Bioassay-guided fractionation of Rubus suavissimus leaf extracts possessing NF-κB inhibitory activities and a separable cytotoxicity

Leaf extract of Rubus suavissimus S. Lee (Rosaceae), a perennial woody shrub indigenous to southern China, was tested for its biological activity in the regulation of transcription factor NF-kappa B (nuclear factor kappa B) A NF-kappa B luciferase reporter cell line was used in this study. The crude extract inhibited NF-kappa B at 100-1000 mu g/ml concentrations with minor cytotoxicity. This finding prompted two more rounds of fractionation toward separating the active fraction from the cytotoxic fraction. Five fractions were obtained in the first round of fractionation, of which RUS-C20 was found to be the most potent in inhibiting NF-kappa B without signs of cytotoxicity up to 1000 mu g/ml concentration. Fraction RUS-C100 showed little inhibition to NF-kappa B and apparent cytotoxicity. Further fractionation of RUS-C20, using Sephadex LH-20 chromatography, resulted in three subfractions that were distinct in chemical constituents as determined by their retention times and UV absorption spectra of each component. NF-kappa B reporter assay indicated that one of the three subfractions of RUS-C20 retained the inhibitory activity, whereas the other two subfractions were inactive. Bioassay-guided fractionation led to a successful separation of efficacy from cytotoxicity and identification of active components. This warrants further isolation for a purified single compound, which can be used as the chemical marker to standardize the extract for inhibition of NF-kappa B.