The antibodies against Atg13 and FIP200 were used according to the manufacturer’s recommendations

K. After influenza A virus infection and Celgosivir Isorhamnetin treatment, we checked the phosphorylation level of ERK in a timedependent manner after virus infection. doi:10.1371/journal.pone.0121610.g005 15 / 21 Antiviral Effect of Isorhamnetin against Influenza 3.5 4.5 Virus titer EID50/ml 108.75 108 108.5 10 Anti-influenza virus activity of the isorhamnetin against influenza A/PR/8/34 in vivo by determining the virus titer in embryonated chicken egg and in mice lung tissues that represented in EID50. P < 0.05, P <0.01. doi:10.1371/journal.pone.0121610.t002 last few decades, flavonols have been a focus of research attention due to their various and biological activities, such as antioxidant activity and anti-cancer activity. Quercetin is a flavonol with various biological effects related to human health, such as antioxidant activity, and anti-influenza virus activity. Kaempferol is another important member of the flavonol subclass with various biological properties, such as antioxidant activity, and antiinfluenza virus activity. Isorhamnetin, a flavonol aglycone found abundantly in herbal plants such as sea buckthorn and Ginkgo bilobaL.has beneficial effects in the treatment of cardiovascular diseases, antioxidant activity, and protective effects against ischemic heart PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19763407 diseases. However, there is no research report showing the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19763871 antiviral activity of isorhamnetin. To explain the biological activities of the flavonoids, structure-activity relationship is used to assess the relationship between their chemical structures and their biological activities. Various research groups have established the link between the chemical structures of the flavonoids and their biological activities. Previously, we revealed that eriodictyol has a protective effect against UV-induced apoptosis, which was attributed to the number and distribution of the hydroxyl groups on the carbon skeleton of the flavonoid. Regarding the connection between the chemical structures of the flavonoids and their inhibitory effects on influenza virus replication, previous reports showed a correlation between the flavonoids structures and their inhibitory effects on influenza virus neuraminidases. Here, our results showed that isorhamnetin has anti-influenza virus activity that is significantly stronger than that of quercetin or kaempferol. Specifically, isorhamnetin possesses a methyl group in the B ring of its carbon skeleton, emphasizing the impact of the methyl group on the biological functions of the flavonoids. Previous studies showed that methylated flavonoids are more lipophilic, and therefore these methylated flavonoids may be more readily transported through biological membranes, leading to cellular uptake better than that of the unmethylated flavonoids. The flavonoids that are methylated on the B ring and A ring are reported to confer potent anticancer activity in vitro and in vivo, and another interesting report revealed that the methylated flavonoids have high hepatic metabolic stability and high intestinal absorption in comparison with the unmethylated flavonoids. Methylation was reported to improve the anticancer potency of the flavonoids via enhancing the entry of these flavonoids into cells and inhibiting their degradation. Taken together, these results suggest that the methyl group on the B ring of the carbon skeleton of isorhamnetin could be the reason that it showed the best antiviral activity among the tested flavonoids against the influenza virus. 16 / 21 Antiviral E