Optimal red clover ethanolic extract by relative aggregated metric increases osteoblastic activity and nuclear factor kappa-B ligand gene expression in SaOS-2 cells

Age-related osteoporosis affects people of both genders at any age especially postmenopausal women. The manifestation of reduced bone strength leads to increased risk of bone fracture. Currently, many have sought for plant-based supplements for the management of bone health. Red clover (Trifolium pratense) contains phytoestrogenic isoflavones which are known to limit osteoclastic activity and reduce bone resorption. Our study aimed to optimize the condition for the preparation of red clover ethanolic extract to obtain highest yield while retaining the desired bioactivity. In this study, relative aggregated metric (RAM) was designed to ease the selection process of extraction formulation based on yield and SaOS-2 cell viability. The selected ethanolic red clover extract obtained from the optimum condition (RC-D) exerted high cell viability in SaOS-2 cells which is comparable to that of 17β-estradiol. RC-D also showed promising dose-dependent alkaline phosphatase activity in SaOS-2 cells. Besides, the RANKL, OPG, ALP and Col-A gene expression analysis revealed that RC-D potentially halts the osteoclastic activity via downregulation of RANKL mRNA expression. In short, RC-D prepared and selected via RAM developed in this study has shown promising anti-osteoporotic characteristics in SaOS-2 cells and might be considered as a functional food for bone quality management.