Study On Decarboxylation Of Ginkgolic Acids In Ginkgo Biloba

Ginkgo resources（Ginkgo seeds）are abundant in China,but its development of application and production is comparatively slow due to the toxicity of Ginkgolic acids in it.In this paper,the quantitative analytical method of Ginkgolic acids in Ginkgo seeds with LC-MS was established by using Ginkgolic acid C15:1 as a standard.What’s more,this study explored the intensification of thermal decarboxlation of Ginkgolic acids by absorpting CO₂ with strong bases and the new biodegradation method for Ginkgolic acids using decarboxlatase and decarboxlating yeast.The main work are as follows:A method for the extraction of Ginkgolic acids from Ginkgo seeds was established using isopropyl alcohol as solvent.And the optimum conditions were obtained from single factor test are 130 r·min^-1 of rotating speed,28℃ of temperature,120 min of extraction time,25/1 of the weight ratio of liquid to solid.The results showed that this method could be used for the pretreatment process of Ginkgolic acids determination in Ginkgo seeds.Compared with the method using Soxhlet Extractor,it simplified the procedure of determination and shortened the time of sample processing.Liquid chromatography-spectrometry mass（LC-MS）was used for quantitative analysis of Ginkgolic acids in Ginkgo seeds under SIM mode.The mobile phase was isopropanol,acetonitrile and acetic acid（42/57/1,v/v/v）.This determination method showed 3.298% of RSD for the precision,102.274% for recovery（RSD=4.518%）,3.689×10^-4 mg·mL^-1 of LOD and 2.452×10^-3 mg·mL^-1 of LOQ for sensitivity.The thermal pressurized degradation of Ginkgolic acids was efficiently intencified by absorbing the produced carbon dioxide with NaOH.In the study,Ginkgo seeds was heated in 135℃ for 120min together with NaOH（10%）existing,and the content of Ginkgolic acid C15:1 can be decreased to 2.097±0.045 mg·kg^-1.A strain obtained from the natural fermentation of Ginkgo seeds at 37℃ for 30 d also showed decarboxylation activity for Ginkgolic acid C15:1.（18.409 ± 0.075）%of Ginkgolic acid C1 5:1 could be degraded in 5 h at 37℃（pH=5.5）with 1 mL cell lysate of the strain.Recombinant plasmid pET21a（+）-sdc was constructed based on the synthetic gene of salicylic acid decarboxylase（sdc）,which was reported by Kirimura et al.And the enzyme was expressed successfully through transfering the plasmid into E.coli BL21（DE3）.Results showed that（39.17± 0.131）%of Ginkgolic acid C15:1 can be degraded in 5 h at 40℃（pH=5.5） with 1mL cell lysate of pET21a（+）-sdc/E.coli BL21（DE3）.The decarboxylation activities of cell lysates of Trichosporon moniliiforme 2.2390 and Trichosporon moniliiforme 2.2665 were determined with Ginkgolic acid C15:1.The results showed that（55.47±0.077）%of Ginkgolic acid C15:1 could be degraded in 16 h at 40℃（pH=5.5）with 1mL cell lysates of Trichosporon moniliiforme 2.2390,but it could not be degraded by Trichosporon moniliiforme 2.2665.