Hydroxysafflor yellow A (HSYA) is the main bioactive ingredient of XBJ injection. At first, the stability of HSYA in solution and in a Xuebijing injection was investigated, then the mechanisms of the increased stability of HSYA in the XBJ injection were investigated to provide useful information on clinical safety. HSYA stability was investigated as a function of pH and temperature in aqueous solution and an XBJ injection, following the guidelines from the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Products were identified by UPLC-MS/MS. HSYA reaction followed first-order kinetics under all conditions. The half-life of HSYA in XBJ was almost 40 times longer than in aqueous solution. The activation energies of HSYA reaction in aqueous solution and XBJ were calculated to be 78.53 and 92.90 kJ∙mol⁻¹ by using Arrhenius equation. The results indicated that HSYA was more stable in XBJ than in aqueous solution. Two products were identified and the mechanism was intra-molecular nucleophilic substitution. The excellent stability of HSYA in XBJ injection partly due to the micelles formed in the injection. The study may provide clues for compatibility in TCM prescription and also provide useful information for further preparation technology research of HSYA and assessment of clinical safety of XBJ.

Introduction: Scutellariae Radix (SR), derived from the root of Scutellaria baicalensis Georgi, is a traditional Chinese medicine (TCM) for clearing heat and cooling blood. It has been used as a traditional herbal medicine and is popular as a functional food in Asian countries today.Methods: In this study, UPLC-Q-TOF-MS was first employed to identify the chemical components in the ethanol extract of SR. Then, the extraction process was optimized using star point design-response surface methodology. Fingerprints of different batches and processed products were established, and chemical markers were screened through a combination of various artificial neural network models. Finally, network pharmacology and molecular simulation techniques were utilized for verification to determine the quality markers.Results: A total of 35 chemical components in SR were identified, and the optimal extraction process was determined as follows: ultrasonic extraction with 80% methanol at a ratio of 120:1 for 70 minutes, with a soaking time of 30 minutes. Through discriminant analysis using various artificial neural network models, the samples of SR could be classified into two categories based on their growth years: Kuqin (dried roots of older plants) and Ziqin (roots of younger plants). Moreover, the samples within each category could be further clustered according to their origins. The four different processed products of SR could also be distinguished separately. Finally, through the integration of network pharmacology and molecular simulation techniques, it was determined that baicalin, baicalein, wogonin, norwogonin, norwogonin-8-O-glucuronide, skullcapflavone II, hispidulin, 8, 8"-bibaicalein, and oroxylin A-7-O-beta-D-glucuronide could serve as quality markers for SR.Discussion: The primary factors affecting the quality of SR were its growth years. The geographic origin of SR was identified as a secondary factor affecting its quality. Processing also had a significant impact on its quality. The selected quality markers have laid the foundation for the quality control of SR, and this research strategy also provides a research paradigm for improving the quality of TCM.