Chemical Constituents From The Male Flowers Of Ginkgo Biloba L. And Their Biological Activities

Ginkgo biloba L.,also named as maidenhair tree,living fossil,is the sole species in the class Ginkgopsida.This plant is native to China and is currently cultivated worldwide for landscaping and medical purposes.The extract of G.biloba,whose primary ingredients were identified as flavonol glycosides,terpene trilactones,phenolic acids,and polyisopentenols,possesses the abilities of antioxidation,anti-inflammation,cardiac-cerebral and peripheral vascular disorders improvement,neuroprotection,antiplatelet aggregation,anti-cancer,and anti-radiation.At present,the research on ginkgo at home and abroad mainly focused on its leaves and seeds.However,no literature is available on the exact chemical constituents of G.biloba flowers or their bioactivity.To further explore the chemical composition and biological activity of G.biloba flowers,this paper firstly systematically studied the chemical composition of G.biloba flowers extract,and carried out related biological activity research based on the characteristics of the isolated compounds.In this paper,the 70%ethanol extract of dried G.biloba flowers was extracted with petroleum ether,chloroform,ethyl acetate and n-butanol respectively.AB-8macroporous adsorption resin column chromatography,silica gel column chromatography,Sephadex LH-20,reversed-phase C18 open column chromatography,preparative HPLC,and recrystallization were applied to separate and purify the extractive parts of ginkgo flowers,which yielded 81 compounds in total.We identified64 compounds,by analyzing the UV,IR,MS and 1D,2D-NMR spectra,including 2new compounds and 59 known compounds,including 9 alcoholic glycosides,ginkgoside A（1）,3-O-β-D-glucopyranosyl-（1→6）-β-D-glucopyranosyl-1-octen-3-ol（3）,hexyl-β-getiobioside（4）,benzylalcohol-O-α-L-arabinopyranosyl-（1→6）-β-D-glucopyranoside（5）,benzylβ-D-xylopyranosyl-（1→6）-β-D-glucopyranoside（6）,dendranthemoside A（8）,dihydrodendranthemoside A（9）,alangionoside A（10）,dihydroalangionoside A（11）;1 phenolic glycoside,7-O-（β-D-glucopyranosyloxy）-5-hydroxy-1（3H）-isobenzofuranone（7）;9 phenylpropanoid glycosides,trans-cinnamic acid-4-O-β-D-glucopyranoside（12）,cis-p-coumaric acid 4-O-β-D-glucopyranoside（13）,p-coumarylalcholglucoside（14）,（Z）-4-hydroxycinnamyl-4-O-β-D-glucopyranoside（15）,coniferin（16）,cis-coniferin（17）,methylconiferin（18）,piperoside（19）,stroside B（20）;4 lignans,olivil-4-O-β-D-glucopyranside（21）,dihydrodehydrodiconiferylalcohol4-O-β-D-glucopyranoside（22）,（+）cyclo-olivil-6-O-β-D-glucopyranoside（23）,（-）isolariciresinol-4-O-β-D-glucopyranoside（24）;4phenylpropanoids,3-（4-hydroxy-3-methoxyphenyl）propane-1,2-diol（25）,trans-caffeic acid（26）,esculetin（27）,p-coumaric acid（28）;2aromatic acids,4-hydroxybenzoic acid（29）,protocatechuic acid（30）;5 biflavonoids,amentoflavone 7’’-O-β-D-glucopyranoside（2）,amentoflavone（31）,sciadopitysin（32）,bilobetin（33）,isoginkgetin（34）;5 flavonoids,iorhamnetin（35）,kaempferol（36）,apigenin（37）,quercetin（38）,gnetifolin B（39）;13 flavonoid glycosides,apigenin-7-O-β-D-glucoside（40）,kaempferol-3-O-α-L-rhamnoside（41）,kaempferol-3-O-rutinoside（42）,quercetin-3-O-β-D-glucopyranoside（43）,quercetin-3-O-α-L-rhamnoside（44）,isorhamnetin-3-O-rutinoside（45）,kaempferol-7-O-β-D-glucopyranoside（46）,kaempferol-3-O-β-D-galactoside-4′-O-β-D-glucoside（47）,isorhamnetin-3-O-β-D-glucopyranoside（48）,rutin（49）,kaempferol-4’-O-β-D-glucopyranoside（50）,kaempferol-3-O-[6′′′-O-p-coumaroyl-β-D-glucopyranosyl-（1→2）-α-L-rhamnopyranoside]（51）,quercetin-3-O-[6′′′-O-p-coumaroyl-β-D-glucopyranosyl-（1→2）-α-L-rhamnopyranoside（52）;2 terpene lactones,ginkgolide B（53）,ginkgolide C（54）;4 nitrogen compounds,4,4′-dihydroxy-3,3′-imino-di-benzoic acid（55）,uracil（56）,nicotinamide（57）,argaminolic A（58）;3 other classes,capilliplactone（59）,daucosterol（60）,β-sitosterol（61）,hexadecanoic acid（62）,ginkgolic acid（63）,glycerin 1-tricosanoate（64）.Compounds 1 and 2 were novel compounds.Twenty four compounds were isolated for the first time from the class Ginkgopsida including compounds 3-15,17-24,55,58 and 59.Griess assay,MTT assay,ELISA,and Real-time PCR techniques were used to evaluate the inhibitory activities of total extract,extractive fractions of the flowers,and the isolated compounds including NO,TNF-α,IL-6,PGE₂ content,iNOS mRNA and COX-2 mRNA levels,as well as cell viability,using LPS-induced RAW 264.7 cells as a model.The ethanol extract of Ginkgo flowers,the chloroform and ethyl acetate fractions can significantly decrease NO,TNF-α,IL-6,PGE₂ production.The CHCl₃-and EtOAc-soluble fractions were demonstrated to be the anti-inflammatory active fractions of Ginkgo flowers.The results showed,at the concentration of 50μM,compounds 1,3,31-34 demonstrated varying degrees of NO inhibition.Notably,bilobetin（33）and isoginkgetin（34）showed inhibition ratios of 80.19%and 82.37%,respectively.Compounds 33 and 34 also exhibited significant dose-dependent inhibitory effects on TNF-α,IL-6,PGE₂,iNOS mRNA and COX-2 mRNA levels.MTT assay,Giemsa staining,PI method,Annexin V-FITC/PI double staining method and Western Blot were used to observe the cell viability and cell morphology of tumor cells treated with biflavonoids.The cell cycle,the proportion of apoptosis,and the expression of apoptosis-related proteins were also studied.Through the above experiments,compounds 33 and 34 were screened out as the antitumor active compounds.Their effects were found to be cell-specific and in a dose and time dependent manner for the most sensitive HeLa cells.They can significantly inhibit the proliferation of HeLa cells with IC₅₀ 14.79μM and 8.38μM,respectively.The significant morphological changes validated their anticancer effects in a dose-dependent manner.They were capable of arresting G2/M phase of cell cycle,inducing the apoptosis of HeLa cells dose-dependently by regulating the apoptosis-related proteins Bcl-2,Bax and caspase-3.The anti-radiation effects of G.biloba leaves extract and G.biloba flowers extract wre studied by using the ⁶⁰Coγ-ray irradiated C57BL/6J mouse model.The anti-radiation effect of G.biloba flowers is better than G.biloba leaves.They can increase the survival rate of irradiated mice by 36.37%and 9.09%respectively.G.biloba flowers extract can prolong the average survival time of irradiated mice by 4.18days and improve the peripheral blood level of mice after irradiation which may be the anti-radiation mechanism of G.biloba flowers.The low dose group（30mg/kg）of G.biloba flowers extract demonstrated to have better anti-radiation effect.