Study On Part Organic Acid And 6-HKA Of Ginkgo Biloba Extract

Objective(1)To establish characteristic spectrum of small molecule organic acid in ginkgo biloba extract(EGB),simultaneously,established a content determination method of 5 kinds of small molecule organic;(2)Explore the content determination method of total organic acid in EGB;(3)To establish the content determination method of 6-hydroxy kynurenic acid(6-HKA)in ginkgo biloba,and make study on transfer rate of 6-HKA in the production process of EGB,clear the loss of 6-HKA in each step of production,to guide process improvement;(4)Study the neuroprotective effect of 6-HKA;(5)To establish a new organic standards.Methods(1)Using the HPLC multi-wavelength switching technology,ZORBAX Eclipse XDB-C8 column(4.6×250mm,5μm),making acetonitrile-phosphoric acid as the mobile phase to gradient elution.Make the shikimic acid,gallic acid,protocatechuic acid,6-HKA,hydroxybenzoic acid as the research object,determined the five kinds of small molecules organic acids in 20 batches EGB,and make the protocatechuic acid as a reference peak(S),calculate the relative retention time of other four peaks,calculate the RSD,and establish characteristic spectrum;(2)Making Determination of total organic acid of 10 batches of EGB by using acid-base titration and UV respectively,meanwhile,combined with HPLC method,make an analysis of the advantages and disadvantages of each method;(3)Making an investigation of extraction solvent,extraction time,extraction temperature,extraction methods,and solid-liquid ratio of 6-HKA,using HPLC method,ZORBAX Eclipse XDB-C8 column(4.6×250mm,5μm),and adopting methanol-acetonitrile-water gradient elutionand detection wavelength of 350nm,to establish a content determination method of 6-HKA in ginkgo biloba,and then,making a determination of 6-HKA in 20 group of ginkgo biloba to calculate the mass production transfer rate;Using the above chromatography conditions,utilizing the simulated test of production to determine the 6-HKA step by step and calculate the fractional and total transfer rate;(4)In vitro,using the PC 12 cell damage induced by glutamic acid test,observe the cells form and calculate cell survival rate;In vivo,using ischemia-reperfusion rats experiment,evaluate the rats mortality,neurobehavioral scores,cerebral infarction area and its possible mechanism of action after intervention by 6-HKA.Using the two test to evaluate nerve protective effect of 6-HKA together.(5)According to the above test results,drafting a new company standards of organic acids of EGB.Results(1)Established the content determination method of five kinds of small molecule organic acids in EGB,and methodological evaluation have been done.Total organic acids content is from 1%to 3%in 20 batches of EGB;Respectively,the relative retention time of each characteristic peak and S peak are:0.2310(shikimic acid),0.4488(gallic acid),1.0000(protocatechuic acid),1.1539(6-HKA),1.9396(p-hydroxy benzoic acid);RSD value are 0.48%,0.11%,0.00%,0.85%,0.10%respectively.(2)The results of the content in total organic acid determined by ultraviolet spectrophotometry(UV)is calculated on the total phenol minus the total flavonoids,but the result showed that part batches of the total phenolic is less than the total flavonoids in EGB,the total organic acid content cannot be calculated by subtracting;The results of total organic acid in EGB determined by Acid-base titration is about 8%,while the results determined by HPLC is from 1%-3%.(3)The content determination method of 6-HKA in ginkgo biloba leaves is suitable by methodology validation;The extraction conditions of 6-HKA from ginkgo biloba are as follows:70%ethanol,60℃ material-liquid ratio is 1:20,ultrasonic extraction,time is 45 min.The total transfer rate of 6-HKA in production is from 20%to 40%,while in small test is 33%-43%,the two results are similar.The major losses of 6-HKA is in resin purification steps,one step loss rate reach 53.7%,total loss rate achieve 44.4%;(4)The in vitro experimental results showed that compared with the ginkgo lactone and ginkgo lactone B of the same dose interve,the cell shape improved situation and cell survival rate by 6-HKA is the best for PC 12 cells injured by glutamate;In vivo experiment results found that the mortality of 6-HKA high dose group rats has a significant decrease after modeled,Neurologic damage symptoms reduced significantly,cerebral infarction area is significantly lower.The neural protection mechanism of 6-HKA is likely to be that it has a relevant to glutamate receptor antagonism action,and it can significantly reduce the content of glutamic acid in brain tissue of ischemia-reperfusion,and inhibite the internal flow of extracellular calcium ions.(5)Drafted the enterprise new standards of EGB contains 6-HKA.Conclusion(1)Thedetermination method of five kinds of small molecule organic acids in EGB builded in this experiment is simple,stable and feasible,and it is suitable for the determination of organic acids in EGB;(2)The UV method in total organic acid content determination had been given up because of the not accurate result;There is a considerable difference in the determinate results between Acid-base titration and HPLC method,the results determinated by HPLC could not be complete,because it is limited to the organic acids which had been quantitated and identified;The results determinated by Acid-base titration is inaccurate,because the results is on the high side;So the content determination method of total organic acid remains to be further explored.(3)The content determination method of 6-HKA in ginkgo biloba builded in this test is stable and feasible,and the pretreatment method is simple;The test result shows that the major loss of 6-HKA is in the process of resin purification,so we can reduce the loss of 6-HKA by optimizing the resin process,and then improving the content of 6-HKA in EGB.(4)The pharmacological and efficacy test results show that the 6-HKA has strong neuroprotective effect;(5)The new standards of enterprises could make further improvement in quality control of EGB.