Identification of anti-SARS-CoV-2 compounds from Qingwen Zhike prescription and exploration of their underlying mechanism by UPLC-Q-Exactive Orbitrap MS, high-throughput screening assays and transmission electron microscopy

Qingwen Zhike prescription (QWZK), a traditional Chinese medicine (TCM) hospital prescription developed in response to the corona virus disease 2019 (COVID-19) pandemic, has demonstrated efficacy in clinical practice. Nevertheless, its specific antiviral components and mechanisms of action remain unclear. This study screened the antiviral compounds against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Qingwen Zhike prescription and explored the underlying mechanism through chemical composition analysis, serum and lung exposure profiles analysis, high-throughput screening, and transmission electron microscopy (TEM) observation. Utilizing the UPLC-Q-Exactive Orbitrap MS system, a total of 279 components were identified from Qingwen Zhike. Among these, 49 components were detected in the serum and lungs of dosed rat, with 26 components distributed abundantly in the lungs. Subsequently, a SARS-CoV-2 pseudovirus-based assay and a main protease (M^pro) enzymatic assay were used to screen for viral entry inhibitors and M^pro inhibitors. The results showed that two alkaloids (ephedrine and pseudoephedrine) and five polymethoxy-flavonoids (3,5,6,7,8,3',4'-heptamethoxyflavone, nobiletin, isosinensetin, tangeretin, and sinensetin) exhibited potent inhibitory effects on viral invasion. Further observation by TEM indicated that these two alkaloids could dissolve the viral envelope, while these five polymethoxy-flavonoids could cause leakage of virus contents, deformation of viral envelope or decomposition of the virus. Collectively, these seven compounds may serve as key antiviral components of QWZK.