Isolation And Purification Of Ginkgolides From Ginkgo Leaves And Preparation Of Ginkgolides Certified Reference Materials

Ginkgo biloba L.is a species of gymnosperms,deciduous trees,since ancient times China's ginkgo leaf is used as medicine and diet records.At the beginning of 1970 s,Ginkgo biloba extract(EGB)for treatment of central peripheral blood disorders and cerebrovascular atherosclerosis was reported,its medicinal value is getting more attention.Ginkgo biloba extract is an active substance extracted from the leaves of Ginkgo biloba by modern extraction technology,and it include two main active chemical components: flavonoids and terpenoids.The Ginkgolides parts of two terpene lactones,including ginkgolide A,B,C,and so on,are PAF(platelet activating factor)antagonist.It can be used in the treatment of thrombosis,acute pancreatitis and cardiovascular disease.In this experiment,the methods of analysis and optimization of extraction process are established,and the separation and preparation of high purity Ginkgolides are found.That is a way to provide theoretical support for further development and utilization of ginkgolides.The main research results are the following.1.The optimization analysis method of ginkgolide bilobalide: detection method using Agilent 1260 and 385-ELSD detector,by YMC-Pack ODS-A(5um,4.6 x 250mm)chromatographic column was analyzed,according to the largest separation degree(measured the degree of separation of R >7.2),the detection time is suitable and safety requirements,select alcohol: water(10:90,v/v)as mobile phase.The flow rate was 1ml/min,as the gas flow rate is 1.60L/min(N2),and the stable drift tube temperature is setting at 90?,so it can be safer and accurate quantitative determination of ginkgolides.2.In the Ginkgolides extraction,the extraction rate is the result,on the basis of single factor experiment,the response surface of four factors and three levels of analysis,two multinomial mathematical models of ethanol extraction of Ginkgolides,and the extraction rate of Ginkgolides as response surface and contour as the response value.The factors are investigated,including the number of extraction,extraction time(h),ethanol concentration(%)and solid-liquid ratio(g/ml)on the effects of ginkgolide extraction.The results showed that the optimum extraction conditions werefollowing: the number of extraction3 times,the extraction time2 h,the concentration of ethanol44%,and the ratio of solid to liquid was 0.04g/ml.Under this condition,the extraction rate was about 0.46%.At the same time compared with subcritical water extraction,in order to improve the extraction rate of ginkgolides and extraction efficiency,single factor experiment was carried out by analysis of four factors: extraction temperature,the number of extraction,extraction time and solid-liquid ratio on Ginkgo biloba lactone extraction rate influence and determine the factors and levels.Response surface methodology(RSM)was used to optimize the extraction of Ginkgolides from Ginkgo Leaves by subcritical water.The results showed that when the extraction temperature180?,the ratio of material to liquid(g/ml)1:25,the extraction time30 min,and the number of extraction was 3,finally the extraction rate was about 0.47%.Subcritical water extraction has the advantages of green environmental protection,no organic solvent residue and high extraction rate,so the purified samples were extracted from subcritical water.3.Ginkgolide from Ginkgolides extraction is pre-purified.After the extraction of Ginkgolides from subcritical water,petroleum ether degreased it nearly colorless,and then extracted with ethyl acetate.The extract of ethyl acetate was added with weak alkali,such as NaHCO3 solution,and the flavonoids were washed and washed it about 3 times.The removal of small molecular impurities with larger polarity by silica gel was better than activated carbon,diatomite and acidic alumina.The Ginkgolide B was purified by recrystallization method.The purity of ginkgolide B was obtained by repeated crystallization of ethanol and water in the experiment.The purity of Ginkgolide B was 95%.4.Purification of ginkgolide by high-speed countercurrent chromatography,solvent system selection for n-hexane: ethyl acetate: methanol: water(4/5/3/5,v/v/v/v),positive transfer,speed of 800r/min,the flow rate was 5ml/min,the operating temperature 25?.Bilobalide,ginkgolide A,B,C preparation purity was more than 99%5.Identify the structure of ginkgolides.The structure and purity of Ginkgolides was identified by TLC,mass spectrometry and nuclear magnetic resonance spectroscopy.6.Ginkgolidescertified reference materials.According toGB/T15000.3-2008 standards,the purity was determinedby8 laboratories.24 bottles of sampleswere selected randomly,each laboratory to send the value of 3 bottles,each bottle of parallel determination 2 times.The value of the laboratory will be a certain concentration of the sample solution,high performance liquid chromatography,the use of peak area normalization method to determine the purity of the results show that the purity of the requirements,can be used as standard.