Influenza virus entry and replication inhibited by 8‐prenylnaringenin from Citrullus lanatus var. citroides (wild watermelon)

(wild watermelon, WWM); however, the active ingredient was unknown. Here, we performed metabolomic analysis to evaluate the ingredients of WWM associated with antiviral activity. Many low-molecular weight compounds were identified, with flavonoids accounting for 35% of all the compounds in WWM juice. Prenylated flavonoids accounted for 30% of the flavonoids. Among the measurable components of phytoestrogens in WWM juice, 8-prenylnaringenin showed the highest antiviral activity. We synthesized 8-prenylnaringenin and used liquid chromatography-mass spectrometry to quantitate the active ingredient in WWM. The antiviral activities of 8-prenylnaringenin were observed against H1N1 and H3N2 influenza A subtypes and influenza B viruses. Moreover, 8-prenylnaringenin was found to inhibit virus adsorption and late-stage virus replication, suggesting that the mechanisms of action of 8-prenylnaringenin may differ from those of amantadine and oseltamivir. We confirmed that 8-prenylnaringenin strongly inhibited the viral entry of all the influenza virus strains that were examined, including those resistant to the anti-influenza drugs oseltamivir and amantadine. This result indicates that 8-prenylnaringenin may activate the host cell's defense mechanisms, rather than directly acting on the influenza virus. Since 8-prenylnaringenin did not inhibit late-stage virus replication of oseltamivir-resistant strains, 8-prenylnaringenin may interact directly with viral neuraminidase. These results are the first report on the anti-influenza virus activity of 8-prenylnaringenin. Our results highlight the potential of WWM and phytoestrogens to develop effective prophylactic and therapeutic approaches to the influenza virus.