key: cord-0785736-16ss45th
authors: Gao, Jiapan; Ding, Yuanyuan; Wang, Yuejin; Liang, Peida; Zhang, Liyang; Liu, Rui
title: Oroxylin A is a severe acute respiratory syndrome coronavirus 2‐spiked pseudotyped virus blocker obtained from Radix Scutellariae using angiotensin‐converting enzyme II/cell membrane chromatography
date: 2021-02-15
journal: Phytother Res
DOI: 10.1002/ptr.7030
sha: 09ec8bed71cd992c39480b7f9e66e90eb9e9368a
doc_id: 785736
cord_uid: 16ss45th

The current worldwide outbreak of the coronavirus disease 2019 (COVID‐19) has been declared a public health emergency. The angiotensin‐converting enzyme II (ACE2) has been reported as the primary host‐cell receptor for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), the causative virus of COVID‐19. In this study, we screened ACE2 ligands from Radix Scutellariae and investigated its suppressive effect on SARS‐CoV‐2 spiked pseudotyped virus in vitro. HEK293T cells stably expressing ACE2 receptors (ACE2 cells) were used to provide the receptor for the ACE2/cell membrane chromatography (CMC) method used for analysis. The SARS‐CoV‐2‐spiked pseudotyped virus was used to examine the anti‐viropexis effect of the screened compounds in ACE2 cells. Molecular docking and the surface plasmon resonance (SPR) assay were used to determine the binding properties. Oroxylin A exhibited an appreciable suppressive effect against the entrance of the SARS‐CoV‐2‐spiked pseudotyped virus into ACE2 cells, which showed good binding to ACE2 as determined using SPR and CMC. Oroxylin A was shown to be a potential candidate in the treatment for COVID‐19 by virtue of its blocking the entrance of SARS‐CoV‐2 into ACE2 cells by specifically binding to the ACE2 receptor.

The current worldwide outbreak of the coronavirus disease 2019 (COVID-19) has been declared a public health emergency. The angiotensin-converting enzyme II (ACE2) has been reported as the primary host-cell receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. In this study, we screened ACE2 ligands from Radix Scutellariae and investigated its suppressive effect on SARS-CoV-2 spiked pseudotyped virus in vitro. HEK293T cells stably expressing ACE2 receptors (ACE2 cells) were used to provide the receptor for the ACE2/cell membrane chromatography (CMC) method used for analysis. The SARS-CoV-2-spiked pseudotyped virus was used to examine the anti-viropexis effect of the screened compounds in ACE2 cells. Molecular docking and the surface plasmon resonance (SPR) assay were used to determine the binding properties. Oroxylin A exhibited an appreciable suppressive effect against the entrance of the SARS-CoV-2-spiked pseudotyped virus into ACE2 cells, which showed good binding to ACE2 as determined using SPR and CMC. Oroxylin A was shown to be a potential candidate in the treatment for COVID-19 by virtue of its blocking the entrance of SARS-CoV-2 into ACE2 cells by specifically binding to the ACE2 receptor. (Huang et al., 2020) . The main symptoms of COVID-19 infection include fever, dry cough, fatigue, myalgia, and diarrhea and this condition could further lead to the severe respiratory syndrome, septic shock, and even death. Similar to other previous outbreaks of newly discovered viral infections, no conventional drugs have been proven to be effective treatment options. According to recent reports, the early use of lopinavir/ritonavir as a therapeutic drug in patients with severe acute respiratory syndrome (SARS) infections could reduce mortality and intubation rates (Jin et al., 2020) .

Ribavirin has also been widely used for SARS based on its broadspectrum antiviral activity. Studies of ribavirin combined with corticosteroids in the early SARS case series have shown that clinical improvement with this combination is still controversial (Lee et al., 2003; Ho et al., 2003) . In addition, β-interferon, glucocorticoid, and supportive treatment with Remdesivir have been reported to be effective in the management of COVID-19 (Jin et al., 2020; Holshue et al., 2020) .

Abbreviations: ACE2 cells, HEK293T cells stably expressing ACE2; ACE2, angiotensinconverting enzyme II; CCK-8, cell counting kit-8; CIB, calcium imaging buffer; CMC, cell membrane chromatography; TCM, traditional Chinese medicine.

In China, most therapies for this pandemic include traditional Chinese medicine (TCM) formulations. Recently, some published studies have shown that TCM preparations shortened the fever reduction time and the average length of hospital stay (Wang & Babina, 2020; Xiong, Wang, Su, Cho, & Xing, 2020; Zhong et al., 2020) . TCM research has provided a promising new strategy for the clinical treatment of this scourge. Radix Scutellariae, known as Huang-Qin in China, has been used as Chinese herbal medicine for more than 2,000 years (Zhao, Chen, & Martin, 2016) .

Scutellaria has been reported to be useful for treating ulcerative colitis (Liang et al., 2019) , fibrosis, and lipid peroxidation in the liver (Wu, Zhi, Lun, Deng, & Zhang, 2018) , and is also known to have antibacterial (Tsai et al., 2018) and antiviral (Kitamura et al., 1998) effects. Various natural products have been isolated from Scutellaria plants including amino acids, flavonoids, essential oils, phenylethanoids, and sterols. More than 30 types of flavones can be found in the roots including oroxylin A, baicalein, baicalin, chrysin, and wogonin (Li, Jiang, & Chen, 2004) . However, no studies have reported the anti-COVID-19 activity of Radix Scutellariae or its mechanism of action.

Cell membrane chromatography (CMC) is a typical highthroughput bioaffinity method that has been used successfully in the screening of potent compounds from TCM preparations. For example, the anti-allergic component of Saposhnikoviae Radix extracts have been screened using mas-related gene X2 (MrgX2)/CMC (Jia et al., 2019) and the antitumor components of Ligusticum wallichii extracts were screened using fibroblast growth factor receptor 4 (FGFR4)/CMC (Zhang, Ding, An, Feng, & Wang, 2015) .

In addition, Fructus Schisandrae Chinensis extracts were screened for anti-hypertensive components using isolated tissues such as the vascular smooth muscle (VSM)/CMC (Yang, Wang, Zhang, Chang, & Li, 2011) . CMC can also be used to investigate the interaction between active compounds and specific receptors and to determine the equilibrium dissociation constant, K D Lin, Lv, Fu, Jia, & Han, 2018; Lv, Fu, Shi, Yang, & Han, 2017) . Hence, this technique is suitable to screen for potential ACE2 ligands.

In this study, we used human embryonic kidney 293T cells highly expressing stable ACE2 receptors (ACE2-HEK293T cells) to screen Scutellaria plants for potential ACE2 blockers that could bind to ACE2.

Then, relevant biological methods were used to confirm the affinity and anti-COVID-19 effects of the screened compounds. 

Approximately 1 × 10 7 ACE2 cells were collected, washed with physiological saline (pH 7.4), and centrifuged at 3,000×g for 10 min at 4 C; this process was performed twice. Then, the cells were disrupted using a sonicator for 30 min on ice, homogenized using an ultrasonic disintegrator according to the set procedure for 3 min, and centrifuged at 1,000×g for 10 min at 4 C. Then, the precipitate was discarded, the supernatant was centrifuged at 12,000×g for 20 min at 4 C, and 10 ml saline was added to resuspend the pellet, followed by centrifugation at 12,000×g.

The ACE2 cell membrane pellet was resuspended with 5 ml saline, which was slowly added to a test tube containing silica gel (50 mg, activated at 105 C for 30 min) under reduced pressure and vibration, and allowed to react for 5 min. The reaction mixture was transferred to a 10-ml centrifuge tube on ice. A magnetic stirrer was used to stir the mixture of the cell-membrane preparation and silica gel at 4 C for 30 min, after which it was allowed to stand overnight to produce a uniformly wrapped ACE2 cell membrane stationary phase.

Lastly, the cell membrane stationary phase was loaded into the CMC column (10 mm × 2.0 mm i.d.).

The cell membrane stationary phase suspension was vortexed, centrifuged at 500×g for 10 min at 4 C. The supernatant was discarded and 5 ml of saline was added to the pellet, followed by vortexing. This process was repeated to remove the excess cell membranes that were not wrapped around the silica gel. Approximately 5 ml of physiological saline was added to the precipitate, followed by vortexing, and then the mixture was poured into a pre-washed column packer. The flow rate of the mobile phase (water) was set to 2.0 ml/min. When the pressure of the column was not more than 10 MPa and the column was loaded for 5 min, the cell membrane stationary phase was loaded into the column core and the packed CMC column was used for liquid chromatography.

Chromatography was performed using an Acchrom instrument (Model 56000, Huapuke Instrument, Beijing Technology Co., Ltd.). Deionized water was used as the mobile phase at a flow rate of 0.2 ml/min. The column temperature was set at 37 C, and 5 μl of the sample was used as the injection volume. A diode array detector was used for full wavelength detection.

For chromatography, the column was first equilibrated with deionized water for 1 hr. Solution B was prepared by dissolving melaleuca glycoside A in solution A at a concentration of 1 × 10 −6 M, which was then further diluted to solutions of 2.5 × 10 −8 M, 5 × 10 −8 M, 1 × 10 −7 M, 2 × 10 −7 M, 4 × 10 −7 M, 6 × 10 −7 M, and 8 × 10 −7 M. Solutions of varying concentrations were injected into the chromatographic column from low to high and when equilibrium was reached, the curve achieved a plateau phase, thereby creating a breakthrough curve.

Then, the mobile phase was then switched to 100% solution A for elution, followed by the next concentration for the adsorption and elution process. This process was continued until eight different breakthrough curves were obtained. In the CMC model, the K D was calculated by the regression curves according to Ma's method (Ma et al., 2017) .

ACE2 and HEK293T cells were cultured in DMEM high-glucose medium containing 10% serum and 1% antibiotics in an incubator at 37 C in an atmosphere of 5% CO 2 . The CCK-8 assay was used to detect the effects of oroxylin A, wogonin, scutellarin, and neobaicalein on cell viability at various concentrations and treatment durations.

The cell suspension was inoculated into a 96-well plate in a 37 C incubator overnight and then treated with oroxylin A, wogonin, scutellarin, and neobaicalein at concentrations of 0.1, 1, 10, 50, 100, 200, 300, 400, 500, 600, and 800 μM in DMEM, followed by incubation at 37 C in an incubator for 24 hr.

Cells cultured in the medium and the medium without any cells were used as the negative control and blank control groups, respectively. After treatment with the compounds, CCK-8 solution was added, followed by incubation at 37 C for 2 hr, and then the optical density (OD) of the samples was measured at a wavelength of 450 nm.

2.7 | Detection of SARS-CoV-2-spiked pseudotyped virus entry into ACE2 cells ACE2 cells (5 × 10 4 /well) were seeded into white 96-well plates in 100 μl DMEM per well and cultured at 37 C in an incubator with 5% CO 2 for 24 hr. The cells were treated with 100 μl of medium containing the corresponding concentration of the test substances for 2 hr and 5 μl of the SARS-CoV-2-spiked pseudotyped virus (Cat PSV001, Sino Biological), followed by incubation for further infection for 10 hr at 37 C in an incubator with 5% CO 2 . Then, the culture medium containing the virus was removed and replaced with 200 μl of fresh DMEM, followed by continuous incubation at 37 C for 48 hr.

The culture medium was subsequently aspirated from the 96-well plate. Then, 20 μl of the cell lysate solution from the Luciferase assay system 100 assays (Promega, E1500) was added to each well, followed by 100 μl of the luminescence solution before the detection of luciferase luminescence. The exposure time was 1 s.

ACE2 cells were seeded in a six-well plate and treated with different concentrations of oroxylin A (0, 20, 40, and 80 μM). A blank control, Annexin V-FITC single staining, and PI single staining groups were also established. The cells were cultured for 24 hr, after which the culture medium was washed out with phosphate-buffered saline (PBS) and the cells were washed once with PBS. Next, the cells were digested with trypsin and the culture medium was collected, blown gently, transferred to a centrifuge tube, and centrifuged at 500×g for 5 min.

After discarding the supernatant, the cells were resuspended in 1 ml of PBS, counted to ensure the number was not less than 10 5 , centrifuged at 500×g for 5 min, and then collected. Next, 400 μl of a 1× binding buffer was added to resuspend the cells, the suspension was centrifuged at 500×g for 5 min. Five microliters of Annexin V-FITC was added to the suspension, mixed well, and incubated for 15 min at room temperature in the dark. Then, 10 μl of PI dye solution was added, mixed well, and the mixture was incubated for 5 min in an ice bath in the dark.

The suspension was examined using flow cytometry within 30 min at excitation and emission wavelengths of 488 and 530 nm, respectively to detect FITC and >575 nm to detect PI. Normal cells showed only very low fluorescence intensity whereas apoptotic cells exhibited strong green fluorescence. Necrotic cells were doublestained with green and red fluorescence.

Cell samples were prepared using 24 mm × 24 mm coverslips, which were sterilized with 75% ethanol and coated with polylysine. Then, 2 × 10 3 ACE2 cells were seeded on the coverslips and cultured in a six-well plate, which was incubated overnight at 37 C in an incubator flushed with 5% CO 2 . Then, the cells were treated with 10, 20, or 40 μM of oroxylin A dissolved in serum-free DMEM for 24 hr. The cells were fixed with 4% paraformaldehyde for 10 min at room temperature, washed with PBS, incubated with 0.5% Triton X-100 for 5 min followed by incubation with 5% BSA for 1 hr at room temperature, and washed three times with PBS. Next, the cells were incubated with the LC3 primary antibody at 37 C for 3 hr, followed by treatment with the fluorescent secondary antibody at room temperature for 2 hr. Then, the cells were treated with TRITC-phalloidin for 30 min at room temperature and mounted with 50 μl of DAPI-containing anti-fluorescence quenching reagent. Cells were visualized using laser confocal fluorescence microscopy.

ACE2 cells were treated with 10, 20, or 40 μM oroxylin A in serumfree DMEM for 24 h, and the total protein were extracted by adding RIPA lysis buffer containing 10% protease inhibitor and a phosphatase inhibitor cocktail (Roche Diagnostics) on ice for 30 min. The insoluble protein lysate was removed by centrifuging the samples at 13,500×g

for 10 min at 4 C and the protein concentration was determined using a bicinchoninic acid protein quantification kit according to the manufacturer's instructions.

The protein in the cell lysates was denatured by boiling the samples with 5× loading sample buffer (Thermo Fisher Scientific, Inc., MA) for 5 min, and equal amounts of protein were separated using 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Shaanxi Pioneer Biotech Co., Ltd.). The separated proteins were transferred onto PVDF membranes (Hangzhou Microna Membrane Technology Co., Ltd., Hangzhou, China), which were blocked using 5% non-fat milk in Tris-buffered saline containing Tween-20 (TBST; Shaanxi Pioneer Biotech Co., Ltd.) for 2 hr at room temperature with continuous agitation.

The membranes were then incubated overnight at 4 C with anti-LC3 (1:1,000) and anti-GAPDH (1:2,000) primary antibodies. The membranes were then washed three times with TBST for 10 min each time, incubated with secondary antibodies (1:20,000) in TBST for 1 hr at 37 C, washed three times with TBST for 10 min each, and developed using an ECL kit. Image-Pro Plus 5.1 software (Media Cybernetics, Inc., Rockville, MD) was used to quantify the protein levels.

All compounds were diluted to the required concentration using CIB.

The incubation buffer contained 4 μM Fluo-3 AM and 0.1% Pluronic F-127. For imaging, cells were washed twice with CIB and imaged at excitation wavelengths of 488 nm. Unless otherwise specified, compounds were added to the well at 10 s after initial imaging, and responses were monitored for an additional 120 s over 1-s intervals.

The cells were identified as responding if [Ca 2+ ] increased by at least 50% after the substances were injected.

2.12 | Surface plasmon resonance assay ACE2 protein with 6× His tag (30 μg/ml) was fixed on the COOH sensor chip using capture-coupling and then, 10, 20, 40, and 80 μM oroxylin A was injected sequentially into the chamber with PBS running buffer.

The interaction of ACE2 with the fixed small molecules was detected using Open SPR™ (Nicoya Lifesciences, Waterloo, Canada) at 25 C. The binding and dissociation times were both 250 s, the flow rate was 20 μl/ s, and the chip was regenerated with hydrochloric acid (pH 2.0). A oneto-one diffusion corrected model was fit to the wavelength shifts corresponding to the various compound concentrations and the data were analyzed using TraceDrawer software (Yang et al., 2018) .

To investigate the interactions between the receptor and ligand, molecular docking studies were conducted using the Sulflex-Dock Mode of Sybyl-X program package (New Tripos International, St. Louis, MO). The docking model of ACE2 used in this study was based on the homology model of ACE2 reported by Dr. Michael W. Pantoliano (Towler et al., 2004) . The predicted ACE2 structure was used to dock oroxylin A and the reported ACE2 ligand MLN-4760.

The data are presented as the means ± SD and were statistically analyzed using a one-way analysis of variance. The differences were considered statistically significant at p < .05. 

CMC is a biological affinity chromatography used to demonstrate receptor-ligand binding. In this study, the ACE2/CMC method was used to screen oroxylin A isolated from Radix Scutellariae extract ( Figure 1A) .

The retention times of oroxylin A, wogonin, scutellarin, and neobaicalein were determined to be 10.90, 5.46, 4.37, and 2.97 min, respectively ( Figure 1B) , which indicated that oroxylin A was the best ACE2 ligand among the four compounds. Next, ACE2/CMC combined with the frontier analysis method was used to calculate the K D of the binding of oroxylin A to ACE2. Regression curves were drawn based on the breakthrough curves of oroxylin A and the K D value was subsequently calculated as the ratio of the intercept to the slope ( Figure 1C,D) .

A CCK-8 assay was used to detect the effects of oroxylin A, wogonin, scutellarin, and neobaicalein on ACE2 cell viability. The half-maximal inhibitory concentration (IC 50 ) values of oroxylin A, wogonin, scutellarin, and neobaicalein were determined to be 164.6, 137.6, 170.9, and 1 83.8 μM, respectively (Table 1) . Treatment with 0, 20, 40, and 80 μM of oroxylin A showed almost no apoptotic effect on ACE2 cells (Figure 2 ).

ACE2 cells were treated with 20 μM oroxylin, wogonin, scutellarin, and neobaicalein to detect their anti-invasive effects on SARS-CoV-2-spiked pseudotype virus. For this experiment, the luciferase luminescence value was defined as 1 for the control group treated with the same solvent without any antagonistic compounds, and the antiinvasive effect was expressed as a ratio to that of the control group. 0.2426 ± 0.0342, and 0.5052 ± 0.0258, respectively ( Figure 3B ). The ability of the SARS-CoV-2-spiked pseudotype virus to enter ACE2 cells was reduced significantly, indicating that oroxylin A could effectively prevent COVID-19 via its interaction with ACE2.

The autophagosome is a spherical structure. LC3, which is an important marker of autophagosomes, is known to be related to its stability.

After treatment with 10, 20, and 40 μM of oroxylin A, ACE2 cells were stained with FITC-LC3, TRITC-phalloidin, and DAPI ( Figure 4A ).

The results indicated that oroxylin A inhibited LC3 expression. LC3

consists of LC3-I and LC3-II, which are located in the cytoplasm and membrane, respectively. LC3-II is converted from LC3-I to initiate the formation and elongation of autophagosomes. Oroxylin A downregulated LC3-II expression and the ratio of LC3-II to LC3-I, indicating the inhibitory effect of LC3-II-mediated autophagy ( Figure 4B ,C).

To confirm the effects of oroxylin A on the Ca 2+ influx, ACE2-HEK293 cells were treated with 20, 40, and 80 μM of oroxylin A. HEK293T cells treated with 80 μM of oroxylin A was used as the control group. Ca 2+ influx was detected using a Ca 2+ mobilization assay with Fluo-3, AM as the Ca 2+ labeling reagent. The results suggested that Ca 2+ influx was not triggered in ACE2-expressing ( Figure 5A -C) and HEK293T cells ( Figure 5D ). (2.00 Å long), and GLU406 (2.11 Å and 1.94 Å long) of ACE2 ( Figure 6A ). Oroxylin A formed a hydrogen bond with the ASN290

(1.95 Å long) and ASP367 (2.19 Å and 2.06 Å long) of ACE2

( Figure 6B ). The ribbon model of oroxylin A and ACE2 shows that the affinity of oroxylin A (4.7562) for hydrogen bonds might be correlated with its affinity for , indicating that oroxylin A showed an appreciable binding ability to ACE2. Surface plasmon resonance (SPR) analysis confirmed that oroxylin A could bind to ACE2;

the K D of oroxylin A was calculated to be 9.72 × 10 −6 M using TraceDrawer™ ( Figure 6D ). The manifestation of COVID-19 ranges from a mild, self-limiting respiratory tract illness to severe progressive pneumonia, multiorgan failure, and death (Huang et al., 2020) . The spike (S) proteins of coronaviruses, including the coronavirus that causes SARS such as COVID-19, are associated with cellular receptors that mediate the infection of their target cells. ACE2, a carboxypeptidase that potently degrades angiotensin II to angiotensin 1-7, has been identified as a functional receptor for SARS-CoV (Li et al., 2003) and a potent receptor for the entrance of this virus into cells (Yan et al., 2020; Zhou et al., 2020) .

ACE2 is currently recognized as a target of SARS-CoV-2 (Skarstein Kolberg, 2020). In this study, an ACE2 high-expression CMC system was established to screen the potential ACE2 ligands and investigate their binding properties. Oroxylin A obtained from Radix Scutellariae had a longer retention time in the ACE2-CMC system than that of neobaicalein, scutellarin, and wogonin. Oroxylin A has been reported to have potential antiviral effects (Jin et al., 2018) ; however, its mechanism against SARS-CoV-2 has not yet been elucidated.

First, we investigated the cytotoxicity of oroxylin A, neobaicalein, scutellarin, and wogonin and found that they all showed less toxicity at lower concentrations. Therefore, follow-up in vitro studies were conducted using a low concentration range of the test compounds.

SARS-CoV-spiked pseudotyped viruses are useful virological tools because of their safety and versatility, especially for emerging and reemerging viruses (Nie et al., 2020) . The SARS-CoV-2-spiked pseudotyped virus experiment showed that oroxylin A had strong antipseudotyped virus activity, which showed a dose-dependent effect in subsequent experiments, whereas neobaicalein, scutellarin, and wogonin did not exhibit this effect.

To further investigate the autophagic and apoptotic effects of oroxylin A, we measured filamentous actin and LC3 expression using immunofluorescence and the expression of autophagy-related proteins LC3-I and LC3-II using western blotting. Autophagy plays a crucial role in immune responses, cellular metabolism, apoptosis, and cell death (Choi, Ryter, & Levine, 2013) . The expression of LC3 protein expression is an important indicator of autophagy (Klionsky et al., 2016) . The conversion of LC3-I to LC3-II plays an important role in the activation of autophagy (Chen et al., 2010) .

Our results demonstrated that oroxylin A decreased LC3mediated autophagy, which suggested that oroxylin A effectively decreased autophagy in ACE2 cells. We found that oroxylin A, at concentrations below 80 μM, had no effect on inducing apoptosis in ACE2-expressing cells. Studies have identified a close relationship between autophagy and the inflammatory response (Zhong, Sanchez-Lopez, & Karin, 2016) . Ca 2+ influx analysis using the intracellular Ca 2+ mobilization assay showed that oroxylin A had no Ca 2+ -influx effect on ACE2 expression and HEK293T cells.

Of late, molecular docking has been widely used for the identification of antiviral compounds effective against SARS-CoV-2. For example, the structure of SARS-CoV-2 3CL pro (3-chymotrypsin-like cysteine protease), which controls coronavirus replication and is essential for its life cycle, has been used to identify anti-COVID-19 components from medicinal plants (Tahir Ul Qamar, Alqahtani, Alamri, & Chen, 2020). The essential residues and ligand-receptor interactions of the peptide-like structure of the main protease inhibitors of SARS-CoV-2 have been identified using the co-crystallized structures of SARS-CoV-2 with the main protease (6Y2F and 6W63) (Pant, Singh, Ravichandiran, Murty, & Srivastava, 2020) . The modeled RNAdependent RNA polymerase (RdRp) of COVID-19 has been targeted using different anti-polymerase ingredients available commercially, which have been approved for use as antivirals (Elfiky, 2020) . In this study, molecular docking studies were used to investigate ligandprotein interactions between ACE2 and oroxylin A. The ribbon model of oroxylin A and ACE2 indicated that the affinity of oroxylin A to ACE2 might be correlated with hydrogen bonds, indicating that they showed good binding ability. SPR analysis further confirmed that oroxylin A binds to the ACE2 protein with a K D of 9.72 × 10 −6 M, which was calculated using TraceDrawer™. These results proved that the binding effect of ACE2 determined using CMC was consistent with the anti-SARS-CoV-2-spiked pseudotyped virus effect identified in ACE2-expressing cells. CMC and the SPR assay demonstrated the binding properties of oroxylin A to ACE2 from different perspectives, and both enabled a consistent conclusion to be drawn. 

The authors declare no competing financial interests.

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