Article pubs.acs.org/JAFC
Collagen Peptides from Crucian Skin Improve Calcium Bioavailability and Structural Characterization by HPLC−ESI-MS/MS Tao Hou,†,‡ Yanshuang Liu,†,‡ Danjun Guo,†,‡ Bo Li,†,‡ and Hui He*,†,‡ †
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, Wuhan 43000, China
‡
ABSTRACT: The effects of collagen peptides (CPs), which are derived from crucian skin, were investigated in a retinoic acidinduced bone loss model. The level of serum bone alkaline phosphatase (BALP) in the model group (117.65 ± 4.66 units/L) was significantly higher than those of the other three groups (P < 0.05). After treatment with 600 and 1200 mg of CPs/kg, the level of BALP decreased to 85.26 ± 7.35 and 97.03 ± 7.21 units/L, respectively. After treatment with 600 mg of CPs/kg, the bone calcium content significantly increased by 22% (femur) and 12.38% (tibia) compared to those of the model group. In addition, the bone mineral density in the 600 mg of CPs/kg group was significantly higher (femur, 0.37 ± 0.02 g/cm2; tibia, 0.33 ± 0.02 g/cm2) than in the model group (femur, 0.26 ± 0.01 g/cm2; tibia, 0.23 ± 0.02 g/cm2). The morphology results indicated bone structure improved after the treatment with CPs. Structural characterization demonstrated that Glu, Lys, and Arg play important roles in binding calcium and promoting calcium uptake. Our results indicated that CPs could promote calcium uptake and regulate bone formation. KEYWORDS: collagen peptides (CPs), retinoic acid, alendronate, calcium uptake, structural characterization
1. INTRODUCTION Calcium is an essential nutrient for humans to maintain their health and the average recommended dietary allowance of calcium for adolescents is approximately 1200 mg/day in China (1). The most common form of calcium for humans is ionized calcium, such as calcium carbonate and calcium lactate (2). However, ionized calcium is difficult to absorb because it is prone to forming precipitation with phytic acid in the intestines (3). The insufficient calcium uptake will cause calcium deficiency. Additionally, as age increases, the older individuals, especially female ones, are susceptible to osteoporosis due to the decrease of estrogen (4). Currently, there are two strategies to prevent calcium deficiency and osteoporosis: (1) improving calcium bioavailability by enhancing the absorption of any calcium present in the diet with calcium promoters, such as prebiotics (5) or bioactive peptides (6); and (2) modulating bone formation, as well as mineralization of the bone matrix (7). Recently, many calcium-chelating peptides, which can promote calcium uptake and bioavailability, have been characterized. For example, casein phosphopeptides (CPPs) can be chelated with calcium to form soluble and stable complexes that are effective for promoting calcium absorption (8). The exact mechanism might be the interaction with calcium channels in the plasma membrane, such as TRPV6 (9) and Cav1.3 (10). However, few reports showed that CPPs could modulate bone formation and bone resorption activity. An in vitro study showed that CPPs could play a role in the modulation of bone cell activity and promote calcium uptake in osteoblast-like cells (11). Nevertheless, an in vivo study declared that CPPs in milk and fermented milk did not acutely affect calcium metabolism in postmenopausal women (12). © 2017 American Chemical Society
Collagen is a cheap and resourceful meat byproduct that is primarily used to obtain gelatin, which is used extensively as a food additive to increase the texture, water-holding capacity and stability of several food products (13). In this study, collagen peptides (CPs) are the processing byproduct hydrolysates of crucian skin and are readily available because large amounts of fish skin are discarded. Several studies have been performed to identify potential antioxidative peptides (14), melanogenesisinhibitory peptides (15) and antihypertensive peptides (16) in fish skin gelatin. In addition, some studies suggest that oral administration of hydrolyzed collagen increase bone mass content and density in rats and mice fed a calcium- or proteindeficient diet17,18. Additionally, in patients with osteoporosis, oral intake of hydrolyzed collagen with calcitonin had a stronger inhibitory effect on bone resorption than calcitonin alone (19). Therefore, the primary aim of this study is to assess the effects of collagen peptides on calcium uptake by measuring the serum calcium content, serum bone alkaline phosphates, and bone parameters in retinoic acid-induced bone loss rats. The second objective is to evaluate the effects of collagen peptides on bone formation by using scanning electron microscopy (SEM) and tartrate-resistant acid phosphatase (TRAP) staining. The third objective is to characterize the amino acid components and sequences of collagen peptides. We hypothesized that collagen peptides could both improve calcium status and bone health, and we predicted several pivotal amino acids and sequences would be observed. Received: Revised: Accepted: Published: 8847
July 3, 2017 September 14, 2017 September 15, 2017 September 15, 2017 DOI: 10.1021/acs.jafc.7b03059 J. Agric. Food Chem. 2017, 65, 8847−8854
Article
Journal of Agricultural and Food Chemistry
Figure 1. Effects of CPs and alendronate on serum parameters: (A) serum calcium content, (B) serum phosphate content, and (C) serum bone alkaline phosphatase (BALP) level. Within a column, the means without a common letter are significantly different [P < 0.05 (Duncan’s multiplerange test at P < 0.05)]. HPLC−ESI-MS system (Agilent Technologies) using the same column and gradient elution that was used for the HPLC analysis. ESI mass spectrometry was performed in positive ionization mode at a drying gas temperature of 325 °C. The dry gas flow rate was 10.0 L/ min, and the capillary voltage was 3500 V. The pressure of the nebulizer was set at 40.00 psi, and the scan spectra range from m/z 100 to 1000. The sequences were identified using LCMSD-Trap Data Analysis software and confirmed by searching the online MS database of the UCSF Mass Spectrometry Facility (http://prospector.ucsf.edu/ prospector/cgi-bin/msform.cgi?form=mspattern). 2.7. Animals and Experimental Design. Fifty 3-month-old Wistar female rats were obtained from Hubei Laboratory Animal Research Center and used in accordance with the National Institutes of Health Guidelines for the Care and Use of Animals and animal ethical approval certificate number MU-2016-001. The animal experiment was performed as described by Hou et al.21 with slight modifications. Briefly, the rats received 80 mg of retinoic acid/kg of body weight once a day for the first 2 weeks and then divided into four groups in which rats received different drugs for an additional 3 weeks: (i) the model control group, 0.9% saline; (ii) the positive control group, 5 mg of alendronate/kg of body weight; (iii) the CPs1 group, 600 mg of CPs/kg of body weight; and (iv) the CPs2 group, 1200 mg of CPs/kg of body weight. The rats in the healthy control group received 0.9% saline for the whole experiment. CPs and other reagents were given by oral gavage. After the experiment, all rats were forced to fast for 12 h and then anesthetized by ether before blood was collected. Next, the rats were sacrificed using cervical dislocation, and the femur and tibia were dissected, cleaned, and stored at −20 °C for further analysis. 2.8. Serum Parameters. The serum calcium level was determined using the methylthymol blue (MTB) assay. The serum bone alkaline phosphatase (BALP) level was measured using enzyme-linked immunosorbent assay kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 2.9. Bone Parameters. Bone biomechanical parameters were measured by a texture analyzer system (TA. XT. PLUS, Stable Micro System, Godalming, Surrey, U.K.) described by Zhao et al.22 The dry weight index was calculated according to the equation DW (103) = dry weight × 1000/body weight. The calcium content was measured by an atomic absorption spectrophotometer (AA6300C, Shimadzu). The bone mineral content (BMC) and bone mineral density (BMD) were determined by scanning the thawed right femurs and tibias with dualenergy X-ray spectrometry (DEXA) (XR-46, Norland, Fort Atkinson, WI). The electron microscopy images and tartrate-resistant acid phosphatase (TRAP) activity of bones were measured according to our previous study.21 2.10. Statistical Analysis. Experimental data are presented as mean values ± the standard deviation (SD). The significant differences of each index among all groups were calculated using one-way analysis of variance in SPSS 18.0 followed by Duncan’s multiple-range test. For the bone test, there was no statistical test performed between the femur and tibia. Differences were considered to be significant at P values of 97% pure) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Acetonitrile [high-performance liquid chromatography (HPLC) grade] was purchased from Thermo Fisher (Waltham, MA). Protamex was purchased from Novozymes (Copenhagen, Denmark). Sodium hydroxide, n-butyl alcohol, H2SO4, citric acid, sodium phosphate, and the other reagents were analytical grade. 2.2. Extraction of Collagen from Crucian Skin. The crucian skin was cut into pieces (1 cm × 1 cm) and later immersed in 10% nbutyl alcohol [1:10 (w/v)] for 24 h. The skins were cleaned three times with distilled water and immersed in a 0.1 M NaOH solution [1:10 (w/v)] for 72 h. Next, the skins were cleaned five times with distilled water, immersed in a 0.2% H2SO4 solution for 30 min, and cleaned three times. Finally, the skins were immersed in 1% citric acid for 3 min. The pretreated crucian’s skins were immersed in distilled water [1:100 (w/v)] at 42 °C for 24 h. The supernatant was collected and then centrifuged at 4000 rpm for 20 min.20 The supernatant was collected, lyophilized, and will be termed fish skin collagen hereafter. 2.3. Preparation of Collagen Peptides from Crucian. Fish skin collagen was denatured for 30 min in boiling water and cooled to 50 °C. Next, the collagen was dissolved in distilled water (3%), and the pH of the collagen solution was adjusted for optimal conditions for the enzyme (Protamex pH 7.0; enzyme concentration of 3 × 103 units/kg) at an optimal temperature (50 °C) for 3.0 h. The degree of hydrolysis of collagen was 10.57%. Next, the hydrolyzed solution was heated in a water bath (100 °C) for 10 min to inactivate the enzyme. The mixture was centrifuged at 3000g for 10 min, and the supernatant was collected. Finally, the supernatant was filtered through a hollow fiber membrane with molecular weight cutoffs of 5, 3, and 1 kDa (PLCC, Millipore, Billerica, MA). Each fraction was collected for a calcium binding capacity study and was lyophilized. 2.4. Calcium Binding Capacity Assay. CPs, CaCl2, and Na2HPO4 were dissolved in deionized water to final concentrations of 1.0 mg/mL, 5 mmol/L, and 0.2 mol/L, respectively. The pH of the solution was maintained at 8.0 and the solution stirred in a water bath (37 °C) for 2 h. Next, the solution was centrifuged at 10000g for 10 min, and the supernatant was collected. The calcium content of the supernatant, defined as the calcium binding capacity, was measured with an atomic absorption spectrophotometer (AA6300C, Shimadzu, Kyoto, Japan). 2.5. HPLC Analysis Instrumentation. CPs ( 0.05) observed in serum calcium content while the serum phosphate level was significantly 8849
DOI: 10.1021/acs.jafc.7b03059 J. Agric. Food Chem. 2017, 65, 8847−8854
Article
Journal of Agricultural and Food Chemistry
Figure 4. Tartrate-resistant acid phosphatase (TRAP) activity in each group. The cytoplasm of osteoclasts (OC) is wine red, and the nuclei are blue. Control: the wine-red section was not evident. Model: the wine-red area increased compared to that of the control group, and osteoclasts (OC) are labeled (arrow). Alendronate: the wine-red area decreased significantly compared to that of the model group. CPs1 and CPs2: the wine-red area decreased compared to that of the model group, and no significant difference was observed between these two treatment groups.
formation or resorption. In the model group, the bone surfaces were scalloped because of the resorption of bone by the increase in the number of osteoclasts. Several lacunae areas were also observed from the bones in the model group. The scalloped regions decreased significantly compared to the model group after the treatment with alendronate and CPs. There was no significant difference in bone structures between the 600 mg of CPs/kg and 1200 mg of CPs/kg treatment groups. 3.4. TRAP Staining of Bones. As seen in Figure 4, the wine-red sections, which indicated the cytoplasm of osteoclasts, in the model group were significantly larger than in the other groups. This result indicated that the activity of osteoclasts in the model group was significantly higher than in other groups. The mechanism of bone loss induced by retinoic acid is due to the decrease in the estrogen level and the increase in the number of osteoclasts. The numbers of osteoclasts decreased after the treatments with alendronate and CPs. Obviously, the effects of alendronate on bone structure were better than the effects of CPs. 3.5. Binding Capacity of Each Fraction. Among all the fractions, the lowest-molecular weight (5 kDa (0.9127 ± 0.008 mg/g) were significantly lower than that of the group without ultrafiltration (1.499 ± 0.084 mg/g). 3.6. Amino Acid Composition of the High Bioactivity of CPs. The amino acid composition of 5%.
Table 2. Structural Summary of CPs (
