Isolation and purification of flavonoids from Ginkgo biloba pollen and their DPPH scavenging activities

Abstract: Ginkgo biloba pollens are pollens from the male Ginkgo biloba trees, and contain many active components represented by flavonoids, terpene lactones, and polysaccharides. The total flavonoid content of Ginkgo biloba pollens was approximately 21.4 mg/g, much higher than that of Ginkgo biloba leaves. Previous analyses of flavonoids in Ginkgo biloba pollens were based on the results from Ginkgo biloba leaves, and the flavonoids of pollens and leaves were found to be very different in type and content. For example, rutin was reported to be abundant in Ginkgo biloba leaves, while the rutin content in Ginkgo biloba pollens was less than 6% of that in Ginkgo biloba leaves. Now the research on chemical constituents of Ginkgo biloba pollens was still in its infancy. Thus, studying the chemical composition of Ginkgo biloba pollens by indirectly measuring the flavonoids in Ginkgo biloba leaves was meaningless, and it was necessary to systematically analyze and purify the flavonoids from Ginkgo biloba pollens. In this study, crude extract of total flavonoids from Ginkgo biloba pollens was obtained by microwave-assisted extraction, and was separated into 4 fractions, petroleum ether, ethyl acetate, n-butanol and water by a fractional extraction method. The extract fraction used for further isolation and purification was selected through an assay of 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activities and an analysis of flavonoid components. The target flavonoids with DPPH scavenging activities in the selected fraction were screened by a 1,1-diphenyl-2-picrylhydrazyl - high performance liquid chromatography - photodiode array detection (DPPH-HPLC-PAD) method, then were isolated and purified by a preparative liquid chromatography (preparative LC), and their chemical structures were identified using ultraviolet-visible spectroscopy, mass spectrometry, hydrogen-1 nuclear magnetic resonance (1H NMR) spectroscopy and carbon-13 nuclear magnetic resonance (13C NMR) spectroscopy. In addition, the DPPH scavenging activities of these components were preliminarily determined. The DPPH scavenging activities of the crude extract as well as the petroleum ether, ethyl acetate, n-butanol and water fractions analyzed by IC50 values were significantly different (P<0.01). The components with DPPH scavenging activities were efficiently enriched in the ethyl acetate and n-butanol fractions. All in all, the ethyl acetate fraction demonstrated the highest DPPH scavenging activity and accumulated most of the target components for purification, so the ethyl acetate fraction was selected for further screening, isolation and purification of the DPPH-scavenging flavonoids. Finally, 6 flavonoid components with DPPH scavenging activities were respectively purified and identified as kaempferol-3,4'- di-O-β-D-glucopyranoside (Compound 1), kaempferol-3-O-β-D-glucopyranoside-7-O-α-L-rhamnoside (Compound 2), kaempferol-3-O-β-D-glucopyranoside (Compound 3), kaempferol-3-O-α-L-rhamnopyranoside (Compound 4), naringenin (Compound 5) and kaempferol (Compound 6), of which Compounds 1 and 2 had not been previously purified from Ginkgo biloba L.. The IC50 values of the 6 compounds from low to high were as follows: Compound 6 < Compound 2 < Compound 3 < Compound 4 < Compound 1 < Compound 5. It meant that the DPPH scavenging ability of kaempferol is the highest among the 6 compounds, followed by kaempferol-3-O-β-D-glucopyranoside- 7-O-α-L-rhamnoside, and no significant differences (P>0.05) were identified between Compound 6, L-ascorbic acid and rutin. This research lays a solid foundation for further study of the functional components of Ginkgo biloba pollens and their biological activities.