Anti‐osteoporosis bioactivity evaluation in zebrafish model of raw and salt‐processed Achyranthes bidentata followed by liquid chromatography–mass spectrometry analysis and correlation analysis

Achyranthes bidentata has been found to possess beneficial effects against osteoporosis, but there is still a lack of comprehensive studies on its anti-osteoporotic compounds. Therefore, in this study, we established a zebrafish osteoporosis model to evaluate the anti-osteoporotic effect of different fractions of raw and salt-processed A. bidentata. Among these fractions, the dichloromethane fraction showed the most promising anti-osteoporotic effect. To further investigate the active compounds responsible for the anti-osteoporosis effects, we prepared and analyzed the dichloromethane fraction of 10 batches of raw and salt-processed A. bidentata using liquid chromatography-mass spectrometry. As a result, we tentatively identified 19 compounds, including 11 saponins, three phenolic amides, three unsaturated fatty acids and two other compounds. To further narrow down the potential active compounds, we employed both orthogonal partial least squares discriminant analysis and gray relationship analysis. Through these analyses, we were able to identify eight compounds that showed a high correlation with the anti-osteoporosis effects of the dichloromethane fraction. Furthermore, we validated the anti-osteoporotic effects of β-ecdysterone, wogonin, ginsenoside Ro, oleanolic acid, linoleic acid and palmitic acid using the zebrafish model. These compounds demonstrated significant anti-osteoporotic effects, further supporting their potential as active compounds in A. bidentata.