ct was evaporated before subjected to liquid-liquid partition for 3 times. The combined EtOAc layer was evaporated to PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19663632 dryness in vacuo to afford EtOAc residue. The residue of EtOAc layer was further subjected to silica gel CC to obtain 8 fractions. In the present study, the polar fraction was selected to do further purification because it was a compound 1-abundant fraction detected by TLC and HPLC. Fr. G was purified by MPLC to obtain 7 fractions. Fr. G6 was resubjected to MPLC to furnish 3 fractions. Fr. G6-1 was finally purified by MPLC to afford compound 1. Chemicals and reagents Absolute ethanol was purchased from Merck. Chloroform and ethyl acetate were bought from RCI Labscan Limited. Methanol and acetronitrile were purchased from Tedia. CD3OD was purchased from Sigma-Aldrich. Sodium methoxide was a product of Sigma-Aldrich. The purity of all isolated compounds for bioassay was more than 95% determined by UHPLC analysis. Mild alkaline hydrolysis of compound 1 A solution of compound 1 in MeOH was treated with sodium methoxide for 48 h at room temperature. The reaction was terminated by adding formic acid after two products of pinoresinol 4-O-b-D-glucopyranoside and a methyl ester of 4hydroxy-3-methoxy benzoic acid were clearly detected by TLC analysis. The influenza viruses were propagated in the allantoic cavities of chicken eggs. The virus titers were determined by 50% tissue MedChemExpress Amezinium metilsulfate culture infectious dose assay. The concentrations of the viral stocks were 105, 105.5, 103.5, 104, 104, 106 and 105.7 TCID50/mL. MDCK and A549 cells were purchased from the ATCC. The above two cell lines were grown in 25 cm2 cell culture flask, then passaged into 96, 48 and 24 well cell culture clusters when used. MDCK and A549 were cultured in monolayer in Dulbecco’s Modified Eagle’s medium and Ham’s F12 medium, respectively, supplemented with 10% fetal bovine serum, penicillin and streptomycin, and incubated at 37uC under 5% CO2 in a humidified atmosphere. with desired concentrations or ribavirin in serum-free Minimum Essential Medium supplemented with 2 mg/ml of L-1- ethyl chloromethyl ketone -treated trypsin. After incubation for 48 h at 37uC under 5% CO2, the CPE induced by the influenza virus was measured microscopically. The concentration required for 50% inhibition of the virusinduced CPE was calculated by the Reed-Muench analysis. Each value was an average from three independent experiments. Selectivity index was calculated by the ratio of TC50/IC50. Plaque reduction assay MDCK Cells were seeded into 24-well culture plates and incubated overnight. The cells were washed with phosphate buffer saline then incubated with 
viruses diluted in serum-free MEM containing 100 U/mL of penicillin and 0.1 mg/mL of streptomycin for 2 h at 34uC at the multiplicities of infection. After viral adsorption, cell monolayer was covered with overlay medium of compound 1 and further cultured at 34uC under 5% CO2 for 48 h. Then, the overlay medium was removed, and the cell monolayer was fixed with 10% formalin, stained with 1% crystal violet, and plaques were counted. Cytotoxicity assay MDCK cells were seeded into 96-well plates at density of 26104 cells/well, and cultured to reach 90% confluence at 37uC under 5% CO2 for 24 h. The medium was replaced with that containing various concentrations of compound 1 or ribavirin and the cells were further incubated at 37uC for 48 h. The 20 mL of MTT -2,5-diphenyltetrazolium bromide) at a concentration of 5 mg/ml was added to each we