Rapid separation method of polymethoxyflavones from citrus using flash chromatography

Abstract

Polymethoxyflavones (PMF’s) are a sub class of flavonoids, present in citrus species. Due to their potential use as a chemopreventive agent based on in vitro studies, a rapid reproducible method for the purification of PMF’s is critical. In the present study, a rapid separation method of PMF’s from Cleopatra mandarin ( Citrus reshni Hort. ex Tan.) and Marrs sweet orange ( Citrus sinensis L. Osbeck) using flash chromatography is developed. Dried peels were powdered and extracted using hexane in a Soxhlet extractor. The extract was concentrated, impregnated with silica gel and subjected for flash chromatography using silica gel column. A gradient solvent system of hexane and acetone was used for the separations. The separation of PMF’s were monitored at wavelengths of 254 nm, 340 nm and all wavelength scan. The separated peaks were collected as individual fractions and pooled after HPLC analysis. Four PMF’s were separated with high purity in gram level quantity. These were identified on the basis of mass spectral and NMR studies as tangeretin, nobiletin, tetramethoxyflavone and sinensitin. The developed method was tested for repeatability with low RSD (%) and also determined as robust. To the best of our knowledge, this is the first report on isolation of PMF’s from Cleopatra mandarin and Marrs sweet orange.