ABSTRACT
A quantitative analysis method for six principal active constituents (acubin, geniposidic acid, chlorogenic acid, pinoresinol di-O-glucopyranoside, geniposide, and pinoresinol 4-O-glucopyranoside) of crude Eucommiae Cortex (EC) and its salt-processed product extracts was developed to investigate and compare their pharmacokinetic behaviors in adenine-induced renal fibrotic rats in vivo. UHPLC-QqQ-MS/MS technology was employed. Scan was conducted in negative ion mode and quantitative determination was carried out by MRM paired ion. The established method was fully validated by specificity, linearity, precision, accuracy, stability, recovery, and matrix effect, and the results of methodological investigation met the requirements of biological sample analysis. Then, a quick, sensitive, and accurate method was successfully established, which could simultaneously measure the contents of six active constituents of crude and salt-processed EC extracts in rat plasma. After a single administration to renal fibrotic rats of crude EC and its salt-processed product extracts, the plasma concentration of each constituent at different time points was measured, the pharmacokinetic parameters were calculated and the concentration time curves were structured. The experiment was approved by the experimental animal ethics committee from Nanjing University of Chinese Medicine (No. 202103A008). The results showed that compared to the crude Eucommiae Cortex group, the tmax of aucubin, pinoresinol di-O-glucopyranoside, geniposide, and pinoresinol 4-O-glucopyranoside in the salt-processed Eucommiae Cortex group rat plasma were significantly lower than those in the crude group (P < 0.05, P < 0.01); the Cmax and AUC0-48 h of chlorogenic acid, the Cmax, AUC0-48 h and AUC0-∞ of pinoresinol di-O-glucopyranoside, and the Cmax of geniposide and pinoresinol 4-O-glucopyranoside were significantly higher than those in the crude group (P < 0.05, P < 0.01). Our investigation found that compared to crude Eucommiae Cortex, a variety of active ingredients could play a role of quick effect with higher peak blood concentration and bioavailability after oral administration of salt-processed Eucommiae Cortex, which were consistent with the traditional Chinese medicine theory of "salt-processing enhancing drug into kidney meridian", providing an experimental basis for the selection of quality control indexes and the in-depth study of processing mechanisms and metabolic rules in vivo of Eucommiae Cortex and its salt-processed product.
ABSTRACT
Plasma metabolomics based on UHPLC-Q-TOF-MS/MS technique was developed for profiling the mechanism on attenuating hepatic fibrosis of Bupleuri Radix (BR) and Paeoniae Radix Alba (PRA) before and after vinegar-processing and compatibility, and to screen potential pharmacodynamic substances by spectrum-effect correlation method in this study. Firstly, SD rats with CCl4-induced hepatic fibrosis were used as an in vivo model. The blood and tissue samples were collected for the analyses of pharmacodynamic indexes and plasma metabolomics after six weeks’ administration of BR, vinegar-processed BR (VPBR), PRA, vinegar-processed PRA (VPPRA), BR-PRA herb-pair, and VPBR-VPPRA herb-pair. The experiment was approved by the experimental animal ethics committee from Nanjing University of Chinese Medicine (No.202103A002). The results of pharmacodynamics indicated that the levels of alanine aminotransferase (ALT, P < 0.01), aspartate aminotransferase (AST, P < 0.01), and hydroxyproline (HYP, P < 0.01) were decreased significantly, while the level of glutathione peroxidase (GSH-Px, P < 0.05) was increased obviously after administration of all treatment groups. Next, UHPLC-Q-TOF-MS/MS was performed to characterize the endogenous metabolites. A total of 20 differential endogenous metabolites related to the pathogenesis of hepatic fibrosis were identified in positive and negative ion modes, mainly involving five metabolic pathways of retinol metabolism, glycerol phospholipid metabolism, glyceride metabolism, fatty acid biosynthesis, and arachidonic acid metabolism. Meanwhile, a concept named correction rate was introduced to evaluate the back-regulation effects of all treatment groups on differential metabolites, and 10 differential metabolites were corrected by all treatment groups. The correction effects of the vinegar-processed herb groups were better than those of the crude ones, and the correction effects of the herb-pair groups were better than those of the single ones. Interestingly, the best correction effect was found in the VPBR-VPPRA herb-pair group, which further verified the efficacy improvement through vinegar-processing and compatibility. Partial least square method and VIP analysis combined with spectrum-effect correlation were applied for screening pharmacodynamic markers, and 38 ingredients with higher correlation with four classical pharmacodynamic indexes (ALT, AST, HYP, and GSH-Px) were identified as pharmacodynamic markers of the anti-hepatic fibrosis effects of BR and PRA before and after vinegar-processing and compatibility. The results of the investigation could not only lay a foundation for clarifying the pharmacodynamic materials and mechanism of vinegar-processing and compatibility of BR and PRA in the treatment of hepatic fibrosis as well as provide a theoretical basis for demonstrating the scientific connotation of processing and compatibility, but also provide a reference for further drug design and development of BR and PRA in clinic.