Study On Purification And Bioactivity Of Gingko Flavonoid

The main active components in the Ginkgo biloba extract are flavonoids and ginkgolides. Flavonoids are natural potent antioxidants; ginkgolides are terpene compound with large value for the heart and central nervous system. Ginkgo biloba extract have a number of biological effects including free radical scavenging, antioxidants, anticancer, expansion of cardiovascular, anti-inflammatory and analgesic effects. Previous researches have been concentrated primarily on the extraction, purification and pharmacological research. In the industry process, the purification of the Ginkgo biloba extract was always operated with the macroporous resin column chromatography procedure and the purity is approximately 24%. This work is mainly concerned with the research on two different purification technology of the flavonoid and the adsorption characteristics and the purification effect of the polyamide resins chromatography procedure, the purification of the flavonoid by metal complexing-dissociation method. The flavonoid aglycone was obtained by the hydrolysis with the high purity flavonoids gained by the purification method. The activity of the two kinds of ginkgo flavonoid was compared by the method of in vivo and in vitro.A process of polyamide resin column chromatography procedure for purifying Ginkgo flavonoid was established. The adsorption characteristic of the Ginkgo flavonoid on the polyamide resin was fit well to the Langmuir equation. The conditions for elution were determined and the eluent was 30% ethanol solution. After one run of the purifying process, the flavonoid content can be raised to more than 50% from 24% and the ginkgolide content was increased to more than 9% from 6%.New purification method was established according to the complex properties of flavonoid with the metal. The complexation-dissociation conditions of the method were optimized and the results were as follows:methanol as reaction solvent, zinc sulfate as complex agent, the pH of the complex solution 9.5,30% of ethanol as the dissociation solution of the flavonoid-metal complex, EDTA as the dissociation agent. After one run of this process, the flavonoid content was increased to more than 55% from 24%, the recovery was higher than 52%. The Ginkgolides compounds cannot be purified by the complex method, but the purified ginkgolides product can be obtained with the solution of the complex using liquid extract method, and the content was up to 80%.The process for hydrolyzing the ginkgo glycoside to make the flavonod aglycone was optimized in the study. The conditions were:temperature was 80℃; the hydrolysis time was 2 hs; the concentration of the hydrochloric acid was 25%; the ratio of the hydrochloric acid to methanol solution was 1:4; the concentration of the material was 1.0 mg/mL; the aglycone recovery was 90%. HPLC and HPLC-MS was used to determine the hydrolysis results and the compounds before and after hydrolysis. The results indicated that before hydrolysis the flavonoid were existed mainly as the glycosides. The glycosidic bond was broken when they were hydrolyzed and the aglycone was obtained. The aglycones were dertermined as quercetin, kaempferol and isorhamnetin and their peaks time in the HPLC chromatography figure were very later than the flavonod glycosides.The activity of the Gingko flavonoid in its two different forms i.e. flavonoid glycoside and aglycone was evaluated in vitro. The DPPH·,OH·,H2O2 and other radicals scavenging activity and the lipid peroxidation inhibiting activity were determined. Results showed that both types of the flavonoids had good radical scavenging activity and lipid inhibiting activity. The capacities of flavonod aglycone were much higher than the flavonod glycoside.Besides the in vitro study for the activity, the activity of the two types of flavonoid was evaluated in vivo with the Kunming rat. The SOD value, MDA value, CAT value and the GSH-Px value of the blood and the tissues were determined. The results revealed that both the two types of the flavonoid had the capacity of anti-aging for the aging induced by the D-galactose. And the activity of the glycosides flavonoid was higher than aglycone flavonoid in blood, kidney and liver.