BACKGROUND/OBJECTIVES: Osteoarthritis (OA) is a major public health issue in Japan and other countries, and foods that prevent or treat OA are in strong demand. Proteins and peptides in chicken meat and bones are known for being rich in functional and nutritional ingredients for the improvement of o...
BACKGROUND/OBJECTIVES: Osteoarthritis (OA) is a major public health issue in Japan and other countries, and foods that prevent or treat OA are in strong demand. Proteins and peptides in chicken meat and bones are known for being rich in functional and nutritional ingredients for the improvement of osteoporosis. We speculated that chicken legs, a food consumed in many regions of the world, may also contain such ingredients. In this study, we aim to (i) evaluate the effect of chicken leg extract (CLE) on the promotion of cartilage matrix production and (ii) identify the active ingredient in CLE that contributes to this function. MATERIALS/METHODS: Artificial CLE digest was prepared, and the acid mucopolysaccharide production-promoting activity of the CLE digest was evaluated by alcian blue staining of ATDC5 cells. CLE was orally administered to rabbits with burr holes in the knee joint of the femur, and the degree of regeneration of cartilage matrix was evaluated. Furthermore, we investigated orally administered CLE-derived peptides in human plasma using LC-MS. From measuring the acid mucopolysaccharide production-promotion activity of these peptides, a molecule considered to be an active ingredient in the CLE digest was identified. RESULTS: CLE digest promoted acid mucopolysaccharide production and facilitated regeneration of cartilage matrix in in vitro and in vivo experiments. Four peptides including phenylalanyl-hydroxyproline (Phe-Hyp) were detected as CLE-derived peptides in human plasma. The effect of CLE was inferred to be due to Phe-Hyp, which was confirmed to be present in the CLE digest. CONCLUSIONS: It was shown that CLE stimulated the production of articular cartilage matrix both in vitro and in vivo, and that CLE could be an effective food for preventing or treating OA. Furthermore, only Phe-Hyp was confirmed as the active compound in the CLE digest, suggesting that the activity of CLE was due to Phe-Hyp.
BACKGROUND/OBJECTIVES: Osteoarthritis (OA) is a major public health issue in Japan and other countries, and foods that prevent or treat OA are in strong demand. Proteins and peptides in chicken meat and bones are known for being rich in functional and nutritional ingredients for the improvement of osteoporosis. We speculated that chicken legs, a food consumed in many regions of the world, may also contain such ingredients. In this study, we aim to (i) evaluate the effect of chicken leg extract (CLE) on the promotion of cartilage matrix production and (ii) identify the active ingredient in CLE that contributes to this function. MATERIALS/METHODS: Artificial CLE digest was prepared, and the acid mucopolysaccharide production-promoting activity of the CLE digest was evaluated by alcian blue staining of ATDC5 cells. CLE was orally administered to rabbits with burr holes in the knee joint of the femur, and the degree of regeneration of cartilage matrix was evaluated. Furthermore, we investigated orally administered CLE-derived peptides in human plasma using LC-MS. From measuring the acid mucopolysaccharide production-promotion activity of these peptides, a molecule considered to be an active ingredient in the CLE digest was identified. RESULTS: CLE digest promoted acid mucopolysaccharide production and facilitated regeneration of cartilage matrix in in vitro and in vivo experiments. Four peptides including phenylalanyl-hydroxyproline (Phe-Hyp) were detected as CLE-derived peptides in human plasma. The effect of CLE was inferred to be due to Phe-Hyp, which was confirmed to be present in the CLE digest. CONCLUSIONS: It was shown that CLE stimulated the production of articular cartilage matrix both in vitro and in vivo, and that CLE could be an effective food for preventing or treating OA. Furthermore, only Phe-Hyp was confirmed as the active compound in the CLE digest, suggesting that the activity of CLE was due to Phe-Hyp.
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가설 설정
In this study, we used animal and cell models to evaluate whether CLE induces the production of articular cartilage matrix and can be an effective food for treating or preventing OA. Since foods are absorbed after digestion, it is important to evaluate the digestion state of ingested foods.
We subsequently found that Phe-Hyp was relevant to increase in acid mucopolysaccharide production in ATDC5 cells, while Pro-Hyp, Leu-Hyp and Glu-Hyp did not increase production. To estimate whether Phe-Hyp is the major active compound in the CLE digest and the proportion of contribution to the effect of CLE, Phe-Hyp and CLE digest were compared in terms of acid mucopolysaccharide production-promoting activity. We found that Phe-Hyp contributed to the majority of the activity of the CLE digest.
제안 방법
The sample was then analyzed by HPLC linked to a 3,200 Q Trap LC-MS/MS system (Biosystems/MDS Sciex, Ontario, Canada) using COSMOSIL 5C18-AR-II Packed Column (2.0 mm I.D. × 250 mm; Nacalai Tesque).
Two rabbits were orally administrated CLE (50 mg/day) and the other two rabbits only water (sham controls) every day for three weeks. After three weeks, the degree of wound healing was evaluated by macroscopic observation and histochemical assessment. Macroscopic observation was evaluated by a four-grade evaluation: score 0 - the burr hole is visible and not filled with matrix; score 1 - the burr hole is slightly difficult to recognize, but not filled with matrix; score 2 - the burr hole is indistinctive or filled with matrix; and score 3 - the burr hole is indistinctive and filled with matrix.
It was reported that in vitro angiotensin-converting enzyme inhibitory activity of whey protein and pea protein was evaluated after artificial digestion [17]. In this study, we conducted in vitro tests using an artificial digest of CLE. Pepsin and pancreatin are commonly used for artificial digestion, but it has been pointed out that digestion with only these two enzymes is not sufficient [18,19].
Because proteins and peptides found in chicken meat and bones are known to be rich in nutritional and functional food ingredients for relieving fatigue, sarcopenia and osteoporosis [9,10], it can be expected that chicken legs also contain such ingredients. We prepared a water-soluble chicken leg extract (CLE), evaluated the acid mucopolysaccharide production-promoting activity of artificially digested CLE in cartilage precursor cells, and examined the matrix regeneration effect of orally administered CLE using rabbits. Moreover, we identified the peptides absorbed in the human body derived from orally administered CLE by LC-MS/MS.
대상 데이터
Wound healing study using rabbits was conducted following previously reported methods [11] with modification. Four SCL-JW rabbits (22-weeks old) were purchased from Japan SLC (Kurume, Japan). Each rabbit underwent surgery to pierce a total of four burr holes in the knee joint of the femur.
데이터처리
In order to compare in vivo activity between the sham control group and CLE ingestion group, the macroscopic observation score and alcian blue-stained area of the injured site was statistically analyzed using the Mann-Whitney U test and Student’s t-test, respectively.
In the in vitro study, statistical analysis between control and other samples was performed using the Dunnett test, while Student’s t-test with Bonferroni correction was used for the comparison among the three samples (control, artificial CLE digest and Phe-Hyp; Fig. 7A).
성능/효과
It was found that only Phe-Hyp at a concentration of 500 μM significantly promoted acid mucopolysaccharide production, while the other three peptides did not show any activity (Fig. 5).
In this study, it was shown that CLE stimulated the production of articular cartilage matrix both in vitro and in vivo, and that CLE could be an effective food for treating or preventing OA. Furthermore, only Phe-Hyp was confirmed as an active compound in the CLE digest, suggesting that the activity of CLE is attributed to Phe-Hyp.
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