Orally administered low-molecular weight agaro-oligosaccharides are absorbed into the plasma of healthy humans.

Agaro-oligosaccharides (AOSs) are known to have biological activities, such as anti-inflammatory, anti-tumor, and anti-obesity effects. Although existing evidence suggests the presence of AOSs in peripheral tissues after oral administration, whether AOSs permeate into the blood circulation remains unknown. Thus, we hypothesized that AOSs with low-molecular weight can permeate the human gastrointestinal tract. To test this hypothesis, the time course of absorption was examined by analyzing plasma samples before and 1, 2, and 4 h after ingestion. Analysis was performed using liquid chromatography/mass spectrometry after labeling with p-aminobenzoic ethyl ester. Our results showed that the plasma concentration of agarobiose (Abi) was higher than that of agarotetraose (Ate); however, agarohexaose was not detected. Additionally, plasma levels of Abi and Ate were proportional to the dose. These results suggest that permeation efficiency is dependent on the molecular weight and that the systemic absorption of Abi via the gastrointestinal tract is better than that of Ate. These findings will contribute to a better understanding of the bioactivity of orally administered AOSs in peripheral tissues.

Intestinal permeability of agaro-oligosaccharides: Transport across Caco-2 cell monolayers and pharmacokinetics in rats.

Agaro-oligosaccharides (AOSs), even-numbered oligosaccharides prepared from agar, are applied to various food, including supplements, drinks, and jellies because of their biological activities. This study aimed to evaluate the AOS permeation in the gastrointestinal tract in vivo and in vitro. Agarobiose (Abi), agarotetraose (Ate), and agarohexaose (Ahe) were detected in rat plasma after oral administration of AOSs. The detection level of agarobiose in the plasma was higher than that of agarohexaose, which was consistent with the permeation study using Caco-2 cell monolayers. Further, the adenosine triphosphate inhibitor (sodium azide) or endocytosis inhibitor (colchicine) did not inhibit AOS permeation through Caco-2 cell monolayers. Conversely, AOS permeation enhanced upon treatment with cytochalasin B, a tight junction disrupter, suggesting that AOSs might have passed mainly through the tight junctions between the intestinal epithelial cells. These results indicate that AOSs, especially agarobiose, can be absorbed as an intact form via the gastrointestinal tract across the intestinal epithelium through the paracellular pathway.

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.

Detection of Balenine in Mouse Plasma after Administration of Opah-Derived Balenine by HPLC with PITC Pre-Column Derivatization.

We examined the absorption of balenine (Bal) in mouse blood after the administration of a high-purity Bal prepared from opah muscle. Using HPLC with phenyl isothiocyanate pre-column derivatization, we successfully isolated imidazole peptides and their constituents. We detected Bal and 3-methylhistidine (3-Me-His) in mouse blood 1 h after the administration of opah-derived Bal. The concentrations of Bal and 3-Me-His significantly increased to 128.27 and 69.09 nmol/mL in plasma, respectively, but were undetectable in control and carnosine (Car)-administrated mice. In contrast, ß-alanine and histidine did not increase in mouse plasma 1 h after the administration of Car and opah-derived Bal. The present study is the first report on the absorption of food-derived Bal in mouse blood and serves as a pilot study for future clinical trials.

Comparison of gelatin and low-molecular weight gelatin hydrolysate ingestion on hydroxyproline (Hyp), Pro-Hyp and Hyp-Gly concentrations in human blood.

This study showed that gelatin ingestion significantly increased prolyl-hydroxyproline (Pro-Hyp) levels in plasma of 9 subjects, with maximum concentrations of 15.5 ± 3.0 nmol/mL 2 h post-ingestion. Hydroxyprolyl-glycine (Hyp-Gly) concentrations were significantly increased and reached a maximal level of 2.3 ± 0.5 nmol/mL 1 h post-ingestion of gelatin. A low molecular weight gelatin hydrolysate (LMW-GH) significantly enhanced concentrations of both peptides, while gelatin hydrolysate ingestion did not significantly enhance the maximum concentration and area under the plasma concentration-time curve (AUC) of Hyp-Gly relative to gelatin. The absorption of free Hyp following gelatin ingestion (94.4 ± 16.4 nmol/mL) was significantly lower relative to GH (150.9 ± 15.3 nmol/mL) and LMW-GH (169.1 ± 32.5 nmol/mL). The present study is the first report demonstrating that Hyp-containing peptides are elevated to µM levels in human plasma after gelatin ingestion. These results suggested that gelatin is useful as a functional food as effectively as GH.

Isolation of balenine from opah (Lampris megalopsis) muscle and comparison of antioxidant and iron-chelating activities with other major imidazole dipeptides.

Balenine (Bal) in opah muscle was extracted using hot water and purified by ion-exchange chromatography and recrystallization to provide 41 g of over 95% pure Bal from 1 kg of opah muscle. The structure of purified Bal was identical to that of an authentic Bal standard by NMR analysis. The antioxidant (ORAC and HORAC values) and Fe(II) ion-chelating abilities of purified Bal were examined by comparison with two major imidazole dipeptides, carnosine (Car) and anserine (Ans). Opah-derived Bal showed significantly higher ORAC and HORAC values and Fe(II) ion-chelating ability at 0.3 mM. In silico molecular simulation revealed that Bal and Car formed hydrogen bonds between the hydrogen atom of the imidazole imino group and the carboxyl carbonyl oxygen, whereas Ans did not. The proposed method for extracting and purifying Bal from opah muscle suggests that opah can be utilized as a functional food or Bal resource.

Improved in Vivo Tracking of Orally Administered Collagen Hydrolysate Using Stable Isotope Labeling and LC-MS Techniques.

Collagen-derived hydroxyproline (Hyp)-containing oligopeptides, known to have various physiological functions, are detected in blood at markedly higher concentrations after oral ingestion of collagen hydrolysate. Monitoring the absorption and metabolism of the bioactive peptides is essential to investigate the beneficial effects of collagen hydrolysate. We previously developed an internal standard mixture by sequential protease digestion of stable isotope-labeled collagen, which enabled highly accurate quantitation of collagen-derived oligopeptides by liquid chromatography-mass spectrometry (LC-MS). However, the use of proteases caused a profound imbalance in the generated peptides. Here, we employed partial acid hydrolysis to achieve more efficient and balanced peptide generation. Various stable isotope-labeled oligopeptides were detected after 0.5 h acid hydrolysis, and marked enhancement of peptide generation compared with the previous enzymatic method was observed, especially for Hyp-Gly (27.8 ± 0.6 ng/µg vs 0.231 ± 0.02 ng/µg). The acid hydrolysate was then heated to generate labeled cyclic dipeptides. Using the novel internal standard mixture in LC-MS, we were able to simultaneously quantitate 23 collagen-derived oligopeptides in human plasma and urine after oral administration of collagen hydrolysate.

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.

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. © 2017 Society of Chemical Industry.

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.

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 [(13)C5,(15)N]Pro and [(13)C5,(15)N]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.

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.

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.

An evaluation of protein intake for metabolic demands and the quality of dietary protein in rats using an indicator amino acid oxidation method.

Currently, protein requirements are generally determined based on nitrogen balance studies, but there are a variety of limitations associated with this method. The indicator amino acid oxidation (IAAO) method, with a theoretical base that differs widely from the nitrogen balance method, was developed as an alternative method for humans. The objective of the present study was to evaluate protein intakes for metabolic demands and protein quality, using protein itself, in rats employing the IAAO technique with L-[1-(13)C]phenylalanine. Male Wistar/ST rats (5-6 wk old) received a graded casein (4.3, 8.6, 12.9, 17.2, 21.5, 25.8%), or a wheat gluten (7.2, 10.8, 14.4, 18.0, 21.6, 25.2%) diet, along with L-[1-(13)C]phenylalanine. An isotopic plateau in breath was achieved 210 min after the start of the (13)C ingestion. The protein intakes for metabolic demands were calculated by applying a mixed-effect change-point regression model to breath (13)CO(2) data, which identified a breakpoint at minimal breath (13)CO(2) in response to graded protein intake. The protein intakes for metabolic demands determined by the IAAO method were 13.1 g/kg BW/d for casein and 18.1 g/kg BW/d for wheat gluten, showing a tendency similar to that determined by the nitrogen balance method. These results demonstrated that the IAAO method could be employed to evaluate not only the protein intakes for metabolic demands, but the dietary protein quality in freely living rats, suggesting that this method might be viable in a clinical setting.

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.