Analysis Of Organic Acids In Ginkgo Biloba Extract And Its Protective Effect Against Cerebral Ischemia

Ginkgo biloba(Ginkgo biloba L.),a rare and precious tree species unique to my country,is one of the most widely used medicinal plants in the world.It first appeared in the Carboniferous period 345 million years ago and is known as the "living fossil" of the earth and the "panda" of the plant world.Ginkgo biloba extract which contains a variety of active ingredients is the main raw material for a series of preparations such as tablets and capsules.It can be used for the treatment of cardiovascular and cerebrovascular diseases,atherosclerosis,fatty liver,diabetes and neurological dysfunction due to the effects of anti-inflammatory,neuroprotective,antioxidant and other pharmacological.Ginkgo biloba preparation was first researched and developed by Dr.Weimar Schuppe Gmb H & Co.KG in the 1960 s.It is one of the most widely used natural medicines for adjuvant treatment of cardiovascular and cerebrovascular diseases in the world and is widely used in medicines,dietary supplements,etc.Although many studies have been carried out on the chemical components of Ginkgo biloba leaves and their extracts,most of them focus on flavonols and terpene lactones and only the contents of flavonol glycosides and terpenoids are regarded as the main quality control indexes in pharmacopoeia standards of various countries.Relevant studies have shown that Ginkgo biloba leaves and their extracts also contain a large number of organic acids.However,there is still a lack of in-depth research on whether organic acids affect the efficacy of Ginkgo biloba extracts and whether they should be included in quality control indicators.Therefore,this paper mainly carries out the following three aspects of research:1.The chemical composition of the raw materials of medicinal ginkgo biloba extract was systematically analyzed by liquid chromatography-mass spectrometry,and its structural categories were clarified.LC-QTOF-MS/MS,nuclear magnetic resonance spectroscopy(1H-NMR,13C-NMR)and other technologies were used to confirm the structures of the five isolated organic acids components.Gallic acid(GA),protocatechuic acid(PA),hydroxy benzoic acid(HBA),6-hydroxykynurenic acid(6-HKA)and shikimic acid(SA)were identified as characteristic constituents in Ginkgo biloba extract.Shikimic acid,HA,and 6-HKA are currently no legal reference substances,moreover 6-HKA is a small molecular organic acids unique to Ginkgo biloba.This study provides a reference for the separation and preparation of these organic acids from Ginkgo biloba.2.Middle cerebral artery occlusion model(MCAO)and photosensitizer rose bengal induced photothrombosis model(PT)in mouse were established to investigate the protective effect of Ginkgo biloba total organic acids on ischemic brain injury.The result showed that Ginkgo biloba total organic acids significantly reduced the volume of cerebral infarction in MCAO mice and improved their neurological symptoms.30mg/kg Ginkgo biloba total organic acids can partially reduce the grid misalignment rate in mouse photochemical embolism model during4-14 days,suggesting the potential therapeutic effect of Ginkgo biloba total organic acids on ischemic brain injury.Further studies revealed that organic acids treatemtn significantly reduced ROS production in cultured neuronal cells that subjected to oxygen-glucose deprivation/reperfusion.However,organic acids fialed to reverse ischemia-induced apoptotic.These results suggested an anti-oxidative role of organic acids underlies the neuroprotection.This study also investigated the potential anti-ischemic brain injury effect of 6-HKA which is characteristic organic acids in Ginkgo biloba.The result demonstrated that 50 mg/kg of 6-HKA could significantly reduce the cerebral infarct volume in MCAO mice,and significantly reduce the grid misalignment rate in PT model mice.In addition,6-HKA can concentration-dependently protect glutamate-damaged primary cultured rat hippocampal astrocytes and reduce LDH leakage rate.However,6-HKA can not improve the survival rate of mouse N2 a cells induced by oxygen-glucose deprivation.All the results suggested that 6-HKA may be the active ingredient in the total organic acids of Ginkgo biloba extract,which has potential anti-ischemic brain injury effects,and its mechanism may be related to the improvement of astrocyte function and survival.This study provided a pharmacological basis for using total organic acids as a quality control standard for Ginkgo biloba extracts.3.A quantitative analysis method for organic acids in ginkgo biloba leaves based on UPLC was established,which realized the rapid,accurate and efficient detection of organic acids.By comparing 25 batches ginkgo biloba extracts at home and abroad and 24 batches of ginkgo biloba preparations from different manufacturers,the contents of various organic acids in ginkgo products from different manufacturers were clarified.The results indicated that the organic acids contents of Ginkgo biloba products from different manufacturers was quite different,the highest was110.13mg/g,the lowest was 1.01mg/g,and the batch-to-batch fluctuation was109.12mg/g.The content of shikimic acid in Ginkgo biloba extract which prepared according to the "European Pharmacopoeia" standard was significantly higher than prepared according to the "Chinese Pharmacopoeia" standard while other organic acids(PA,6-HKA,etc.)indexes showed a clear grouping trend.Above products significantly divided into high-abundance organic acid and low-abundance organic acid groups,with a relative ratio of about 63% and 37% by cluster analysis and principal component analysis.Furthermore,the pharmacodynamic activities of highabundance and low-abundance organic acids in anti-ischemic brain injury were compared.The results showed that high-abundance organic acids had protective effects on neuron-like cells after differentiation of oxygen-glucose-deprived N2 a cells,while low-abundance organic acids did not.Organic acids have no protective effect.The results of this study provide scientific data for further improving the quality standards of medicinal ginkgo biloba extracts.In summary,this paper confirmed the characteristic components of organic acids in Ginkgo biloba extract,further evaluated the potential therapeutic effects of Ginkgo biloba total organic acids and 6-HKA on ischemic brain injury and established a quantitative analysis method of organic acid by UPLC evaluates the content and activity of commercially available products and proposes the necessity of organic acid quality control.