Collagen Tripeptide Research Articles

Orally Available Collagen Tripeptide: Enzymatic Stability, Intestinal Permeability, and Absorption of Gly-Pro-Hyp and Pro-Hyp.

Collagen-derived small peptides, such as Gly-Pro-Hyp (GPH) and Pro-Hyp (PH), play a role in various physiological functions. Although collagen degrades in the gastrointestinal tract randomly and easily, it is not readily cleaved into bioactive peptides. To increase the bioavailability of bioactive peptides, a collagen tripeptide (CTP) was prepared from fish scales by the digestion method using collagenase from nonpathogenic Bacillus bacteria. It was demonstrated that Hyp-containing peptides-GPH and PH-were better absorbed and reached higher plasma levels after the oral administration of CTPs in rats compared to high molecular weight collagen peptide (H-CP). GPH and PH were stable in gastrointestinal fluid and rat plasma for 2 h, and GPH was able to be transported across the intestinal cell monolayer. These results suggest that the ingestion of CTP is an efficient method for taking bioactive peptides orally due to the enzymatic stability and intestinal permeability of GPH and PH.

Production Method for Cyclic Dipeptide Derived from Native Collagen

In this study, a new method for producing cyclo(-Gly-Pro) using collagen as a raw material was examined. First, collagen was enzymatically hydrolysed and purified to obtain collagen tripeptide (CTP), rich in “Gly-X-Y” tripeptides. After heating this product under atmospheric pressure in an aqueous solution at 95°C for 24 h, purification was achieved by reverse-phase column chromatography. The isolated component was confirmed to be cyclo(-Gly-Pro) through structural analysis by MS and NMR spectroscopies. Purity was determined to be 93.6%, and the recovery rate from CTP was 22%, indicating that much Gly-Pro-Y in CTP contributed to cyclization. The cyclization rate from Gly-Pro-Hyp or Gly-Pro-Ala was much higher than that of Gly-Pro, suggesting that cyclo(-Gly-Pro) was efficiently generated from the Gly-Pro-Y sequence. In summary, this is a simple, practical manufacturing method for producing cyclo(-Gly-Pro) from collagen at low cost with high efficiency.

Absorption and Urinary Excretion of Peptides after Collagen Tripeptide Ingestion in Humans

Collagen tripeptide (CTP) is a collagen hydrolysate containing a high concentration of tripeptides with a Gly-X-Y sequence, such as Gly-Pro-Hyp. To test the effects of this preparation, we compared the absorption of peptides in humans after ingestion of a tripeptide fraction of CTP (CTP-100), a CTP preparation containing ca. 50% Gly-X-Y tripeptides (CTP-50), and a collagen peptide that did not contain tripeptides (CP). The postprandial levels of Gly-Pro-Hyp and Pro-Hyp in the plasma increased in those subjects who ingested CTP-100 and CTP-50, and were higher with greater Gly-Pro-Hyp ingestion. This demonstrated that collagen hydrolysates were efficiently absorbed when the collagen was ingested in the tripeptide form. Gly-Pro-Hyp and Pro-Hyp were also found in the urine after ingestion of CTP-100 or CTP-50. Similar to the results for the plasma concentration, the urinary excretion of Gly-Pro-Hyp and Pro-Hyp was also dependent on the amount of Gly-Pro-Hyp ingested. This indicates that ingested Gly-Pro-Hyp and generated Pro-Hyp were relatively stable in the body and were transported to the urine in the peptide form. The concentration of Hyp-Gly in the plasma was low after the ingestion of CP and CTP-100 but higher after the ingestion of CTP-50. Overall, our results suggest that tripeptides derived from collagen are absorbed efficiently by the body.

Absorption and plasma kinetics of collagen tripeptide after peroral or intraperitoneal administration in rats.

Collagen tripeptide (CTP) is a collagen-derived compound containing a high concentration of tripeptides with a Gly-X-Y sequence. In this study, the concentrations and metabolites of CTP were monitored in rat plasma after its administration. We performed a quantitative analysis using high-performance liquid chromatography tandem mass spectrometry according to the isotopic dilution method with stable isotopes. We confirmed that the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp were transported into the plasma. Dipeptides, which are generated by degradation of the N- or C-terminus of the tripeptides Gly-Pro-Hyp, Gly-Pro-Ala, and Gly-Ala-Hyp, were also present in plasma. The plasma kinetics for peroral and intraperitoneal administration was similar. In addition, tripeptides and dipeptides were detected in no-administration rat blood. The pharmacokinetics were monitored in rats perorally administered with Gly-[(3)H]Pro-Hyp. Furthermore, CTP was incorporated into tissues including skin, bone, and joint tissue. Thus, administering collagen as tripeptides enables efficient absorption of tripeptides and dipeptides.

Effects of oral administration of tripeptides derived from type I collagen (collagen tripeptide) on atherosclerosis development in hypercholesterolemic rabbits

Digestion of type I collagen with a collagenase-type protease yields a collagen tripeptide (Ctp) fraction comprising Gly-X-Y sequences that exhibit diverse biological activities. We previously demonstrated that Ctp inhibits the proliferation and migration of cultured aortic smooth muscle cells (SMCs) in vitro. These cells contribute to the pathogenesis of atherosclerosis and other cardiovascular diseases. In order to evaluate the effects of Ctp on atherosclerosis development in vivo, here we used the Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbit model of familial hypercholesterolemia to determine the effects of oral administration of Ctp for three months. Ctp induced a significant decrease in the area occupied by atherosclerotic plaques in the aorta and in the level of total serum cholesterol. The components of atherosclerotic plaques underwent distinct changes, including reduction in the populations of macrophages and SMCs and a significant decrease in the proportion of macrophages to SMCs. Ctp administration decreased the number of cells in plaques that expressed proliferating cell nuclear antigen and the number of cells with oxidative damage to DNA as indicated by 8-hydroxy-2'-deoxyguanine detection. These findings are the first to define the mechanism underlying the inhibitory effects of Ctp on atherosclerosis development in hypercholesterolemic rabbits, and suggest that Ctp provides an effective therapy for treating atherosclerosis.

Effect of high advanced-collagen tripeptide on wound healing and skin recovery after fractional photothermolysis treatment

Collagens have long been used in pharmaceuticals and food supplements for the improvement of skin. We evaluated the efficacy of high advanced-collagen tripeptide (HACP) on wound healing and skin recovery. Using an in vitro model, we performed HaCaT cell migration assays and collagen gel contraction assays using HACP concentrations of 1, 10 and 100μg/mL. In this pilot study, eight healthy volunteers were randomly divided into two groups. Both the control and experimental groups received fractional photothermolysis treatment, but in the experimental group, four subjects received 3g/day of oral collagen peptide (CP) for 4weeks. To assess transepidermal water loss in each patient before and after the treatment, we used a Corneometer and a Cutometer, and we also assessed the patient's Erythema Index. The cell migration assay showed that HACP enhanced wound closure, but not in a dose-dependent manner. The collagen gel contraction assay showed increased contractility when patients were treated with 100μg/mL HACP, but the results were not significantly different from those of controls. We found that post-laser erythema resolved faster in the experimental group than in the control group (P<0.05). In addition, the recovery of skin hydration after fractional laser treatment was greater in the experimental group than in the control group by day 3 (P<0.05), and the experimental group showed significantly improved post-treatment skin elasticity compared with the controls by day 14 (P<0.05). Collagen tripeptide treatment appears to be an effective and conservative therapy for cutaneous wound healing and skin recovery after fractional photothermolysis treatment.

Inhibitory effect of collagen-derived tripeptides on dipeptidylpeptidase-IV activity

The collagen tripeptide fragments Gly-Ala-Hyp, Gly-Pro-Ala and Gly-Pro-Hyp were generated by hydrolyzing collagen from pig-skin, cattle-skin, fish-scales and chicken-feet, respectively, with Streptomyces collagenase. Collagenase treatment increased the concentration of tripeptides in the hydrolysates by 13–15% (w/w). Of the three peptides, Gly-Pro-Hyp was a true peptidic inhibitor of dipeptidylpeptidase-IV (DPP-IV), because DPP-IV could not hydrolyze the bond between Pro-Hyp. This tripeptide was a moderately competitive inhibitor (Ki = 4.5 mM) of DPP-IV, and its level in the collagen hydrolysates could be greatly increased (4–9% [w/w]) using Streptomyces collagenase.

Effects of collagen tripeptide supplement on skin properties: A prospective, randomized, controlled study

Background: Experimental and clinical trials have indicated that dietary supplements can have beneficial effects on skin health. Objective: We investigated to evaluate the effect of daily collagen peptide (CP) supplement on skin properties. Methods: Thirty-two healthy volunteers were randomized to receive either no supplement (Group A), CP 3 g (Group B), CP 3 g, and vitamin C 500 mg (Group C), or vitamin C 500 mg (Group D) daily for 12 weeks. Skin properties evaluated included hydration, transepidermal water loss (TEWL), and elasticity using a corneometer, tewameter, and cutometer, respectively. Results: Changes from baseline in the corneometer were statistically significant between Groups A and B (p = 0.011) and Groups A and C (p = 0.004). There were statistically significant differences in cutometer from baseline between Groups A and B (p = 0.005) and Groups A and C (p = 0.015). There was no significant difference from baseline in the corneometer and cutometer between Groups B and C. The greatest changes in TEWL from baseline were seen in Group B, and the second greatest changes were seen in Group C. Conclusions: Daily CP supplementation may improve skin hydration and elasticity, but concomitant intake of low-dose vitamin C did not enhance the effect of CP on skin properties.

The effect of a collagen tripeptide fragment (GER) on fibroblast adhesion and spreading depends on properties of an adhesive surface

The effect of collagen tripeptide fragment GER on the adhesion and spreading of mouse embryonic fibroblasts STO to different substrates (polystyrene plastic, poly-L-lysine, fibronectin, gelatin) has been studied. It was found that tripeptide GER was involved in fibroblast adhesion and spreading. The cell response depended both on the mode of tripeptide addition to culture medium and the substrate type. Coincubation of fibroblasts with tripeptide stimulated the cell attachment and spreading to untreated plastic and plastic coated with fibronectin or gelatin but did not change cell adhesion to immobilized poly-L-lysine. Preincubation of cells with tripeptide resulted in partial inhibition of fibroblast adhesion and spreading on fibronectin- and gelatin-coated substrata. It was shown that activation and inhibition of adhesive processes after tripeptide treating was higher on fibronectin than gelatin. The data obtained support the assumption about concerted action of tripeptide GER (activity was dependent both on the used concentration of the tripeptide and the mode of tripeptide addition to culture medium) and chemical characteristics of substrate (polymers of styrene and L-lysine, ECM proteins in native (fibronectin) or partly denatured (gelatin) form) on the cell adhesion and spreading. The main targets that GER peptide may affect during the formation of cell-substrate interactions are discussed.

Periodic knee injections of collagen tripeptide delay cartilage degeneration in rabbit experimental osteoarthritis

IntroductionCollagen peptides have been reported to possess various biological activities for various cell types. The purposes of this study were, first, to examine the therapeutic effects of collagen tripeptide (Ctp) in rabbit osteoarthritis and, second, to explore a synergetic effect with hyaluronan (HA).MethodsOsteoarthritis was induced by anterior cruciate ligament transection of the right knee in 72 Japanese white rabbits and they were divided into four groups (control, Ctp, HA and Ctp/HA). Each material was injected weekly into the knee, and knee joint samples were collected 5, 10 and 15 weeks after surgery. Macroscopic and histomorphological analyses of cartilage were conducted. Expression of type II collagen and matrix metalloproteinase-13 was also analyzed immunohistochemically. A Tukey's honestly significant difference test was used to evaluate the statistical significance of difference in the macroscopic, histological and immnohistochemical results.ResultsAll treatment groups exhibited slightly higher resistance to the progression of osteoarthritis than the control group macroscopically 15 weeks after surgery. Histologically, intra-articular injection of Ctp significantly reduced cartilage degradation 10 weeks after surgery, and Ctp/HA significantly reduced it 5 weeks after surgery in comparison with the control. Immunohistochemically, both Ctp-treated and Ctp/HA-treated groups had significantly increased type II collagen-positive chondrocytes at the fifth week after the surgery, although the numbers of matrix metalloproteinase-13-positive chondrocytes were not affected.ConclusionPeriodical injections of Ctp and Ctp/HA delayed progression of cartilage degeneration of early osteoarthritis induced by anterior cruciate ligament transection in rabbits. This effect appears to be exerted by promotion of type II collagen synthesis predominantly.

Effects of Collagen Tripeptide Supplement on Photoaging and Epidermal Skin Barrier in UVB-exposed Hairless Mice

Collagen tripeptide (CTP) is a functional food material with several biological effects such as improving dry skin and wound and bone fracture healing. This study focused on the anti-photoaging effects of CTP on a hairless mouse model. To evaluate the effects of CTP on UVB-induced skin wrinkle formation in vivo, the hairless mice were exposed to UVB radiation with oral administration of CTP for 14 weeks. Compared with the untreated UVB control group, mice treated with CTP showed significantly reduced wrinkle formation, skin thickening, and transepidermal water loss (TEWL). Skin hydration and hydroxyproline were increased in the CTP-treated group. Moreover, oral administration of CTP prevented UVB-induced MMP-3 and -13 activities as well as MMP-2 and -9 expressions. Oral administration of CTP increased skin elasticity and decreased abnormal elastic fiber formation. Erythema was also decreased in the CTP-treated group. Taken together, these results strongly suggest that CTP has potential as an anti-photoaging agent.

Oral administration of collagen tripeptide improves dryness and pruritus in the acetone-induced dry skin model

Collagen tripeptide (CTP) is a highly purified, non-antigenic, low-allergenic collagen fraction that is known to have various biological effects. This study was aimed at investigating the therapeutic effects of CTP for dryness and pruitus in acetone-induced dry skin model mice.ICR mice were treated with acetone followed by oral administration of CTP (80 or 500mg/kg/day) for 3 days. As a result, oral administration of CTP in acetone-induced dry skin model mice significantly decreased Transepidermal water loss (TEWL) and suppressed scratching behavior.

Oral administration of collagen tripeptide improves dryness and pruritus in the acetone-induced dry skin model

Dry skin causes pruritus and discomfort in patients with xerosis and atopic dermatitis. General treatment for skin dryness involves the topical application of an emollient. However, more effective, simpler therapies are desired. Collagen tripeptide (CTP) is a highly purified, non-antigenic, low-allergenic collagen fraction that is known to have various biological effects. To clarify the therapeutic effects of CTP for dry skin using acetone-induced dry skin model mice. ICR mice were treated with acetone followed by oral administration of CTP (80 or 500mg/kg/day) for 3 days. Hyaluronic acid production induced by CTP was assessed using human dermal fibroblasts in vitro and in an acetone-induced dry skin model mice in vivo. Transepidermal water loss (TEWL) and scratching behavior were evaluated. Furthermore, the effects of CTP on intraepidermal nerve fibers and expression of semaphorin 3A (Sema3A) and nerve growth factor (NGF) were examined by immunohistochemistry and quantitative RT-PCR. CTP enhanced hyaluronic acid production in human dermal fibroblasts in vitro and in murine skin in vivo. Oral administration of CTP in acetone-induced dry skin model mice significantly decreased TEWL and suppressed scratching behavior. Intraepidermal nerve growth was dramatically inhibited in CTP-treated mice. Quantitative PCR analysis and immunohistochemical study revealed that CTP abolished the increased NGF and decreased Sema3A levels induced by acetone treatment. Oral administration of CTP improves dry skin and normalizes axon-guidance factors in the epidermis in addition to reducing pruritus. CTP may be used in a new therapeutic strategy against dry skin and pruritus.

皮膚浸透性コラーゲン・トリペプチド (CTP) の機能性と有用性

皮膚浸透性コラーゲン・トリペプチド (CTP) の機能性と有用性

Comparison between the effect of collagen tripeptide and sodium hyaluronan to prevent knee osteoarthritis: a preliminary in vivo study

Comparison between the effect of collagen tripeptide and sodium hyaluronan to prevent knee osteoarthritis: a preliminary in vivo study

Effects of topical gluco‐oligosaccharide and collagen tripeptide F in the treatment of sensitive atopic skin

Sensitive skin is a dermatological problem of increasing incidence in western countries and is sometimes associated with atopic condition and bacterial sovrainfection. The purpose of this study is to evaluate in a double blind, randomized, placebo controlled trial the efficacy of gluco-oligosaccharide and collagen tripeptide F in controlling the signs and symptoms of sensitive atopic skin. Forty female subjects (age, 30-59 years) affected by non-lesional atopic sensitive skin entered the study. Skin sensitivity was determined by a dermatologist on the basis of medical history, stinging test, dermatological examination and a questionnaire. A treatment with the test products (active and placebo) was carried out for 4 weeks. Measurements and clinical evaluation were carried out at baseline and at the end of the study. The following objective parameters investigated were bacterial count, skin pH and colour, transepidermal water loss (TEWL), stratum corneum hydration, skin roughness and mechanical properties. Clinical assessment included also a scoring system for dryness, desquamation, irritation, erythema and papules. Significant differences were found in the active treated group when compared with the placebo and in particular for instrumental parameters of roughness (P < 0.02), volume (P < 0.01), TEWL (P < 0.02), erythema (P < 0.0006) and clinical parameters of dryness, desquamation and irritation (P < 0.001). Moisturization levels and skin colour improved significantly in both the active and placebo groups. In conclusion, the study shows that the modulation of bacterial proliferation and normalization of skin barrier properties and stratum corneum moisturization can improve the symptoms of sensitive skin.

Role of collagen tripeptide fragment GER on activation of adhesion and modification of fatty acid composition in membrane phospholipids of CHO-K1 cells

It has been found that the multiply repeated tripeptide fragment GER (Gly-Glu-Arg) from different collagen types stimulates the nonspecific adhesion of CHO-K1 cells. Activation of cell adhesion is accompanied by modifications to the fatty acid composition in the phospholipids of the cell membrane. Cell incubation with the synthetic GER peptide increases the unsaturation index of phosphatidylcholin (PC), phosphatidylethanolamine (PEA), and phosphatidylinositol (PI). Arachidonic (C20:4ω6) acid is mainly contributed to the increased index of PI. Not only arachidonic acid but other unsaturated fatty acids, such as docosatetraenoic (C22:4ω6), docosapentaenoic (C22:5ω3), and docosahexaenoic (C22:6ω3), are responsible for the increased index of PC and PEA. In addition, the elevation of the relative content of polyenoic fatty acids in PI is concomitant with a reduced amount of monoenoic fatty acids, mainly due to decline in the oleic (C18:1) acid level. The role of GER peptide in (1) the activation of cell adhesion as a regulator of active or inactive states of integrin receptors; (2) modification of fatty acid composition in major classes of phospholipids as a modulator of the fluidity in annular lipid zones surrounding to the adhesive molecules is discussed.

Promotion by Collagen Tripeptide of Type I Collagen Gene Expression in Human Osteoblastic Cells and Fracture Healing of Rat Femur

Peptides produced by the enzymatic degradation of collagens are reported to have various activities of biological and medical interest. The mechanisms underlying their actions are, however, poorly understood. We have produced, by collagenase digestion of type I collagen, a highly purified, non-antigenic, and low allergenic tripeptide fraction (collagen tripeptide, Ctp). We report here the effects of Ctp on the in vivo bone fracture healing and in vitro calcification of osteoblastic cells. An oral administration of Ctp to rats with a femur fracture accelerated the fracture healing. Ctp apparently stimulated the calcification of human osteoblastic cells in culture. This osteotrophic effect was accompanied by a significant increase in type I collagen protein production and its mRNA levels. DNA microarray and quantitative RT-PCR analyses demonstrated that Ctp upregulated the bone-specific transcription factor, Osterix, suggesting that the induction of type I collagen gene expression by Ctp was mediated by upregulation of this factor.

Conformational analysis of polytripeptides (Gly‐Pro‐Ala)n, (Gly‐Ala‐Hyp)n, and (Gly‐Ala‐Ala)n in connection with the problem of collagen structure

AbstractConformational analysis of triple helics of a type of collagen was performed with typical collagen tripeptide sequences based on Gly‐Pro‐Ala, Gly‐Ala‐Hyp, and Gly‐Ala‐Ala. During energy minimization, the possibility of continual deformation of the pyrrolidine cycle was taken into account in order to achieve better accuracy in the resulting structure. The (Gly‐Pro‐Ala)n structure is almost isomorphic to the (Gly‐Pro‐Hyp)n structure obtained in the previous work [Tumanyan, V. G. &amp; Esipova, N.G. (1982) Biopolymers 21, 475–497]. For a collagen‐type structure, the optimal conformation of (Gly‐Ala‐Hyp)n tends to have a decreased unit twist (t = 15°), although the energy advantage with respect to the conformation with t = 45° is not so significant. A similar situation is observed for (Gly‐Ala‐Ala)n. In this case, the energy decrease during unwinding to t = 15° from t = 45° is quite small. The conformations of (Gly‐Ala‐Hyp)n and (Gly‐Ala‐Ala)n with t = 15° exhibit a similarity with a triple complex of polyproline II helices—a noncoiled coil such as (Gly‐Pro‐Hyp)n and (Gly‐Pro‐Ala)n. A similar structure may be postulated for subcomponent cq1 of the first component of a human complement containing substantial Gly‐X‐Pro and Gly‐X‐Y tripeptide derivatives in the primary structure (X, Y = any amino acid). The results suggest that the observed helical symmetry of collagen (t = 36°) is a consequence of superposition of diffraction patterns (for sufficiently long segments) from various helices (t varies from ∼15° for Gly‐X‐Hyp and Gly‐X‐Y to ∼56° for Gly‐Pro‐Ala). For short alternating segments, some unification of different helical structures is possible.

Some Stereochemical Implications of the Molecular Conformation of Collagen

AbstractA space‐filling model of collagen has been constructed in accordance with the conformation found for (Gly.Pro.Pro)n. The model shows that leucyl and phenylalanyl residues can be easily accommodated at position X of the collagen tripeptide sequence Gly.X.Y, but are severely constrained by steric hindrance at position Y. Examination of the model has also led to the suggestion that hydrogen bonding of hydroxyproline OH through a water molecule to glycyl CO on the same polypeptide chain might account for the stabilisation of the triple helix conformation by hydroxyproline.