Yasutaka Shigemura

Effect of Prolyl-hydroxyproline (Pro-Hyp), a Food-Derived Collagen Peptide in Human Blood, on Growth of Fibroblasts from Mouse Skin

We examined the effect of prolyl-hydroxyproline (Pro-Hyp), which occurs in human peripheral blood after ingestion of collagen peptide, on the migration and growth of mouse skin fibroblasts. Mouse skin discs were cultured on a 24-well plastic plate in a fetal bovine serum (FBS)-free medium. Addition of Pro-Hyp (200 nmol/mL) significantly increased the number of fibroblasts migrating from the skin to the plate after incubation for 72 h. This effect of Pro-Hyp was abolished by the addition of mitomycin C. The fibroblasts that had migrated from the mouse skin were collected and cultured on collagen gel. The growth of fibroblasts on the collagen gel was suppressed even in the presence of FBS, while rapid fibroblast growth was observed on the plastic plate. Addition of Pro-Hyp (0-1000 nmol/mL) to the medium containing 10% FBS enhanced the growth of fibroblasts on the collagen gel in a dose-dependent manner. These results suggest that Pro-Hyp might stimulate the growth of fibroblasts in the skin and consequently increase the number of fibroblasts migrating from the skin.

Identification of a novel food-derived collagen peptide, hydroxyprolyl-glycine, in human peripheral blood by pre-column derivatisation with phenyl isothiocyanate

Peptides in the blood of subjects before and after collagen hydrolysate ingestion were fractionated by ion exchange and size-exclusion chromatographies and then derivatised with phenyl isothiocyanate. The derivatives were characterised by reserved phase (RP)-HPLC. Prolyl-hydroxyproline (Pro-Hyp), which has been identified in the previous studies, was detected as a major food-derived collagen peptide in the blood of all subjects (n=5). Another major peptide was identified as hydroxyprolyl-glycine (Hyp-Gly) in the blood of four subjects, which has not been detected in previous studies. The ratio of Hyp-Gly to Pro-Hyp depended on subjects and ranged from 0.00 to 5.04. Hyp-Gly was less susceptible to human serum peptidase than Pro-Hyp. Hyp-Gly enhanced the growth of mouse primary fibroblasts on collagen gels in a higher extent than Pro-Hyp. These findings suggest that Hyp-Gly plays a significant role in exerting the biological effects by ingestion of collagen hydrolysate.

Dose-dependent changes in the levels of free and peptide forms of hydroxyproline in human plasma after collagen hydrolysate ingestion

The presence of hydroxyproline (Hyp)-containing peptides in human blood after collagen hydrolysate ingestion is believed to exert beneficial effects on human health. To estimate the effective beneficial dose of these peptides, we examined the relationship between ingested dose and food-derived Hyp levels in human plasma. Healthy volunteers (n=4) ingested 30.8, 153.8 and 384.6 mg per kg body weight of collagen hydrolysate. The average plasma concentration of Hyp-containing peptides was dose-dependent, reaching maximum levels of 6.43, 20.17 and 32.84 nmol/ml following ingestion of 30.8, 153.8 and 384.6-mg doses of collagen hydrolysate, respectively. Ingesting over 153.8 mg of collagen hydrolysate significantly increased the average concentrations of the free and peptide forms of Hyp in plasma. The Hyp absorption limit was not reached with ingestion of as much as 384.6 mg of collagen hydrolysate. These finding suggest that ingestion of less than 30.8 mg of collagen hydrolysate is not effective for health benefits.

Identification of Food-Derived Elastin Peptide, Prolyl-Glycine (Pro-Gly), in Human Blood after Ingestion of Elastin Hydrolysate

Elastin hydrolysate has apparent beneficial effects, and the food-derived peptide prolyl-glycine (Pro-Gly) is present in human blood after oral ingestion. Following ingestion of elastin hydrolysate (10 g/60 kg body weight) by healthy human volunteers, peripheral blood was used to prepare plasma samples from which peptides were extracted by solid phase extraction and fractionated by size-exclusion chromatography (SEC). Peptides in the SEC fractions were derivatized with phenyl isothiocyanate (PITC) and resolved by reversed phase (RP)-HPLC. Pro-Gly was the major food-derived elastin peptide, reaching a maximum (18 μM) at 30 min after ingestion, and decreasing to approximately 20% at 4 h after ingestion. Finally, in cell culture, levels of Pro-Gly in the medium above 0.1 μg/mL significantly enhanced elastin synthesis of normal human dermal fibroblasts (NHDF) without affecting the rate of cell proliferation.

Detection of endogenous and food-derived collagen dipeptide prolylhydroxyproline (Pro-Hyp) in allergic contact dermatitis-affected mouse ear

Generation of collagen dipeptides and deposition of orally administered prolylhydroxyproline (Pro-Hyp) in local inflammatory sites were examined in mice with hapten (2,4-dinitrofluorobenzene)-induced dermatitis in the ear. Pro-Hyp content in the hapten-treated ear was significantly higher in the chronic phase of contact dermatitis than the vehicle control. In contrast, hydroxyprolylglycine contents remained at lower levels in all cases compared to Pro-Hyp. Four hours after the ingestion of [13C5,15N]Pro and [13C5,15N]Pro-Hyp, labeled-Pro-Hyp and Pro, respectively, appeared only in the ear with dermatitis. Thus, Pro-Hyp is generated and degraded as part of the rapid synthesis and degradation of collagen in the ear with dermatitis. In addition to the endogenously generated Pro-Hyp, the orally administered Pro-Hyp was deposited in the ears. [1] Pro-Hyp is generated and degraded as part of the rapid synthesis and degradation of collagen in the ear with dermatitis. [2] The orally administered Pro-Hyp was deposited in the ears.

Changes in composition and content of food‐derived peptide in human blood after daily ingestion of collagen hydrolysate for 4 weeks

BACKGROUND Daily ingestion of collagen hydrolysate for a long period improves skin and joint conditions. It has been speculated that the beneficial effects are exerted by food-derived hydroxyproline (Hyp) peptides, which are detected in human blood after single ingestions. In the present study, to investigate the effect of long-term ingestion of collagen hydrolysate on Hyp peptides profile in blood, the concentrations of Hyp-peptides in human blood before and after daily ingestion for a long period were examined. RESULTS Hyp-peptides increased to a maximum level at 1 h after ingestion and reverted to their initial levels within 24 h during experimental period. Pro-Gly and Hyp-peptides such as Pro-Hyp-Gly, Pro-Hyp, Ile-Hyp, Leu-Hyp, Hyp-Gly, Glu-Hyp and Ala-Hyp were identified in the blood after ingestion of collagen hydrolysate at 4.5 g day-1 for 4 weeks. For the whole period, Pro-Hyp was the leading compound. The compositional rate of Hyp-Gly showed a tendency to increase, while that of Pro-Hyp tended to decrease after daily ingestion. CONCLUSION The present results indicate that daily ingestion of collagen hydrolysate for a long period can change compositional rate of Hyp peptides in human blood. This fact suggests that long-term ingestion of collagen hydrolysate might change exo- or endo-type protease activity in the digestive tract, which may consequently promote beneficial effects.

A Pilot Study for the Detection of Cyclic Prolyl-Hydroxyproline (Pro-Hyp) in Human Blood after Ingestion of Collagen Hydrolysate

Levels of short linear hydroxyproline (Hyp)-containing peptides, such as prolyl-hydroxyproline (Pro-Hyp), increase in human blood after the ingestion of collagen hydrolysate, which has been associated with beneficial effects for human skin and joints. The present study demonstrates the presence of a novel food-derived collagen peptide, cyclic Pro-Hyp, in human blood after the ingestion of collagen hydrolysate. The cyclic Pro-Hyp levels in plasma samples were estimated by liquid chromatography mass spectrometry (LC-MS). Cyclic Pro-Hyp levels significantly increased in the plasma after ingestion of collagen hydrolysate, reaching a maximum level after 2 h and then decreasing. The maximum level of cyclic Pro-Hyp in plasma ranged from 0.1413 to 0.3443 nmol/mL, representing approximately 5% of linear Pro-Hyp in plasma after ingestion of collagen hydrolysate. Addition of cyclic Pro-Hyp in medium at 7 nmol/mL significantly enhanced the growth rate of mouse skin fibroblasts on collagen gel more extensively compared to linear Pro-Hyp.

Aroma Leakage from Orange Juice Packed in Gable-Top Paper Containers for Chilled Distribution

We conducted a study to examine aroma leakage from orange juice packed in gable-top paper containers for chilled distribution. Limonene, an aromatic component of orange juice, was considered as an index compound of aroma leakage, and its seepage on the surface of the container and concentration in the orange juice were measured by GC-MS for 12 commercial samples. After 3 days of storage, limonene was detected on the surface of 8 orange juice containers, and the concentration of limonene in the orange juice was found to have decreased. Thus, limonene leaked through the container within a few days, and the extent of leakage differed between containers, presumably depending upon their barrier properties. In addition, limonene was detected in green tea and milk that was stored together with the unopened orange juice containers at 4℃. The transference of orange aroma into milk was significant, because the contamination of the milk was confirmed by subjective sensory evaluation. This study suggests the possibility of transfer of aroma compounds through paper containers to other beverages.