One-step isolation of γ-oryzanol from rice bran oil by non-aqueous hydrostatic countercurrent chromatography

The value-added gamma-oryzanol was purified in one step from crude rice bran oil (RBO) using a preparative hydrostatic countercurrent chromatography (hydrostatic CCC) method, operating in the dual mode. The fractionation was performed using a non-aqueous biphasic solvent system consisting of heptane-acetonitrile-butanol (1.8:1.4:0.7, v/v/v), leading rapidly to the target compounds. Transfer of the analytical CCC method to large-scale isolation was also carried out yielding a high quantity-high purity fraction of gamma-oryzanol. In addition, a fraction of hydroxylated triterpene alcohol ferulates (polar gamma-oryzanol) was clearly separated and obtained. Furthermore, a fast HPLC-APCI(+/-)-HRMS method was developed and applied for the identification of gamma-oryzanol as well as the polar gamma-oryzanol in RBO and the resulting fractions. The purity of gamma-oryzanol fraction was estimated as 97% based on HPLC-APCI-HRMS analysis.