Exploration of bioactive constituents and immunoregulatory mechanisms of a hanshi-yufei formulation for treating COVID-19

Abstract

Objective: The objective of this study was to characterize the chemical compounds of a Hanshi-Yufei formulation (HSYF; a modified formulation of a traditional Chinese medicine used for treating COVID-19) to elucidate the mechanism of action and to evaluate potential anti-inflammatory effects of HSYF. Materials and Methods: The chemical constituents of HSYF extract were characterized using UPLC-Q-TOF/MS. Subsequently, a set of TCM network pharmacology methods was applied to identify disease-associated genes and to predict target profiles and pharmacological actions associated with the constituents of HSYF. Then, the antiviral effects of HSYF on H1N1 were assessed in RAW264.7 cells using MTT assays. Expression levels of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α following infection of RAW264.7 cells with H1N1 were measured using an enzyme-linked immune sorbent assay (ELISA), and expression levels of inflammatory-related factors were detected using western blotting. Results: In total, 165 chemical constituents (including glycosides, tannins, volatile oils, amino acids, triterpenoids, polyphenols, phenylpropanoids, sesquiterpenes, alkaloids, and flavonoids, among others) were tentatively identified in HSYF. Network pharmacology demonstrated that HSYF can regulate immunomodulatory- and anti-inflammatory-related targets of multiple pathways through its active ingredients, suggesting potential anti-COVID-19 effects. Furthermore, cell viability assays and ELISA showed that HSYF significantly inhibited H1N1 replication in RAW64.7 cells and markedly reduced expression of pro-inflammatory cytokines TNF-α and IL-6 at the proteins level. Conclusions: The results of the present study help improve our understanding of the therapeutic effects of HSYF in COVID-19 treatment from multi-level perspectives.