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

Abstract

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 ColA 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.

Trifolium pratense ethanolic extract alters the gut microbiota composition and regulates serum lipid profile in the ovariectomized rats

“…The extract was standardized and managed by two functional indicator components: formononetin and biochanin A (Additional file 2 ). HPLC quantification of the indicative compounds (formononetin and biochanin A) in TPEE was conducted and reported in our previous study [ 24 ].…”

“…MS were taken at 70 eV. HPLC quantification of the indicative compounds (formononetin and biochanin A) in TPEE was conducted and reported in our previous study [ 24 ].…”

“…To allow direct comparison among the data obtained from different analysis in our study, a metric system was constructed. The data obtained in each analysis were normalized using Min-Max normalization [ 24 ] to ensure the output was bounded between 1 and 5. Specifically, the NC was scored as 1 and the Sham as 5.…”

Serum biomarker-based osteoporosis risk prediction and the systemic effects of Trifolium pratense ethanolic extract in a postmenopausal model

“…BCA and FMT are the potential active ingredients in TPEE and they were commonly known as the indicative compounds in red clover extract [ 28 , 42 , 43 ], therefore BCA and FMT in the sample were confirmed using HPLC analytical method. In this study, TPEE was prepared in accordance with GMP standards and the Food Item Manufacturing Report in this study.…”

“…The representative chromatogram and spectra were shown in Additional file 1 . HPLC quantification of the indicative compounds, biochanin A (BCA) and formononetin (FMT) in TPEE was carried out and reported in our previous study [ 28 ].…”