Simultaneous separation of six pure polymethoxyflavones from sweet orange peel extract by high performance counter current chromatography

Successful isolation of polymethoxyflavones (PMFs) from citrus peels has led to numerous evaluations of PMFs in a broad spectrum of biological activities, such as inhibition of chronic inflammation, cancer prevention and anti-atherogenic properties. Recent reports associated with the health promoting properties of PMFs in citrus fruits have dramatically increased. However, the limiting factor in animal and human study of PMFs is still the supply of pure PMFs, such as tangeretin, nobiletin, sinensetin and 3,5,6,7,3′,4′-hexamethoxyflavone. Herein, we introduce the newly developed efficient separation method using high-performance counter-current chromatography (HPCCC) in isolating multiple pure single PMFs simultaneously in one cycle process. With the smallest preparation loop on the semi-preparative HPCCC instrument, the optimized solvent system of hexanes/ethyl acetate/methanol/water resulted in the isolation of pure sinensetin, tangeretin, nobiletin, 3,5,6,7,3′,4′-hexamethoxyflavone, 5,6,7,4′-tetramethoxyflavone and 3,5,6,7,8,3′,4′-heptamethoxyflavone directly from crude sweet orange peel extract in one cycle of separation process by HPCCC in the mode of reverse phase. The purity of each of the six isolated PMFs is greater than 96.6% analyzed by high-performance liquid chromatography and proton nuclear magnetic resonance. Scale-up and high purity of individual PMFs can be separated by using a large separation loop in preparative HPCCC model. The renovated HPCCC methodology can be practically used in natural product isolation and consequent biological property evaluation.