Study On The Chemical Constituents Of Ginkgo Biloba Leaf And Branch

Ginkgo biloba L.is a perennial deciduous tree of the genus Ginkgo in the family Ginkgo.It has a history of nearly 200 million years and is called "living fossil".The main chemical components of Ginkgo biloba include flavonoids,terpene lactones,phenolic acids,polyisoprenols,etc.,which have the characteristics of anti-ischemia,anti-oxidation,anti-convulsant and anti-platelet activating factor activities,as well as free radical scavenging,improving blood flow and protecting blood vessels.This study further explored the chemical components of Ginkgo biloba leaf and branch,and conducted qualitative and quantitative analysis of the chemical components of Ginkgo biloba leaves in different varieties.In addition,the anti-PAF-induced platelet aggregation activity of ginkgolide,the main component of ginkgo biloba,was evaluated.The main research methods,contents and results are as follows:1.In this paper,a new method for rapid identification and separation of ginkgolide compounds was adopted.Firstly,an analytical strategy based on characteristic ions and neutral lost ions was developed,which can be used to comprehensively analyze compounds in Ginkgo biloba leaf that have a similar ginkgolide substructure.Secondly,the fractions of ginkgo biloba extract separated by neutral alumina and silica gel columns were detected using UHPLC-QTOF-HR-MS/MS,and new ginkgolides were identified through characteristic ions and neutral loss.Then,the new ginkgolide was targeted and isolated.Finally,new and known ginkgolides were identified using spectral data,physicochemical properties of the compounds,and X-ray single crystal diffraction crystallography.Using the above method,a total of 41 ginkgolides were first discovered in the separated fraction,including 30 potentially new ginkgolides and 11 known ginkgolides.Subsequently,the fraction was targeted and separated by silica gel column chromatography,ODS column chromatography,Sephadex LH-20 column chromatography,and semi preparative HPLC,and a total of 18 ginkgolides were obtained and identified,they are 10 new ginkgolides(compounds 12,13,18,19,21,26,27,6,9 and 20)and 8 known ginkgolides(ginkgolide A,ginkgolide B,ginkgolide C,ginkgolide J,ginkgolide P,ginkgolide Q,ginkgolide K,bilobalide).While separating ginkgolide compounds,40 non-ginkgolide compounds were isolated,and 37 of them were identified by spectral data and chemical and physical properties analysis.They were:Amentoflavone(L1),Sciadopitysin(L2),Apigenin(L3),Epicycloolivil(L4),Lasiandrans A(L5),Lasiandrans B(L6),Dibutyl phthalate(L7),Di(2-ethylhexyl)phthalate(L8),Isorhamnetin(L9),Quercetin(L10),Hydroquinone(L11),Rutin(L12),Kaempferol(L13),Isorhamnetin-3-O-glucose(L14),Hovetrichoside C(L15),5-hydroxymethylfurfural(L16),Ginkgoside B(L17),Idaeusinol A(L18),Ginkgoside A(L19),(7S,8R)-Dihydro-3-hydroxy-8-hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1-benzofuran propanol(L20),Ethyl-βrutinosides(L21),Trans-3-(4’-hydroxyphenyl)acryloyl ethyl ester[(E)-3-(4hydroxyphenyl)acrylic acid ethyl ester](L22),Dihydroconiferol(L23),pHydroxyacetophenone(L24),(+)-Cycloolivil(L25),(+)-Epinoresinol(L26),(-)Epinoresinol(L27),3-(4-hydroxy-3-methoxyphenyl)propane 1,2-diol(L28),pHydroxycinnamic acid(L29),N-(2-hydroxyphenyl)-acetamide(L30),(-)-Eugenol(L31),(-)-Eugenol(L32),(+)-Eugenol(L33),3-hydroxy-4-methoxyphenylethanol(L34),p-Hydroxybenzaldehyde(L35),Caffeic acid(L36),Ferulic acid(L37).Among the above compounds,there are 8 flavonoids,13 lignin compounds,1 furanic acid compound,1 glycoside compound,3 alkaloids,6 phenylpropanoid compounds,and 5 other compounds.Among them,compounds L4,L5,L6,L7,L8,L11,L15,L16,L18,L20,L21-L37 are isolated from Ginkgo biloba leaf for the first time.2.The total extract of Ginkgo biloba branch was separated by macroporous resin chromatography,silica gel column chromatography,ODS column chromatography,Sephadex LH-20 column chromatography and semi-preparative HPLC.A total of 54 compounds were obtained.Through the spectral data and physicochemical property analysis of the compounds,51 compounds were identified,among which 21 compounds were repeated with Ginkgo biloba leaf,and 30 compounds were not repeated,respectively:Apigenin-7-O-β-D-glucuronide(Y1),Apigenin 7-O-glucuronic acid(Y2),4,5-O-dicaffeoyl quinic acid(Y3),3-O-methylsoamericanic acid A(Y4),cis-3-[2-[1-(3,4-dihydroxy-phenyl)-1-hydroxymethyl]-1,3-ben-zodioxol-5-yl]-(E)-2propenoic acid(Y5),Gallic acid(Y6),Rosmarinic acid(Y7),(Y8),(Y9),Ginkgetin(Y10),Isoginkgetin(Y11),Genkwanin(Y12),Ginkgolic acid(C15:1,Y13),Bisbenzopyran(Y14),Kerinol(Y15),Evofolin B(Y16),Cerberalingans L(Y17),Argignan F(Y18),Luteolin-7-O-β-D-glucuronide(Y19),Catechin(Y20),Kaempferol 3-O-rutin(Y21),Protocatechuic acid(Y22),Protocatechuic acid(Y23),Benzoic acid(Y24),Vanillic acid(Y25),Isovanillic acid(Y26),Kaempferol-3-rhamnoside(Y27),Bilobetin(Y28),Indol-3-ylacetylaspartic acid(Y29),Stearic acid(Y30).Among the above compounds,there are 2 ginkgolides,10 flavonoids,9 phenolic acids,5 lignin,1 alkaloid and 3 other compounds.Among them,compounds Y8 and Y9 are new ginkgolides,and compounds Y3,Y4,Y5,Y6,Y7,Y14,Y15,Y16,Y17,Y18,Y19,Y22,Y23,Y24,Y25,Y26 and Y29 are isolated from the branch of Ginkgo biloba for the first time.3.In order to study the difference of chemical components of different varieties of Ginkgo biloba leaf,this paper firstly analyzes the chemical components of Ginkgo biloba leaf based on UHPLC-QTOF-MS/MS combined with diagnostic ion and neutral loss strategy,and then analyzes the different varieties of Ginkgo biloba leaf based on the quantitative results of various components combined with statistical analysis methods.The specific steps of this method are as follows:(a)DPI spectral simplification and NL filtering;(b)The compounds in Ginkgo biloba leaf were analyzed based on precursor ions,diagnostic ions and neutral loss strategies;(c)Quantitative analysis of multiple components in different varieties of Ginkgo biloba leaf;(d)The differential varieties were screened by principal component analysis(PC A);(e)Heat map visualization is used to visualize the content distribution of various compounds in ginkgo leaf varieties,and VIP score analysis is used to find out the main compounds that cause differences between different varieties.Results:A total of 110 compounds were identified in Ginkgo biloba leaf,including 17 organic acids,3 alkaloids,15 ginkgolides and 75 flavonoids,including 42 compounds identified in Ginkgo biloba leaf for the first time.Of the 42 compounds identified for the first time,6 are potential new compounds.In addition,multi-component quantitative analysis and statistical analysis successfully screened two special varieties,namely Tancheng 231 and Wuxi Huaye,and another variety with regional characteristics:Jiangsu Taixing variety.From the VIP score,the 10 compounds(VIP>1)that mainly lead to the difference between ginkgo leaf varieties are compounds 74,10,78,25,56,16,32,57,49 and 17.The content of these 10 compounds has an important impact on the quality of Ginkgo biloba leaf.Compounds 10,25,16,32 and 57 are the first compounds found and reported in Ginkgo biloba leaf.4.The anti-PAF-induced platelet aggregation activity of 12 new ginkgolides(compounds 12,13,18,19,21,26,27,6,9,20,Y8 and Y9)and 3 known ginkgolides(ginkgolide A,31,ginkgolide B,33,ginkgolide C,32)were evaluated.In the experiment,PAF was used as the platelet aggregation inducer,gingko.diterpene lactone was added to the collected rabbit blood respectively,and the inhibitory effect of each compound on platelet aggregation was detected by turbidimetry,and its structureactivity relationship was preliminarily discussed.The results showed that compounds 31,33,32,12,13,18,19,21,26,6,9 and 20 had inhibitory activity in a dose-effect relationship,while compounds 27,Y8 and Y9 had no activity.Based on the comparison of the activity results of ginkgolide A,ginkgolide B and ginkgolide C with compounds 6,9 and 20,and the comparison of the activity results of compounds 12 and 13,it was found that the isomerization of the C-14 position of ginkgolide(The methyl group at C-14 position is in the β configuration)may reduce the PAF-induced antiplatelet aggregation activity.When the activities of compounds 12 and 18,13 and 19,and 21 and 26 were compared,it was found that when the C-7 position was linked with hydroxyl,PAF-induced antiplatelet aggregation activity might be reduced.Conclusion:(1)In this study,a total of 112 compounds were isolated from Ginkgo biloba leaf and branch,and 106 of them were identified.After removing 21 repetitive compounds,85 compounds were identified,including 12 new ginkgolides,and 44 compounds were first reported.This paper enriches the pharmacodynamic material basis of Ginkgo biloba.Before this work,only 11 ginkgolides were reported internationally,and in this paper,the number of ginkgolides was elevated to 23.(2)This paper proposes a new method for identifying ginkgolides for the first time,laying the foundation for the discovery of ginkgolides.(3)The chemical components of Ginkgo biloba leaf were analyzed based on UHPLC-QTOF-MS/MS combined with diagnostic ion and neutral loss strategies,and the quantitative results of various components were combined with statistical analysis methods to analyze different varieties of Ginkgo biloba leaf,providing a basis for screening different quality varieties of Ginkgo biloba leaf,also providing an assistance to the safety and quality control of clinical Chinese medicine.(4)The anti PAF induced platelet aggregation activity of ginkgolides was studied,and their structure-activity relationship was preliminarily discussed,providing a foundation for the development of new drugs of ginkgolides.