Nutr Res Pract.  2019 Dec;13(6):480-487. 10.4162/nrp.2019.13.6.480.

Study of the cartilage matrix production-promoting effect of chicken leg extract and identification of the active ingredient

• Yamada H
• Nakamura U
• Nakamura T
• Uchida Y
• Yamatsu A
• Kim M

• KMID: 2464127
• DOI: http://doi.org/10.4162/nrp.2019.13.6.480

Abstract

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.

Keyword

Osteoarthritis; phenylalanyl-hydroxyproline; cartilage; chicken leg

MeSH Terms

Alcian Blue
Cartilage*
Cartilage, Articular
Chickens*
Femur
Humans
In Vitro Techniques
Japan
Knee Joint
Leg*
Meat
Osteoarthritis
Osteoporosis
Peptides
Plasma
Public Health
Rabbits
Regeneration
Alcian Blue
Peptides

Figure

• 

  Fig. 1 Size Exclusion Chromatography profiles. (A) CLE and (B) artificial CLE digest and (C) molecular weight standards were detected at 214 nm. Elution time of standards were the following: a - Aprotinin (MW: 6,512, elution time: 22.314 min); b - angiotensin II (MW: 1,046, elution time: 32.504 min); c - triglycine (MW: 189, elution time: 33.664); and d - glycine (MW: 75, elution time: 35.529 min).

• 

  Fig. 2 Acid mucopolysaccharide production by artificial CLE digest in ATDC5 cells. Artificial CLE digest was added at concentrations of 100, 500, 1,000 and 2,000 µg/mL. Values represent mean ± SD. Alcian blue-stained area is expressed as a relative value with control as 100%. **P < 0.01 compared with control, n = 3.

• 

  Fig. 3 Anatomy of the knee joint of the animals after 3 weeks CLE administration. (A) Macroscopic observation at burr holes in the sham control. (B) Macroscopic observation at burr holes in the CLE ingestion group. (C) Alcian blue staining of the burr holes in the sham control. (D) Alcian blue staining of the burr holes in the CLE ingestion group.

• 

  Fig. 4 Mass spectrometry chromatograms of artificial CLE digest in SEC fractions 36–39 ((A); m/z = 120–200, (B); m/z = 200–250, (C); m/z = 250–300, (D); m/z = 300–400, (E); m/z = 400–1,000). Peaks marked with Single-Letter Amino Acid Code and “Hyp” represent amino acids. Peaks marked with lowercase represent peptides. Peaks marked with asterisk are compounds that could not be estimated as peptides with any combination of amino acids.

• 

  Fig. 5 Acid mucopolysaccharide production in ATDC5 cells cultured with CLE-derived peptides identified from human plasma. Each sample was added at a concentration of 500 µM. Values represent mean ± SD. Alcian blue-stained area is expressed as a relative value with control as 100%. * P < 0.05 compared with control, n = 3.

• 

  Fig. 6 Acid mucopolysaccharide production by Phe-Hyp in ATDC5 cells. Phe-Hyp was added at concentrations of 12.5, 25, 50 and 75 µM. Values represent mean ± SD. Alcian blue-stained area is expressed as a relative value with control as 100%. * P < 0.05 and ** P < 0.01 compared with control, n = 3.

• 

  Fig. 7 Comparison of acid mucopolysaccharide production in ATDC5 cells. (A) Comparison of artificial CLE digest (2,000 µg/mL) and Phe-Hyp (21 µg/mL) in consideration of material balance. * P < 0.05, n.s., not significant. (B) Comparison of Pro-Hyp (2.5 mM) and Phe-Hyp (50 µM) in consideration of the abundance ratio in human plasma. Values represent mean ± SD. Alcian blue-stained area is expressed as a relative value with control as 100%. * P < 0.05 compared with control, n = 3.

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