Purification and Identification of Genistein in Ginkgo biloba Leaf Extract

The Ginkgo biloba leaf extract was hydrolyzed under acidic conditions and neutralized with 5% aqueous NaOH. The hydrolysate was extracted using a mixture of ethyl acetate and tetrahydrofuran (7:3, v/v) and was concentrated by the vacuum evaporation of organic solvent. Genistein was isolated from the hydrolysate using silica gel column chromatography with a gradient elution of dichloromethane and ethyl acetate and further recrystallized in dichloromethane. The whole process was monitored using high performance liquid chromatograph, equipped with photodiode array detector and electrospray ionization mass spectrometer/mass spectrometer (HPLC-PDA-ESI-MS/MS). The HPLC chromatogram with UV at 260 nm showed that the purity of genistein was greater than 98%. The structure of genistein was identified using UV absorption curves, nuclear magnetic resonance (1H NMR, 13C NMR) spectra, ESI-MS in negative mode, and tandem MS spectra. Also, the fragmentation mechanism for genistein is discussed in detail.