Effect of buffalo casein-derived novel bioactive peptides on osteoblast differentiation

Purpose Epidemiological and intervention studies show that milk consumption in childhood and during adolescence is related to higher bone mineral density. Milk and milk products prevent the bone loss in pre- and postmenopausal women. Apart from calcium, there are other biologically active compounds in milk such as bioactive peptides which may play a role in promoting bone health. Casein is the major protein in milk which has also been reported to have numerous biological active peptides within it. The hypothesis of the present study was to identify the key peptides behind osteoanabolic nature of the milk protein, which further can be used to prepare functional foods to alleviate bone diseases like osteoporosis. Hence, this study was carried out to investigate osteogenic nature of four novel bioactive peptides [PEP1 (EDVPSER), PEP2 (NAVPITPTL), PEP3 (VLPVPQK) and PEP4 (HPHPHLSF)] derived from buffalo casein by in vitro osteoblast differentiation model. Methods Calvaria cells were isolated from 3-day-old rat pups, cultured under in vitro conditions till confluence and further used for experiments. Calvarial osteoblast cells were cultured in the presence or absence of peptides including positive controls up to 21 days. Effect of peptides was checked at regular intervals by quantifying osteoblast differentiation marker genes (ALP, OCN and COL-1) expression, alkaline phosphatase activity, osteocalcin level in culture supernatants, mineral deposition by alizarin red staining and caspase-3 and 9 assays. Results The osteoblast differentiation marker genes (ALP, OCN and COL-1) expression was significantly [(p < 0.01) (p < 0.001)] up-regulated in the presence of these peptides. The peptides also significantly induced alkaline phosphatase activity, osteocalcin level and mineral deposition in comparison with the control. It was also observed that all the four peptides did not show any cytotoxic effect during 21-day treatment period. Conclusion All peptides enhanced osteoblast differentiation along with the positive controls. These results hold an immense scope to use peptides as preventive measure for reducing incidence of osteoporosis. These peptides can also be used as drugs and can be utilized as functional ingredients in functional foods preparation for osteoporosis therapy, but in vivo studies are required for further confirmation.