Screening for Q-markers of Antiviral Granules based on neuraminidase inhibitors and Effect-constituent index

Antiviral granules (AG) is a classic traditional Chinese medicine commonly employed for influenza treatment. However, there is an urgent need to improve its quality control standards. The quality markers (Q-markers), guided by the Effect-constituent Index (ECI), were screened through in vitro and in vivo component identification, molecular docking, and neuraminidase (NA) inhibition experiments. The chemical components in AG underwent analysis using UPLC-Q-Orbitrap HRMS and GC-MS, and serum pharmacochemistry was employed to analyze the components absorbed into the bloodstream. Molecular docking was used to evaluate the affinity of the prototype components to NA, and the inhibitory activity of the candidate components was later confirmed through NA inhibitor experiments. Following this, a UHPLC-MS/MS method was developed to screen and quantify the Q-markers in 20 sample batches. Lastly, the ECI was formulated using a multi-index comprehensive evaluation method that combines composition, content, and activity. A total of 246 compounds were identified in AG, and 89 prototype components and 19 metabolic components were detected in the serum of the rats. Molecular docking revealed that the binding energy of 35 candidate active ingredients was better than that of the original ligand and the target protein. Among these candidate active ingredients, 12 candidate Q-markers demonstrated significant inhibitory effects in NA inhibition experiments, with IC50 values ranging from 0.954 to 3.408 mM. A UHPLC-MS/MS content determination method was established for the quantification of 11 Q-markers, including Forsythiaside A, Pogostone, Verbascoside, Mangiferin, 5-methoxy-8-hydroxypsoralen, Curcumenol, Luteolin-7-glucuronide, Kaempferol-3-O-rutinoside, Baicalin, Rutin, and Bergaptol, in 20 batches of samples. The ECI was calculated based on activity and content of these Q-markers. Pearson's correlation analysis of the NA inhibitory effect of the samples and the ECI resulted in an R value of 0.859, indicating a good correlation. Furthermore, this correlation coefficient surpassed those observed between the content of each Q-marker and the NA inhibitory effect. The Q-markers for treating influenza with AG have been successfully screened, and the ECI of AG has been constructed. These findings will provide beneficial strategies for improving the quality control level of AG.