| Ginkgolic acids(GAs)are 6-alkyl or 6-alkenyl derivatives of salicylic acid.Their carbon atoms number of side chain is from 13 to 17 with side chain's double bond number 0?2.They mainly exist in Ginkgo biloba leaves,fruit and sarcotesta,especially in the sarcotesta of Ginkgo biloba.There are five main ginkgolic acids found in the current research:ginkgolic acid(13:0),ginkgolic acid(15:1),ginkgolic acid(17:2),ginkgolic acid(15:0),ginkgolic acid(17:1).Ginkgolic acids are regarded as harmful components in the extract of Ginkgo biloba leaves since it has various toxicities such as sensitization,embryotoxicity,immunotoxicity and cytotoxicity.However,in current study,it has been found that ginkgolic acids have a variety of pharmacological activities,such as anti-tumor,anti-bacterial,insecticidal(or its host),anti-inflammatory,ete,which have shown wide application prospect.The ginkgo resources are abundant in our country.According to statistics,about 100,000 tons of ginkgo seed coats are discarded as waste every year,which pollutes the environment and wastes a lot of resources.Studying the biological activity of ginkgolic acid can lay a theoretical foundation for the further development and utilization of Ginkgo biloba exocarp,and increase the utilization rate of Ginkgo biloba exocarp,turning waste into treasure,which not only can bring huge economic benefits,but also can do outstanding contributions to China's social environment.This study improved the extraction method of ginkgolic acids based on the relevant literature.The method is simple and practicable,and toxic solvent such as cyclohexane or chloroform was avoided,which is suitable for extracting ginkgolic acids from sarcotesta of Ginkgo biloba.Ginkgolic acids were isolated from the extract of Ginkgo biloba exocarp,and five ginkgolic acid monomers were further isolated,purfied and identified for later biological activity research.The experimental content of this study is as follows:1.S eparation and identification of ginkgolic acid monomersObjective:To separate the ginkgolic acid monomers from Ginkgobiloba exocarpMethods:Ginkgobilobaexocarp was dried and pulverized,and then extracted with absolute ethanol.The obtained extract was evaporated to dryness and suspended in distilled water,and then extracted with equal volume of petroleum ether and ethyl acetate successively,and each was extracted twice respectively.The extract was evaporated under reduced pressure,and then the petroleum ether layer and ethyl acetate layer extract were obtained respectively.The similar chromatographic behavior was observed at a UV detection wavelength of 254 nm by TLC,and then the petroleum ether and ethyl acetate extracts were combined and separated by open silica gel column with gradient elution of petroleum ether,petroleum ether-ethyl acetate(9:1,8:2,7:3,1:1;containing 1%glacial acetic acid),and finally the column was eluted with methanol which obtained 21 eluting fractions.They were observed at a UV detection wavelength of 254 nm by TLC,and the eluted fractions with similar chromatographic behavior were combined and evaporated under reduced pressure to obtain the total ginkgolic acids.It was repeatedly purified by semi-preparative RP-HPLC to obtain five monomeric compounds of ginkgolic acid Fr.A-1-1-1(35 mg),Fr.A-2-2-2(68 mg),Fr.A-3-3(15 mg),Fr.B-1-1(20 mg),Fr.B-2-2-2(17.5 mg),respectively.Results:The purity analysis of the five ginkgolic acid monomeric compounds was carried out by HPLC-DAD,each ginkgoic acid monomer compound has a purity of over 95%,and the identification of their chemical structures was carried out by 1H-NMR,13C-NMR and HR-MS,and then Fr.A-1-1-1,Fr.A-2-2-2,Fr.A-3-3,Fr.B-1-1,Fr.B-2-2-2 were identified as ginkgolic acid(13:0),ginkgolic acid(15:1),ginkgolic acid(17:2),ginkgolic acid(15:0)and ginkgolic acid(17:1),respectively.2.Inhibitory activities of ginkgolic acid monomeric compounds on XOD in vitroObjective:Evaluation of the inhibitory effect of ginkgolic acid monomer compounds on XOD in vitroMethods:In the enzyme inhibition reaction,the product uric acid in the reaction system is an indicator of XOD,and WST-1 is added to detect the amount of superoxide anion formation in the XOD superoxide anion formation system.The inhibitory effects of ginkgolic acid monomeric compounds on xanthine oxidase were evaluated by microplate method.In the reaction system,the final reaction volume was 200 ?L,the final concentration of the XOD solution was 4 mU/mL,the final concentration of the XA solution was 250 ?mol/L,and the final concentration of the WST-1 solution was 100 ?mol/L.Five concentrations were set for each testing sample,each concentration was paralleled with four replicate wells,and the experiment was repeated for three times.The absorbance of each well was measured at 295 nm using a microplate reader.The average inhibition rate was calculated and the IC50 was calculated using GraphPad Prism 5.0 software.Results:In the enzyme inhibition experiment,the IC5O of ginkgolic acid(13:0)and ginkgolic acid(15:1)against XOD were 31.87± 1.87 and 25.48±0.66 ?mol/L(n=3,(?)±s),respectively,showing better inhibition of XOD activity in vitro;In the superoxide anion removal experiment,the IC50 of ginkgolic acid(13:0)and ginkgolic acid(15:1)were 27.39±1.65 and 17.63±1.36 ?mol/L(n=2,(?)±s),respectively.Comparing the IC50 of ginkgolic acid(13:0)and ginkgolic acid(15:1)against XOD in vitro and superoxide anion scavenging experiments,the IC50 of the latter was found to be smaller than the former,which meant ginkgoic acid(13:0)and ginkgolic acid(15:1)not only had inhibitory effects on XOD in vitro,but also had certain scavenging effect on superoxide anion.The above results can provide a basis for further study on the anti-oxidation mechanism of ginkgolic acids and the evaluation of the intervention effects of ginkgolic acids on gout and oxidative stress injury.3.Cytotoxicity of ginkgolic acid monomeric compounds on PC 12 cellsObjective:Study the toxicity of ginkgolic acid monomeric compounds on PC 12 cellsMethods:PC12 cell suspensions in logarithmic growth phase were seeded in 96-well plates,and cell viability was measured by MTT assay.Five concentration were set for each testing sample,each concentration was paralleled with five replicate wells,and the experiment was repeated for three times.The absorbance was measured at 490 nm using a microplate reader to calculate the average cell proliferation inhibition rate,and the IC50 was calculated by GraphPad Prism 5.0 softwareResults:The IC50 of ginkgolic acid(13:0),ginkgolic acid(15:1),ginkgolic acid(17:2),ginkgolic acid(15:0),and ginkgolic acid(17:1)were 9.42± 1.08,10.82±0.80,7.55±0.34,5.85±0.92 and 5.54±0.47 ?mol/L(n=3,(?)±s),respectively.The inhibitory effects of five ginkgolic acid monomeric compounds on PC 12 cells in vitro are as follows:ginkgolic acid(17:1)>ginkgolic acid(15:0)>gingkolic acid(17:2)>ginkgolic acid(13:0)>ginkgolic acid(15:1).PC12 cells are rat adrenal medullary chromoblast-differentiated cell lines and have the general characteristics of neuroendocrine cells.The inhibitory effects of five ginkgolic acid monomeric compounds on PC 12 cells can preliminarily indicate that the ginkgolic acid monomer ic compounds have certain neurocytotoxicities.In summary,the existing extraction methods of ginkgolic acids was improved in the study,and five ginkgolic acid monomers with high purities were isolated.It was found that ginkgolic acid monomers had certain inhibitory activities on XOD and superoxide anion scavenging effects in vitro,but possessed certain neurocytotoxicity in further biological activity researches.Therefore,the above results provide a theoretical basis for the development and application of ginkgolic acids. |
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