• BMB Reports
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• v.34 no.3
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• pp.219-224
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• 2001

To identify the antioxidative peptides in the gelatin hydrolysate of bovine skin, the gelatin was hydrolyzed with serial digestions in the order of Alcalase, pronase E, and collagenase using a three-step recycling membrane reactor. The second enzymatic hydrolysate (hydrolyzed with pronase E) was composed of peptides ranging from 1.5 to 4.5 kDa, and showed the highest antioxidative activity, as determined by the thiobarbituric acid method. Three different peptides were purified from the second hydrolysate using consecutive chromatographic methods. This included gel filtration on a Sephadex G-25 column, ion-exchange chromatography on a SP-Sephadex C-25 column, and high-performance liquid chromatography on an octadecylsilane chloride column. The isolated peptides were composed of 9 or 10 amino acid residues. They are: Gly-Glu-Hyp-Gly-Pro-Hyp-Gly-Ala-Hyp (PI), Gly-ProHyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly (PII), and Gly-ProHyp-Gly-Pro-Hyp-Gly-Pro-Hyp (PIII), as characterized by Edman degradation and fast-atom bombardment mass spectrometry. The antioxidative activities of the purified peptides were measured using the thiobarbituric acid method, and the cell viability with a methylthiazol tetrazolium assay The results showed that PII had potent antioxidative activity on peroxidation of linoleic acid. Moreover, the cell viability of cultured liver cells was significantly enhanced by the addition of the peptide. These results suggest that the purified peptide, PII, from the gelatin hydrolysate of bovine skin is a natural antioxidant, which has potent antioxidative activity.

• Journal of Preventive Medicine and Public Health
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• v.44 no.1
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• pp.9-13
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• 2011

Objectives: Although increased reactive oxygen species (ROS) is caused by stress accelerates collagen degradation, there was no data on the relationship between stress and urinary hydroxyproline (Hyp) and proline (Pro), a good marker of collagen degradation. The purpose of this study was to evaluate the relationship between depression, anxiety, and stress (DAS) and concentrations of urinary Hyp and Pro. Methods: 97 hospital employees aged 20 to 58 were asked to fill out comprehensive self-administrated questionnaires containing information about their medical history, lifestyle, length of the work year, shit-work and DAS. Depression Anxiety Stress Scale (DASS) was applied to evaluate chronic mental disorders. Urine samples were analyzed using High Performance Liquid Chromatography (HPLC) with double derivatization for the assay of hydroxyproline and proline. Results: The mean value of Hyp and Pro concenturation in all subjects was $194.1{\pm}113.4\;{\mu}mol/g$ and $568.2{\pm}310.7\;{\mu}mol/g$. DASS values and urinary Pro concentrations were differentiated by sex (female > male, p < 0.05) and type of job (nurse > others, p < 0.05). In the stepwise multiple linear regressions, urinary Hyp and Pro concentrations were influenced by stress (Adjusted $r^2$ = 0.051) and anxiety and job (Adjusted $r^2$ = 0.199), respectively. Conclusions: We found that stress and anxiety were correlated with urinary Hyp and Pro concentrations. To identifying a definite correlation, further study in large populations will be needed.

• Korean Journal of Food Science and Technology
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• v.40 no.4
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• pp.436-442
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• 2008

Hydroxyproline and Pro-Hyp dipeptide are the digestive products of collagen hydrolysate called collagen peptide. Some suggested that collagen peptides could improve aged or damaged skins, however, the effects of collagen peptides on the skin have not been known. In this study, we investigated the effects of digestive products of collagen peptides, hydroxyproline and Pro-Hyp dipeptide on skin quality using the UV-damaged dorsal skin of hairless mouse as a model system. Female SKH hairless mice were pre-irradiated with UV for 7 weeks, and then hydroxyproline, Pro-Hyp dipeptide were orally administered for 7 weeks with UV irradiation. Wrinkle formation (by replica image), skin elasticity, barrier status (by TEWL, transepidermal water loss), epidermis thickness, and biophysical changes in the stratum comeum (by hematoxylin & eosin staining) were examined. With the oral peptide treatment, effects such as skin barrier maintenance, anti-skin thickening, and recovery of the stratum corneum were observed. These results indicate that oral intake of collagen peptides may have beneficial effects on damaged skin cells.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.630-633
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• 2003

The mussel adhesive protein Mefp-1 is a natural, strong and durable adhesive that is stable under corrosive, saline conditions. Mefp-1 is found in the marine mussel Mytilus edulis and it has a molecular weight of ca. 130,000. The primary structure is mainly composed of repeating decapetides: Ala-Lys-Pro -Ser-Tyr Hyp-Hyp-Thr-DOPA-Lys. To elucidate the mechanism by which Mefp-1 bonds to metal surfaces, we have used surface-enhanced Raman spectroscopy to study the interactions of peptides related to the Mefp-1 decapeptide repeat with gold surfaces. We have concluded that the tyrosine residue and the carboxyl terminus interact strongly with the gold surface, and that proline and hydroxyproline constrain the conformations of the peptides, thereby limiting the types of possible interactions of the functional groups with the gold surface.

• Food Science of Animal Resources
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• v.33 no.4
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• pp.474-480
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• 2013

This study was performed to investigate the effects of high pressure/high temperature (HPHT) treatment on the recovery efficiency and characteristics of porcine placenta hydrolysates. The placenta hydrolysates were characterized by solubility, free amino acid contents, gel electrophoresis, gel permeation chromatography (GPC) and amino acid composition. Placenta was treated at 37.5 MPa of pressure combined with various temperatures (150, 170, and $200^{\circ}C$) or various holding times (0, 30, and 60 min at $170^{\circ}C$). Insoluble raw placenta collagen was partially solubilized (> 60% solubility) by the HPHT treatment. Free amino group content of placenta collagen was increased from 0.1 mM/g collagen to > 0.3 mM/g collagen after HPHT treatment, reflecting partial hydrolysis of collagen. The molecular weight ($M_w$) distribution showed evidence of collagen hydrolysis by shifting of $M_w$ peaks toward low molecular weight when treated temperature or holding time was increased. Alanine (Ala), glycine (Gly), hydroxyproline (Hyp), and proline (Pro) contents increased after the HPHT treatments compared to a decrease in the others. In particular, the increase in Gly was obvious, followed by Hyp and Pro, reflecting that placenta hydrolysates were mainly composed of these amino acids. However, increasing temperature or holding time hardly affected the amino acid compositions. These results indicate that the HPHT treatment is advantageous to hydrolyze collagen derived from animal by-products.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.47 no.2
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• pp.147-153
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• 2021

Collagen hydrolysate (CH) is known to prevent skin aging by stimulating skin dermal fibroblasts to promote synthesis of extracellular matrix such as collagen and elastin. Recently, among the various factors that cause skin aging, advanced glycation end products (AGEs) have received particular attention. However, the effect of CH on AGE accumulation has not been studied. Since CH could affect AGE accumulation by promoting production of skin structural proteins, clinical trial was performed using low molecule collagen peptide (LMCP), which were CH containing 25% tripeptide and 4% Gly-Pro-Hyp. Skin autofluorescence (SAF) values were measured using an AGE reader to evaluate accumulation of AGE in skin. As a result of applying 0.5% and 1.0% LMCP solutions to the subject's forearm for 8 weeks, the SAF value at the test site significantly decreased compared to the control site. Additionally, in vitro test was performed using CCD-986sk to evaluate the promotion of collagen synthesis in skin fibroblasts by LMCP. As a result, 800 ㎍/mL of LMCP significantly increased synthesis of human pro-collagen Iα1 (COL1A1) in CCD-986sk. Through this study, we have confirmed that tropical LMCP applications can promote collagen synthesis to help anti-glycation effects, suggesting that LMCP has potential as an anti-aging cosmetic material.

• Journal of Microbiology and Biotechnology
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• v.6 no.1
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• pp.7-11
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• 1996

MR-387Al (ARPA-Val-Pro) and A2 (AHPA-Val-Hyp) were prepared as aminopeptidase N inhibitors through the synthesis of peptide MR-387A and B analogues which contained 3-amino-2-hydroxy-4-phenyl butanoic acid (ARPA) as a zinc-chelating moiety. They are competitive inhibitors of aminopeptidase N with inhibition constants(Ki) of 4.1 $\times 10^{-7}\;and 1.1 \times 10^{-6}$ M, respectively. MR-387Al also strongly inhibited aminopeptidase B of human myelogenous leukemia K-562 cell with $IC_50$ of 0.35 $\mu$ M. Inhibitions of aminopeptidase N activity by ARPA-bearing inhibitors of various peptide chain lengths also have been studied. $IC_ 50$ values of AHPA-Val (bestatin), ARPA-Val-Pro (MR-387Al) and ARPA-Val-Pro-Leu (MR-387C) compared against porcine kidney aminopeptidase N were 20.1, 0.60 and 0.08 $\mu$ M, respectively. These results support that a multiple interaction between the $S_1\to S'_3$ sites of aminopeptidase N and the $P_1\to P'_3$ of the inhibitor plays a crucial role in stabilizing strongly the enzyme-inhibitor complex.

• Preventive Nutrition and Food Science
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• v.13 no.3
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• pp.196-203
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• 2008

Hwangtae, dried Alaska pollack, is a major storage product in the fish processing industry. Hwangtae is prepared by removing the internal organs and drying outdoors during the cold witner months by allowing it to thaw during the daytime and re-freeze at night under sub-zero ($-10^{\circ}C$) conditions and gradually dry from December until the next April for around 5 months from Myungtae. In this study, ground Hwangtae was hydrolyzed using two proteolytic enzymes (pepsin and alcalase) which produced five soluble active peptides from Hwangtae (yellowish dried Pollack, Theragra chalcogramma) protein. Two different peptides with strong antioxidative activity were isolated from the hydrolysate using consecutive chromatographic methods of Sephadex G-25 gel, ion-exchange chromatography on a Sepharose-Sephadex C-25 gel, and high-performance liquid chromatography. The isolated peptides, APO1 and APO2, were composed of 16 and 13 amino acid residues, respectively. Both peptides contained a Gly residue at the C-terminus and the repeating motif Gly-Pro-Hyp. The peptide with a molecular weight less than 1,000 Daltons (APACE) obtained from enzymatic hydrolysates of Hwangtae exhibited the highest ACE inhibitory activity. The APACE peptides was composed of 4 amino acid residues (Gly-Leu-Leu-Pro). These results suggest that Hwangtae hydrolysates could be a good source of peptides with ACE inhibitory activity. Biochemical analysis indicated that two 70 kDa peptides (APG1 and APG2) isolated from the hydrolysate had gelatinoytic activity, which was shown to be a calcium dependent protease type as showed by gelatin SDS PAGE.

• Food Science of Animal Resources
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• v.34 no.1
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• pp.14-19
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• 2014

This study investigated the effects of protease treatments (trypsin, chymotrypsin, and pepsin) under various pressure levels (0.1-300 MPa) for the characteristics of porcine placenta hydrolysates. According to gel electrophoretic patterns, the trypsin showed the best placental hydrolyzing activity followed by chymotrypsin, regardless of the pressure levels. In particular, the peptide bands of tryptic-digested hydrolysate were not shown regardless of applied pressure levels. The peptide bands of hydrolysate treated chymotrypsin showed gradual decreases in molecular weights ($M_w$) with increasing pressure levels. However, the pepsin did not show any evidences of placental hydrolysis even though the pressure levels were increased to 300 MPa. The gel permeation chromatography (GPC) profiles showed that the trypsin and pepsin had better placental hydrolyzing activities under high pressure (particularly at 200 MPa), with lower $M_w$ distributions of the hydrolysates. Pepsin also tend to lower the $M_w$ of peptides, while the major bands of hydrolysates being treated at 300 MPa were observed at more than 7,000 Da. There were some differences in amino acid compositions of the hydrolysates, nevertheless, the peptides were mainly composed of glycine (Gly), alanine (Ala), hydroxyproline (Hyp) and proline (Pro). Consequently, the results indicate that high pressure could enhance the placental hydrolyzing activities of the selected proteases and the optimum pressure levels at which the maximum protease activity is around 200 MPa.

• Journal of Microbiology and Biotechnology
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• v.31 no.10
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• pp.1401-1408
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• 2021

This study examined whether the oral administration of low-molecular-weight collagen peptide (LMCP) containing 3% Gly-Pro-Hyp with >15% tripeptide (Gly-X-Y) content could ameliorate osteoarthritis (OA) progression using a rabbit anterior cruciate ligament transection (ACLT) model of induced OA and chondrocytes isolated from a patient with OA. Oral LMCP administration (100 or 200 mg/kg/day) for 12 weeks ameliorated cartilage damage and reduced the loss of proteoglycan compared to the findings in the ACLT control group, resulting in dose-dependent (p < 0.05) improvements of the OARSI score in hematoxylin & eosin (H&E) and Safranin O staining. In micro-computed tomography analysis, LMCP also significantly (p < 0.05) suppressed the deterioration of the microstructure in tibial subchondral bone during OA progression. The elevation of IL-1β and IL-6 concentrations in synovial fluid following OA induction was dose-dependently (p < 0.05) reduced by LMCP treatment. Furthermore, immunohistochemistry illustrated that LMCP significantly (p < 0.05) upregulated type II collagen and downregulated matrix metalloproteinase-13 in cartilage tissue. Consistent with the in vivo results, LMCP significantly (p < 0.05) increased the mRNA expression of COL2A1 and ACAN in chondrocytes isolated from a patient with OA regardless of the conditions for IL-1β induction. These findings suggest that LMCP has potential as a therapeutic treatment for OA that stimulates cartilage regeneration.