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 (<i>Trifolium pratense</i>) 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.

年龄相关性骨质疏松症（Age-related osteoporosis）可累及各年龄段的两性人群，尤以绝经后女性为甚。骨强度下降会导致骨折风险升高。目前已有诸多研究尝试采用植物源性补充剂来管理骨骼健康。红车轴草（*Trifolium pratense*）含有植物雌激素类异黄酮，这类物质可抑制破骨细胞活性、减少骨吸收。本研究旨在优化红车轴草乙醇提取物的制备工艺条件，在保留目标生物活性的前提下获得最高提取率。本研究设计了相对聚合评分法（Relative Aggregated Metric，RAM），以提取率与SaOS-2细胞活力为依据，简化提取配方的筛选流程。从最优工艺条件下获得的红车轴草乙醇提取物（RC-D），在SaOS-2细胞中展现出较高的细胞活力，与17β-雌二醇（17β-estradiol）的效果相当。RC-D在SaOS-2细胞中还表现出良好的剂量依赖性碱性磷酸酶活性。此外，对RANKL、OPG、ALP及Col-A基因的表达分析结果显示，RC-D可通过下调RANKL mRNA的表达，潜在抑制破骨细胞活性。综上，通过本研究开发的RAM筛选并制备得到的RC-D，在SaOS-2细胞中展现出良好的抗骨质疏松特性，有望作为骨健康管理的功能性食品加以应用。