies, USA) equipped with an Agilent SB-C18 RRHD column (1.8 μm, 2.1 × 50 mm) at a flow rate of 0.2 mL/min. A gradient elution was employed consisting of mobile phase A (acetonitrile) and B (H20, 0.1% formic acid), as follows: 0–1 min, A 0% v/v to 15% v/v; 1–4 min, A 15% v/v to 85% v/v; 4–8 min, A 85% v/v; 8–9 min, A 85% v/v to 15% v/v; 9–10 min, A 15% v/v. The electrospray ionization mass spectrometry was carried out on a maXis impact instrument (Bruker Co., Germany). Scans were acquired in positive ion mode (ESI+) from 50 to 1500 m/z with sodium formate solution as an internal standard to correct molecular weight. The selected peptides were subjected to tandem mass spectrometry sequencing. High purity N2 was used for drying (7.0 L/min), and nebulization (1.0 Bar). Peptide sequences were identified by processing the MS/MS spectra using Compass Data Analysis software (Version 4.1, Bruker Daltonics, Germany) as well as manual calculation. The identified peptides and YGFGG were synthesized using the Fmoc strategy for further study.

2.6.3. Flow cytometric assay for mitochondrial membrane potential assay MC3T3-E1 cells were grown in a 6-well plate at a density of 6 × 104 cells/well. The cells were then incubated with the identified peptides (0.5 mM) for 48 h, after which the complete medium was replaced with the same medium containing 3.2 μM Cd(Ac)2 together with 0.5 mM synthetic peptides. The changes in mitochondrial membrane potential of the treated cells were detected by JC-1 assay. In accordance with the manufacturer’s instructions, the cells were incubated with JC-1 (10 μg/ ml) at 37 °C for 20 min in the darkness. Then the cells were washed twice with cold JC-1 staining buffer, resuspended in 1.0 mL of the buffer and analyzed by flow cytometry (CytoFLEX, Beckman Coulter, USA). Each assay was repeated in triplicate. 2.6.4. DAPI staining of nuclei MC3T3-E1 cells were seeded into a 48-well plate at a density of 6 × 103 cells/well. After treatment with 0.5 mM of the identified peptides for 48 h, the cell model of osteoporosis was established by incubating with 3.2 μM Cd(Ac)2 for 48 h. The treated cells were fixed with 4% paraformaldehyde at a room temperature for 20 min, washed with PBS three times, and then stained with 100 ng/mL DAPI stain for 10 min. In the final step, images of the stained cells were captured by means of a fluorescence microscope (Olympus IX73, Carl Zeiss, Germany), using the 40 × objective.

2.5. Establishment of a cell model of osteoporosis The widely used cell line MC3T3-E1 was expanded in ɑ-MEM with 10% FBS. The cells were cultured to 70–80% confluency and then collected using 0.25% EDTA-trypsin. Generally, MC3T3-E1 were maintained at 37 °C in a humidified atmosphere with 5% CO2. The cadmium acetate [Cd(Ac)2] solution was prepared as follows: 1.2 mg Cd (Ac)2 was first dissolved in 10 μL DMSO, then 10 mL complete medium was added to make a 0.5 mM cadmium acetate stock solution, which was further diluted to 20 μM for use. MC3T3-E1 cells were seeded separately at a density of 104 cells/well into a 96-well microplate. After 24 h postseeding, the growth medium was removed. Triplicate wells were treated with serial dilutions of Cd(Ac)2 (1, 2, 4, 5, 6, 8 and 10 μM) for 24 and 48 h, respectively. In the control group, the cadmium solution was replaced by the complete medium. The cell viability of each group was analyzed using the MTT assay. Briefly, 20 μL of MTT (5 mg/ mL) was added to each well and incubated for 4 h at 37 °C. Then, the medium was removed and replaced with 150 μL of DMSO to dissolve the formazan crystals. The absorbance was measured at 570 nm by a microplate reader (Biotech, Inc., USA). Relative cell viability was determined by dividing the absorbance of the Cd(Ac)2-treated group by that of the control group. The half-maximal inhibitory concentration (IC50) of Cd(Ac)2 was calculated using the Probit regression model procedure in SPSS 22.0 (IBM Corp., USA). The calculated concentration of Cd(Ac)2 was finally experimentally to the cells for verification. YOYO-1 (591/509) dye at a final concentration of 100 nM was added together with Cd(Ac)2, then incubated for 48 h. The morphology of the dying cells was observed under a fluorescence microscope (Olympus IX73, Zeiss, Germany) using the 20 × objective. Finally, the viability of the Cd-treated cells was measured using the CCK-8 assay, according to the manufacturer’s instructions.

2.6.5. Flow cytometric annexin V-FITC/PI assay MC3T3-E1 cells were seeded into a 6-well plate at a density of 6 × 104 cells/well. The cells were then treated with the identified peptides at a 0.5 mM concentration for 48 h, after which the medium was exchanged for the complete medium which contained 3.2 μM Cd (Ac)2 together with 0.5 mM of the identified peptides and the cells incubated for a further 48 h. The apoptosis rate was quantified using the Annexin V-FITC/PI Apoptosis Detection Kit. Briefly, the cells were harvested and resuspended in 100 μL of binging buffer, and then coincubated with Annexin V-FITC and PI for 15 min at room temperature in the dark. Finally, 400 μL of binding buffer was added and the cells were analyzed by flow cytometry (CytoFLEX, Beckman Coulter) within 1 h. Each assay was repeated in triplicate. 2.7. Statistical analysis All the results were expressed as the means ± standard deviations of three independent experiments. Statistical significance (p < 0.05, 0.01 and 0.001) was calculated using one-way ANOVA followed by the least significant difference (LSD) procedures using in SPSS statistics 22 software (IBM Corp., USA). In addition, a Pearson correlation test was 132

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Fig. 1. Enzymatic hydrolysis of chicken sternal cartilage to produce peptides (CSCP) under different industrially relevant parameters. (A) Hydrolytic processing of chicken sternal cartilage; (B) Protein content of each CSCP; (C) Degree of hydrolysis (DH) of each CSCP; (D) Molecular weight distribution (MW < 1 kDa) of each CSCP; (E) DPPH· scavenging capacity of each CSCP; (F) ORAC values of each CSCP; (G) Correlation analysis between DH and ORAC; (H) Correlation analysis between molecular weight distribution (MW < 1 kDa) and ORAC; (I) Correlation analysis between molecular weight distribution (MW < 1 kDa) and DPPH· scavenging capacity; (J) Correlation analysis between DH and DPPH· scavenging capacity. Significant differences between CSCP: *p < 0.05.

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Fig. 2. Chromatograms at various purification steps of chicken sternal cartilage and the antioxidant activity of each sub-fraction. (A) Elution profile of chicken sternal cartilage hydrolyzed for 9 h and separated by anion-exchange chromatography on a DEAE-52 cellulose column. The gradient line (gray) shows the concentration of NaCl in the eluting solvent. The elution was performed at a flow rate of 0.5 mL/min and peptides were detected at 214 nm; (B) ORAC values of subfractions (F1 to F5); (C) Additional purification of fraction F1 by Sephadex G-15 chromatography. The elution was performed at a flow rate of 0.5 mL/min with distilled water and peptides were detected at 214 nm. The gel permeation chromatography results are shown in the top right corner of the figure; (D) ORAC values of sub-fractions (F1-a to F1-c). Significant difference of the ORAC values among each sub-fraction are indicated with asterisks: as * p < 0.05 and *** p < 0.001.

E/S ratio of 0.25% (w/w) had a satisfactory DH value (29.20 ± 0.01%), as well as a DPPH· scavenging IC50 value of 3.32 ± 0.12 mg/mL, the remaining protein content remained was only 34.59 ± 2.39%. Therefore, this E/S ratio does not meet the criteria for large-scale production. An E/S ratio of 0.75% seemed optimal due to its highest ORAC value. However, the protein content and the DPPH· scavenging capacity were both lower than that of the hydrolysates under an E/S ratio of 0.50%. As no significant difference of the molecular weight distribution (MW < 1 kDa) was found between the peptides hydrolyzed at the E/S ratios of 0.50 and 0.75% (w/w). Thus, an E/ S ratio of 0.50% was chosen for further hydrolysis of small peptides. The pH value is also an important parameter for protein hydrolysis. In order to obtain soluble hydrolysates without a bitter taste, pH ranging from 6.0 to 7.0 was applied for the hydrolysis of chicken sternal cartilage. The chicken sternal cartilage that was hydrolyzed at pH 7.0 showed higher DH (22.02 ± 0.45%) with a percentage of small peptides (MW < 1 kDa) up to 46.52%. The IC50 values of the DPPH· scavenging activity dropped gradually with increasing pH. The ORAC values of CSCPs ranged from 0.34 ± 0.02 to 0.36 ± 0.01 μmol TE/mg peptides (p > 0.05) when hydrolyzed at different pH values. Consequently, pH 7.0 was used for the preparation of CSCP. When the impact of temperature on the hydrolysis was assessed, it was clear that a relatively low temperature (50 °C and 55 °C) should be chosen, since the protein content at these temperatures reached 55.08 and 56.22%, respectively, which was significantly higher than what was observed at high temperature (60 °C) (p < 0.05). In order to obtain more small peptides coupled with high ORAC values, 55 °C was suitable for the production of CSCP. With the extension of the hydrolysis time, the DH as well as the ORAC values were gradually increased. Long term hydrolysis (up to 9 h)

conducted to determine the correlation between variables.

3. Results and discussion 3.1. Optimal hydrolysis conditions for the preparation of chicken sternal cartilage peptides (CSCP) With the aim to provide a guidance for the large-scale production, widely used industrial parameters should be first tested on a laboratory scale to select favorable hydrolysis conditions for the preparation of hydrolysates (Fig. 1.A). The crude protein content of chicken sternal cartilage was detected as 63.59 ± 0.19% on a dry weight basis, indicating that it is a rich potential source for the production of peptides. As shown in Fig. 1B-F, trypsin, alcalase and papain showed a favorable extraction efficiency, with protein contents above 50% in all case. A higher degree of hydrolysis (DH) was observed with nonspecific enzymes, such as alcalase and flavourzyme. Since chicken sternal cartilage contains abundant glycine and proline, the hydrolysis using trypsin and papain with specific enzyme cutting sites was restrict to a certain extent. Previous studies have demonstrated that the antioxidant activity of peptides was closely related to their anti-osteoporosis ability (Mada, Reddi, Kumar, Kapila, & Kapila, 2017; S.B. Mada et al., 2017). Thus, in vitro antioxidative tests were used to evaluate the potential anti-osteoporosis effect of the hydrolysates. Alcalase converted the greatest proportion of the chicken sternal cartilage into small peptides (MW < 1 kDa) with a DPPH· scavenging IC50 values of 4.66 ± 0.09 mg/mL and an ORAC value of 0.48 ± 0.02 μmol TE/mg peptides. Thus, alcalase was selected as the best enzyme for further study. Although chicken sternal cartilage hydrolyzed using alcalase at an 134

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Fig. 3. Amino acid sequence of the purified peptides (F1-c) from chicken sternal cartilage. The identification was performed using UPLC system coupled to an ESIQUAD-TOF mass spectrometer. The MS/MS spectrum was acquired over an m/z range of 50–1500, and the peptide sequences were calculated manually using the Compass Data Analysis software. (A) Base peak chromatogram of the F1-c fraction; (B) LC MS/MS spectrum of GGAP (m/z = 301.1488); (C) LC MS/MS spectrum of QLGPA and QIGPA (m/z = 485.2349 for both) (D) LC MS/MS spectrum of MPKYA (m/z = 609.2654); (E) LC MS/MS spectrum of QGPAN (m/z = 508.2659).

and ORAC (r = 0.534, p = 0.004) (Fig. 1H). These results suggested that the exposure of abundant antioxidant residues was responsible for the ORAC capacity of chicken sternal cartilage hydrolysates. Although no significant correlation was found between the molecular weight distribution (MW < 1 kDa) and DPPH· scavenging activity (r = 0.313, p = 0.112) (Fig. 1I), a significant negative correlation was observed between DH and the DPPH· scavenging activity (r = -0.460, p = 0.016) (Fig. 1J). Thus, DH is a vital parameter during the hydrolysis processing. Based on the correlation analysis, both DH and the molecular weight distribution (MW < 1 kDa) were deduced as important factors for the quality control of CSCP in large-scale production.

produced more smaller peptides compared with the hydrolysates obtained after 4 h of hydrolysis. The fraction of the CSCP (MW < 1 kDa) reached up to 61.62 ± 2.78% when the material was hydrolyzed at 55 °C for 9 h. Furthermore, the hydrolysates obtained after 9 h had the best DPPH· scavenging activity (IC50 = 5.14 ± 0.19 mg/mL). Consequently, the hydrolysis time of as 9 h was selected for further experiments. Correlations among the hydrolysis parameters were investigated using Pearson’s analysis. One previous study has demonstrated that peptides with smaller molecular weights have stronger antioxidant activities (Sun, Chang, Ma, & Zhuang, 2016). Interestingly, a very significant correlation was found between DH and ORAC (r = 0.737, p < 0.001) (Fig. 1G). Generally, a high DH usually related to the thorough hydrolysis of proteins, leading to a small molecular weight of the resulting peptides. Accordingly, a significant moderate correlation was found between the molecular weight distribution (MW < 1 kDa)

3.2. Purification of the chicken sternal cartilage peptides (CSCP) Chicken sternal cartilage hydrolyzed under the optimal conditions (alcalase at an E/S ratio of 0.50%, pH 7.0, 55 °C and 9 h) was subjected 135

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Fig. 4. Establishment of a cell model of osteoporosis. (A) Dose-response of Cd(Ac)2-challenged MC3T3-E1 cells. The cells were challenged with various doses of Cd (Ac)2 (0–10 μM) for 24 and 48 h, respectively. Cell viability was measured using the MTT assay; (B) Plot of log-transformed dose and Probit-transformed cell viability for Cd(Ac)2 toxicity in MC3T3-E1 cells; (C) Images of MC3T3-E1 cells in complete medium (control group) and with 3.2 μM Cd(Ac)2 (model group). Apoptotic and necrotic cells were identified using the green fluorescent nuclear dye YOYO-1; (D) Cell viability of MC3T3-E1 cells when challenged with 3.2 μM Cd(Ac)2 as determined using the CCK-8 assay. *p < 0.05, compared with control for 24 h incubation and #p < 0.05, compared with control for 48 h incubation.

spectrometer (Fig. 3). According to the base peak chromatogram, five compounds with UV absorbance at 214 nm (ranging from 0.2 to 3 min, Fig. 3A) were finally identified to be peptides with the sequences including Gly-Gly-Ala-Pro (GGAP, Peak 5 at 1.01 min), Gln-Leu-Gly-ProAla (QLGPA, Peak 7 at 1.03 min), Gln-Ile-Gly-Pro-Ala (QIGPA, Peak 7 at 1.03 min), Met-Pro-Lys-Tyr-Ala (MPKYA, Peak 8 at 1.54 min), and GlnGly-Pro-Ala-Asn (QGPAN, Peak 10 at 1.83 min). The detected molecular masses of 301.15 Da ([M+H]+), 485.23 Da ([M+H]+), 485.23 Da ([M+H]+), 609.27 Da ([M+H]+), and 508.27 Da ([M+Na]+) were in good agreement with the theoretical mass values of 300.31 Da, 484.55 Da, 484.55 Da, 608.76 Da, and 485.5 Da, respectively (Fig. 3BE). According to the manual calculations, most of the b- and y-ions were also consistent with their theoretical value. Since leucine and isoleucine have the same m/z, QLGPA and QIGPA both had m/z values of 485.23. The most prominent feature of collagen is its tripeptide repeat sequence, depicted as –(G-P-X) n–, where X is any amino acid residue. Here, the identified three peptides (QLGPA, QIGPA, and QGPAN) containing glycine (G) and proline (P) were in excellent agreement with the collagen characteristic. Additionally, the presence of glutamine (Q) in these peptides might contribute to their antioxidant activity. Recent results have demonstrated that peptides containing P residues are generally resistant to degradation by digestive enzymes due to their ability to form peptide bonds with cis isomerism (Nimalaratne, Bandara, & Wu, 2015). Therefore, it seems that these novel peptides might be recalcitrant to gastrointestinal digestion.

to further purification. Recently, S. Mada et al. (2017) reported that peptides derived from casein with antioxidative activity had an osteoporosis-reversing effects in osteoblasts. The prevention of osteoporosis might be attributed to the antioxidant activity of the peptides. In this study, an in vitro ORAC assay was carried out to isolate the potentially pharmacologically active fractions. Ion-exchange chromatography is typically used to purify amphiphilic peptides based on their affinities to the anion exchange resins. As shown in Fig. 2A, five fractions (F1-F5) were separated from CSCP using DEAE-52 cellulose column and eluted with pure distilled water or different concentrations of NaCI (0.01, 0.05, 0.1 and 0.2 M). The ORAC value of F1 (1.60 ± 0.13 μmol TE/mg peptides) was significantly higher than that of the other four fractions (Fig. 2B). The molecular weight distribution of F1 was then analyzed using a TSK column with phosphate buffer as the mobile phase. As shown in the top right corner of Fig. 2C, nearly 63% of the components in the F1 fraction had with molecular weights below 1 kDa. Thus, the Sephadex G-15 gel filtration with a recommended separation range from 100 to 1500 Da was used to further purify F1, which yielded and three subfractions (F1-a, F1-b and F1-c) with good resolution. The ORAC values of fraction F1-c (3.23 ± 0.15 μmol TE/mg peptides) was significantly higher than those of the other two fractions (Fig. 1D), supporting the hydrolysis that peptides with smaller molecular weight might generally have better antioxidative activity.

3.3. Identification of the chicken sternal cartilage peptides 3.4. Establishing a cell model of osteoporosis In order to identify the potential peptides from chicken sternal cartilage, the F1-c fraction with the highest antioxidant activity was subjected to RP-UPLC analysis coupled on-line to an ion trap mass

A cell model of osteoporosis, induced by Cd(Ac)2, was established for further screening of potential therapeutic peptides from chicken 136

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investigated. Compared with the control group, a significantly decrease of cell confluence as well as a definite change in the morphology of the model group [treated with 3.20 μM Cd(Ac)2] was observed (Fig. 4C). The cells were detached and became markedly thinner, whereby some of them exhibited shrinkage or lost their normal shape and became round. These phenomena are indicative apoptosis. Interestingly, no green fluorescence was found in the control group, while some was observed in the model group. The YOYO-1 dye is a cell-impermeable fluorescent DNA staining dye, which can only enter the cells without a complete enclosing membrane, and thus dead cells will be labeled with green fluorescence. The treatment with Cd(Ac)2 can cause apoptotic cell death. According to the results of the CCK-8 assay, the viability of the MC3T3-E1 cells exposed to 3.20 μM Cd(Ac)2, at 49.24 ± 8.64%, was much lower than that of the control, and the difference was extremely significant (p < 0.001). Therefore, 3.2 μM Cd(Ac)2 was chosen as the optimal concentration for the establishment of a Cd-induced cell model of osteoporosis. Previous research has demonstrated that the EC50 value of CdCl2 was 5 and 7 μM for Saos-2 and MG-63 cells after 48 h of exposure, respectively (Ha, Burwell, Goodwin, Noeker, & Heggland, 2016). Moreover, a dose-dependent decrease in the viability of these two cell lines was also observed. These results were in good agreement with the observations made with MC3T3-E1 cells in this study. Nevertheless, there was a small difference in IC50 values among the different osteoblast cell lines. 3.5. Effects of the identified peptides on the cell viability of Cd-treated cells With the aim of screening anti-osteoporosis peptides from chicken sternal cartilage, the cytotoxicity together with the proliferation effects of these peptides on MC3T3-E1 cells were analyzed first. OGP (10-14) (YGFGG) is the active motif of osteogenic growth peptide(Pigossi, Medeiros, Saska, Cirelli, & Scarel-Caminaga, 2016). The protective effect of this C-terminal pentapeptide on osteoblastic lineage cells, including stimulating the proliferation, differentiation, and matrix mineralization, has been validated (Stakleff et al., 2013). In this paper, the YGFGG peptide was used as the positive control (P.C.) to evaluate its reversal effect on the cell model of osteoporosis. MC3T3-E1 cells were treated with 0.1 and 0.5 mM synthetic peptides for 48 h, and the cell viability was assessed using the MTT assay. As shown in Fig. 5A, QIGPA, QLGPA, MPKYA and QGPAN (0.1 mM) marginally increased the proliferation of MC3T3-E1 cells by 39.34, 21.44, 21.23 and 37.06% at 48 h, respectively. Low bone mass is a crucial factor in the development of osteoporosis. Therefore, the results indicated that these four peptides at a concentration of 0.1 mM have the potential for protection against osteoporosis. At a higher dose (0.5 mM), there were still no negative effects on cell proliferation, and this concentration was used for the subsequent experiments. After 48 h of pre-treatment by the peptides, the same peptides were applied together with 3.2 μM Cd(Ac)2 for another 48 h. At the end of the 48 h Cd-treatment period, the cell viability of the model group was 48.05 ± 4.29%. As shown in Fig. 5B, the cell viability of the positive control treated with YGFGG at 0.1 mM was significantly higher than that of the model group (p < 0.05). It is worth mentioning that MPKYA showed no protective effect on the Cd-induced osteoporosis model at any tested dose. However, the other three peptides, QIGPA, QLGPA and QGPN, promoted the proliferation of the cells even at 0.1 mM. Therefore, it is possible that the proliferation of the cells during the first 48 h helped reduce the cytotoxicity of the cadmium treatment. Thus, peptides with the ability to promote osteoblast proliferation might be beneficial for the treatment of osteoporosis. To estimate the false positive effect caused by the proliferation induced by peptides, the results obtained with high concentrations (0.5 mM) of peptides were taken into account. The efficacy of the other four synthetic peptides were in the order of QGPAN < GGAP < QIGPA < QLGPA, with the cell viability ranging from 61.94 to 90.96%. The cell viability of the group treated with QLGPA was even comparable to that of the control group. These

Fig. 5. The protective effect of the identified peptides against Cd-induced osteoporosis model. (A) Viability of MC3T3-E1 cells after exposure to the identified peptides for 48 h; (B) Cell viability in the Cd-induced osteoporosis model pre-treated with or without identified peptides. The cell viability was assessed using the MTT Assay and the CCK-8 Assay, respectively. *p < 0.05, compared with the control group and #p < 0.05, compared with the model group. P.C. is the positive group that was pre-treated with YGFGG.

sternal cartilage. As presented in Fig. 4A, Cd(Ac)2 treatment caused a decrease in cell viability in a dose- and time- dependent manner. After 24 h of treatment with Cd(Ac)2, no significant differences of cell viability were found between the MC3T3-E1 cells treated at a concentration of 1or 2 μM, respectively. Conversely, the cell viability gradually decreased as the Cd(Ac)2 concentration was increased from 4 to 10 μM, reaching 50.98 ± 7.00% when the cells were treated with the highest dose of Cd(Ac)2 (10 μM) for 24 h. A dose-dependent reduction in cell viability was observed when the cells were treated with Cd(Ac)2 for 48 h (p < 0.05). A marked cytotoxic effect was observed with 10 μM Cd(Ac)2, with only 19.42% viable cells remaining after 48 h. With the aim of simulating the osteoporosis injury through long-term treatment with the lowest dose of Cd(Ac)2, 48 h incubation was used for the model. As shown in Fig. 4B, the concentration-viability relationship suitably fitted by the Probit model with goodness-of-fit tests providing low χ2-values (8.895) and high p-values (0.919). The 50% cell viability dose (IC50 values) was calculated based on the established Probit model. The results indicated that the IC50 value was 3.09 μM, with 95% lower- and upper fiducial limits of 2.84 and 3.34 μM. In order to verify whether the fitted concentrations are accurate, 3.20 μM Cd(Ac)2 was applied to the MC3T3-E1 cells and the morphology and viability of the cells were 137

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Fig. 6. Analysis of mitochondrial membrane potential in pre-treated Cd-induced cell model of osteoporosis following pre-treatment with the indicated peptides from chicken sternal cartilage. (A) Representative scatter plots of flow cytometric analysis showing red/green (aggregation/monomer) JC-1 dye fluorescence in MC3T3-E1 cells; (B) Percentage of the cells with depolarized mitochondrial membranes (cells displaying green fluorescence); (C) Quantification of mitochondrial membrane potential as a ratio of JC-1aggregate to JC-1 monomer (red/green) fluorescence intensity. The identified peptides were used at a concentration of 0.5 mM. *p < 0.05, versus control group; #p < 0.05, versus model group. P.C. is the positive group that was pre-treated with YGFGG.

cadmium. It was somewhat surprising that a significant decrease in the percentage of cells with green fluorescence and increase red fluorescence was observed in all groups pre-treated with the peptides. The percentage of cells with depolarized mitochondrial membranes is shown in Fig. 6B. There was no statistically significant difference between the QIGPA treated group (10.94 ± 0.12%), QGPAN treated group (9.42 ± 0.74%) and control group. As shown in Fig. 6C, the red/ green fluorescence ratio of the cadmium-administered group was decreased by 46.02% compared with that of the control group (p < 0.05), while those of the QLGPA, MPKYA, YGFGG, QIGPA and QGPAN treated groups markedly increased by 7.29, 9.14, 12.50, 15.71 and 25.95% compared with model group, respectively. Among these, the red/green fluorescence ratio of the groups treated with QIGPA and QGPAN was even comparable to that of the control group. The results suggested that peptides from chicken sternal cartilage were able to reverse the decrease in ΔΨm and attenuate the disruptive effects of cadmium on the mitochondrial membrane potential in MC3T3-E1 cells.

results indicated the four identified peptides from chicken sternal cartilage showed a favorable protective effect in the Cd-induced osteoporosis model at 0.5 mM concentration. 3.6. The identified peptides increased the mitochondrial membrane potential (ΔΨm) of the Cd-treated cells ΔΨm is a vital parameter which reflects the mitochondrial functional status, and is thus closely related to overall cell function. Here, ΔΨm was assessed using JC-1 method and was quantified using flow cytometry. The JC-1 probe will spontaneously form aggregates with intense red fluorescence in healthy cells. Conversely, it is present as monomers with green fluorescence in apoptotic or damaged cells whose membrane potential is over 140 mV (Cottet‐Rousselle, Ronot, Leverve, & Mayol, 2011). The red/green fluorescence ratio is widely used to estimate the changes in ΔΨm. After the cells were treated with 3.2 μM Cd(Ac)2, a shift of fluorescence from red to green was observed (Fig. 6A), indicating that mitochondrial depolarization was induced by 138

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Fig. 7. The protective effect of synthetic peptides against Cd-induced osteoporosis in MC3T3-E1 cells. (A) DNA condensation (white arrow) was measured by DAPI staining and analyzed under a fluorescence microscope with 200 × magnification; (B). Representative flow cytometry profile with the signal for annexin-V FITC staining on the x axis and PI on the y axis. The lower right quadrant, upper right quadrant and upper left quadrant represent early apoptotic, late apoptotic and necrotic cells, respectively; (C) Percentage of the events in different stages under various peptide-treatments. △ indicates the positive control group (YGFGG); # indicates significance at p < 0.05 versus the model group.

3.7. The identified peptides reduced the apoptosis rate of the cells exposed to Cd

staining. As shown in Fig. 7A, the nuclei of the apoptotic cells in the model group were characterized by size reduction, nuclear condensation and fragmentation. By contrast, only a few apoptotic cells were found in the groups treated with QIGPA and QLGPA. However, more nuclei of apoptotic cells were dyed bright blue in the group treat with

Because mitochondrial damage is generally followed by alternations of the nucleus, the nuclear morphology was investigated using DAPI 139

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Universities [017ZD079] and the 111 Project [B17018].

MPKYA. These results further suggested that the observed apoptotic effect caused by cadmium was reversed by pre-treatment with QIGPA and QLGPA rather than that with MPKYA. In order to investigate the mode of cadmium-induced cell, Cd-induced apoptosis of MC3T3-E1 cells was quantified by annexin V-FITC/ PI double staining after peptide treatment for 48 h. Apoptotic cells externalize phosphatidylserine to the outer layer of the cell membrane, to which annexin-V binds (Schutte, Nuydens, Geerts, & Ramaekers, 1998). The percentages of cells in the four quadrants are representative of live cells (Q1-LL, Annexin-V-FITC-/PI-), early apoptotic cells (Q1-LR, Annexin-V-FITC+/PI-), late apoptotic cells (Q1-UR, Annexin-V-FITC-/PI +) and necrotic cells (QI-UL, Annexin-V-FITC+/PI+) (Fig. 7B). The percentages of these four forms of cells in the various treatment groups were calculated and shown in Fig. 7C. Flow cytometric analysis demonstrated that cadmium-challenged MC3T3-E1 cells had a relatively high early apoptotic rate, at 35.55%, while the percentage of late apoptotic and necrotic cells reached to 17.17%. No differences were found in the percentage of apoptotic cells in the group treated with MPKYA (0.5 mM), suggesting that this peptide could not protect MC3T3-E1 cells from cadmium-induced damage. Although the positive control (YGFGG) significantly increased the percentage of live cells, it promoted the transformation from early apoptosis to late apoptosis, and even induced cell death. Notably, it can be clearly seen that when the cells were treated with GGAP, QIGPA, QLGPA and QGPAN at a concentration of 0.5 mM, the percentages of early apoptotic cells were 13.96, 25.48, 13.04 and 8.20%, respectively, while the late apoptotic and necrotic cells respectively made up 14.01, 8.40, 3.16 and 12.04% of the total. Consistently with the results of the MTT assay and mitochondrial membrane potential assay, QLGPA was proved to be more effective than the other peptides in the protection against cadmium-induced apoptosis. Furthermore, peptides containing the GPA amino acid sequence exhibited more a favorable protective effect against cadmium-induced osteoporosis.

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4. Conclusion In this study, the hydrolysis conditions were optimized for the preparation of small molecular weight (MW < 1 kDa) chicken sternal cartilage peptides with high antioxidant activity. The optimal conditions, entailing alcalase treatment at an E/S ratio of 0.50%, at 55 °C and pH 7.0 for 9 h, can be used as a basis for production scale-up. Five novel peptides (GGAP, QIGPA, QLGPA, MPKYA and QGPAN) were successfully identified from the sub-fraction with high ORAC value using LC MS/MS. Among these, peptides with the GPA amino acid sequence exhibited a more favorable reversal effect in the Cd-induced cell model of osteoporosis than the other two peptides. They increased the viability and mitochondrial membrane potential, as well as reduced the apoptosis rate of Cd-challenged cells. Therefore, these identified peptides may have potential as functional foods for the prevention of osteoporosis. However, further studies are required to investigate the cellular mechanism by which peptides pre-treatment exerts its effect in the Cdinduced osteoporosis model, followed by in vivo studies to confirm the effect in animal models. Conflict of interest None. Acknowledgements The authors gratefully acknowledge the National Natural Science Foundation of China [No. 31671804], Science and Technology Program of Guangzhou [201604020047], Guangdong Special Funding for Outstanding Young Scholars [2014TQ01N645], Guangdong Science and Technology Planning Project [2017B090901063 and 2015B020230001], the Fundamental Research Funds for the Central 140

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