2.1 Materials

Sennoside A, sennoside B, DPD, protocatechuic acid, sennoside C, and D-glucuronic acid were purchased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). Delanzomib, niclosamide, hesperidin, luteolin 7-O-glucuronide, gallic acid, delphinidin, GC376, and vitamin K3 were obtained from MedChemExpress (Monmouth Junction, USA). The chromatograms of the active ingredients delphinidin and DPD are provided in the supplementary materials.

Granules were obtained from the Department of Traditional Medicine, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine. To prepare the stock solution, 100 mg of each granule was accurately weighed and transferred into a volumetric flask. One milliliter of ultrapure water was added to the granules, followed by vigorous shaking to facilitate dissolution. The mixture was then subjected to ultrasonication for 1 to 2 h using an ultrasonic processor (frequency: 35 kHz, power: 200 W) to ensure complete dissolution. Subsequently, the mixture was centrifuged at 15,000 rpm for 10 min at 4 °C to separate the supernatant. The collected supernatant was used as the stock solution with a concentration of 100 mg/mL.

2.2 Cell culture and viability assay

HEK293T cells were sourced from ATCC (Manassas, Virginia, USA), while Vero-E6 cells, originating from African green monkey kidney epithelial tissue, were acquired from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Both cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) (Yeasen Biotechnology Co., Ltd., Shanghai, China), 100 U/mL penicillin, and 100 µg/mL streptomycin, and maintained at 37 ℃ in a humidified environment with 5% CO2.

HEK293F cells, also provided by the Cell Bank of the Chinese Academy of Sciences, were grown in OPM-293 CD05 medium (OPM Biosciences, Shanghai, China) under shaking conditions (125 rpm) at 37 ℃ with 8% CO2.

Cytotoxicity in HEK293T cells stably expressing hACE2 (hACE2-HEK293T) was assessed using the Cell Counting Kit-8 (CCK-8) from Meilunbio (Dalian, China). A 50 µL suspension of cells was plated in 96-well plates and incubated for 12 h. After pretreatment with 10 µL of a 10 × solution of the test compound at 37 ℃ for 1 h, 40 µL of fresh medium was added. The cells were incubated for another 24 h, after which the medium was refreshed and incubation continued for an additional 24 h. Following this, the medium was replaced with a basic medium containing 10% CCK-8 reagent, as per the manufacturer’s protocol.

Vero-E6 cells were seeded into 96-well plates at a density of 2 × 104 cells per well and cultured for 8 h at 37 ℃. The test compounds, diluted in DMEM with 2% FBS, were added, and the cells were incubated for 24 h. Subsequently, the medium was exchanged with DMEM containing 10% CCK-8 solution, and the cells were incubated for 2 h. The optical density (OD) at 450 nm was measured using a BioTek microplate reader (Winooski, USA).

2.3 Production of SARS-CoV-2 pseudovirus

SARS-CoV-2 pseudovirus was produced following a previously established method [29, 30]. Briefly, HEK293T cells were co-transfected with pNL4.3-Luc-R.E and an optimized SARS-CoV-2 S protein sequence provided by Prof. Lu Lu from Fudan University using the LipoFiter 3.0 transfection reagent (Hanheng Bio, Shanghai, China). The total transfection amount was 15 µg, maintaining a 1:4 ratio between the envelope and skelemin protein plasmids. Additionally, codon-optimized expression plasmids for SARS-CoV-2 Omicron (GenScript, Nanjing, China) and plasmids expressing Delta (#172320) and Gamma (#170450) spike proteins (Addgene, Watertown, USA) were similarly transfected. Six hours post-transfection, the medium was replaced with fresh DMEM. Two days later, the pseudovirus-containing supernatant was collected, filtered through a 0.45 µm membrane, and stored at − 80 ℃ in 1 mL aliquots.

2.4 Pseudovirus neutralization assay

To assess the inhibition of pseudovirus binding by the compounds, we followed a previously reported method [29, 30]. hACE2-HEK293T cells were seeded at 2.5 × 105 cells per well in 96-well plates (50 µL/well) and cultured for 24 h. Compounds were pre-diluted to 10 × concentrations, and 10 µL of each was added to the wells. After a 1-h incubation, 40 µL of pseudovirus was added, and the mixture was incubated at 37 ℃ for 24 h. Luciferase activity was measured using a luciferase assay kit (Meilunbio, Dalian, China), with luminescence detected by Cytation 5 (BioTek, Winooski, USA). IC50 values were calculated using nonlinear regression analysis in GraphPad Prism 8.0 (California, USA).

2.5 Ligand database preparation

A total of 837 active ingredients from ephedra, liquorice, rhubarb and Platycodon grandiflorum were obtained from TCMSP [31] (https://tcmsp-e.com/tcmsp.php). After removing duplicate components and checking the charge states, 810 candidate compounds were retained. The initial three-dimensional structures were used for the virtual screening.

The crystal structure of ACE2 in complex with the RBD of SARS-CoV-2 S protein (PDB code: 6M0J) was chosen as the receptor structure for virtual screening. The protein was prepared with Protein Preparation Wizard in Maestro [32], with a pH of 7.0 ± 2.0, and the water molecules and other solvent molecules were removed. The bond orders were assigned and the hydrogens were appended. Other parameters were set as the default. Then, the hydrogen bond network of the protein was optimized and the protein structure was further energy-minimized by the OPLS4 force field. Finally, chain E of 6M0J, i.e., the S-RBD region ranging from T333 to G526 was extracted, which was used as the receptor for subsequent virtual screening. The crystal structure of SARS-CoV-2 3CLpro (PDB code: 7SI9) was chosen to be the receptor for exploring the interactions between delphinidin and DPD with 3CLpro. The protein was prepared with the same procedure as the above SARS-CoV-2 S-RBD.

2.6 Docking-based virtual screening

The docking-based virtual screening was accomplished by Glide [32]. In our previous study, R403 and Y505 of S-RBD were demonstrated to play an important role in the specific recognition of ACE2 [29]. Meanwhile, Q493 and Y449 are important residues that bind to ACE2 and form two hydrogen bonds with ACE2, respectively [33]. Therefore, the four residues R403, Y449, Q493 and Y505 were taken as the grid center. The grid box was set to 30 Å × 30 Å × 30 Å. The SP [34] mode of Glide was used to dock the compound library into the S-RBD binding site. The docking scoring function was used for ranking all outputs. Finally, the top 30 docking results were retained for further analysis and processing.

For 3CLpro, the center of the grid box was defined as the centroid of the co-crystallized ligand in 7SI9 (x = − 10.525, y = 39.901, z = − 18.324), and the size of the grid box was set to be similar to the ligand. Docking was performed using standard precision (SP), and docking poses were ranked based on the docking scoring function. The pose with the best docking score was selected for further analysis.

2.7 Protein expression and purification

Plasmids encoding recombinant SARS-CoV-2 S-RBD with a His tag (His-S-RBD), generously provided by Prof. Chunhe Wang from the Shanghai Institute of Materia Medica, were transfected into HEK293F cells using PEI (Polysciences, Warrington, USA) as described previously [29, 30]. After five days of culturing, Ni–NTA resin (Smart-Lifesciences, Changzhou, China) was used to purify the His-S-RBD protein. The purified product was then analyzed by SDS-PAGE to confirm its purity and molecular weight.

2.8 Flow cytometry

A mixture of 25 µL of 2 µM His-S-RBD and 25 µL of each compound at the required concentration was preincubated for 1 h before incubating with 50 µL of Vero-E6 cells at a density of 3 × 106 for 1 h at room temperature. The cells were washed twice with PBS, followed by the addition of 100 µL CoraLite® Plus 647-conjugated anti-His-tag monoclonal antibody (Proteintech, Wuhan, China) at a 1:400 dilution and incubated at 4 °C for 30 min. Flow cytometry analysis was performed after two PBS washes (Beckman, California, USA).

2.9 Surface plasmon resonance assay (SPR)

Binding affinities between compounds and ACE2 or S-RBD were evaluated using a Biacore T200 instrument (Cytiva, UK). ACE2 or S-RBD proteins were immobilized on a CM5 chip via amine-coupling chemistry, with ACE2 at pH 4.0  and S-RBD at pH 5.5. A mixture of 50 mM NHS and 200 mM EDC was injected to activate the surface, after which 20 µg/mL ACE2 was immobilized to achieve 15,020 RU, and 30 µg/mL S-RBD reached 7193 RU. Surface blocking was performed using 1 M ethanolamine at pH 8.5. Delphinidin with 5% DMSO was injected into the flow system and analyzed for 120 s, followed by 300 s of dissociation. Glycine–HCl at pH 3.0 was used for surface regeneration between analyte injections. Data were processed using double reference subtraction and solvent corrections. Binding affinities were determined using a Langmuir 1:1 binding model in Biacore Evaluation software (Cytiva, UK).

2.10 Enzymatic inhibition assay

SARS-CoV-2 3CLpro was expressed and purified following previously established methods [35,36,37]. For enzyme inhibition assays, recombinant 3CLpro of SARS-CoV-2 and SARS-CoV (final concentration: 120 nM) was incubated with test compounds in 0.1 M PBS (pH 7.4) containing 1 mM EDTA for 30 min. The reaction was initiated with the addition of a 20 µM FRET-based peptide substrate (Dabcyl-KNSTLQSGLRKE-Edans) (GenScript, Nanjing, China), and fluorescence was measured after 20 min using a Cytation 5 plate reader (BioTek, Winooski, USA), with excitation at 340 nm and emission at 490 nm.

2.11 Host protease inhibitory activity assay

The activities of cathepsin B (CTSB), cathepsin L (CTSL), and chymotrypsin C (CTRC) were assessed following established methods [35, 36]. CTSB (Sino Biological, Beijing, China) was diluted to 100 nM in sodium acetate buffer (pH 5.5) containing EDTA and DTT, then activated for 30 min at 30 °C. After further dilution to 500 pM, CTSB was incubated with the test compounds for 30 min, followed by the addition of 5 µM Z-Phe-Arg-AMC (NJPeptide Biotech, Nanjing, China) to initiate the reaction. Fluorescence was recorded using a Cytation 5 reader at excitation/emission wavelengths of 360/460 nm.

Similarly, CTSL (Sino Biological, Beijing, China) was diluted, activated, and incubated with the test compounds before Z-Phe-Arg-AMC was added, with fluorescence measurements taken under the same conditions. For CTRC (Novoprotein, Suzhou, China), a 5 µg/mL solution was incubated with the test compounds in Tris buffer, then activated with trypsin (Sigma, Saint Louis, USA). The reaction was initiated using 100 µM Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (NJPeptide Biotech, Nanjing, China), and absorbance was measured at 405 nm.

2.12 Anti HCoV-229E activity determination in vitro2.12.1 Cell culture and determination of maximum non-toxic concentration

BEL-7402 cells were initially seeded into culture vessels at an optimal density and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO2 to ensure robust growth and complete adherence. Subsequently, the cells were treated with varying concentrations of the test compound. The cytopathic effect (CPE) was continuously monitored to determine the maximum non-toxic concentration (MNTC) of the compound for BEL-7402 cells, which is a crucial parameter for the safe conduct of subsequent experiments.

2.12.2 Viral infection and antiviral activity assay

After establishing the MNTC, the cells were infected with HCoV-229E virus. The infected cells were then treated with sample solutions diluted to concentrations below the MNTC to evaluate the antiviral activity of the sample solutions at different concentrations. The characteristic morphological changes indicative of HCoV-229E infection (CPE) were closely monitored daily under a light microscope. To quantify the extent of CPE, direct imaging and specialized image analysis software were employed. Specifically, the percentage of cells exhibiting CPE was determined by counting the affected cells under a microscope. The collected data were analyzed to calculate the proportion of cells displaying characteristic morphological changes of viral infection, thereby providing a reliable and objective basis for assessing antiviral efficacy.

2.12.3 RNA extraction and quantitative PCR

To facilitate downstream molecular analyses, total RNA was extracted from treated cells 24 h post-treatment. RNA extraction was performed using kits from Shandong Sparkjade Biotechnology Co., Ltd. (Jinan, China) according to the manufacturer's protocol. Briefly, cells were lysed in Trizol reagent, followed by phase separation with chloroform and precipitation with isopropanol. The RNA was washed with ethanol and dissolved in RNase-free water. Reverse transcription and quantitative PCR (qPCR) were performed using kits from Beyotime Biotechnology (Shanghai, China) and Abclonal (Wuhan, China) respectively. GAPDH was used as the reference gene. Primer sequences are detailed in Table 1.

Table 1 Primer information