Antiviral Agents/pharmacology* ; COVID-19 ; Coronavirus 3C Proteases ; Humans ; Lactams ; Leucine ; Molecular Docking Simulation ; Molecular Dynamics Simulation ; Nitriles ; Proline ; Protease Inhibitors/pharmacology* ; SARS-CoV-2

OBJECTIVE: Jingfang Granules have been recommended for the prevention and treatment of corona virus disease 2019 (COVID-19). Through chemical analysis and bioactivity evaluation, this study aims to elucidate the potential effective components of Jingfang Granules. METHODS: The inhibitory acti-vities of Jingfang Granules extract against 3-chymotrypsin-like protease (3CL^pro), papain like protease (PL^pro), spike protein receptor-binding domain (S-RBD) and human cyclooxygenase-2 (COX-2) were evaluated using enzyme assay. The antitussive effects were evaluated using the classical ammonia-induced cough model. The chemical constituents of Jingfang Granules were qualitatively and quantitatively analyzed by liquid chromatography-mass spectrometry (LC/MS). The 3CL^pro and PL^pro inhibitory activities of the major compounds were determined by enzyme assay, molecular docking, and site-directed mutagenesis. RESULTS: Jingfang Granules exhibited 3CL^pro and PL^pro inhibitory activities, as well as COX-2 inhibitory and antitussive activities. By investigating the MS/MS behaviors of reference standards, a total of fifty-six compounds were characterized in Jingfang Granules. Sixteen of them were unambiguously identified by comparing with reference standards. The contents of the 16 major compounds were also determined, and their total contents were 2 498.8 μg/g. Naringin, nodakenin and neohesperidin were three dominating compounds in Jingfang Granules, and their contents were 688.8, 596.4 and 578.7 μg/g, respectively. In addition, neohesperidin and naringin exhibited PL^pro inhibitory activities, and the inhibition rates at 8 μmol/L were 53.5% and 46.1%, respectively. Prim-O-glucosylcimifugin showed significant inhibitory activities against 3CL^pro and PL^pro, and the inhibitory rates at 8 μmol/L were 76.8% and 78.2%, respectively. Molecular docking indicated that hydrogen bonds could be formed between prim-O-glucosylcimifugin and amino acid residues H163, E166, Q192, T190 of 3CL^pro (binding energy, -7.7 kcal/mol) and K157, D164, R166, E167, T301 of PL^pro(-7.3 kcal/mol), respectively. Site-directed mutagenesis indicated amino acid residue K157 was a key active site for the interaction between prim-O-glucosylcimifugin and PL^pro. CONCLUSION Prim-O-glucosylcimifugin, neohesperidin, and naringin as the major compounds from Jingfang Granules could inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus proteases 3CL^pro and PL^pro. The results are valuable for rational clinical use of Jingfang Granules.
Amino Acids ; Ammonia ; Antitussive Agents ; COVID-19 ; Chymases ; Coronavirus 3C Proteases ; Cyclooxygenase 2 ; Cyclooxygenase 2 Inhibitors ; Cysteine Endopeptidases/metabolism* ; Humans ; Molecular Docking Simulation ; Papain ; Peptide Hydrolases ; SARS-CoV-2 ; Spike Glycoprotein, Coronavirus ; Tandem Mass Spectrometry

As a representative drug for the treatment of severe community-acquired pneumonia and sepsis, Xuebijing (XBJ) injection is also one of the recommended drugs for the prevention and treatment of coronavirus disease 2019 (COVID-19), but its treatment mechanism for COVID-19 is still unclear. Therefore, this study aims to explore the potential mechanism of XBJ injection in the treatment of COVID-19 employing network pharmacology and molecular docking methods. The corresponding target genes of 45 main active ingredients in XBJ injection and COVID-19 were obtained by using multiple database retrieval and literature mining. 102 overlapping targets of them were screened as the core targets for analysis. Then built the PPI network, TCM-compound-target-disease, and disease-target-pathway networks with the help of Cytoscape 3.6.1 software. After that, utilized DAVID to perform gene ontology (GO) function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to predict the action mechanism of overlapping targets. Finally, by applying molecular docking technology, all compounds were docked with COVID-19 3 CL protease(3CLpro), spike protein (S protein), and angiotensin-converting enzyme II (ACE2). The results indicated that quercetin, luteolin, apigenin and other compounds in XBJ injection could affect TNF, MAPK1, IL6 and other overlapping targets. Meanwhile, anhydrosafflor yellow B (AHSYB), salvianolic acid B (SAB), and rutin could combine with COVID-19 crucial proteins, and then played the role of anti-inflammatory, antiviral and immune response to treat COVID-19. This study revealed the multiple active components, multiple targets, and multiple pathways of XBJ injection in the treatment of COVID-19, which provided a new perspective for the study of the mechanism of traditional Chinese medicine (TCM) in the treatment of COVID-19.
Angiotensin-Converting Enzyme 2/metabolism* ; Biological Availability ; COVID-19/virology* ; Coronavirus 3C Proteases/metabolism* ; Drugs, Chinese Herbal/therapeutic use* ; Humans ; Medicine, Chinese Traditional/methods* ; Molecular Docking Simulation/methods* ; Protein Interaction Mapping/methods* ; SARS-CoV-2/physiology* ; Signal Transduction/drug effects* ; Spike Glycoprotein, Coronavirus/metabolism*