The samples were analyzed in negative ion mode with the linear configuration

e control treatment. Western blot analysis To prepare whole-cell extracts, cells were washed 3 times using cold PBS, scraped from the dishes, and suspended in protein extraction buffer PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19762596 containing 1% Triton X-100, 100 mM Tris-HCl, 10 mM NaCl, 10% glycerol, 1 mM sodium orthovanadate, 50 mM sodium fluoride, and 1 mM phenylmethylsulfonyl fluoride. After incubation on ice for 30 min, lysates were centrifuged and proteins in the supernatants were quantified using the Bradford Protein Assay Reagent. Protein samples were separated via 10% SDS-PAGE and transferred to nitrocellulose membranes. The ML-128 membranes were blocked using 5% nonfat dry milk and 0.1% Tween-20 in Tris-buffered saline and probed with the primary antibody. Western blotting was performed using antibodies against t-ERK, p-ERK, actin, and LC3B, as well as antimouse, anti-rabbit, and anti-goat IgG-peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence kit. In vivo antiviral assay Six-week-old female C57BL/6 mice purchased from KOATECH infected with the PR8 influenza strain. After anesthesia, mice were inoculated with 4.25 log10EID50/mL in 30 L sterile PBS via the intranasal route. Two hours post-infection, each group received the following compounds: Tamiflu capsules were dissolved in PBS at a concentration of 10mg/kg and then centrifuged at 1,000rpm/ 10minutes for removal of the filler. From this solution 200 L was administered per day orally for 5 days, or flavonoid was initially dissolved in 0.2% DMSO and then diluted in PBS to a final concentration of 1 mg/kg and less than 0.2% DMSO. This mixture mixed will by vortex and 30 L of this mixture was administered per day via the intranasal route for 5 days. One week after infection, the lungs of 3 mice per group were collected for the virology analysis. Lung tissues were excised, homogenized using a homogenizer, and centrifuged. Ten-fold serial dilutions of the supernatant from the lung homogenate samples were subjected to the virology assays for virus titer determination. Lung homogenates were injected into the allantoic sac of 10-day-old embryonated eggs, which were incubated at 37C for 48 hr, after which the allantoic fluid was harvested. The virus titer in the allantoic fluid was assessed using the HA assay. The virus titer in the fluid was calculated based on the method of Reed and Muench. For the assessments 6 / 21 Antiviral Effect of Isorhamnetin against Influenza of body weight and survival rate, mice were observed daily for 14 days and monitored for clinical signs. Statistical analysis Each experiment was repeated a minimum of 3 times, and data are presented as mean standard deviation. For the analysis of the significance of differences, we used one-way analysis of variance or the two-tailed Student’s t-test. P values equal to or less than 0.05 and 0.01 were considered statistically significant. Results The antiviral potency of the methylated flavonol isorhamnetin The main goal of this study was to identify the flavonoid compound with the lowest cytotoxicity and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19763404 the highest antiviral activity among the tested flavonoids, which were diosmetin, eriodictyol, kaempferol, isorhamnetin, and quercetin. These flavonoids possess 2 benzene rings linked by a 3-carbon chain that forms a closed pyran ring with variously distributed hydroxyl and methyl groups. We investigated the influence of flavonoids treatment on the cell viability of MDCK cells. The addition of the flavonoid compounds did not produce significant c