Extraction And Identification Of Flavonoids From Cultured Cell Of Ginkgo Biloba

Ginkgo biloba is precious species in our country for its specific active substancesof flavonoids, which have some sanitarian function as antioxidant, bacteriostat,antiphlogistic, anticarcinogen, preventing cardiovascular and cerebrovascular diseasesand so on. Prospect of exploitation of Ginkgo biloba is perspective.It is an effectiveway to use cell culture technique for large-scale production of Ginkgo bilobaflavonoids to provide raw material for industrial production in attention of the furtherprocessing industry development of Ginkgo biloba. Based on the the studies ofpredecessors, the induction and subculture conditions of callus were investigated inthis article.The suspendsion cultural conditions ofGinkgo biloba cell wereexhaustivllyresearched. Fluorescence spectrum method was established for flavonoidsdetection. Extracting technological parameters offlavonoids glycosideswere optimized,and thereafter the model equation of significantly factors on extraction wasestablished. The kinetic mechnism of flavonoids glycosides extraction was alsoapproached. The identification of flavonoids glycosides was also studiedby UV,HPLC and MS. The anti-oxidation and the inhibition bacteria activity of flavonoidsfrom suspension cultural cell in vitro were verified. The main results from studies areachieved as following:In the callus induction experiment, DCR medium and hormone BA do well thanMS medium and KT hormone. But MS subculture medium and hormone KT has apositive effect in promoting callus growth. Active carbon as adsorbent can inhibit cellgrowth, but play a promoting role in the accumulation of flavonoids.Sucrose was the favourite carbon source for the suspension cultural cell. Andglucose was most suitable for flavonoids synthesis. The mixture of nitrate-nitrogenand ammoniacal nitrogen can promote cell growth, but nitrate-nitrogen was in favourof flavonoids synthesis. The optimal cultural condition was of inoculum40g/L celland phenyl alanine precursor in100mL liquid medium with film sealing in24h lightper day for high yield flavonoids. Citric acid can promote cell growth, while Vitaminc was conducive to flavonoids synthesis. And the inhibition of cell brown wasperformed by Vitamin C.The method of fluorospectrophotometry for total flavonoids detection wasestablished with standard rutin. Results indicated that fluorescence intensity and rutinconcent was linear relationship with regression equation for y=29.92x+36.49, （R2=0.986）. Linear range was1.810^-63.210^-5mol/L. Standard recovery of101.3%was achieved. The analysis results obtained from suspension cultured cell of Ginkgobiloba suggested that the development method is convenient for flavonoids detection.Ethanol extraction condition was optimized by response surface methodology.Quadratic polynomial model about significant factors, extraction time, temperatureand ethanol concent on flavonoids yield was obtained. The optimal extractionconditions was ethanol concent72.05%for3.285h at76.85℃. In this condition, theyield of flavonoids was1.8101%, which was consistent with the predictive value ofthe model. It was demonstrated that the model was accurate and reliable for flavonoidglycosides yield prediction.The regression equation was deduced by quadratic regression orthogonal rotationcombination design experiments.The optimal condition of ultrasonic-assisted methodwas ethanol concent100%and ultrasonic power500W at80℃for50min. The yieldof flavonoids in revification tests was3.0345%, consistent with the predicted value ofthe model. Repeatability and reliability were performed well by the model.The extraction dynamics model for flavonoids from suspension cultured cell wasdeduced on the base of Fick’ first law. This model was adapted to the extraction ofactive components of medicine plants, which belongs to an apparent first order kineticequation. Revification tests showed that the theoretic value of the content offlavonoids and relative raffinate rate basically consistent with experimental value in0-360min. The established dynamic model can be used to describe the actualextraction process of flavonoid glycosides from Ginkgo biloba cell.The fragmentation regularity of quercetin, kaempferol, isorhamnetin wasstudied using HPLC-UV-ESI-MSnmethod in350nm wavelength, at the range of50-1000m/z scanning scope. In the same conditions, flavonoidsfrom the extraction ofcultured cells were identified. Eightflavonoidglucosides were presumed respectivelyfor3-O［2-O,6-O-bis （α-L-rhamnosyl）-β-D-glucosyl］quercetin;3-O［2-O,6-O-bis（α-L-rhamnosyl）-β-D-glucosyl］kaempferol;3-O［6-O-（α-L-rhamnosyl）-β-D-glucosyl］myricetin;3-O［2-O-（α-L-rhamnosyl）-β-D-glucosyl］quercetin;3-O［6-O-（α-L-rhamnosyl）-β-D-glucosyl］isorhamnetin;3-O［6-O-（α-L-rhamnosyl）-β-D-glucosyl］ kaempferol；3-O［2-O-（6-O-p-coumaroyl-β-D-glucosyl）-α-L-rhamnosyl］ quercetin；3-O ［2-O-（6-O-p-coumaroyl-β-D-glucosyl）-α-L-rhamnosyl］kaempferol.The anti-oxidation and bacteriostasis of flavonoids in vitro from suspension cultured cell of Ginkgo biloba were studied. The results showed that the clearance forhydroxy radical and hydrogen peroxide by flavonoids significantly better than citricacid, phytic acid and Vitamin C. But the clearance for superoxide anion by flavonoidswas more below than Vitamin C. The chelation ratio with Fe2+was significantlyhigher than active control. Antioxidative capacity of flavonoids on plant oil was alsostronger than active control in experimental content. The results of bacteriostasisexperiment in vitro showed that bacteriostatic activitywas enhanced with the increaseof flavonoids concentration. The maximum of inhibition zone diameter was2.2cmand minimum for1.1cm in the experimental content. The minimum of bacteriostaticconcentration on Escherichia coli was0.141mg/mL. The bacteriostatic ratio wouldachived81%with the condition for flavonoids concentration2.25mg/mL and actingtime10h.