L. pterodonta (10 kg) was collected manually during October 2015 in Yunnan (China) and subsequently stored in a dry, ventilated environment. The herbarium specimen was authenticated by Professor Rongping Zhang (Kunming Medical University, Kunming, China) and deposited in the College of Pharmaceutical Sciences (Kunming Medical University).

Madin-Darby canine kidney (MDCK) and A549 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were grown in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% heat-inactivated fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.). A/PR/8/34 was purchased from ATCC. A/Guangzhou/GIRD/07/09 (H1N1), A/Guangzhou/GIRD/02/09 (H1N1) and influenza B virus were isolated from routine clinical throat swab specimens of infected patients treated in the First Affiliated Hospital of Guangzhou Medical University (Guangzhou, China). Several strains of avian influenza virus, including A/Duck/Guangdong/2009 (H6N2), A/Duck/Guangdong/1994 (H7N3) and A/Chicken/Guangdong/1996 (H9N2); were provided by Dr Jianxin Chen (South China Agricultural University, Guangzhou, China) and subsequently stored in State Key Laboratory of Respiratory Disease, Guangzhou Medical University. The influenza viruses were grown in the allantoic cavity of embryonated chicken eggs for 48 h at 35°C, followed by 12 h at 4°C (9). Following this, the harvested viruses were preserved at 80°C prior to further experimentation.

Ultra-high-performance liquid chromatography/quadrupole-time of flight-mass spectrometry (UHPLC/Q-TOF-MS) was performed using Agilent 1290 UHPLC (Agilent Technologies, Inc., Santa Clara, CA, USA) and Bruker maXis impact Q-TOF-MS (Bruker Corporation, Billerica, MA, USA) systems. Column chromatography (CC) was performed using silica gel (200–300 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Thin layer chromatography was performed on pre-coated silica gel GF₂₅₄ plates. Spots were visualized under UV light (254 or 356 nm) or using iodine fuming. All solvents used were of analytical grade and were purchased from Guangzhou Chemical Reagents Factory. HPLC grade acetonitrile was purchased from Merck KGaA (Darmstadt, Germany). HPLC grade methanol was purchased from RCI Labscan, Ltd. (Bangkok, Thailand). HPLC grade formic acid was purchased from Merck KGaA (Darmstadt, Germany).

Pterodontic acid and pterodondiol were isolated from L. pterodonta in the laboratory, and were identified by MS and nuclear magnetic resonance spectroscopy analysis (purity, >98%). Pterodontic acid (1.25 mg) and pterodondiol (0.99 mg) were accurately weighed and dissolved in 1 ml methanol to give individual stock solutions. Pterodontic acid stock solution was subsequently diluted to 6.25 µg/ml and pterodondiol stock solution 4.95 µg/ml. All solutions were stored at 4°C prior to UHPLC-Q-TOF-MS analysis.

Powdered plant material (1 kg) was extracted with methanol by percolation, followed by the collection and vacuum concentration of 40 l eluate to yield 135 g methanol extract. The extract was suspended in H₂O (800 ml) and subjected to liquid-liquid partition by the addition of petroleum ether. The residue (48 g) of the petroleum ether layer was subjected to silica gel CC (petroleum ether-ethyl acetate, 10:1) to obtain the C8 fraction (38 g) (10).

C8 (0.01 g) was accurately weighed into a 5-ml volumetric flask and dissolved in methanol, and additional methanol was subsequently added to give a final volume of 5 ml. The sample solution was filtered through a 0.22-µm polytetrafluoroethylene filter and diluted 20 times for UHPLC/Q-TOF-MS analysis.

UHPLC was performed with the Agilent 1290 ultra-high performance liquid chromatography system (Agilent Technologies, Inc.). The chromatography was performed on an Agilent Poroshell 120 EC-C18 column (150×3.0 mm, 2.7 µm; Agilent Technologies, Inc.). The mobile phase consisted of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The following gradient elution procedure was used: 0–8 min, 30–70% B; 8–13 min, 70–100% B; 13–16 min, 100% B. The flow rate was 0.35 ml/min, the injection volume was 2 µl, and the column temperature was maintained at 30°C. Eluted compounds were detected with an Agilent diode array detector (Agilent Technologies, Inc.) over a wavelength range of 200–400 nm.

Mass spectrometry was performed on a Q-TOF-MS with an electrospray ionization interface (Bruker Corporation) operating in the positive mode. The Q-TOF-MS source parameters were as follows: End plate offset, −500 V; capillary voltage, 4,000 V; collision energy, 7 eV; nebulizing gas (N₂) pressure, 2.0 bar; drying gas (N₂) flow rate, 8.0 l/min; drying gas temperature, 200°C; and mass range, m/z 100–1,300.

The 50% toxic concentration (TC₅₀) of C8 was determined. MDCK cells (2×10⁴ cells/well) were seeded into 96-well plate for 24 h at 37°C and subsequently washed with PBS. The cells were treated with the indicated amounts of C8 (0, 12.5, 25, 50, 100 and 200 µg/ml) and cultured at 37°C for 48 h. The cytotoxicity of the C8 was measured with an MTT assay, as previously described (10). The TC₅₀ was calculated using the Reed-Muench method (11).

MDCK cells (1.0×10⁴ cells/well) were seeded in 96-well plates and grown to 90% confluence at 37°C for 24 h. To clearly observe the anti-influenza activity of C8, MDCK cells were washed with PBS and infected with 100 median tissue culture infective dose (TCID₅₀) of A/PR/8/34 (H1N1) at 37°C for 2 h. Following medium removal, different concentrations of C8 (two-fold dilution) in serum-free Minimum Essential Medium (MEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 2 µg/ml tosyl phenylalanyl chloromethyl ketone-trypsin. Following incubation for 48 h at 37°C, the cytopathogenic efficiency (CPE) of the influenza virus was measured microscopically using a MI12 inverted phase contrast microscope (Micro-shot Technology Limited, Guangzhou, China; magnification, ×200). The concentration required for 50% inhibition of the virus-induced CPE (half-maximal inhibitory concentration; IC₅₀) was calculated by the Reed-Muench method (11). The selection index was calculated by the ratio of TC₅₀/IC₅₀ (12).

A549 cells (2×10⁴ cells/well) were cultured at 37°C under 5% CO₂ for 24 h. Following this, cells were washed with PBS and subsequently incubated with A/PR/8/34 virus [multiplicity of infection (MOI)=0.1], diluted in PBS, for 30 min at 37°C. Following this, the inoculums were discarded and cells were incubated with MEM in the absence and presence of different concentrations (100 and 150 µg/ml) of C8 for 24 h at 37°C. Cell lysis and western blot analysis was performed as previously described (13). Cells were lysed on ice for 10 min with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with a phosphatase inhibitor cocktail (Beyotime Institute of Biotechnology). Protein concentration was determined with the bicinchoninic protein assay kit. Proteins (30 ng/lane) were separated using 10% SDS-PAGE and subsequently electrotransferred onto polyvinylidene fluoride (PVDF) membranes. The PVDF membrane was blocked with 5% bovine serum albumin (BSA; 9048-46-8; GBCBIO Technologies Inc., Guangzhou, China)/TBS-Tween 20 for 1 h at room temperature prior to incubation at 4°C overnight with antibodies against Toll-like receptor 7 (TLR7; cat. no. 2633), myeloid differentiation primary response protein 88 (MyD88; cat. no. 4283), tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6; cat. no. 8028), phosphorylated-p65 (cat. no. 3033), p65 (cat. no. 8242) and GAPDH (cat. no. 2118); all at a dilution of 1:1,000 and purchased from CST Biological Reagents Co., Ltd. (Shanghai, China). Following this, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (cat. no. 7074; 1:5,000; CST Biological Reagents Co., Ltd.) for 60 min at room temperature. The complexes were detected using a western lighting chemiluminescence system (Thermo Fisher Scientific, Inc.).

A549 cells were seeded into 48-well plates at 37°C with 5% CO₂. When the cell culture reached 50–70% confluence, cell were incubated with /PR/8/34 (H1N1; MOI=5) virus or TNF-α (20 ng/ml; 300-01A; PeproTech China, Suzhou, China) for 2 h at 37°C. The supernatant was subsequently aspirated, cells were washed twice with PBS and C8 (75, 100 and 150 µg/ml) was added to wells. After 9 h, cells were washed three times with PBS and fixed with 4% paraformaldehyde in PBS for 15 min at 4°C. Cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min at room temperature and blocked with 3% BSA in PBS for 30 min at 37°C, followed by incubation with anti-p65 antibody (1:50; cat. no. 8242; CST Biological Reagents Co., Ltd.) overnight at 4°C. Following a further wash, cells were incubated with fluorescein isothiocyanate-conjugated secondary antibody (1:100; cat. no. SA00003; ProteinTech Group, Inc., Chicago, IL, USA) at 37°C for 1 h. The nuclei were stained with DAPI (5 µg/ml; cat. no. 10236276001; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 30 sec at room temperature, and fluorescence was visualized using a Zeiss Axiovert 135 fluorescence microscope (Zeiss AG, Oberkochen, Germany; magnification, ×400) (14).

A549 cells were cultured in 96-well plates at 37°C with 5% CO₂ for 24 h, and subsequently infected with A/PR/8/34 virus (MOI=0.1) for 2 h at 37°C. The inoculums were discarded and the cells were treated with various concentrations of C8 (100 and 200 µg/ml) for 24 h at 37°C. Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA (1 µg) was reverse transcribed into cDNA using the Prime-Script RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China) at 50°C for 30 min. qPCR was performed using an ABI7500 Real-time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the following thermocycling conditions: 95°C for 30 sec, followed by 35 cycles of 95°C for 5 sec and 60°C for 40 sec (15). Relative gene expression levels of C-C motif chemokine ligand 2 (MCP-1), interleukin (IL)-1β, IL-6, IL-8 and GAPDH were calculated using the 2^−∆∆Cq method (16). The RT-qPCR primers and probes for analyses are listed in Table I.

A549 cells (1×10⁵ cells/well) were grown in 6-well plates at 37°C with 5% CO₂ for 24 h and subsequently washed with PBS twice. A/PR/8/34 virus (MOI=0.1) was incubated with the cells for 2 h, followed by treatment with various concentrations of C8 (100 and 150 µg/ml) at 37°C for 24 h. The supernatants were collected after 24 h treatment and centrifuged at 16,000 × g at 4°C to remove cell debris. IL-6, IL-8, TNF-α, C-X-C motif chemokine 10 (IP-10), MCP-1 and C-C motif chemokine 5 (RANTES) were detected using the Bio-Plex liquid phase chips kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with the Bio-Plex 200 system (Bio-Rad Laboratories, Inc.) (17).

Statistical analyses were performed using SPSS 18.0 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance followed by Fisher's Least Significant Difference post-hoc test was used to calculate statistical significance. Data are presented as the mean ± standard deviation. Experiments were performed in triplicate. P<0.05 was considered to indicate a statistically significant difference.

The presence of pterodontic acid and pterodondiol in C8 was determined by UHPLC/Q-TOF-MS in the positive ion mode. Base peak chromatograms of sample C8 are presented in Fig. 1. Extraction ion chromatograms of the two standards and C8 are presented in Fig. 2. The results (Table II) obtained from UHPLC-Q-TOF-MS analysis determined that the pterodontic acid and pterodondiol content in C8 was 107.98 and 111.3 mg/g, respectively. These results provided essential data required for the identification and quality control of C8 obtained from L. pterodonta.

C8 was examined for its cytotoxic ability in confluent MDCK cell cultures. No significant cytotoxic effects were observed at >200 µg/ml. To evaluate the anti-influenza activity of C8, MDCK cells were infected with influenza virus (100 TCID₅₀) and C8 was added at increasing concentrations. Following treatment for 48 h, the antiviral effect of C8 was evaluated. C8 exhibited an antiviral effect on a number of influenza virus strains, with IC₅₀ values of 19.9–91.4 µg/ml (Table III).

TLR7 recognizes influenza single-stranded RNA and subsequently combines with the adapter protein MyD88, which induces the phosphorylation of IL-1 receptor-associated kinase 1 (IRAK) through IRAK4. Following this, IRAK1 interacts with TRAF6, which is able to activate the NF-κB signaling pathway (18,19). Western blot analysis indicated that C8 inhibited TLR7, MyD88, TRAF6 and p-p65 phosphorylation levels at 100 and 150 µg/ml (Fig. 3).

p65-p50 constitutes the typical NF-κB inhibitor (IκB) proteins, the p50 and p65 complex translocates to the nucleus to promote the inflammatory response (20,21). The results revealed that C8 inhibited p65 nuclear translocation induced by TNF-α (Fig. 4) and influenza virus (Fig. 5).

The effects of C8 on inducing cytokine production were determined. The results revealed that the IL-1β, IL-6, IL-8 and MCP-1 mRNA expression was significantly reduced in C8-treated cells after 24 h (P<0.05; Fig 6). The results of the Bio-Plex assay demonstrated that the protein expression of IL-6, IL-8, TNF-α, IP-10, MCP-1 and RANTES was inhibited (P<0.001; Fig. 7).