Potential anti-dengue activity of three Faramea species (Rubiaceae) and their common active new flavanone glycoside

Dengue virus infection is a neglected disease prevalent in most tropical and subtropical areas. Currently there is no vaccine or any antiviral drug indicated for the routine treatment of dengue patients. As a part of our ongoing search for potential anti-dengue virus agents we have investigated the MeOH extracts from the leaves of three Brazilian species of the genus Faramea (Rubiaceae). The in vitro cytotoxic and anti-dengue virus serotype 2 effects were measured in human hepatocarcinoma cell lineage (HepG2). The HepG2 cells were infected at a multiplicity of infection (MOI) of 1 with DENV-2 16681. After adsorption period, the cells were cultured in alpha-MEM medium, treated with 50 µg of the samples and then incubated for 48h at 37 °C in 5% of CO₂ atm. Cell viability was assessed by MTT assay and viral replication was determined by virus titration by plaque assay using mouse fibroblast kidney cell line (BHK-21) of conditioned medium. The results were expressed as pfu/mL and normalized by the control. The extracts were non-cytotoxic and a marked reduction on viral load (ranged from 90% to 100%) was observed. Bioassay-guided fractionation of the bioactive crude extracts led to active flavonoid-rich MeOH/H₂O 9:1 fractions (reduction on viral load ranged from 77% to 100%). Sephadex LH-20 and reverse phase (C18) CC of these active fractions allowed isolation of a common bioactive new flavanone glycoside: 5-hydroxy-4'-methoxy-flavanone-7-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (reduction on viral load of 83%). Structural determination was made by 1D and 2D NMR techniques, UV, OR and CD.