Purification Of Ginkgolide B And Its Derivatives And Their Effects On Viability Of Induced SH-SY5Y Cells

Ginkgolide is an important active ingredient of Ginkgo biloba,which has anti-inflammatory,anti-oxidative and anti-apoptotic effects.Ginkgolide B(GB)has significant neuroprotective effect.There are abundant and good quality Ginkgo biloba leaves in Qinba Mountains and it is great significance to improve the level of deep processing and further extend the industrial chain.In this paper,the preparation of extract of Ginkgo biloba leaves by enzymatic pretreatment,the separation and purification of ginkgolides,the preparation of GB derivatives and their effects on the activity of induced SH-SY5Y cells were preliminary studied.(1)The enzymatic hydrolysis process of Ginkgo biloba leaves was optimized by single factor experiment and orthogonal experiment.The optimum process was as follows:the dosage of compound enzyme(cellulase:pectinase=1:1)was 1/300(6.7×10~3U)of Ginkgo biloba leaves,the temperature was 50?,the time was 4.0 h,and the initial pH was 5.0.Under this condition,the enzymatic hydrolysis was the best.The extraction rates of flavonoids and lactones were 92.38%and 91.74%respectively.The contents of total flavonoids and total lactones from extract of Ginkgo biloba leaves were 30.32%and 8.85%,which meet the requirement of Chinese Pharmacopoeia,American Pharmacopoeia,European Pharmacopoeia and International Business Standard about extract of Ginkgo biloba leaves that requires the total flavonoids content>24.0%and the total lactones content>6.0%.(2)Using the extract of Ginkgo biloba leaves(700g)as raw material,we obtained five Ginkgolides by extraction,decolorization,crystallization and preparation chromatography purification.They are Ginkgolide A,10.14 g,purity 98.36%,yield 97.69%;Ginkgolide B,3.11 g,purity 98.24%,yield 96.26%;Ginkgolide C,7.38 g,purity 99.01%,yield 95.39%;Ginkgolide J,0.98 g,purity 98.57%,yield 95.19%;Bilobalide,6.70 g,purity 98.47%,yield97.51%.Compared with the existing literatures,the preparation efficiency and yield of Ginkgolides monomer were obviously improved,having a good industrial application prospects.(3)Four Ginkgolides B derivatives were synthesized by C-10 ester bond condensation reactions of GB with cinnamic acid,4-methoxycinnamic acid,4-hydroxy cinnamic acid and3,4-dimethoxycinnamic acid respectively and high pressure preparative chromatography purification.They are the compound GBHC-1,48.6 mg,purity 98.23%,yield 17.2%;the compound GBHC-2,54.2 mg,purity 98.38%,yield 18.3%;the compound GBHC-3,45.7 mg,purity 97.12%,yield 15.6%;the compound GBHC-4 39.9 mg,purity 98.26%,yield 12.8%.The four derivatives of Ginkgolides B were confirmed by NMR and looked up in the SciFinder database,it's found that they are new compounds and haven't been reported in literatures before.(4)The cell tests showed that 10?mol/L GB,10?mol/L GBHC-1,10?mol/L GBHC-2,1?mol/L GBHC-3 and 10?mol/L GBHC-4 have significant proliferation effects on6-hydroxydopamine(6-OHDA)-induced SH-SY5Y cells,the protective effect of GBHC-4 on SH-SY5Y cells was slightly better than other compounds,but there was no significant difference among GB,GBHC-1,GBHC-2,GBHC-3 and GBHC-4.