Recycling preparative isolation of six bicyclol active metabolites from SD rat urine using macroporous resin, offline 2D LPLC/HPLC, and prep-HPLC combined with pharmacodynamic evaluation of two active metabolites

Abstract

In our previous study, several bicyclol (BIC) metabolites were found to possess higher solubility, security, and efficacy than the parent drug. However, further research can’t be conducted without monomeric metabolites. In the current study, a highly efficient preparative approach for six BIC-active metabolites from Sprague-Dawley (SD) rat urine was developed. First, 1000 mL of urine was purified and concentrated to 50 mL using microporous resin. Second, middle chromatogram isolated (MCI) GEL®CHP20P adsorbent was used to create a low-pressure liquid chromatography (LPLC) column, which was combined with high-performance liquid chromatography (HPLC) to build an offline 2D system to visualize the separation process. Samples were segmented into 25 tubes and merged into three fractions. Then, recycling preparative HPLC was applied to the monomeric preparation to improve the efficiency. The prepared metabolites possessed high purity (greater than98%), and were verified by nuclear magnetic resonance (NMR). Finally, an isoniazid (INH)-induced liver injury zebrafish model was established to evaluate the efficiency of the BIC, M7, and M8 metabolites. The M7 metabolite exhibited a higher efficiency than BIC in histopathology, gene expression, and aminotransferase levels. Consequently, this study provided a strategy that integrating modern analytical techniques to prepare metabolites for discovering high value candidate compounds from biological metabolism.