Administration Of Collagen Hydrolysate Research Articles

Substrate specificity of exopeptidases in small intestinal mucosa determines the structure of food-derived collagen peptides in rat lumen and blood

Gly-Pro-Hyp-Gly is the most common motif in collagen. Ingestion of collagen hydrolysate (CH) has been reported to significantly increase Pro-Hyp and Hyp-Gly in human peripheral blood. However, the elevation of Gly-Pro content remains negligible. This study seeks to elucidate the underlying reasons behind the bioavailability of CH-derived peptides using in vivo and in vitro digestion. After oral administration of CH (800 mg/kg body weight) to rats, Pro-Hyp significantly increased in the lumen, small intestinal tissue, and blood from the abdominal vein, while only negligible amounts of Gly-Pro were detected. In vitro digestion of CH by digestive exopeptidases, including aminopeptidase N, leucine aminopeptidase, and carboxypeptidase A predominantly generated Gly-Pro. Interestingly, the digestion of CH with crude extract of small intestinal mucosa predominantly generated Pro-Hyp and negligible amounts of Gly-Pro. Furthermore, synthetic Gly-Pro was rapidly degraded by crude extract of the intestinal mucosa, while Pro-Hyp showed higher resistance. These facts indicate prolidase activity in the mucosa plays a crucial role in determining collagen-derived peptides in the body.

Pharmacokinetics of collagen dipeptides (Gly-Pro and Pro-Hyp) and tripeptides (Gly-Pro-Hyp) in rats.

Although systemic exposure to peptides, such as Gly-Pro-Hyp, Pro-Hyp, and Gly-Pro, has been reported following administration of collagen hydrolysates from fish scale and porcine skin in vivo, the individual peptide pharmacokinetics remain unknown. We administered the three peptides individually to rats via the intravenous (5mg/kg) and intragastric (100mg/kg) routes and then monitored systemic exposure and urinary excretion. The peptides in biological samples were analyzed via liquid chromatography/tandem mass spectrometry. Gly-Pro-Hyp tended to exhibit higher first-pass metabolism than Pro-Hyp; the absolute oral bioavailabilities of Gly-Pro-Hyp and Pro-Hyp were 4.4% and 19.3%, respectively. Gly-Pro levels were very low in the systemic circulation. Pro-Hyp biotransformed from Gly-Pro-Hyp behaved similarly to Pro-Hyp alone when administered orally. Flip-flop kinetics (elimination rate ≫ absorption rate) were evident, probably reflecting transporter-mediated slow absorption. A double-peak phenomenon was observed for Gly-Pro-Hyp and Pro-Hyp when administered orally, and 5.9%±2.6% and 1.9%±0.3% of each dose were excreted in urine after intravenous administration, respectively. Urinary recovery of Gly-Pro was limited to 0.4%±0.5% of the intravenous dose. This work represents the first individual pharmacokinetics of Gly-Pro-Hyp, Pro-Hyp, and Gly-Pro in vivo.

Identification of a highly stable bioactive 3-hydroxyproline-containing tripeptide in human blood after collagen hydrolysate ingestion

There are increasing reports demonstrating high bioavailability of 4-hydroxyproline (4Hyp)-containing oligopeptides after oral ingestion of collagen hydrolysate and their bioactivity. In contrast, no study investigates the fate of another collagen-specific but minor amino acid, 3Hyp. Here, we identified Gly-3Hyp-4Hyp tripeptide in human blood at high concentrations, comparable to other 4Hyp-containing oligopeptides, after ingesting porcine skin collagen hydrolysate. Additionally, Gly-3Hyp-4Hyp uniquely maintained the maximum concentration until 4 h after the ingestion due to its exceptionally high resistance to peptidase/protease demonstrated by incubation with mouse plasma. In mice, oral administration of collagen hydrolysate prepared from bovine tendon, which contains a higher amount of 3Hyp, further increased blood Gly-3Hyp-4Hyp levels compared to that from bovine skin. Furthermore, Gly-3Hyp-4Hyp showed chemotactic activity on skin fibroblasts and promoted osteoblast differentiation. These results highlight the specific nature of the Gly-3Hyp-4Hyp tripeptide and its potential for health promotion and disease treatment.

Novel Hyp-Gly-containing antiplatelet peptides from collagen hydrolysate after simulated gastrointestinal digestion and intestinal absorption.

Bioactive components causing the antiplatelet activity upon collagen hydrolysate (CH) ingestion have not been clarified yet. This study aimed to identify antiplatelet peptides from CH after simulated gastrointestinal digestion and intestinal absorption. Four antiplatelet peptides containing the Hyp-Gly (OG) sequence including OG, Hyp-Gly-Glu (OGE), Pro-Gly-Glu-Hyp-Gly (PGEOG) and Val-Gly-Pro-Hyp-Gly-Pro-Ala (VGPOGPA) were successfully identified. All four peptides exhibited antiplatelet activity, but OGE and PGEOG exerted stronger activity than OG and VGPOGPA. The IC50 value of OGE and PGEOG was 1.076 mM and 1.167 mM, respectively. These four antiplatelet peptides could survive simulated gastrointestinal digestion and be absorbed intact by Caco-2 cells. Furthermore, plasma stability experiments showed that OG and OGE showed a good stability in human plasma, but PGEOG and VGPOGPA showed a relatively poor stability. In vivo studies indicated that OG and OGE were present in blood after the oral administration of CH. Meanwhile, OGE exerted significant in vivo anti-thrombotic activity after its ingestion. The present study clarifies the antiplatelet components causing the CH activity and highlights the potential application of CH or these four peptides as functional foods to combat thrombosis by inhibiting platelet aggregation.

Absorption and metabolism of orally administered collagen hydrolysates evaluated by the vascularly perfused rat intestine and liver in situ.

A number of studies have shown that oral administration of collagen hydrolysate (CH) results in the absorption of di- and tri-peptides. In order to understand the dynamics of CH absorption and metabolism, molecular profiles of hydroxyproline (Hyp) and Hyp-containing peptides (HCPs) were analyzed by in situ perfusion of rat intestine and liver. The total amount of absorbed HCPs during 1 h of perfusion was 16.6 μmol, which was significantly higher than that of free Hyp (6.6 μmol). In addition, HCPs were also reliably detected in hepatic perfusate at the level higher than free Hyp. Thus, the results demonstrated that CH is absorbed predominantly as peptides, which subsequently enter systemic circulation. Size exclusion chromatography showed that perfusates include significant amount of HCPs larger than tripeptides, leading us to analyze these peptides in detail. Mass spectrometric analysis of intestinal perfusate finally identified three CH-derived peptides, which are surprisingly large as food-derived circulating peptides. Peptide quantitation by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that di- and tri-peptides, which are previously identified as major peptides in circulating blood, comprise only a part of HCPs in intestinal and liver perfusate. Finally, analysis of portal vein blood revealed that the larger peptides, such as pentadecapeptide identified in this study, could be absorbed in vivo. Taken all together, this study showed that peptides which are larger than tripeptide could reach to the circulation system after administration of CH, revealing previously unknown dynamics of absorption of CH.

Fish Collagen Hydrolysates Modulate Cartilage Metabolism.

Osteoarthritis is a degenerative joint disease in which interleukin-1β plays a major role in the inflammatory process. Administration of collagen hydrolysate was an optional treatment of osteoarthritis. Fish has become an interesting source of collagen hydrolysate because of religious reason and there is no risk from mad cow disease. However, the effects of different sizes of fish collagen hydrolysate on cartilage and chondrocyte metabolism have not been well studied yet. This study examined the effect of different sizes of fish collagen hydrolysate on cartilage metabolism. Three different sizes of fish collagen hydrolysate were prepared by size exclusion using centrifugation, which composed of small fraction (<3 kDa), medium fraction (3-10 kDa) and large fraction (>10 kDa). Using porcine cartilage explant, in physiological condition, all the three fractions had no effect on cartilage metabolism, but they could induce pro-MMP3 and pro-MMP13 secretions through activation of p-ERK and p-p38. In pathological condition induced by interleukin-1β and oncostatin-M, small and medium fractions showed additive effect with interleukin-1β and oncostatin-M on cartilage degradation, whereas large size had no effect. In addition, the effect of small size occurred through further activation of p-p65, which resulted in further induction of active-MMP13, while medium size had a different mechanism. In conclusion, all three fractions fish collagen hydrolysate had no effect on cartilage metabolism in physiological condition, but small and medium fractions had adverse effect on cartilage in pathological condition. Taken together, various sizes of fish collagen hydrolysate showed different effects on cartilage metabolism. Therefore, different sizes of fish collagen hydrolysates play different roles on cartilage metabolism, especially in the pathological condition.

Oral Administration of Collagen Hydrolysates Improves Glucose Tolerance in Normal Mice Through GLP-1-Dependent and GLP-1-Independent Mechanisms.

The aim of this study was to evaluate the antidiabetic properties of collagen hydrolysates (CHs). CHs exhibited dipeptidyl peptidase-IV inhibitory activity and stimulated glucagon-like-peptide-1 (GLP-1) secretion in vitro. We also determined whether CHs improve glucose tolerance in normal mice. Oral administration of CHs suppressed the glycemic response during the oral and intraperitoneal glucose tolerance tests (OGTT and IPGTT), but the effects were weaker in IPGTT than in OGTT. CHs had no effect on the gastric emptying rate. A pretreatment with the GLP-1 receptor antagonist, exendin 9-39 (Ex9), partially reversed the glucose-lowering effects of CHs, but only when coadministered with glucose. CHs administered 45 min before the glucose load potentiated the glucose-stimulated insulin secretion. This potentiating effect on insulin secretion was not reversed by the pretreatment with Ex9, it appeared to be enhanced. These results suggest that CHs improve glucose tolerance by inhibiting intestinal glucose uptake and enhancing insulin secretion, and also demonstrated that GLP-1 was partially involved in the inhibition of glucose uptake, but not essential for the enhancement of insulin secretion.

Herb extracts and collagen hydrolysate improve skin damage resulting from ultraviolet-induced aging in hairless mice.

We examined the effect of the daily ingestion of herb extract from Eucommia ulmoides leaves and Korean ginseng on skin damage induced by repeated UV irradiation of hairless mice. The herb extract was orally administered to mice at a dose of 1000 mg/kg/day. The hydration of mice dorsal skin decreased significantly with repeated UV irradiation, but did not decrease when the herb extract was administered for seven weeks. Transepidermal water loss (TEWL) increased with UV irradiation, but decreased with the administration of dietary herb extract. These effects were more pronounced when combined with the administration of collagen hydrolysate. Geniposidic acid from E. ulmoides leaves and ginsenoside Rg1 from Korean ginseng reduced TEWL and increased the skin moisture content of UV-damaged skin on hairless mice, respectively. We concluded that this dietary herb extract reduced the skin damage caused by UV-induced aging, with geniposidic acid and ginsenoside Rg1 detected in the blood.

P199 DISPARATE EFFICACY OF COLLAGEN HYDROLYSATE AND GLUCOSAMINE ON THE EXTRACELLULAR MATRIX METABOLISM OF ARTICULAR CHONDROCYTES

Purpose: Glucosamine has been used in the treatment of osteoarthritis (OA) for several years. More recently, collagen fragments were shown to provide a positive effect on joint health. The objective of this study was to investigate the efficacy of a specific type of collagen hydrolysate (CH) on the biosynthesis of ECM macromolecules in comparison with glucosamine sulfate (GS) and glucosamine hydrochloride (GH) in a chondrocyte culture model. Methods: Primary porcine articular chondrocytes and human femoral head chondrocytes were cultured under reduced oxygen conditions. The culture medium was supplemented with various concentrations of CH. In parallel experiments chondrocytes were treated with either GS or GH in various concentrations up to 2.5mM. At the end of the culture period the amount of cellassociated proteoglycans (PGs) and the extent of PG synthesis were quantified by measuring 35S-sulfate incorporation and by colorimetric assay. The amount of aggrecan accumulated in the ECM was determined by Western Blotting. In addition, type II collagen biosynthesis was quantified by means of ELISA technique. The results were confirmed by immunocytochemical detection of type II collagen and by analyzing the incorporation of 14C-proline into matrix proteins. Results: Supplementation of the culture medium with CH resulted in a statistically significant (p<0.05) increase of PG synthesis. The amount of cell-associated PGs was almost doubled after CH treatment, compared with the control cells. Administration of CH was also associated with increased aggrecan expression and a statistically significant (p<0.05) 1.5-fold increase of type II collagen biosynthesis. In contrast, the administration of GS or GH had no stimulatory effect on the type II collagen biosynthesis of the chondrocytes. Moreover, although slight differences could be observed between GS and GH, supplementation of glucosamine had no significant effect on the amount of cell-associated PGs, or total PG synthesis, compared to the controls. Conclusions: These results indicate a stimulatory effect of CH on the synthesis of PG and type II collagen. In contrast, GS and GH failed to stimulate the synthesis extracellular matrix (ECM) macromolecules by chondrocytes. These data suggest that CH may help reduce degenerative changes of the ECM by stimulating anabolic processes in cartilage tissue.