Purification of Curcumin, Demethoxycurcumin, and Bisdemethoxycurcumin by High-Speed Countercurrent Chromatography

Curcuminoids are substances of great interest because of their important pharmacological activities, particularly anti-inflammatory, anticarcinogenic, and anti-Alzheimer's activities. In this study, we report the first procedure and effect of processing for the high, efficient, and useful purification of curcumin, demethoxycurcumin, and bisdemethoxycurcumin from turmeric powder. Purification involves highspeed countercurrent chromatographic (HSCCC) separation of these curcuminoids using a simple two-phase solvent system composed of n-hexane/chloroform/methanol/water (5/10/7.5/2.5, v/v). The HSCCC-fractionated effluent peaks indicated that the peak resolutions were 1.7 between curcumin and demethoxycurcumin and 2.1 between demethoxycurcumin and bisdemethoxycurcumin for 25 mg of loaded turmeric powder. These purified substances were analyzed by liquid chromatography-tandem mass spectrometry with scan and daughter scan negative modes, and the wide absorbance from 200 to 500 nm was monitored by photodiode array detection. The separation yielded 1.1 mg of curcumin, 0.6 mg of demethoxycurcumin, and 0.9 mg of bisdemethoxycurcumin (> 98% purity). Moreover, the antioxidant effect of curcuminoids was measured by a 1,1-diphenyl-2-picrylhydrazil assay. The order of antioxidant activity was purified curcumin > purified demethoxycurcumin > purified bisdemethoxycurcumin > turmeric powder. Curcumin, demethoxycurcumin, and bisdemethoxycurcumin can be used for various evaluations of their pharmacological activities.