Ginkgolic Acid Monomer Separation,Analysis And Application

Ginkgo biloba is China’s peculiar rare tree species, known as the "living fossil of the plant kingdom". It is classified as a national secondary protection plant. In our country, the resources of ginkgo biloba account for70%of the total world. Both at home and abroad, there are many researches about the effective components of ginkgo biloba, mainly focused on flavonoids and lactones.Ginkgolic acids (GAs) are important active components of ginkgo biloba in addition to flavonoids and lactones. They mainly exist in leaves (about1-2%of dry weight), fruit and testa (about3-5%of dry weight), especially in the testa of ginkgo biloba. Ginkgolic acids are6-alkyl or6-alkenyl salicylic acid’s derivatives. Its carbon atom number of side chain is from13to19with side chain’s double bond number0-3. Ginkgolic acids mainly consist of ginkgoneolic acid, ginkgolic acid, hydroginkgolic acid, hydroginkgolinic acid and bilobols, etc.Modern toxicology studies show that ginkgolic acids have biological toxicity such as allergic reaction, embryo toxicity, immune toxicity and cell toxicity. They are considered to be the main toxical components in EGb. Therefore, at home and abroad, content of ginkgolic acids is the important monitoring index in ginkgo biloba extract and preparations. Since1960s, ginkgolic acids have been studied. In pharmacopoeia standards of Europe, the United States, Japan and China ginkgolic acids in ginkgo biloba extract and preparations are strictly limited. In European pharmacopoeia and American pharmacopoeia, content of ginkgolic acids (mass fraction) are required below 5ppm, and some enterprise internal standard even demand less than1ppm. Chinese pharmacopoeia2010also demand ginkgolic acids limit as below10ppm. Therefore, it seems particularly important to deeply study the ginkgolic acid composition.In this dissertation, the following study on ginkgolic acids was carried out.A, Separation of ginkgolic acids monomersObjective:To separate the ginkgolic acids monomers from Ginkgo exopleuraMethod:Pulverized Ginkgo exopleura was extracted with4fold of petroleum ether (30-60℃) for2times and2h per time. The extraction solutions were collected together and concentrated to obtain crude extract. The crude extract was purified to remove part of pigment impurity with microporous resin, and then fraction Ⅰ(mainly containing ginkgolic acids C13:0, C15:1, also a few C17:2), and fraction Ⅱ(mainly containing ginkgolic acids C17:1and a few C15:0) were obtained by separation with the reversed-phase silica gel column. The fraction I was concentrated to get ginkgolic acid monomer C13:0after repeated recrystallization with acetonitrile-water, then the ginkgolic acids C15:1was concentrated in mother liquid. The crystal C13:0was dissolved in mathanol, and ginkgolic acid monomer C13:0(ginkgoneolic acid) was purified by prepatative liquid chromatography with the mobile phase of mathanol-water. The ginkgolic acid monomers C15:1and C17:2from mother liquid and C17:1and C15:0from fraction Ⅱ were also prepared by prepatative liquid chromatography with the mobile phase of mathanol-water.Results:The ginkgolic acid monomers of C13:04.98g, C15:111.5g, C17:20.45g C15:00.87g and C17:19.29g were separated from1kg of Ginkgo testa.Their structures were confirmed by HPLC-DAD detection, HPLC-MS,’H-NMR and13C-NMR analysis, and all of their purities were≥98%.Conclusion:Ginkolic acid monomers (purity≥98%) were extracted and separated by reflux and prepatative liquid chromatography. This study solved the problem of lacking standard and low purity standard, and it has laid the foundation for the further study of ginkgolic acids. B、Development of analytical method for ginkgolic acids and its application to quality control of manufacturing process of ginkgo biloba extractObjective:To establish a new analytical method for ginkgolic acids by high performance liquid chromatograpgy, and apply this method to quality control of ginkgo biloba extract for preparing the low acid ginkgo biloba extract in large scale production.Method:The ginkgo biloba extract was dissolved in methanol. Separation was carried out on an Agilent C18column with mobile phase of acetonitrile-0.1‰trifluoroacetic acid at a flow rate of1ml-min-1. The dection wavelength was210nm. Then, the developed method was applied to the quality control of intermediates in the key steps of large scale production.Results:There was a good linear relationship between peak area and concentration of ginkgolic acid. The recoveries were99.4%for C13:0,99.7%for C15:1,98.6%for C17:2,98.0%for C15:0and98.8%for C17:1, respectively, with RSD<2.0%. The density and time in water precipitation process and alcohol concentration and elution volume in resin purification process were main factors influencing the content of ginkgolic acids.Conclusion:The results demonstrated that the established method for determining ginkgolic acids is simple, accurate and reliable, and it is suitable for the limit detection of the low acid ginkgo biloba extract. This method can also be applied to control the quality of intermediates in the large scale production.