Inhibitory Effect of Baicalin on 2019-NCOV Invasion

Objective To study the the inhibitory effect of baicalin on novel Coronavirus (2019-NCOV) entry process in vitro. Methods The pseudovirus system of SARS-CoV-2 S protein constructed with luciferase reporter gene was used in the study. A luciferase kit was used to detect the changes of luciferase expression in Huh-7 cells, and then the virus inhibition curve was plotted. Results Baicalin can signi�cantly inhibited the infection rate of pseudovirus. There was no signi�cant difference in the virus inhibition curve between the baicalin&virus pre-incubation group and co-incubation at different concentrations, indicating that baicalin could not directly bind to virus, but inhibited the virus S protein mediated cell fusion process. We futher found that, the inhibition rate of baicalin to virus decreased signi�cantly in the 4h group, but had no signi�cant difference in the 0h and 2h groups at the concentration of 0.125mg/ mL, indicating that baicalin may have an inhibitory effect on virus invasion stage rather than adsorption stage, and the mediated inhibition stage occurred within 4h. Conclusion Baicalin may mediate the fusion of SARS-CoV-2 S protein with cell surface receptor and exert anti-novel coronavirus activity by playing a role in inhibitting virus invasion in non-adsorption stage.


Introduction
In December 2019, a new infectious respiratory disease was named "COVID-19(Coronavirus Disease 2019)" by the World Health Organization (WHO) was reported 1 .The causative agent causing this disease has been successfully identi ed as SARS-COV-2 2 .Until now, more than 260 million people have been diagnosed with COVID-19 of worldwide scope, resulted in more than 5.2 million deaths and posed a signi cant threats to to international health and the economy.SARS-CoV-2 includes two open reading frames ORF1a and ORF1b, each of which can be translated as two viral multiprotein pp1a and pp1ab by host ribosomes after entering the host cells.Then, polymeric protein precursors such as RNA-dependent RNA polymerase (RdRp) and helicase can be cutted by under main protease (Mpro) and Papain-like protease (PLpro), to produce multiple non-structural proteins, which are indispensable in viral transcription and replication 3 .Any blocking of key enzymes or proteins can inhibit the replication of SARS-CoV-2 and be one of the good targets to nd antiviral agents before vaccines are available.Recently, Coronavirus drug targets have been reported include structural proteins (S protein, M protein, N protein and E protein), major proteases, papain like proteases and RNA polymerases.
Baicalin is the main component isolated from Scutellaria baicalensis Georgi (Huangqin in Chinese) roots.
According to previous studies, Baicalin have the e cacy of antibacterial, antiviral, anti-in ammatory, antioxidant, anti-tumor, anti-diabetes and its complications, etc 4,5 , and It have shown antiviral activities against viral pathogens by directly killing viruses, inhibiting virus replication, regulating the expression of functional proteins in host cells and other mechanisms.Baicalin was reported that not only have signi cant an antiviral activity on in uenza virus, hepatitis virus, Coxsackie virus, respiratory syncytial virus [6][7][8][9] , but also was identi ed as the rst noncovalent, nonpeptidomimetic inhibitor of SARS-CoV-2 3CL protease(3CLpro) in the SARS-CoV-2 infected cells.Recent report indicated that three phenolic hydroxyl groups of baicalein binds with 3CLpro at Leu141/Gly143 as well as Ser144/His163 through a series of direct and indirect hydrogen bonds, x the free oxygen anion ring structure and prevent peptide substrates from accessing the active site so as to achieve the anti-virus action 10 .The result of molecular docking showed that baicalin and bromocryptostine can bind with N-terminal and C-terminal active sites in homology model of nonstructural protein (NSP) 14 protein of novel Coronavirus respectively.What's more, baicalin may play an antiviral role as an inhibitor of papain like protease in SARS-CoV-2.All of the studies above are focused on post-invasion replication phase, whether baicalin can plays an antiviral role in the adsorption stage has not been reported yet.In order to solve this problem, we used lentivirus-based SARS-CoV-2 spike pseudotyped virions containing a re y luciferase gene as a reporter to mimic SARS-CoV-2 spike-driven cell entry to explore the inhibition effect of baicalin on SARS-CoV-2 and predict the possible new target protein of baicalin.These study provides data support for further study on the antiviral role of baicalin for SARS-CoV-2.

Cell viability assay
The Huh-7 cell line was purchased from National Collection of Authenticated Cell Cultures and maintained in DMEM High sugar medium supplemented with 10% fetal bovine serum in a humid incubator with 5% CO 2 at 37°C.The standard solution of baicalin with different concentrations was prepared and Huh-7 SARS-COV-2 susceptible cells were incubated in the culture medium contains baicalin of for 24h.The cell viability was detected by MTT assay, and the non-toxic dose of baicalin was obtained.

SARS-CoV-2 spike pseudovirus infectivity assay
Pseudotyped entry representing viral infectivity was evaluated by measuring luciferase activity in cell lysates.The experiment was divided into blank control, negative control, positive control and test substances groups.In the 96-well plate, 50µL of pseudovirus solution diluted to 1.3×10 4 titer, 100µ L of Huh-7 cell culture solution at a density of 2×10 5 cells/ml and 50µl baicalin solution with different concentrations or equal volumes of complete medium were added into per ori ce respectively for test substances group or positive control group.What'more, the negative control group cells were incubated with equal test substance solution without pseudovirus solution, and the blank control group was supplemented with complete medium only.The virus inhibition curve was determined by luciferase kit after incubation for 24h.
Comparison test between pre-incubation group and coincubation group 96-well plates were laid, and besides blank control, negative control and positive control group, two test article groups were set at certain concentration according to the cell survival test results.In one test article group, baicalin solution with different concentrations was incubated with 1.3×10 4 pseudovirus solution at 37℃ for 2h, and then the mixed solution were added into Huh-7 cell suspension for infection.In the other group, baicalin and pseudovirus solution at the same concentration were added into huh-7 cell suspension at the same time.After incubation for 24h, luciferase was detected both to draw the virus inhibition curve.
Pseudovirus infectivity assay at different time points 50µL of pseudovirus solution diluted to 1.3×10 4 titer and 100µL of Huh-7 cell culture solution were added into 96-well plate, then cells were incubated at 37℃with 5% CO 2 .2h, 4h and 8h post-infection, 50µL of 0.5mg/mL baicalin solution were supplemented respectively at certain concentration according to the cell survival test results.Blank group was supplemented with complete medium.Fluorescein detection was performed after post-infection 24h to draw virus inhibition curve.

Cell culture and transfection
Huh-7 cells were cultured in high-glucose Dulbecco's modi ed Eagle's medium supplemented with 5% heat-inactivated FBS and 1% penicillin and streptomycin and grown at 37℃ in a humidi ed atmosphere containing 5% CO 2 .Cells were transfected with the indicated plasmids using Lipofectamine 3000 according to the manufacturer's speci cations.

Western blotting analysis
Cells were harvested and lysed in RIPA buffer,and protein were separated on SDS polyacrylamide gels and transferred to PVDF memebranes.The membranes were blocked with 5% BSA, then was incubated with ACE2 or Actin primary antibodies at 4℃ overnight.Membranes were washed three times with TBST, then were incubated with appropriate secondary antibodies at room temperature for 1h.Membranes were subsequently developed with ProSignal Pico ECL reagent and chemiluminescent signals were acquired using Bio-Rad ChemiDoc imager Results 1 Baicalin is an effective inhibitor of SARS-CoV-2 pseudovirus Baicalin can signi cantly inhibit the infection rate of huh-7 cell pseudovirus (Fig. 1A), and according to the result of cell viability assay, it can still produce signi cant inhibitory effect at concentration with no cytotoxicity.(conc 0.2mg/ml) (Fig. 1B).
2 Baicalin is not a virus neutralizer and play a role in the early stage of virus infection Two groups were construted with (group a) or without (group b) pre-incubation of baicalin and pseudovirus solution prior to the process that huh-7 cells were incubated with baicalin and pseudovirus solution for 24h.The inhibitory effect of baicalin on pseudovirus infection was observed at different concentrations.The results showed that there was no signi cant difference in the infection rate curves between the two groups (Fig. 2A), indicating that baicalin could not bind to virus directly.According to the results of cell survival test, we choosed 0.125mg/mL as the maximum concentration in follow-up experiment.In the study, Huh-7 cells were incubated with 0.125mg/mL baicalin at different times after infection with SARS-CoV-2 pseudovirus.We found the virus inhibition rate was signi cantly reduced in the 4h group, and there was no signi cant difference in the 0h and 2h groups (Fig. 2B,C).These results suggest that baicalin may inhibit virus entry into cell (non-adsorption), and the mediated entry into the inhibition phase occurred within 4h.
3 The role of ACE2 in the anti-invasion effect of Baicalin Since the rst step SARS-CoV-2 infection is binding of the virus to cell surface receptors such as angiotensin converting enzyme 2 (ACE2) on the airway epithelium, we induced ACE2 overexpression in Huh-7 cells using transduction with an ACE2-lentivirus construct.The protein level of ACE2 was successfully upregulated as shown in Fig. 3A.The effect of ACE2 overexpression on the increasing virus inhibition rates induced by Baicalin at different concentration was further investigated.The assay indicated that overexpression of ACE2 can increase the viral infection rate, which is represented by negative virus inhibition rates, and can inhibit the elevated inhibition rates induced by Baicalin especially at low concentration (Fig. 3B).

Discussion
SARS-COV-2 enters cells mainly through membrane fusion and endocytosis, which is completed by the interaction of S protein on the cell surface with receptors on the cell surface.Untill now, several cell receptors of coronaviruses have been identi ed, such as Amino Peptidase N (APN), Angiotensinconverting enzyme 2 (ACE2), Dipeptidyl Peptidase 4 (DPP4), etc. 11 Among them, ACE2 is the earliest con rmed cell surface receptor of SARS-COV-2 with high a nity.Studies have con rmed that the binding force of ACE2 with SARS-COV-2 virus is 10 20 times compared to SARS virus 12 .We provide an in vitro study on antiviral effect of baicalin in pseudovirus system.From the results of this study, we can conclude that baicalin signi cantly inhibited the cell invasion of the pseudovirus system, which was due to the interaction between baicalin and the cell surface receptor, rather than the direct polymerization of baicalin and the virus surface S protein.In addition, the in vitro test con rmed that the virus inhibition rate had no difference with 2h and increased at 4h with the extension of the co-incubation time of virus cells, which also supported the rst point of view of this study.Therefore, we boldly speculated that the inhibitory effect of baicalin on the invasion process of pseudovirus system found in this study might be caused by the effect of Baicalin on the binding process of S protein and ACE2.In order to con rm the hypothesis, we construct a ACE2 high expression cell lines and investigated the vital function of ACE2 in the anti-invasion effect of baicalin.Recently, some researchers have found that baicalin may have strong binding ability with ACE2 through molecular docking 13 , which may support our ndings futher.But due to laboratory security level restrictions, this study only provides a preliminary discussion and new idea for development of anti SARS-CoV-2 drug only, the speci c mechanism in live virus is required for further study .