荆防颗粒中抑制新型冠状病毒蛋白酶3CLpro及PLpro的活性成分

目的：荆防颗粒是新型冠状病毒肺炎预防及轻症治疗的推荐用药，本文结合化学成分分析及活性检测，阐明其潜在的活性成分。方法：采用酶学方法测定荆防颗粒提取物对新型冠状病毒3-chymotrypsin-like protease (3CL^pro)、papain like protease (PL^pro)、spike protein receptor-binding domain (S-RBD)及人cyclooxygenase-2 (COX-2)的抑制活性，利用氨水引咳小鼠模型测试其止咳作用；基于liquid chromatography-mass spectrometry(LC/MS)技术对荆防颗粒进行化学成分定性、定量分析，阐明其化学组成；采用酶学实验、分子对接、定点突变等方法测定荆防颗粒中抑制3CL^pro、PL^pro的主要活性成分并阐明可能的作用机制。结果：荆防颗粒提取物对新型冠状病毒3CL^pro、PL^pro蛋白酶具有一定的抑制作用，且具有COX-2抑制活性及止...

Bioactive compounds of Jingfang Granules against SARS-CoV-2 virus proteases 3CLpro and PLpro

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.