Study On The Inhibition Of SARS-CoV-2 And Inflammation By The Extract Of Thesium Chinense Turcz And Its Complex

In 2021,the novel coronavirus（SARS-CoV-2）is still spreading around the world,and the prevention and control of the epidemic and related drug research and development are urgent.For the novel pneumonia（COVID-19）,there are no specific anti-coronavirus drugs,and the drugs recommended by the guidelines are still limited and ineffective.Thesium Chinense Turcz is a dried whole plant of the Santalaceae plant.There are about30 genera and more than 400 species,widely distributed.In China,35 species and 6varieties have been identified,with substantial production occurring everywhere.T.chinense is listed in the book of"Tujing Herbal Medicine".It has the functions of"clearing heat and detoxifying"and"regulating qi".The taste of T.chinense is gentle,slightly bitter,astringent and cold,so it is known as"plant antibiotic".T.chinense contains flavonoids,organic acids,alkaloids,sterols,phenols,volatile oils and other chemical components.As a kind of broad-spectrum antibacterial herbal medicine with good application prospects,the whole herb has the functions of antibacterial and anti-inflammatory,clearing heat and detoxifying,tonifying kidney and astringent essence.It has been reported that Sanghuangprous vaninii has anti-influenza A（H1N1and H3N2）and influenza B viruses.Therefore,in the experimental design,the combination of S.vaninii and T.chinense in different proportions was used to form the composite solution,which fully took into account the strong antiviral effect of T.chinense and the strong anti-inflammatory effect of S.vaninii.Objective:The purpose of this study is to study the inhibition of SARS-CoV-2 and inflammation by the extract of T.chinense and its complex,and further complement the current research on the novel coronavirus of traditional Chinese medicine.Methods:The inhibitory effects of different polar fractions（ethanol,petroleum ether,ethyl acetate,n-butanol and water extract）T.chinense and S.vaninii（ethanol and water extract）on the replication of SARS-CoV-2 were initially screened.Using African green ape kidney epithelial cells（Vero）as host cells,nucleocapsid protein（NP）expression was detected by real-time quantitative PCR.In order to further study the anti-entry effects of the ethanol extract of T.chinense and the water extract of S.vaninii on SARS-CoV-2 virus,the expression level of NP was detected by real-time quantitative PCR and Western blotting.We evaluated the inhibitory activity of different polar extracts（ethanol,petroleum ether,ethyl acetate,n-butanol,and water）of T.chinense and S.vaninii on the production of inflammatory cytokines in lipopolysaccharide（LPS）-stimulated mouse monocyte macrophage leukemia cells（RAW264.7）.The expression levels of tumor necrosis factor（TNF-α）and interleukin-6（IL-6）were detected by ELISA and PCR.Finally,the expression level of NP was detected by PCR for the inhibition of SARS-CoV-2 replication and entry ability of the complex（the ethanol extract of T.chinense and the water extract of S.vaninii in the ratio of 1:1,1:2and 2:1）.The inhibitory activity of TNF-αand IL-6 in RAW264.7 cells stimulated by LPS was determined by ELISA and PCR.Results:The ehanol extract of T.chinense inhibited the replication(EC₅₀:259.3μg/mL)and entry(EC₅₀:364.2μg/mL)of SARS-CoV-2 in Vero cells and significantly reduced the levels of IL-6 and TNF-αproduced by LPS-stimulated RAW264.7 cells.The petroleum ether(EC₅₀:163.6μg/mL),ethyl acetate(EC₅₀:22.92μg/mL)and n-butanol(EC₅₀:56.80μg/mL)extracts showed a weak inhibitory effect on the replication of SARS-CoV-2 in Vero cells,while the water extract showed no activity at all.In the anti-inflammatory effect,the petroleum ether fraction,ethyl acetate fraction and n-butanol fraction showed significant concentration dependence in the specific drug concentration range,and the expression levels of IL-6 and TNF-αinduced by LPS gradually decreased with the increase of concentration.The aqueous extract only slightly inhibited the expression of TNF-αinduced by LPS at the low concentration of160μg/mL.Ethanol extract(EC₅₀:690.3μg/mL)and water extract of S.vaninii had certain inhibitory effect on the replication of SARS-CoV-2,but they had no antiviral entry effect.All parts of S.vaninii showed good anti-inflammatory effect,and there was a significant concentration dependence with the increase of concentration,and the water extract of S.vaninii showed a very significant anti-inflammatory effect.Complex 1(T.chinense:S.vaninii=1:1,EC₅₀:500.6μg/mL),complex 2(T.chinense:S.vaninii=1:2,EC₅₀:522.9μg/mL),complex 3(T.chinense:S.vaninii=2:1,EC₅₀:410.8μg/mL)inhibited the replication of SARS-CoV-2,and the entry phase complex 1(EC₅₀.593.9μg/mL)against SARS-CoV-2 inhibited the virus better than complex 2 and complex 3.All the three compounds had extremely significant inhibitory effects on the release of cytokines TNF-αand IL-6,and showed an obvious trend.Conclusion:The ethanol extract of T.chinense and its complex 1 significantly inhibited the replication and entry of SARS-CoV-2 in Vero cells,and significantly reduced the levels of IL-6 and TNF-αproduced by RAW264.7 cells stimulated by LPS.