Discovery and verification of Q-markers for promoting blood circulation and removing stasis of raw and wine-steamed Vaccaria segetalis based on pharmacological evaluation combined with chemometrics

Dried and mature seeds of Vaccaria segetalis (Neck.) Garcke ex Asch. (VS) are known for their therapeutic effects, as they stimulate blood circulation, promote menstruation and diuresis and eliminate gonorrhoea. However, due to its hard shell, the dissolution of its active ingredients is often improved by steaming and frying in clinical applications. Among the processed products, wine-steamed Vaccaria segetalis (WVS) is one of the commonly used ones. Numerous historical records have shown that wine steaming can enhance the efficacy of drugs to promote blood circulation and remove blood stasis. However, the differences in the efficacy of VS and WVS in promoting blood circulation and removing blood stasis have not been thoroughly studied, and the possible reasons for these differences have not been reported.The objective of this study was to identify quality markers (Q-markers) that could differentiate the efficacy of promoting blood circulation and removing blood stasis of VS and WVS, which could serve as a basis for the rational application of VS and WVS in clinical settings.A pharmacodynamic comparison between the water extracts of VS and WVS was carried out based on a mouse acute blood stasis model (ABS) and thrombus zebrafish model. The potential bioactive substances of WVS were screened by investigating the correlation between common peaks identified for 10 batches of WVS by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) and their rate of thrombosis inhibition in zebrafish. Furthermore, multivariate statistical analysis of chemical components between VS and WVS was conducted to speculate the Q-markers combined with the results of the bioactive components. Based on the efficacy verification of Q-markers, the content of Q-markers in 10 batches of WVS was evaluated.The results of efficacy comparison assays demonstrated that the efficacy of WVS was more prominent than VS at the same dose. Five components were screened as effective components of WVS for promoting blood circulation and removing blood stasis by correlation analysis. Furthermore, a total of 24 common ingredients were identified in VS and WVS extracts, and 9 of them showed increased dissolution rate after wine steaming, including 4 active ingredients, Hypaphorine, Vaccarin, Saponarin, and Isovitexin-2″-O-arabinoside, which were screened out by correlation analysis. The monomer test suggested that these 4 components could activate blood circulation and remove blood stasis in a dose-dependent manner. Consequently, Hypaphorine, Vaccarin, Saponarin, and Isovitexin-2″-O-arabinoside were selected as Q-markers to distinguish between VS and WVS. The content determination showed that the total contents of 4 Q-markers of WVS from 10 batches with different origins ranged from 0.478% to 0.716%.This study compared the efficacy of VS and WVS in promoting blood circulation and resolving stasis and revealed Q-markers that reflected the difference in efficacy between them for the first time, which laid the foundation for establishing quality standards for WVS.