Efficacy evaluation and metabolomics analysis of raw and salt-processed Achyranthes bidentata Radix in zebrafish larvae for osteoporosis treatment

Osteoporosis, characterized by reduced bone density and the deterioration of bone tissue, poses a significant health challenge. The mechanisms underlying the protective effects of both raw and salt-processed Achyranthes bidentata Radix in osteoporosis remains unclear. This study endeavors to unravel and analyze the therapeutic mechanisms of these two forms of Achyranthes bidentata Radix in osteoporotic zebrafish larvae, utilizing GC/MS-based metabolomics. Zebrafish larvae were categorized into five groups: blank control, model, positive control, and groups treated with raw and salt-processed Achyranthes bidentata Radix. Following drug administration, notable enhancements were observed in both mineralized bone area and cumulative optical density. Various data mining techniques were employed, encompassing principal component analysis, orthogonal projections to latent structures discriminant analysis, and metabolic pathway analysis. These analyses unveiled 26 differential endogenous metabolites with significant biological implications in the zebrafish osteoporosis model. Among these metabolites, 12 (including acetamide, L-lactic acid, threonine, glycerol, rhamnose, azelaic acid, palmitic acid, inositol, stearic acid, hexadecane, sucrose, and glyceryl monostearate) were validated using standard compounds, exhibiting strong linear correlation coefficients (R²) ranging from 0.9917 to 0.9999. Furthermore, the method demonstrated excellent repeatability, as evidenced by relative standard deviation (RSD) values below 7.37%. The average spiked recoveries of the standard compounds fell within the range of ± 15%, ranging from 85.45% to 114.28%. Additionally, the stability of the standard compounds was confirmed after three freeze-thaw cycles, with RSD values remaining below 14.40%. Collectively, the metabolomic analysis unearthed potential biomarkers that could serve as indicators of the therapeutic effects of raw and salt-processed Achyranthes bidentata Radix on osteoporosis. This research offers valuable insights into the potential utilization of these herbal remedies as natural interventions for osteoporosis.