Article pubs.acs.org/JAFC
Bioavailability and Bioavailable Forms of Collagen after Oral Administration to Rats Lin Wang,*,† Qing Wang,† Jing Qian,† Qiufang Liang,† Zhenbin Wang,† Junmin Xu,‡ Song He,§ and Haile Ma† †
School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, People’s Republic of China Zhenjiang Kehua Aquaculture Development Company Limited, Zhenjiang 212134, People’s Republic of China § Lianyungang Haideyi Food Company Limited, Lianyungang 222113, People’s Republic of China ‡
ABSTRACT: The bioavailability and bioavailable forms of collagen after oral administration to rats were investigated in this study. The relative and absolute bioavailability of collagen were 57.8% and 49.6%, respectively, which was indirectly evaluated by the bioavailability of Hyp in collagen using a pharmacokinetic method. The amino acid profile of plasma showed that more than 63.4% of the collagen was absorbed from the intestine in the form of peptide, and there was a good linear correlation between the absorbed amount of an amino acid and its content in collagen (R2 = 0.9225). The collagen peptides in plasma were purified by Sephadex G10 and Eclipse XDB C18 chromatography and further indentified (Ala-Asn, Ala-Hyp-Gly, Asp-Glu, Glu-Asn, GluAsp, Glu-Met, Gly-Pro-Hyp, Leu-Hyp, Leu-Met, Phe-Gly-Asn, Pro-Gly-Leu, Pro-Leu, Ser-Gly-Met, Ser-Hyp, Ser-Pro-Gly, TyrMet) with UPLC−ESI-MS. These results may help to speculate about the molecular mechanism behind the physiological effects of collagen after oral administration. KEYWORDS: collagen, bioavailability, bioavailable form, pepetide, pharmacokinetic
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INTRODUCTION Collagen is the most abundant protein in the vertebrate body, comprising about one-third of the total protein content. Its hydrolysates have long been used to improve blood circulation, arrest bleeding, and improve joint condition by reducing pain in traditional medicine.1 Recent animal experiments and human trials have also suggested that oral administration of collagen or its hydrolysates had beneficial effects on skin aging;2 osteoarthritis and osteoporosis;3 rheumatoid arthritis;4 and the properties of nails, hair, and the Achilles tendon,5,6 etc. Regretfully, the mechanism behind the physiological effects of collagen has not yet been entirely elucidated. To speculate about the mechanism of the physiological effects, the question must be clarified as to whether collagen can be absorbed and, furthermore, in what form. It has been reported that the hydroxyproline (Hyp) concentration in human blood increased significantly and reached a maximum level within 0.5−2 h after the oral ingestion of collagen hydrolysates.1,7,8 Oesser et al. also found that the radioactivity in plasma reached a maximal concentration 6 h after the oral administration of 14C-labeled collagen hydrolysates to mice.9 These indicated that collagen could be quickly absorbed from the intestine after oral ingestion. However, there were little data about the bioavailability of collagen, which is a high-molecularweight scleroprotein (about 300 kDa) with a special triple-helix structure and unbalanced amino acid profile.10,11 Furthermore, several collagen peptides (Pro-Hyp, Pro-HypGly, Ala-Hyp, Ala-Hyp-Gly, Ser-Hyp-Gly, Leu-Hyp, Ile-Hyp, Phe-Hyp, Hyp-Gly) have been identified from blood after oral ingestion of collagen hydrolysates.1,7,8 Ohara et al. also reported that the structure of the collagen peptide is different depending on the collagen type and source.1 In our previous study, it was © 2015 American Chemical Society
found that collagen could be hydrolyzed into a large number of peptides under simulated gastrointestinal digestion in vitro.12 Consequently, we hypothesized that there must be more peptides unobserved in blood. Tilapia is the second most important group of farmed fish after carps and is usually processed as fillets accompanied by large quantities of byproducts.13 As an alternative source to land-based animals, collagen is largely produced from the byproducts and widely used in the food, cosmetic, biomedical, and pharmaceutical industries. In this paper, the oral bioavailability and bioavailable forms of collagen from Nile tilapia (Oreochromis niloticus) were investigated using a pharmacokinetics method and UPLC−ESI-MS. The results may help to speculate about the molecular mechanism behind the physiological effects of collagen.
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MATERIALS AND METHODS
Chemicals. The collagen was prepared from the skin of Nile tilapia (O. niloticus) as in our previous report and then dissolved in normal saline (NS).14 Hyp was purchased from Sigma Chemicals (St. Louis, MO). Amino acids mixture standard solution was obtained from Wako Chemicals (Osaka, Japan). Sephadex G10 was provided by GE Healthcare (Little Chalfont, UK). Animals. Healthy female SD rats (500−600 g, 6-month-old) were obtained from the Laboratory Animal Research Center, Jiangsu University, China. The rats were housed at controlled temperature (23 ± 2 °C), humidity (55 ± 10%), and light (12 h light/12 h darkness). Received: Revised: Accepted: Published: 3752
November 27, 2014 March 24, 2015 March 31, 2015 March 31, 2015 DOI: 10.1021/jf5057502 J. Agric. Food Chem. 2015, 63, 3752−3756
Article
Journal of Agricultural and Food Chemistry
Statistical Analysis. The data were presented as mean value ± standard deviation. Statistical analyses were performed using one-way analysis of variance. Multiple comparisons of means were done by the LSD test. A P value of 500 Da), and the F3 was free amino acids. The observation was not in agreement with Iwai’s report that no collagen peptide larger than Pro-Hyp-Gly could be found in plasma.7 From the F2, 16 kinds of small peptides were identified: AlaAsn, Ala-Hyp-Gly, Asp-Glu, Glu-Asn, Glu-Asp, Glu-Met, GlyPro-Hyp, Leu-Hyp, Leu-Met, Phe-Gly-Asn, Pro-Gly-Leu, ProLeu, Ser-Gly-Met, Ser-Hyp, Ser-Pro-Gly, Tyr-Met (Figure 4). These were quite different from previous reports,1,7,8 which could be due to the differences of the collagen source, experimental animal, and detection method. It is known that oligopeptides are frequently degraded in a short time by peptidase in blood. For example, the half-lives of Ala-Tyr, GlyTyr, Ala-Gln, Gly-Gln, N-acetyl-Ser-Asp-Lys-Pro, and Pyr-HisPro-NH2 in blood were less than 10 min.20,21 However, combined with the high percentage of collagen peptides in plasma (63.4%) 2 h after oral ingestion, it indicated that these collagen peptides might have a high resistance to digestion by the enzymes in plasma and the gastrointestinal tract. It has been demonstrated that Pro-Hyp and Pro-Hyp-Gly had chemotactic activity to the human fibroblast, peripheral blood neutrophils, and monocytes.22 This showed that collagen peptides acted as a biological messenger and triggered the synthesis of new collagen fibers and reorganization of the extracellular matrix by stimulating fibroblast cells. Pro-Hyp and Hyp-Gly had a stimulatory activity on the proliferation, migration, and hyaluronic acid synthesis of the skin fibroblast,8,23,24 which indicated that collagen peptides modulated the cells and extracellular matrix in the skin. Asp-
Mean value ± standard deviation from three separate determinations.
plasma significantly increased after ingestion of collagen, which indicated that the digested collagen could be absorbed in the forms of both free amino acid and peptide. When we compared the increased amount of peptide-bound amino acid with that of total amino acid, it was found that more than 63.4% of the amino acid residues in collagen were absorbed in the form of a peptide. The Glu absorbed in peptide form had the highest proportion (more than 97.5%), and there was a relatively high proportion of Asp, Phe, His, and Arg, whereas Hyp, Pro, Tyr and Hyl were very low. Referring to the amino acid profile of collagen,13 it was observed that there was a good linear correlation between the increased amount of an amino acid in plasma and its content in collagen (R2 = 0.9225) (Figure 2), which showed that the absorbed amount of an amino acid from the intestine depended on its content in collagen. Furthermore, Hyp was the closest to the regression curve among all the amino acids, which indicated that it was reasonable to indirectly evaluate the bioavailability of collagen by determining the bioavailability of Hyp in collagen. Separation of Collagen Peptides. The plasma filtrate was first fractionated into three fractions (F1, F2, F3) by a Sephadex 3754
DOI: 10.1021/jf5057502 J. Agric. Food Chem. 2015, 63, 3752−3756
Article
Journal of Agricultural and Food Chemistry
Figure 3. Chromatographic profiles of plasma filtrate before and after ingestion of collagen, using Sephadex G10 (a) and Eclipse XDB-C18 columns (b). Red, before; blue, after. Figure 4. Chromatographic profiles of plasma filtrate fraction (F2) with a BEH130 C18 column.
Gly-Glu-Ala could stimulate osteoblast-related gene expression of bone marrow cells.25 Furthermore, Ala-Hyp and Gly-Pro-Val were potential inhibitors of angiotensin-converting enzyme, and Gly-Pro-Hyp was suggested to be involved in platelet aggregation,26,27 suggesting their beneficial effects on hypertension and thrombosis. However, only a small part of the collagen peptides and their physiological effects are currently known about. It is necessary to obtain more detailed information on the physiological effects and molecular mechanisms of the collagen peptides in further investigation. In conclusion, the bioavailability and bioavailable forms of collagen after oral administration to rat were investigated in this study. The bioavailability of collagen was lower than animal protein, but was comparable to cereal protein. Most of the digested collagen was absorbed in the form of a peptide rather than a free amino acid, and the absorbed amount of an amino
acid depended on its content in collagen. Moreover, some collagen peptides in the plasma were purified and identified, but further studies are needed to investigate their physiological effects and molecular mechanisms.
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AUTHOR INFORMATION
Corresponding Author
*Phone/fax: +86 511 88780201. E-mail:
[email protected]. Funding
This research was supported by the Natural Science Foundation of Jiangsu Province (BK20140545), Science and Technology Support Program of Jiangsu Province 3755
DOI: 10.1021/jf5057502 J. Agric. Food Chem. 2015, 63, 3752−3756
Article
Journal of Agricultural and Food Chemistry
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(BE2014339), and Science and Technology Support Program of Zhenjiang City (NY2014023). Notes
The authors declare no competing financial interest.
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DOI: 10.1021/jf5057502 J. Agric. Food Chem. 2015, 63, 3752−3756
