The medicinal herbs produced in the Sichuan province of China, Coptis chinensis Franch., Ranunculaceae (batch no. 20170501), and Zingiber officinale Roscoe, Zingiberaceae (batch no. 20150702), were purchased from Zhejiang Chinese Medical University Decoction Pieces Co., Ltd., Hangzhou, China. The specimens were deposited in the School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, and accession numbers were assigned as follows: Coptis chinensis Franch., Ranunculaceae (no. 20190917), and Zingiber officinale Roscoe, Zingiberaceae (no. 20191209). The plants were identified by Professor Shui-li Zhang, School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, China.

“Huanglian” and “ganjiang” (3 g each) were refluxed in water (60 ml) for 2 h. After filtration, the residue was refluxed with water (48 ml) for 1 h again. Filtering and mixing the solutions, the resulting H2O-soluble extract corresponded to 0.5 g/ml, which was stored at 4 °C.

HPLC was used for quality control of the total extract. The analysis was performed in an Agilent 1260 system, including G1315D diode array detector and ChemStation chromatography workstation. Chromatographic separation was performed with an Agilent Hypersil BDS C18 column (250 mm × 4.6 mm, 5 μm) and monitored at 280 nm at 30 °C. The mobile phase was a mixture of water containing 0.3% phosphoric acid (triethylamine controlled pH 4) (A) and acetonitrile (B), and the elution method was as follows: 0–18 min (30–40% B), 18–25 min (40–57% B) with a flow rate of 1 ml/min. The injection volume was 10 μl (0.5 g/ml). Berberine hydrochloride (RT 8.18 min; purity  >98%; lot: 165230–201203) and 6-gingerol (RT 22.65 min; purity  >98%; lot: 0713–9906) were purchased from National Institutes for Food and Drug Control, China, and used as chemical markers.

Influenza A virus strain A/PR/8/34 (H1N1) and MDCK cells were kindly supplied by Virus Research Institute, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, China.

Institute Cancer Research mice (120 male individuals; 18–22 g) were supplied by the Animal Experimental Center, Zhejiang Academy of Medical Sciences, China (ZSLL-2016–191). Experiments on infected animals were carried out in Zhejiang Provincial Center for Disease Control and Prevention. Mice (120) were randomly divided into 6 groups, healthy group, infection group, oseltamivir group (23 mg/kg), H2O-soluble plant extracts high dose group (Extract-H, 18 g/kg), plant extract middle dose group (Extract-M, 9 g/kg), and plant extract low dose group (Extract-L, 4.5 g/kg), 20 mice for each. Except for the healthy group, after being anesthetized with ether, the mice in each group were intranasally infected with 50 μl of 10 LD50 virus, and viral pneumonia was induced. After 2 h, oseltamivir or plant extract was administered intragastrically for 1 week. The healthy group and the infection group were fed the same volume of water.

The mice were sacrificed on the 7th day and lungs (Cui et al. 2022), and spleens and thymus were collected (Du et al. 2020). After washing in saline and dried, the samples were weighted, and the following formulae were used for index calculation:

$$\mathrm\;\mathrm\;\left(\mathrm/\mathrm\right)=\frac\;\mathrm\;\left(\mathrm\right)}\;\left(\mathrm\right)}$$

$$\mathrm\mathrm\mathrm\mathrm\mathrm\;\mathrm\mathrm\mathrm\mathrm\mathrm\;\mathrm\mathrm\mathrm\mathrm\mathrm\mathrm\mathrm\mathrm\mathrm\mathrm\;\mathrm\mathrm\mathrm\mathrm\;\%=\frac\;\mathrm\;\mathrm\;\mathrm\;\mathrm-\mathrm\;\mathrm\;\mathrm\;\mathrm\;\mathrm\;\mathrm}\;\mathrm\;\mathrm\;\mathrm\;\mathrm\;\mathrm}\;\times\;100\%$$

Lung tissues were fixed with 10% formalin and dehydrated in gradient ethanol, permeabilized in xylene, and embedded into paraffin. The lung tissues were cut into 4-μm-thick sections, stained with hematoxylin/eosin, and observed under a microscope.

RNAs from lung tissues were extracted and amplified by RT-PCR. The primers were designed by Sangon Biotech (Shanghai) Co., Ltd. (Table S2). The relative expression level of the M1 gene (viral load of influenza virus) was calculated by the formula 2−△△Ct.

Mouse blood was collected from the mice orbit, and the serum was obtained by centrifugation at 1369.55 × g for 15 min at 4 °C. IL-2 and IL-6 levels in serum were detected by ELISA kit.

TLR3, TLR7, MyD88, RIG-I, MAVS, TRAF-3, NF-κB p65, and GAPDH primers (Table S2) were designed by JRDUN Biotechnology (Shanghai) Co. Ltd., China, and synthesized by Shanghai Generay Biotech Co. Ltd., China. Lung tissue was homogenized at 4 °C by the application of Trizol total RNA extraction protocol. Total RNA was extracted from the homogenate. Reverse transcription of RNAs was performed by the First Strand cDNA Synthesis Kit (Thermo, America). Then, the cDNA was amplified in the RT-PCR system (ABI-7300 Real-Time Detector, ABI, America). Amplification conditions were pre-denaturation at 95 °C for 2 min, 95 °C for 15 s, 55 °C for 35 s, and circulation for 40 times. The relative expression level of the target gene was calculated by the formula 2−△△Ct.

Six groups were set as the control group, the virus model group, and the positive control group (10 mmol/ml 3-methyladenine (3-MA), 2 h pretreated cells), and plant extracts high, middle, and low dose groups (3.125, 1.56, and 0.78 mg/ml) in MDCK cell. Each group was cultured for 16 h in DMEM medium with RFP-GFP-LC3B double fluorescence plasmid. Then, the cells were inoculated with 100 median tissue culture infective doses (TCID50) of the H1N1 virus for 1 h, 100 μl/well, except for the control group (Moradi et al. 2017). Plates were washed twice with PBS. Then plant extracts were added, and cells cultivated and observed after 24 h.

Tissues and cells were lysed in RIPA lysis buffer and quantified by BCA protein assay kit. After SDS-PAGE electrophoresis and semi-dry membrane transfer, 1 h blocking, TLR3 (1:500), TLR7 (1:1000), MyD88 (1:1000), RIG-I (1:1000), MAVS (1:1000), TRAF3 (1:1000), NF-κB p65 (1:1000), LC3 (1:1000), and GAPDH (1:5000) antibodies were incubated overnight, and the secondary antibodies (1:2000) were incubated in 1 h at room temperature. Immunoblotting signals were detected by ECL Western blot detection system and analyzed by Image-J software.

All statistical computations were performed on SPSS 21.0. ANOVA, and multiple comparisons were applied. The p < 0.05 were considered statistically significant. The charts were shown in Graphpad Prism 8.