Yak (Bos grunniens) bones collagen‐derived peptides stimulate osteoblastic proliferation and differentiation via the activation of Wnt/β‐catenin signaling pathway

As the world's population is transitioning gradually to an ageing stage, the incidence of osteoporosis is increasing annually. Yak bone is one of the major components of Tibetan medicine and it has mainly been associated with an improvement in bone health, for example against osteoporosis. However, the functional bioactive ingredients and the underlying mechanisms are still unclear.Sequential purification of yak-bone hydrolysates was achieved by ultrafiltration, size exclusion chromatography, and semi-preparative reverse-phase high-performance liquid chromatography. After this, 35 novel peptides were identified by mass spectrometry analysis, of which peptide GPAGPPGPIGNV (GP-12) displayed the highest osteoblast proliferation-promoting activity, with an increase of 42.7% in cell growth. An in vitro stability study demonstrated that GP-12 was digested into smaller peptides (GP-9, GV-9, AV-10 and GP-11) after simulated gastrointestinal digestion and absorption (Caco-2 cell monolayers) experiments. However, some of them still can be absorbed intact through the (Caco-2 cell monolayers by a paracellular route (Papp: 5.36 ± 0.34 cm s-1 ). Flow cytometry results indicated that GP-12 enhanced osteoblastic proliferation by inducing the alteration of the cell-cycle progression both from the G0/G1 to the S phase and from the S to the G2/M phase. Quantitative real-time polymerase chain reaction (PCR) and western blot results revealed that GP-12 induced osteoblastic proliferation and differentiation in a dose-response manner through the activation of a Wnt/β-catenin signaling pathway.These findings highlighted that such peptides hold the promise of discovering candidates for functional and health-promoting foods, which could be potentially used for the treatment of osteoporosis. © 2020 Society of Chemical Industry.