Ginkgo leaf organic acid extract, preparation method therefor and use thereof

Abstract

Provided is a ginkgo leaf organic acid extract, which comprises the following components in percentage by weight: 60%-68% of protocatechuic acid, 10%-14% of 6-hydroxykynurenic acid, 5%-8% of gallic ac

Description

TECHNICAL FIELD The invention relates to the field of pharmaceutical chemistry, specifically relating to a Ginkgo leaf organic acid extract, its preparation method and application in the preparation of medicine for preventing and / or treating cerebral ischemic injury. BACKGROUND OF THE INVENTION Ginkgo leaf extract preparation is a commonly used plant extract preparation for cardiovascular and cerebrovascular diseases, and is also one of the most widely used natural medicinal preparations in the world. The pharmacopoeia standard has undergone many revisions since Ginkgo leaf extract was launched in China. The 2020 edition of Chinese Pharmacopoeia stipulates that Ginkgo leaf extract should contain Ginkgo flavonol glycosides content >24% (w / w); Ginkgo terpene lactones content >6% (w / w); content of Ginkgo terpene lactones (calculated by the total amount of Ginkgolide A, Ginkgolide B, Ginkgolide C and bilobalide) should not be less than 0.2% (w / w); Ginkgolic acid content <5 mg / kg; High-Performance Liquid Chromatography (HPLC) peak area ratio of quercetin to kaempferol should be 0.8 ~ 1.2; HPLC peak area ratio of isorhamnetin to quercetin should be higher than 0.15; quercetin content should not exceed 1.0% (w / w); kaempferol content should not exceed 1.0% (w / w); isorhamnetin content should not exceed 0.4% (w / w). The Chinese Pharmacopoeia suggests that Ginkgo flavonol glycosides and Ginkgo terpene lactones are main medicinal ingredients in Ginkgo leaf extract, although their corresponding component content is less than 40% (w / w). Many of other medicinal substances, such as organic acids, procyanidins, polyprenols, polysaccharides and protein compounds, possess significant pharmacological activities and can be used as indictors to improve efficacy and quality of Ginkgo leaf extract, and even as new plant extracts with remarkable efficacy. Organic acids in Ginkgo biloba include alkyl phenolic acids and strong polar organic acids. In Ginkgo leaf extract, strong polar organic acids include shikimic acid, 6-Hydroxykyinircnic acid (6-HKA) , protocatechuic acid, quinic acid, chlorogenic acid and other active components. Shikimic acid is a synthetic raw material for the anti-influenza medicine Oseltamivir, which has various pharmacological activities such as antibacterial, antiinflammatory, antiplatelet and antithrombotic effects. 6-HKA is a derivative of kynurenic acid and has a high affinity for glutamate receptors. Inhibition of glutamate receptors can avoid excessive activation of some receptors which are harmful to neurons. 6-HKA can inhibit glutamate receptors to make a certain neuroprotective effect. Protocatechuic acid has anti-inflammatory, antioxidant, anti-tumor and some other pharmacological activities, which can improve neurocognitive function impairment caused by chronic intermittent hypoxia, enhance the learning and memory ability of chronic intermittent hypoxia rats, and reduce oxidative stress and cell apoptosis. OH 6-HKA Protocatechuic acid Cerebral stroke, also known as "zhong feng" in Chinese medical science, is a cerebrovascular disease that can result in death or permanent nemologic deficits by interrupting brain blood supply. It is the second leading cause of death over the world and a leading cause of adult disability. Cerebral stroke can be divided into ischemic and hemorrhagic, wherein ischemic stroke cases account for 85% of all strokes. Among the preparations derived from Ginkgo biloba, Ginkgo leaf extract and dipyridamole injection, Shuxuening injection and Ginkgolide injection are used to treat acute ischemic stroke; orally taken Ginkgo leaf extract preparations, yinxingtongzhi preparations are used to treat chronic ischemic stroke. However, the main components of existing Ginkgo biloba derived preparations are Ginkgo total flavones, Ginkgolides, and Ginkgo leaf extract. The medicinal and market value of organic acids of Ginkgo biloba leaves have not been fully explored. In the prior art, there are many researches on organic acids from Ginkgo biloba leaves as active ingredients of extract, but there are few researches on organic acid extracts for ischemic stroke. Patent CN108524531A with the title "‘Pharmaceutical composition”, filed on March 1, 2017 and publicated on September 14, 2019, discloses a pharmaceutical composition for treating ischemic cerebral stroke, which comprises 18.1% (w / w) ~ 50.9% (w / w) of lactone component, 48.8% (w / w) ~ 81.8% (w / w) of flavonoid component and 0.07% (w. w) ~ 6.83% (w / w) of organic acid component, wherein the organic acid component contains chlorogenic acid and protocatechuic acid. However, mam active ingredients of the invention are lactone and flavonoid. The invention can reduce cerebral infarction area of acute cerebral ischemia-reperfusion model mice and reduce neurological function score by limiting the proportion of each flavonoid component and proportion of each lactone component. Patent CN114366761A with the title “Preparation method of Ginkgo leaf extract rich in organic acid”, filed on January 18, 2022 and publicated on April 19, 2022, provides an extract preparation method which comprises following steps: adding an ethanol aqueous solution into Ginkgo leaves for extraction; purifying extract solution through cation exchange column, macroporous resin column and anion exchange resin column in sequence; removing Ginkgolic acid of extract to obtain a Ginkgo leaf extract with total acid content >17% (w / w). In the Ginkgo leaf extract, organic acid composition comprises shikimic acid content >15% (w / w), quinic acid content >1% (w / w), protocatechuic acid content >1% (w / w) and 6-HKA content >0.3% (w / w). What’s more, the extract also has Ginkgo flavonol glycosides content between 24% (w / w) and 27% (w / w). terpene lactone content between 6.0% and 6.6%. However, pharmacological data is not disclosed in the invention, and medicinal effect of the extract is unknown. Furthermore, Ginkgo biloba derived organic acid extracts that are mainly composed of shikimic acid and 6-HKA have been disclosed on the prior art. Patent CN103784448A with the title "‘Preparation method of natural 6-hydroxykynurenic acid extract and application thereof’, filed on Januarv 7, 2014 and publicated on November 18, 2015, discloses a preparation method of extract with 50% (w / w) ~ 90% (w / w) 6-HKA that is separated from macroporous resin eluation water of Ginkgo leaf extract. Administrated with the extract at a dose of 3 mg / kg, cerebral infarction area of acute ischemia-reperfusion stroke mice was reduced from 31.5±3.8% (in the model group) to 20.1±2.1%. The extract has a good protective effect on acute ischemia-reperfusion stroke. Patent CN107353201A with the title “A high-content natural shikimic acid extract product and a preparing method thereof’, filed on May 9, 2016 and publicated on November 17, 2017, discloses an extract with shikimic acid content more than 98% that is separated and prepared from waste water generated in a Ginkgo leaf extract production process. The extract didn’t disclose any pharmacological or medicinal data. As discussed above, the prior art discloses extracts of Ginkgo leaf organic acids as active ingredients in extract, but the organic acid content of those extracts is low, the compositions are unclear, and the efficacy in chronic ischemic stroke is not obvious. SUMMARY OF THE INVENTION An object of the present invention is to provide a plant extract with significant medicinal effect and clear composition for treating ischemic cerebral stroke. Developing this extract into medicine can meet the market demand of traditional Chinese medicine preparations for chronic ischemic cerebral stroke. The invention provides a Ginkgo leaf organic acid (GLOA) extract, which contains protocatechuic acid, 6-HKA, gallic acid and 4-Hydroxybenzoic acid. It shows significant effects in treating cerebral ischemic injury, especially in chronic ischemic cerebral stroke. The technical scheme of the present invention is as follows: a GLOA extract, wherein the extract has medicinal components, including 60% ~ 68%(w / w) protocatechuic acid, 10%~ 14%(w / w) 6-HKA, 5%~ 8%(w / w) gallic acid and 3% ~ 8%(w / w) 4-Hydroxybenzoic acid, wherein the extract has a total organic acid content of more than 80%(w / w). The present invention further limits the content ratio of the organic acid, wherein content of protocatechuic acid, 6-HKA, gallic acid and 4-Hydroxybenzoic acid conform to one of following conditions: content ratio of protocatechuic acid to 6-HKA is between 4 and 6, content ratio of protocatechuic acid to gallic acid is between 9 and 12, content ratio of protocatechuic acid to 4-Hydroxybenzoic acid is between 8 and 14. Gallic acid, also named 3, 4, 5-trihydroxybenzoic acid, is a natural polyphenol with a kind of the simplest chemical structures, widely exists in various plants, vegetables and fruits. It has high medicinal and health care value. Because gallic acid has some phenolic hydroxyl groups in molecular structure, it is a natural antioxidation and has a strong antioxidant activity. 4-Hydroxybcnzoic acid, also named P-hydroxybenzoic acid, is a widely used organic synthesis raw material.lt has some pharmacological effects such as antibacterial, hypoglycemic and anti-inflammatory effects. OH 4-Hydroxybenzoic acid The present invention provides a GLOA extract with significant effect on treating cerebral ischemic injury, especially chronic ischemic stroke, through controlling the content of protocatechuic acid, 6-HKA, gallic acid and 4-Hydroxybenzoic acid, as well as content ratio of protocatechuic acid and the others. In the extract, protocatechuic acid, 6-HKA, gallic acid and 4-Hydroxybenzoic acid can achieve synergistic effect and enhance medicinal effect. The second object of the present invention is to provide a preparation method of the above-mentioned extract. The method selects the Ginkgo leaf extract that is in accordance with the standards of Chinese Pharmacopoeia, then combines the Ginkgo leaf extract dissolving, polyamide column chromatography purifying, macroporous resin column chromatography purifying, concentrating and drying to obtain the GLOA extract. It comprises the following steps: (1) selecting Ginkgo leaf extract as raw material, wherein Ginkgo leaf extract meets the standards of Chinese Pharmacopoeia, Preferably, Ginkgo leaf extract has high organic acid component content satisfying high organic acid component indicators, which is screened according to high organic acid component indicators by an organic acid content detection method. Preferably, the standard of high organic acid component indicators include: 6-HKA greater than 2 mg / g, 4-Hydroxybenzoic acid greater than 1 mg / g, and gallic acid greater than 1 mg / g. (2) dissolving the raw material from step (1) in boiling water or ethanol solution to obtain a crude extract solution I, wherein ethanol solution having less than or equal to 10% (v / v) concentration. (3) purifying the crude extract solution I through a polyamide column, eluting with 3 bed volume (BV) ~ 5 BV of 5% ~ 10%(v / v) ethanol at 1 BV / h ~ 3 BV / h flow rate, collecting eluate and concentrating to 1 BV~2 BV, filtering and collecting filtrate to obtain crude extract solution II. Preferably, polyamide column has pore size of 0.55 mm- 1.7 mm or 0.25 mm~ 0.55 mm. (4) purifying the crude extract solution II through a macroporous resin column, eluting with 3 BV - 5 BV of 10% ~ 15%(v / v) ethanol at 1 BV / h ~3 BV / h flow rate, collecting ethanol eluate, concentrating and drying to obtain the GLOA extract. Preferably, macroporous resin column is selected from D101 or AB-8, and ethanol eluate is concentrated and dried at 50 € ~70'C. The third object of the present invention is to provide a use of the above extract in the preparation of medicine for preventing and / or treating cerebral ischemic injury, including chronic ischemic stroke. Technical advantages of the present invention: (1) The GLOA extract has clear components in present invention, which can achieve synergistic effect and enhance medicinal effect on the treatment of cerebral ischemia injury, especially chronic ischemic stroke disease. Result of the test on monomeric compounds showed that efficacy of 6-HKA was better than that of the other three organic acids. For person of ordinary skill in the art, increasing proportion of the other three organic acids and reducing proportion of 6-HKA in mixture will make its neuroprotective effect lower or equal to that of 6-HKA monomer under the same concentration. The experiment results of oxygen-glucose deprivation-reoxygenation (OGD / R) injury model on mouse neuroblastoma N2a cells suggested that cell survival rate of samples 1 ~ 3 in the present invention was within a range between 73.6% to 77.6%, which was significantly higher than that of 6-HKA monomer ( 64.2%), as shown in Table 1. What’s more, total organic acid content of samples 1~ 3 were similar to that of sample 5, but N2a cells protective effect of samples 1~3 were better than sample 5, which ruled out a speculation about the total organic acid content affecting the neuroprotective effect. Therefore, it can be inferred that neuroprotective effect of samples 1 ~ 3 are determined by content of protocatechuic acid, 6-HKA, gallic acid, 4-Hydroxybenzoic acid, and content ratio thereof. The extract with specific organic acid content range corresponding to samples 1 3 has neuroprotective synergy effect, wherein the content ratio of protocatechuic acid to 6-HKA is between 4 and 6, the content ratio of protocatechuic acid to gallic acid is between 9 and 12, and the content ratio of protocatechuic acid to 4-Hydroxybenzoic acid is between 8 and 14. Table 2 shows composition of organic acid extracts protected by the present invention. Table 1 Results of the OGD / R model test of the protected organic acid extract of the present invention on mouse neuroblastoma N2a cells Group Name N Cell survival rate %( Mean±SD) Blank Blank 4 98.9%+6.8% OGD / R model OGD / R 5 51.8%±7.7%&aa OGD / R+ Butylphthalide OGD / R+NBP 4 62.7% ± 12.1% OGD / R+ Edaravone OGD / R+EDA 4 65.8%+16.2% OGD / R+ Ginkgo leaf extract OGD / R+GBE 4 76.7%+11.0% OGD / R+ Protocatechuic acid OGD / R+PA 4 63.0%+12.0% OGD / R+ 6-Hydroxykynurenic acid OGD / R+6-HKA 4 64.2%+7.3% OGD / R+ Gallic acid OGD / R+GA 4 59.1%±12.1% OGD / R+ 4-Hydroxybenzoic acid OGD / R+HBA 4 61.4%±13.5% OGD / R+ Sample 1 OGD / R+EOA1 4 73.6%+10.1%* OGD / R+ Sample 2 OGD / R+EOA2 4 77.6%+8.9%* OGD / R+ Sample 3 OGD / R+EOA3 4 75.9+9.8%* Compared to blank group, △△△ p<0.01. Compared to OGD / R model group, *p<0.05. Table 2 Organic acid extract component content protected by the present invention Organic acid Sample 1 Sample 2 Sample 3 Protocatechuic acid 61.92% 66.40% 67.78% 6-HKA 10.75% 12.68% 11.74% Gallic acid 6.11% 5.94% 6.27% 4-Hydroxybenzoic acid 7.68% 6.65% 5.80% Sum 86.46% 91.67% 91.59% (2) The invention combines raw material control, separation and extraction to protect preparation method of the GLOA extract. The preparation method comprises: controlling the raw material by preferred Ginkgo leaf extract; dissolving raw material into boiling water or ethanol below 10% (v / v); purifying crude extraction solution through polyamide column chromatography and collecting 5%~ 10% (v / v) ethanol eluent; purifying eluent through macroporous resin column chromatography; and concentrating eluent to obtain the GLOA extract. Comparing preparation method of sample 2 with that of sample 5, they have the same preparation method except using different raw materials. The 6-HKA content in the raw material of sample 5 is 1.24 mg / g, the content of 6-HKA in sample 5 is 6.62%. Both kinds of 6-HKA content in sample 5 are half of those in sample 2, respectively. It can be seen that control of the raw material, Ginkgo leaf extract, has an impact on the organic acid content of the finished product. The invention limits content of organic acid in Ginkgo leaf extract to make quality of the raw material under control, just as follows: the 6-HKA content is higher than 2 mg / g, both content of the gallic acid and 4-Hydroxybenzoic acid are higher than 1 mg / g. Samples 2 and 4 use raw materials with the quality control above, and select different eluent of polyamide column and macroporous resin column in each preparation method. The total organic acid content, protocatechuic acid content, and 6-HKA content of sample 4 are respectively lower than sample 2. It can be seen that samples 1 ~ 3 can achieve neuroprotective synergy effect under specific raw material control, specific separation and extraction conditions. The raw materials, preparation processes and finished products of samples 2, 4 and 5 are shown in Table 3, and results of drug efficacy are shown in Table 4. Table 3 Comparison of raw materials, preparation processes and finished products of samples 2,4 and 5 Process Content / Preparation method Content / Preparation method Sample 2 Sample 4 Sample 5 Raw material detection Organic acid content of Ginkgo leaf extract (mg / g) Protocatechuic acid 14.24 15.3 19.5 6-HKA 2.75 2.15 1.24 Gallic acid 1.38 1.47 1.65 4-Hydroxybenzoic acid 1.17 1.41 0.65 Subtotal 1 19.54 20.33 21.84 Preparation method Crude extract solution I preparation Solvent 8%(v / v) ethanol boiling water 8%(v / v) ethanol Polyamide column Pore size 0.25mm~ 0.55mm 0.55mm~1.7mm 0.25mm~ 0.55mm Elution solvent 5%(v / v) ethanol 10%(v v) ethanol 5%(v / v) ethanol Elution volume 5BV 3 BV 5 BV Elution rate 3 BV / h 2 BV / h 3 BV / h Macroporous resin column Type D101 Elution solvent 15%(v / v) ethanol 8%(v / v) ethanol 15%(v / v) ethanol Elution volume 5BV Elution rate 3BV Finished product detection Organic acid content (%) Protocatechuic acid 66.40 59.11 70.40 6-HKA 12.68 8.00 6.62 Gallic acid 5.94 7.39 3.88 4-Hydroxybenzoic acid 6.65 6.92 5.17 Subtotal 2 91.67 81.42 86.07 Table 4 Comparison of samples 2, 4 and 5 in the results of OGD / R model test on mouse neuroma blast cells Group Name n Cell survival rate %(Mean±SD) Blank Blank 4 98.9%±6.8% OGD / R model OGD / R 5 51.8%t7.7%^ OGD / R+ Butylphthalide NBP 4 62.7%±12.1% OGD / R+ Edaravone EDA 4 65.8%±16.2% OGD / R+ Ginkgo leaf extract GBE 4 76.7%±11.0% OGD / R+ Sample2 EOA2 4 77.6%±8.9%* OGD / R+ Sample 4 EOA4 4 68.6%±11.6% OGD / R+ Sample 5 EOA5 4 65.4%±10.1% Compared to blank group, △△△ p<0.01. Compared to OGD / R model group, *p<0.05. (3) The present invention prepares the GLOA extract through some steps: selecting Ginkgo leaf extract which is in accordance with the standards of Chinese Pharmacopoeia as raw material, dissolving raw material into boiling water or ethanol below 10% (v / v), purifying crude extraction solution through polyamide column chromatography, collecting 5%~ 10% (v / v) ethanol eluent, purifying eluent through macroporous resin column chromatography, and concentrating to obtain the Ginkgo leaf organic acid extract. The present invention is based on the preparation method of patent CN202010732010.X "A kind of Ginkgo biloba flavonoid extract and its preparation method and characteristic map construction method" which is invented by our research and development team before. The present invention is built upon step 1) and step 2) of patent CN202010732010.X, macroporous resin column chromatography and concentration to obtain GLOA extract. Wherein, the sample solution is eluted through polyamide column by purified water or ethanol below 10% (v / v) to collect organic acid and lactone component in step (2) in the present invention, which will not interfere with flavonoids collecting during 0.1%~0.5% (v / v) sodium carbonate solution eluting of patent CN202010732010.X. The technical scheme of the present invention achieves reuse of production waste liquid and reduces the production costs. In addition, the preparation process adopts low-concentration ethanol as solvent and elution reagent to achieve green production. (4) Ginkgolic acid of GLOA extract is not detected, which meets the requirement about Ginkgolic acid being less than Ippm in Ginkgo leaf extract in Chinese Pharmacopoeia. The GLOA extract is safe to use. DESCRIPTION OF DRAWINGS Fig.l shows the HPLC chromatograms of Ginkgo leaf extract, wherein GA is gallic acid, PA is protocatechuic acid, HBA is 4-Hydroxybenzoic acid, 6-HKA is 6-hydroxykynurenic acid. Fig.2 shows the cytotoxicity experiment results of GLOA extract on mouse neuroblastoma cells, in which Blank is blank control, NBP is positive control butylphthalide, EDA is positive control edaravone, GBE is raw material Ginkgo leaf extract, PA is protocatechuic acid, GA is gallic acid, HBA is 4-Hydroxybenzoic acid, E0A1 is sample 1, E0A2 is sample 2, E0A3 is sample 3, E0A4 is sample 4, and E0A5 is sample 5. The higher the cell survival rate, the lower the cytotoxicity. Fig.3 shows the OGD / R model experiment results of GLOA extract on mouse nemoblastoma cells, in which Blank is blank control, OGD / R is model group, OGD / R+NBP is positive control butylphthalide group, OGD / R+EDA is positive control edaravone group, OGD / R+GBE is the raw material Ginkgo leaf extract group, OGD / R+PA is protocatechuic acid group, OGD / R+GA is gallic acid group, OGD / R+HBA is 4-Hydroxybenzoic acid group, 0GD / R+E0A1 is sample 1 group, 0GD / R+E0A2 is sample 2 group, 0GD / R+E0A3 is sample 3 group, 0GD / R+E0A4 is sample 4 group, 0GD / R+E0A5 is sample 5 group. The higher cell survival rate, the better neuroprotective effect. Fig.4 shows chronic ischemic stroke experiment results of GLOA extract on mice. Figure 4-1 is the brain slice of the mouse middle cerebral artery occlusion (MCAO) experiment. Model is the model group, and E0A2 is the sample 2 group. The larger light-colored area at slice right side, the higher damage degree of the right brain tissue. Figure 4-2 shows tire cerebral infarction volume rate of MCAO mice; Figure 4-3 shows the score of cerebral neurological symptoms on mice. The lower neurological symptoms score, the better neuroprotective effect.Fig.5 shows photochemical embolization experiment results of GLOA extract on mice, in which Model is the model group, and EOA2 is the sample 2 group. The lower foot fault rate, the better inhibition effect of photochemical embolization in mice. DETAILED DESCRIPTION OF THE INVENTION The following further describes the present invention with reference to examples. The examples below further illustrate the invention, rather than limit the scope thereof. The Ginkgo leaf extract whose manufacturer is Zhejiang Conba Pharmaceutical Co., Ltd., used in the following examples is prepared according to the 2020 edition of Chinese Pharmacopoeia. Production batch numbers of Ginkgo leaf extract are 191106, 190301, 190109, 180307, 180807 and 180912. Example 1 Organic acid composition detection method The detection method for organic acid components and content in the Ginkgo leaf extract and GLOAE in the present invention is not limited to methods such as HPLC, UPLC, LC-MS. The UPLC detection method of the present invention was optimized by research team based on our existing patent ZL201510826946.8 "Method for determining content of organic acid in Ginkgo leaf extract". The detection method accepted UPLC rather than HPLC, deleted pretreatment step about removing procyanidins from bovine serum albumin, and shortened detection time within 14 minutes to make the quantitative detection of five organic acids realized. The specific detection method was as follows: 1. Preparation of test solution A proper amount of Ginkgo leaf extract sample was added into 10 mL volumetric flask, and diluted with a small amount of purified water. The mixture was processed with ultrasound for 20 min, and cooled down, after which purified water was added to metered volume. Then the flask should be shaken well, and the filtration process was applied to collect subsequent filtrate as the test solution. 2. Preparation of reference substance solution A proper amount of protocatechuic acid, gallic acid and 4-Hydroxybenzoic acid reference substance were added into three 50 mL volumetric flasks, and diluted with 40% methanol to metered volume, respectively. And then, all of flasks should be shaken well to obtain 3 kinds acid stock solution. A proper amount of 6-HKA reference substance was added into 100 mL volumetric flask and diluted with methanol. And then, flask should be shaken well to obtain 6-HKA stock solution. 3. UPLC detection conditions Chromatographic conditions and system adaptability test: octadecylsilane chemically bonded silica gel column; acetonitrile and 0.1% trifluoroacetic acid solution as mobile phase; flow rate 0.4 mL / min; UV detection wavelength at 254 nm; column temperature 30 °C. The mobile phase of gradient elution procedure was shown in Table 5: Table 5 Mobile phase of gradient elution procedure T (min) 0.1% Trifluoroacetic acid solution (%) Acetonitrile (%) 0-1.2 100 0 1.20-2.0 100-95 0—5 2.0-7.0 95—30 5—70 7.0-14.0 30 70 Operation method: applicant precisely measured 5 pL of the reference substance solution and the test solution respectively, and injected them into UPLC machine for detection. 4. Experiment results 4.1 Detection linear range Protocatechuic acid reference substance, 6-HKA reference substance, gallic acid reference substance and 4-Hydroxybenzoic acid reference substance were prepared into different concentrations reference substance stock solutions, and analyzed date in the UPLC detection conditions above. According to detection results, applicant took the peak area as vertical coordinate (y), reference substance concentration (pg / mL) as abscissa (x) to draw standard curve and perform linear regression. The results were shown in Table 6. Table 6 Linear regression equation of organic acid in UPLC detection Organic acid Regression equation Linear range (pg / mL) Protocatechuic acid y=44730.9338x+4525.3107, R2=l .0000 40.04-200.2 6-HKA y=122148.1405x-9249.5026, R2=1.0000 7.776-38.88 Gallic acid y=l 16010.1 L3x-17159.2753. R2=L0000 5.896-29.48 4-Hydroxybenzoic acid y=76795.6281x+10988.6000,R2=0.9997 5.896-29.48 4.2 Content of organic acids in Ginkgo leaf extract Different Ginkgo leaf extract test solution were analyzed according to the UPLC detection conditions above, and the experiment results were shown in Table 7. Table 7 Organic acid content of Ginkgo leaf extract raw material (unit: mg) Batch NO. 1 Batch NO. 2 Batch NO. 3 Batch NO. 4 Batch NO. 5 Protocatechuic acid 12.42 14.24 16.17 15.3 19.5 6-HKA 3.27 2.75 2.85 2.15 1.24 Gallic acid 2.51 1.38 1.46 1.47 1.65 4-Hydroxybenzoic acid 1.75 1.17 1.26 1.41 0.65 Grand total 19.95 19.54 21.74 20.33 23.04 Example 2 Preparation of organic acid extract sample 1 Organic acid extract sample 1 was prepared by following steps: (1) Batch NO. 1 Ginkgo leaf extract 500g was selected as raw material; (2) the raw material from step (1) was dissolved in boiling water to obtain a crude extract solution I; (3) the crude extract solution I was purified through a polyamide column whose pore size is 0.55mm~1.7mm, eluted with 3 BV of 10% ethanol at 1 BV / h flow rate, collected eluate and concentrated to 2 BV, filtered and collected filtrate to obtain crude extract solution II; (4) the crude extract solution II was purified through a D101 macroporous resin column, eluted with 5 BV of 10% ethanol at I BV / h flow rate, collected ethanol eluate, concentrated and dried at 500-70^0 to obtain the GLOA extract sample 1. Example 3 Preparation of organic acid extract sample 2 Organic acid extract sample 2 was prepared by following steps: (1) Batch NO. 2 Ginkgo leaf extract 2kg was selected as raw material; (2) the raw material from step (1) was dissolved in 8% ethanol solution to obtain a crude extract solution I; (3) the crude extract solution I was purified through a polyamide column whose pore size is 0.25mm~ 0.55mm, eluted with 5 BV of 5% ethanol at 3 BV / h flow rate, collected eluate and concentrated to 1 BV, filtered and collected filtrate to obtain crude extract solution II; (4) the crude extract solution II was purified through a D101 macroporous resin column, eluted with 5 BV of 15% ethanol at 3 BV / h flow rate, collected ethanol eluate, concentrated and dried at 50°C~70°C to obtain the GLOA extract sample 2. Example 4 Preparation of organic acid extract sample 3 Organic acid extract sample 3 was prepared by following steps: (1) Batch NO. 3 Ginkgo leaf extract 500g was selected as raw material; (2) the raw material from step (1) was dissolved in 10% ethanol solution to obtain a crude extract solution I; (3) the crude extract solution I was purified through a polyamide column whose pore size is 0.25mm~ 0.55mm, eluted with 4 BV of 5% ethanol at 2 BV / h flow rate, collected eluate and concentrated to 2 BV, filtered and collected filtrate to obtain crude extract solution II; (4) the crude extract solution II was purified through a AB-8 macroporous resin column, eluted with 3 BV of 15% ethanol at 2 BV / h flow rate, collected ethanol eluate, concentrated and dried at 50°C~70°C to obtain the GLOA extract sample 3. Example 5 Preparation of organic acid extract sample 4 Organic acid extract sample 4 was prepared by following steps: (1) Batch NO. 4 Ginkgo leaf extract 500g was selected as raw material; (2) the raw material from step (1) was dissolved in boiling water to obtain a crude extract solution I; (3) the crude extract solution I was purified through a polyamide column whose pore size is 0.55mm~1.7mm, eluted with 3 BV of 15% ethanol at 2 BV / h flow rate, collected eluate and concentrated to 2 BV, filtered and collected filtrate to obtain crude extract solution II; (4) the crude extract solution II was purified through a D101 macroporous resin column, eluted with 5 BV of 5% ethanol at 3 BV / h flow rate, collected ethanol eluate, concentrated and dried at 50°C~70“C to obtain the GLOA extract sample 4. Example 6 Preparation of organic acid extract sample 5 Organic acid extract sample 5 was prepared by following steps: (1) Batch NO. 5 Ginkgo leaf extract 500g was selected as raw material; (2) the raw material from step (1) was dissolved in 8% ethanol solution to obtain a crude extract solution I; (3) the crude extract solution I was purified through a polyamide column whose pore size is 0.25mm~ 0.55mm, eluted with 5 BV of 5% ethanol at 3 BV / h flow rate, collected eluate and concentrated to 1 BV, filtered and collected filtrate to obtain crude extract solution II; (4) the crude extract solution II was purified through a D101 macroporous resin column, eluted with 5 BV of 15% ethanol at 3 BV / h flow7 rate, collected ethanol eluate, concentrated and dried at 50°C~70°C to obtain the GLOA extract sample 5. The transfer rate of organic acid extracts in Examples 2- Examples 6 ranges from 55% to 65%. Example 7 Determination of organic acid component content Ginkgo leaf extract prepared in Examples 2~6 w ere determined by the assay method in Example 1. The experiment results w ere shown in Table 8: Table 8 The content of the organic acid in sample 1 ~ sample 5 Organic acid Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Protocatechuic acid 61.92% 66.40% 67.78% 59.11% 70.40% 6-HKA 10.75% 12.68% 11.74% 8.00% 6.62% Gallic acid 6.11% 5.94% 6.27% 5.39% 3.88% 4-Hydroxybenzoic acid 7.68% 6.65% 5.80% 6.92% 5.17% Grand total 86.46% 91.67% 91.59% 79.42% 86.07% Example 8 Effects of different organic acid samples on the OGD / R mouse neuroblastoma N2a cells survival rate 1. Medicine preparation A commercially available Ginkgo leaf extract, 5 kinds of organic acid compound, GLOA extract sample 1 ~ sample 5, positive controls butylphthalide and edaravone were dissolved in DMSO solution to prepare 100 mg / mL stock solution, respectively. All of stock solutions were stored at -20 °C and diluted to 1 pg / mL with medium immediately before use. 2. Culture of mouse neuroblastoma cells Mouse neuroblastoma N2a cells were routinely cultured at 37°C in 5% CO2 incubator with DMEM medium which contained 10% fetal bovine serum, 100 U / mL penicillin and 0.1 mg / mL streptomycin. When cell confluence reaching 75%, the cells were passaged. 3. Cytotoxicity evaluation of medicine Mouse neuroblastoma N2a cells were treated with 1% N-2 supplement DMEM medium (without bovine serum) for 24 hours to differentiate into neuron-like cells, replaced the medium with normal medium and incubated for 2 hours after prophylactic administration in sequence. All of neuron-like cells were divided into 12 groups and added with Ginkgo leaf extract, 4 kinds of organic acid compounds, GLOA extract sample 1 ~ sample 5, positive controls butylphthalide and edaravone at 1 pg / mL, respectively. Evers' group had 3 wells. All the groups were detected OD value at 490 mn in microplate reader by CCK-8 method, and cells toxicity evaluation of all medicine were detected. 4. Protective effect of medicines on OGD / R injury model of N2a cells The primary mouse neurons were treated with OGD / R model to simulate ischemia environment in vitro, which was used to test neuroprotective effects of organic acids. N2a cells were seeded into 96-well plate with 3000 cells / well and 100 pL of regular DMEM per well, and adhered overnight, followed by being treated with 100 pL of 1% N-2 supplement DMEM medium (without bovine serum) per well for 24 h and differentiated into neuron-like cells to establish OGD / R model. The medium of neuron-like cells was replaced with 100 pL conventional DMEM medium per well. All of neuron-like cells were divided into 12 groups and added with 1 pg mL of Ginkgo leaf extract, 4 kinds of organic acid compounds, GLOA extract sample 1~ 5, positive control butylphthalide and edaravone to pretreat 2h, respectively. After being washed with 100 pL / well PBS once, the medium was changed to 100 pL / well DMEM medium without glucose and serum. The cell plate was placed in CO2 incubator that was added with 5%CO2 and 95%N2 at flow rate 25 L / min for 6 min, and incubated at 37°C for 4h. And then cells were treated with Ginkgo leaf extract, 4 kinds of organic acid compounds, GLOA extract sample 1~ sample 5, positive control butylphthalide and edaravone respectively for 24 h. On the contrary, the neuron-like cells were treated with 100 uLw ell conventional DMEM medium and then placed in CO: incubator which was added with 5% CO: and 95% air, and incubated at 37° C for 4 h as the OGD / R model group. All the groups were detected OD value at 490 nm in microplate reader by CCK-8 method, and cells protective effect of all medicine were detected. 5. Experiment results 5.1 Cytotoxicity evaluation The experiment results were shown in Table 9. Table 9 Organic acid extract cytotoxicity test results Group Name N Cell survival rate % (Mean + SD) Blank Blank 5 100%±5% Butylphthalide NBP 3 90.5%±4.3% Edaravone EDA 3 91.9%±5.4% Ginkgo leaf extract GBE 3 94.2%±6.8% Protocatechuic acid PA 3 101.8%±8.5% 6-Hydroxykynurenic acid 6-HKA 3 94.9%±10.9% Gallic acid GA 3 98.1%±9.3% 4-Hydroxybenzoic acid HBA 3 99.7%±7.5% Sample 1 EO Al 3 96.6%±5.2% Sample 2 EOA2 3 97.3%±8.4% Sample 3 EOA3 3 95.7%±7.2% Sample 4 EOA4 3 97.6%±5.6% Sample 5 EOA5 3 98.4%±7.1% The test results showed that the Ginkgo leaf extract, each organic acid compound and sample 1 ~ sample 5 had preferable safety at the administration concentration of 1 pg / mL. 5.2 Results of medicines’ protective effect on N2a cells against OGD / R injury When the oxygen deprivation model was established successfully, N2a cells survival rate with the 4 organic acid compounds were higher than that with the OGD / R model group, which indicated that the 4 monomeric compounds above had protective effects on N2a cells against OGD / R injury. The efficacy of 6-HKA was better than that of the other 3 organic acids and the positive control butylphthalide. As shown in Table 10. Table 10 N2a cytoprotective effect results of organic acid extracts Group Name N Cell survival rate % (Mean+SD) Blank Blank 4 98.9%±6.8% OGD / R model OGD / R 5 51.8%±7.7%AAA OGD / R+ Butylphthalide OGD / R+NBP 4 62.7%±12.1% OGD / R+ Edaravone OGD / R+EDA 4 65.8%±16.2% OGD / R+ Ginkgo leaf extract OGD / R+GBE 4 76.7%±11.0% OGD / R+ Protocatechuic acid OGD / R+PA 4 63.0%±12.0 OGD / R+6-HKA OGD / R+6-HKA 4 64.2%±7.3% OGD / R+ Gallic acid OGD. / R+GA 4 59.1%±12.1% OGD / R+4-Hydroxybenzoic acid OGD / R+HBA 4 61.4%±13.5% OGD / R+ Sample 1 OGD / R+EOA1 4 73.6%±10.1%* OGD / R+ Sample 2 OGD / R+EOA2 4 77.6%±8.9%* OGD / R+ Sample 3 OGD / R+EOA3 4 75.9±9.8%* OGD / R+ Sample 4 OGD / R+EOA4 4 68.6%±11.6% OGD / R+ Sample 5 OGD / R+EOA5 4 65.4%±10.1% Note: AAA represents the comparison with the blank group, p<0.01; * represents the comparison with the OGD / R model group, p<0.05. The survival rate of N2a cells in sample 1 ~ 3 was at range of 73.6% to 77.6%, which was significantly higher than that of 6-HKA monomer (64.2%). What’s more, total organic acid content of sample 1~ sample 3 was at range of 86.46% to 91.67%, which was similar to that of sample 5 (87.61%). It is ruled out a speculation about the total organic acid content affecting the neuroprotective effect. Therefore, it can be inferred that specific organic acid content range corresponding to sample 1 ~ sample 3 has neuroprotective synergy effect, wherein the content ratio of protocatechuic acid to 6-HKA is between 4 and 6; the content ratio of protocatechuic acid to gallic acid is between 9 and 12; and the content ratio of protocatechuic acid to 4-Hydroxybenzoic acid is between 8 and 14. The present invention is developed from the preparation method of patent CN202010732010.X "A kind of Ginkgo biloba flavonoid extract and its preparation method and characteristic map construction method" which is invented by our research and development team before. The present invention is built upon step 1) and step 2) of patent CN202010732010.X, macroporous resin column chromatography and concentration to obtain Ginkgo leave organic acid extract. Wherein, the crude extract solution is eluted through polyamide column by purified water or ethanol below 10% (v / v) to collect organic acid and lactone component in step (3) in the present invention, which will not interfere with flavonoids collecting during 0.1% ~0.5% (v / v) sodium carbonate solution eluting of patent CN202010732010.X. It can be seen that the present invention fully enriches the active ingredients of neuroprotection in Ginkgo leaf extract, realizes the reuse of production waste liquid and reduces production costs, and provides a Ginkgo leaf organic acid extract with clearer components and remarkable neuroprotective effect. Comparing preparation methods of sample 2 with that of sample 5, they have the same preparation method except using different raw materials. The 6-HKA content in the raw material of sample 5 was 1.24 mg / g, and the content of 6-HKA in sample 5 was 6.62%. Both kinds of 6-HKA content in sample 5 were half of those in sample 2, respectively. When transfer rate of the above organic acid extracts is between 55%~65%, control of the raw material Ginkgo leaf extract components has an impact on the organic acid content of the finished product. Combined with the composition range of the raw material Ginkgo leaf extract of sample 1 to 3 and excluded large differences data in the raw material of sample 5, the organic acid content in the raw material Ginkgo leaf extract was limited that the 6-HKA content was higher than 2 mg / g, both content of the gallic acid and 4-Hydroxybenzoic acid were higher than 1 mg / g. Limiting content of organic acid in raw material Ginkgo leaf extract can make quality of the raw material under control. Sample 2 and sample 4 used raw materials with the quality control above, and selected different eluent of polyamide column and macroporous resin column in each preparation method. The total organic acid content, protocatechuic acid and 6-HKA content of sample 4 were lower than sample 2. It can be seen that sample 1 ~ sample 3 can achieve neuroprotective synergy effect under specific raw material control, specific separation and extraction conditions. Combined with the preparation process parameters of sample 1 to 3 and excluded individual process parameters in sample 4, the preparation process of the present invention is as follows: (1) selecting Ginkgo leaf extract as raw material, wherein the 6-HKA content is higher than 2 mg / g, both content of the gallic acid and 4-Hydroxybenzoic acid are higher than 1 mg / g; (2) dissolving the raw material from step (1) in boiling water or ethanol to obtain a crude extract solution I, wherein ethanol having content of less than 10% (v / v); (3) purifying the crude extract solution I through a polyamide column, eluting with 3 ~5 BV of 5%~10%(v / v) ethanol at 1 ~3 BV / h flow rate, collecting eluate and concentrating to 1 ~2 BV, filtering and collecting filtrate to obtain crude extract solution II; (4) purifying the crude extract solution II through a macroporous resin column, eluting with 3 ~5 BV of 10%~15%(v / v) ethanol at 1~3 BV / h flow rate, collecting ethanol eluate, concentrating and drying to obtain the GLOA extract. Example 9 Effects of organic acid extracts on chronic ischemic stroke mice According to the cell experiment results in Example 8, we further verified the drug efficacy of sample 2 on chronic ischemic stroke mice. 1. Experimental animals 8~1 O-week-old C57BL / 6 male mice were provided by Shanghai Slac Laboratory Animal Co., Ltd., whose animal certificate number was SCXK (Shanghai) 2017-0005. During the experiment, operations such as feeding, administration, surgery and material collection of animals all followed the regulations of the management of experimental animals. 2. Medicine preparation and administration mode Sample 2 was dissolved in physiological saline to prepare low concentration (1 mg / mL) solution, medium concentration (3 mg / mL) solution and high concentration (10 mg / mL) solution, and the pH was adjusted to neutral bylM NaHCOs before ready to use. In this study, sample 2 was administered by gavage at doses of 10 mg / kg, 30 mg / kg and 100 mg / kg, respectively. 3. Establishment of MCAO mouse model 8~10-week-old male C57BL / 6 mice were adaptively reared for 3 days, and randomly divided into model group (physiological saline, 10 mL / kg, i.g.) and administration group according to their body weight. Mice were fasted for 12 hours with free access to water, pre-administered 5 Days. Before the pMCAO operation, model group and administration group were respectively given physiological saline solvent and different doses of medicines which was dissolved in physiological saline to prepare clear solution and the pH was adjusted to neutral by IM NaHCOs before ready to use. Then mice were anesthetized with isoflurane, monitored cortical cerebral blood flow of middle cerebral artery (MCA) by Doppler laser flowmetry. The skin of mouse head was separated, bregma was exposed, and then the optical fiber front end of the laser Doppler flowmeter was fixed on 2 mm behind the bregma and 6 mm on the right side of the midline in the animal’s skull. The mouse was placed supine, then the right common carotid artery was exposed and isolated, the distal end of carotid artciy was ligated and the proximal end was clamped with hemostatic forceps in sequence. Another microscissors was used to make a small cut in right internal carotid artery for inserting about 10 mm length of blunt-tip No. 6-0 nylon monofilament suture to block the origin of the MCA and establish a MCAO model. Date of blood flow was collected through observation. If the blood flow value decreases by more than 80%, the model is established successfully; if the blood flow value decreases by less than 80%, the animal should be excluded. 4. Experimental operation Mice were randomly divided into model group (physiological saline, 10 mL / kg, i.g.) and sample 2 group (10 mg / kg, 30 mg / kg, 100 mg / kg, i.g.) according to their body weight. Model group was given physiological saline, sample 2 group were given different doses before surgery. The neurological symptom scores and cerebral infarction volume statistics of mice in each group were evaluated 24 hours after MCAO operation. The brains of the mice in each group were harvested after operation 24 hours, stained by 2,3.5-triphcnyltctrazolium chloride (TTC), and counted the infarct volume in sequence. The neurological symptom was scored 24 hours after ischemia, and specific score was determined as follows: 0 point, no symptom; 1 point, when tail of mouse is lifted, the mouse rotates to the left; 2 points, when mouse is placed on a flat surface, the mouse cannot walk in a straight line and rotate to the left; 3 points, the mouse cannot walk or rotate, and roll to one side; 4 points, no spontaneous activity; 5 points, death. Mice were euthanized 24 hours after surgery, and brain tissue was removed and sliced at 1 mm intervals to obtain coronal slices. Coronal slices were stained with 0.25% TTC (Sigma, T8877) to determine cerebral infarct volume. Cerebral infarct volume was calculated by software Image-Pro Plus 7.0 through an indirect method which could correct cerebral edema deviation. Cerebral infarction volume rate in mice= (sum of left uninfarcted arcas-sum of right uninfarcted areas) / sum of left uninfarcted areas* 100%. 5. Experiment results The experiment results were shown in Table 11 and Figure 4. To compare with the model group, sample 2 group (100 mg / kg) could significantly reduce the cerebral infarction volume rate (p<0.01). According to the symptoms scores of mice, it was found that different doses of sample 2 had a tendency to reduce the neurological symptom score. Compared with the model group, 30 mg / kg of sample 2 could significantly reduce the neurological symptom score of mice. The above results indicated that GLOA extract sample 2 exhibited a potential anti-chronic ischemic stroke effect in the MCAO model. Table 11 Results of MCAO test on mice (Mean±SD, n=6) Group Cerebral infarction volume rate % Neurological symptom score Model 47.5%±6.4% 3±0.6 Sample 2 10 mg / kg 34.3%±9.2% 2.7±0.5 30 mg / kg 37.0%±12.4% 1.8±0.8 100 mg / kg 28.4%±11.4%** 2.2±0.8* Example 10 Effects of organic acid extracts on photochemical embolization mice 1. Experimental animals To prepare the same experimental animals with reference to example 9. 2. Medicine preparation and administration mode To prepare and administrate medicine with reference to example 9. 3. Establishment of photochemical embolization mouse model 8~1 O-week-old male C57BL / 6 mice were adaptively reared for 3 days, and randomly divided into model group (physiological saline, 10 mL / kg, i.g.) and administration group according to their body weight. Mice were fasted for 12 hours with free access to water, pre-administered 7 Days and performed embolism surgery. Mice were anesthetized with isoflurane, used small animal stereotaxic instrument to immobilize their heads after 3-5 min of anesthesia, administrated 11.875 mg / mL rose bengal at a dose of O.lmL / lOg by intraperitoneal injection and waited for more than 5 minutes to make sure that rose bengal was distributed in the cerebral blood vessels through the circulatory system in sequence. Mice were cut scalp, then placed the light source on the right side of the frontal suture 1.5mm to irradiate with a 16000 lux cold light source for 15 min, and covered with sanitary napkins on their eyes during the process. After irradiation, mice were sutured scalp and disinfected with iodophor, then placed on electric blankets and irradiated with heating lamps. 24 hours after the operation, mice were weighed and respectively administrated with physiological saline solvent and medicine continuously for 14 days in model group and administration group, and behavioral tests were performed on alternate days (1, 3 . . . 15 days). The medicine was dissolved in physiological saline to prepare clear solution and the pH was adjusted to neutral by IM NaHCOs before ready to use. 4. Experimental operation Mice were randomly divided into model group (physiological saline, lOmL / kg, i.g.) and sample 2 group (lOmg / kg, 30mg / kg, i.g.) according to their body weight. Mice were administered with physiological saline and sample 2 respectively and continuously for 14 days after photochemical embolization surgery. The effect of organic acids on photochemical embolization-induced focal cerebral ischemia injury in mice was detected by analyzing the foot fault score of mice in grid test on alternate days (1,3...15 days). Mice were subjected to grid test according to the method reported in literature. The test mice were placed on a 32cm*20cm steel grid (1.2cm* 1.2cm grid inside) to make them walk freely for more than 3 min, and the walking process was recorded by a camera under the grid. The video was played at 0.5 times speed and number of foot fault of the mouse’s forelimb in 100 steps was recorded. Foot fault rate =number of foot fault / 100*100%. 5. Experiment results The experiment results were shown in Table 12 and Figure 5. Compared with the model group, sample 2 group (10 mg / kg and 30 mg / kg) could reduce foot fault rate in mice. The sample 2 could significantly reduce foot fault rate on the 9th Day and 13th Day at dose of 10 mg / kg (p<0.05) and significantly reduce foot fault rate on the 13th Day at dose of 30 mg / kg (p<0.05), which suggested that GLOA sample 2 could partially alleviate motor sensory function of the post-cerebral ischemia mice in the photochemical embolization model. Table 12 Results of foot fault rate in photochemical embolization test on mice (Mean±SD, n=9) Day Model Sample 2 10 mg / kg 30 mg / kg 0 6.7%±1.5% 7.0%±1.8% 6.8%±1.7% 1st 22.8%±4.0% 19.5%±4.1% 17.0%±4.4% 3rd 18.63%±4.9% 15.0%±4.8% 13.8%±3.9% 5th 21.29%±4.1% 16.4%±5.4% 13.0%±3.2% 7th 17.89%±5.5% 12.7%±5.3% 15.1%±4.3% 9th 19.20%±6.1% 12.9%±3.9%* 13.4%±4.6% 11th 16.94%±6.1% 13.8%±4.5% 10.8%±3.3% 13 th 16.28%±3.6% 12.9%±4.3%* 12.0%±3.7%* 15 th 13.00%±7.6% 11.4%±3.9% 8.0%±2.2% Note: * indicates the comparison with the model group, p<0.05.

Claims

14 11 241. A Ginkgo leaf organic acid extract, wherein the preparation method of the Ginkgo leaf organic acid extract comprises the following steps:(1) selecting a Ginkgo leaf extract that meets standards of Chinese Pharmacopoeia as raw material;(2) dissolving the raw material from step (1) in boiling water or ethanol solution with a concentration of less than or equal to 10% (v / v) to obtain a crude extract solution I;(3) purifying the crude extract solution I through a polyamide column, eluting with 3~5 BV of 5%-10%(\ \) ethanol at a flow rate of 1-3 BV / h, collecting eluate and concentrating to 1-2 BV, filtering and collecting filtrate to obtain crude extract solution II;(4) purifying the crude extract solution II through a macroporous resin column, eluting with 3-5 BV of 10%~15%(v / v) ethanol at a flow rate of 1-3 BV / h, collecting ethanol eluate, concentrating and drying to obtain the Ginkgo leaf organic acid extract;wherein a content of 6-Hydroxykynurenic acid in the Ginkgo leaf extract obtained by step (I) is greater than 2 mg / g, and the content of the gallic acid and 4-Hydroxybenzoic acid are higher than 1 mg / g.

2. The Ginkgo leaf organic acid extract according to claim 1, wherein the polyamide column of step (3) has pore size of 0.55mm-1,7mm or 0.25mm~ 0.55mm.

3. The Ginkgo leaf organic acid extract method according to claim 1, wherein in step (4) macroporous resin column is selected from D101 or AB-8, and ethanol eluate is concentrated and dried at 50°C —70 °C.

4. A Ginkgo leaf organic acid extract of extract according to claim 1 for use in preventing and / or treating cerebral ischemic injury.

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