Virucidal activity of Cannabis sativa L. (hemp) root and stem extracts against Japanese encephalitis virus: role of stigmasterol.

Japanese encephalitis virus (JEV) poses a significant public health risk due to the lack of effective antiviral therapies. To identify novel antiviral agents, we evaluated the antiviral activity of ethanol extracts and organic solvent fractions derived from the roots and stems of hemp (Cannabis sativa L.). Noncytotoxic concentrations of the extracts and fractions were determined using in vitro cytotoxicity assays. At these concentrations, several fractions demonstrated potent virucidal activity, with the hexane and chloroform fractions showing the strongest effects. Post-treatment of virus-infected cells with these fractions significantly suppressed viral replication, as evidenced by reduced JEV mRNA and E protein expression. In contrast, pre-treatment or co-treatment did not yield notable antiviral effects. GC-MS analysis revealed the presence of multiple known hemp-derived compounds in the active fractions. Among them, stigmasterol exhibited strong virucidal and antiviral activity. It inhibited viral entry and growth when applied during or after infection and significantly decreased viral mRNA and E protein levels in infected cells. These findings suggest that stigmasterol contributes to the antiviral effects of hemp extracts and may be one of the active compounds responsible for inhibiting JEV replication. This study highlights the potential of hemp-derived natural products, particularly stigmasterol, as promising candidates for the development of antiviral agents against JEV infection.