SEPARATION AND PURIFICATION OF ANTIOXIDANT GLYCOGLYCEROLIPIDS FROM FRUIT OF CUCURBITA MOSCHATA BY HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY COUPLED WITH PREPARATIVE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Glyceroglycolipids, widely distributed in edible plants, possess potent antithrombotic, antiviral, antitumor, and anti-inflammatory activities. In this paper, a simple method based upon high-speed counter-current chromatography (HSCCC) coupled with preparative high-performance liquid chromatography (Pre-HPLC) was developed for the separation and purification of antioxidant glycoglycerolipids from the fruits of Cucurbita moschata (pumpkin). HSCCC separation was performed on a two-phase solvent system composed of dichloromethane-light petroleum (60-90 degrees C)-ethanol-H2O (6:2:4:4, v/v). Semi-purified peak fractions from HSCCC separation were further purified by Pre-HPLC to yield six glycoglycerolipid monomers with purities of over 98%. Their structures were elucidated by electrospray ionization mass spectrometry (ESI-MS), H-1-NMR, and C-13-NMR. Their antioxidant activities in vitro were also evaluated through 1,1-diphenyl-2-picrylhydrazyl assays. Tetrasaccharide-containing glycoglycerolipids exhibited stronger antioxidant activity compared to that of butylated hydroxytoluene, and it can be assumed to be potentially useful as a safe antioxidant in food processing industries. The results indicate that the method is simple, fast, and efficient.