Isoflavones, such as Genistein (Ge) and Daidzein (Dz) are widely studied Phytoestrogens with potent anti-osteoporotic and good antioxidant activity. Cowpea is one such legume having high isoflavone content and hence we aimed at studying the beneficial effects of the isoflavones isolated from cowpea as it is widely accepted staple food in India. Previously, we reported the effect of Cowpea isoflavones (CP) and Vitamin D (VD) owing to its ability of improving the osteoporotic condition in a diet induced osteoporotic rat model. In the present study, we tried to explore the underlying mechanism of CP and VD along with positive controls Dz and Ge in influencing the functions of human osteoblasts at cellular level.Initially, MG-63 cells were assessed for the expression of genes involved in BMP-2 signaling pathway, like Bone morphogenic protein (BMP-2), transcription factor Osterix (OSX), total and phosphorylated Smad 1/5/8 levels and osteoblast specific genes levels namely Alkaline phosphatase (ALP), Osteopontin (OPN), and Collagen by immunoblot, flow cytometry, and quantitative RT-PCR studies. All the levels that were upregulated with the initial exposure of the compounds got inhibited after Noggin exposure a specific BMP-2 antagonist both at protein level and m-RNA level, except OSX where the expression was totally hindered in CP and Ge treated groups alone. Hence, CP and VD activate BMP-2/Smad signaling pathway and promote further proliferation and differentiation of osteoblasts. Therefore, results prove that isoflavones isolated from cowpea could be used in treating bone-related disorders. K E Y W O R D SALP, BMP, collagen, MG-63 Osteoblasts, Noggin, Osteopontin, Smad 1/5/8

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