CN115006435B - Ginkgo leaf organic acid extract and preparation method and application thereof - Google Patents
Ginkgo leaf organic acid extract and preparation method and application thereof Download PDFInfo
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- CN115006435B CN115006435B CN202210705631.8A CN202210705631A CN115006435B CN 115006435 B CN115006435 B CN 115006435B CN 202210705631 A CN202210705631 A CN 202210705631A CN 115006435 B CN115006435 B CN 115006435B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/16—Ginkgophyta, e.g. Ginkgoaceae (Ginkgo family)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a ginkgo leaf organic acid extract, which comprises the following medicinal components, by weight, 60% -68% of protocatechuic acid, 10% -14% of 6-hydroxykynurenic acid, 5% -8% of gallic acid, 3% -8% of p-hydroxybenzoic acid, and more than 80% of total organic acid. According to the invention, through controlling the content of protocatechuic acid, 6-hydroxykynurenic acid, gallic acid and p-hydroxybenzoic acid and controlling the content ratio of protocatechuic acid to other 3 organic acids, the obtained ginkgo leaf organic acid extract has a remarkable effect of treating chronic ischemic cerebral apoplexy, and realizes synergistic effect. The invention also provides a preparation process of the extract, which comprises dissolving ginkgo leaf extract, polyamide column chromatography, macroporous resin column chromatography and concentrating and drying. The preparation process realizes the reutilization of production waste liquid, reduces the production cost, and adopts low-concentration ethanol as a solvent and an eluting reagent to realize green production.
Description
Technical Field
The invention belongs to the field of pharmaceutical chemicals, and in particular relates to a ginkgo leaf organic acid extract, a preparation method and application thereof in preparing a medicament for preventing and/or treating cerebral ischemia injury.
Background
The ginkgo leaf extract preparation is a plant extract preparation commonly used for heart and cerebral vessels, and is also one of the most widely used natural pharmaceutical preparations worldwide. The ginkgo leaf extract is subjected to multiple pharmacopoeia standard modification after being marketed in China, and the standard requirement of the ginkgo leaf extract in Chinese pharmacopoeia of 2020 edition is that the total flavonol glycoside is more than or equal to 24 percent, the terpene lactone is more than or equal to 6 percent, and the peak area ratio of quercetin/kaempferol is 0.8 to 1.5; the terpene lactone content is not less than 0.2% based on total amount of bilobalide A, bilobalide B, bilobalide C and bilobalide; ginkgolic acid is less than or equal to 5mg/kg; the peak area ratio of the quercetin to the kaempferol is 0.8-1.2, and the peak ratio of the isorhamnetin to the quercetin is more than 0.15; the content of quercetin and kaempferol is not more than 1.00%, and that of isorhamnetin is not more than 0.40%. As can be seen from the pharmacopoeia standard of 2020 edition, ginkgo flavone and lactones are the main effective components of ginkgo leaf extract, but the content of the corresponding components is less than 40%. Other substances, such as organic acids, procyanidins, polyprenols, polysaccharides and proteins, have great part of better pharmacodynamic activity, can be used as indexes for improving the pharmacodynamic effect and quality of the ginkgo leaf extract, and even provide a new plant extract with obvious pharmacodynamic effect.
The organic acid component in semen Ginkgo comprises alkyl phenolic acid compound and large polar organic acid component. The major polar organic acids in folium Ginkgo extract comprise shikimic acid, 6-hydroxykynurenic acid (6-Hydroxykynurenic acid, 6-HKA), protocatechuic acid (Protocatechuic acid), quinic acid, chlorogenic acid, etc. Wherein shikimic acid is the synthetic raw material of oseltamivir which is an anti-avian influenza drug, and has various pharmacological activities such as antibiosis, anti-inflammatory, antiplatelet, antithrombotic and the like. 6-HKA has higher affinity to glutamate receptor as canine uric acid derivative, and inhibiting glutamate receptor can avoid overactivation of multiple receptors harmful to neurons, thereby playing a certain neuroprotective role. Protocatechuic acid has various pharmacological activities such as anti-inflammatory, antioxidant and anti-tumor, can improve neurocognitive function damage caused by chronic intermittent hypoxia, enhance learning and memory ability of chronic intermittent hypoxia rats, and relieve oxidative stress and apoptosis.
Cerebral apoplexy, also known as "stroke", is a cerebrovascular disease in which the blood supply to the brain area is interrupted, resulting in death or permanent neurological impairment. The disease is the second leading cause of death worldwide and is also the leading cause of physical disability in adults. Cerebral apoplexy can be classified into ischemic or hemorrhagic cerebral apoplexy, and ischemic cerebral apoplexy accounts for 85% of the total cerebral arterial thrombosis. In the ginkgo-derived preparation, ginkgo dipyridamole injection, shuxuening injection and bilobalide injection are used for treating acute ischemic cerebral apoplexy; an oral preparation of folium Ginkgo extract and ginkgo ketoester for treating chronic ischemic cerebral apoplexy. However, the main components of the existing ginkgo source preparation are ginkgo total flavone, bilobalide and ginkgo leaf extract, and the medicinal value and market value of the ginkgo leaf organic acid are not fully mined.
In the prior art, researches on organic acid extract of ischemic cerebral apoplexy are less reported that organic acid of ginkgo leaves is not used as an effective component of the extract.
Patent CN108524531a "a pharmaceutical composition" (filing date 2017.03.01, publication date 2018.09.14) defines a pharmaceutical composition for treating ischemic stroke, which comprises: 18.1 to 50.9 percent of lactone component; 48.8 to 81.8 percent of flavonoid component; organic acid component 0.07% -6.83%, wherein the organic acid is chlorogenic acid and protocatechuic acid. However, the main active ingredients of the technical scheme are lactone and flavone, and the content ratio of the lactone compounds is limited by limiting the content ratio of the flavone compounds, so that the cerebral infarction area of the mice with acute cerebral ischemia reperfusion models is reduced, and the neurological score of the mice is reduced.
Patent CN114366761A "preparation method of ginkgo leaf extract rich in organic acid" (application date 2022.01.18, publication date 2022.04.19) defines a method for obtaining the total content of organic acid which is more than or equal to 17% after the ginkgo leaf is sequentially subjected to ethanol extraction by a cation exchange resin column, a macroporous resin column and an anion exchange resin column and the ginkgo acid is removed. The shikimic acid content in the organic acid is more than or equal to 15%, the quinic acid content is more than or equal to 1%, the protocatechuic acid content is more than or equal to 1%, and the 6-HKA content is more than or equal to 0.3%. In addition, the content of the total flavonol glycoside is 24-27%, and the content of the terpene lactone is 6.0-6.6%. However, pharmacological data are not disclosed in the above-mentioned techniques, and the effect of the pharmacological effects is not known.
In addition, the prior art also discloses an organic acid extract of ginkgo biloba source which takes shikimic acid and 6-HKA as main components. Patent CN103784448A 'A preparation method and application of natural 6-hydroxykynurenic acid extract' (application date 2014.01.07, publication date 2015.11.18) defines an extract with 50% -90% of 6-HKA content, which is prepared by separating from ginkgo leaf extract macroporous resin aqueous solution, and the cerebral infarction area of acute ischemic reperfusion cerebral apoplexy mice is reduced from 31.5+/-3.8% of model group to 20.1+/-2.1% under the dosage of 3mg/kg, so that the extract has better acute ischemic reperfusion cerebral apoplexy protection effect.
Patent CN107353201a "a high-content natural shikimic acid extract and its preparation method" (filing date 2016.05.09, publication date 2017.11.17) defines an extract with shikimic acid content of more than 98% separated and prepared from waste water in the production process of ginkgo leaf extract, the extract does not disclose pharmacological data, and the pharmacodynamic effect is unknown.
From the above, the prior art discloses an extract containing ginkgo leaf organic acid as an active ingredient, but the content of the organic acid is low, the ingredient is not clear, and the drug effect of chronic ischemic cerebral apoplexy is not obvious.
Disclosure of Invention
The invention aims to solve the technical problems that: provides a plant extract with obvious medicinal effect and definite components for treating ischemic cerebral apoplexy. The extract can be developed into a medicine, and can meet the market demand of traditional Chinese medicine preparations for chronic ischemic cerebral apoplexy. The invention provides a ginkgo leaf organic acid extract, which comprises protocatechuic acid,6-HKA, gallic acid and p-hydroxybenzoic acid, and has remarkable effect in treating cerebral ischemia injury, especially in treating chronic ischemic cerebral apoplexy.
The technical scheme of the invention is as follows: the ginkgo leaf organic acid extract comprises, by weight, 60% -68% of protocatechuic acid, 10% -14% of 6-HKA, 5% -8% of gallic acid, 3% -8% of p-hydroxybenzoic acid, and the total organic acid content of the extract is more than 80%.
The invention further defines the content proportion of organic acid, and the content of protocatechuic acid,6-HKA, gallic acid and parahydroxybenzoic acid meets one of the following conditions: wherein the content ratio of protocatechuic acid/6-HKA is 4-6; or the content ratio of protocatechuic acid/gallic acid is 9-12; or the content ratio of protocatechuic acid/p-hydroxybenzoic acid is 8-14.
Gallic acid (Gallic acid), 3,4, 5-trihydroxybenzoic acid, also known as Gallic acid or Gallic acid, is the simplest natural polyphenol substance in chemical structure, widely exists in various plants, vegetables and fruits, and has good medicinal and health care values. Because of the existence of a plurality of phenolic hydroxyl groups in the structure, the gallic acid has good antioxidation effect and is a natural antioxidant.
P-hydroxybenzoic acid (P-hydroxy benzoic acid), also known as nipagin acid, is a widely used organic synthetic raw material. The p-hydroxybenzoic acid has the pharmacological effects of resisting bacteria, reducing blood sugar, resisting inflammation and the like.
According to the invention, through controlling the content of protocatechuic acid,6-HKA, gallic acid and p-hydroxybenzoic acid and controlling the content ratio of protocatechuic acid to other 3 organic acids, the obtained ginkgo leaf organic acid extract has the remarkable effect of treating cerebral ischemia injury, especially in chronic ischemic cerebral apoplexy, and realizes synergistic effect.
The second object of the present invention is to provide the preparation method of the above extract, which is prepared by selecting ginkgo leaf extract meeting national pharmacopoeia standards, combining ginkgo leaf extract dissolution, polyamide column chromatography, macroporous resin column chromatography, concentration and drying. The method is characterized by comprising the following steps of:
(1) Selecting ginkgo leaf extract meeting national pharmacopoeia standard as raw material.
Preferably, the ginkgo leaf extract with high organic acid is used as a raw material, and screening is carried out by an organic acid content detection method and high organic acid component indexes;
preferably, the high organic acid content index comprises a 6-HKA content of greater than 2mg/g, a parahydroxybenzoic acid content of greater than 1mg/g, and a gallic acid content of greater than 1mg/g.
(2) Dissolving the raw materials in the step (1) with boiling water or ethanol with concentration of less than 10% to prepare a sample loading liquid 1.
(3) The sample loading liquid 1 passes through a polyamide column, 3 BV-5 BV is eluted by 5% -10% ethanol, the flow rate is 1 BV/h-3 BV/h, the effluent liquid and the ethanol eluent are collected, the column volume is concentrated to 1-2 times, and the filtrate is taken as the sample loading liquid 2.
Preferably, the polyamide column is 10-30 mesh, 30-60 mesh.
(4) The sample liquid 2 passes through a macroporous resin column, 3 BV-5 BV is eluted by 10% -15% ethanol, the flow rate is 1 BV/h-3 BV/h, the ethanol eluent is collected, concentrated and dried, and the ginkgo leaf organic acid extract is obtained.
Preferably, the macroporous resin column in the step (4) is D101 or AB-8, and the concentration temperature is 50-70 ℃.
A third object of the present invention is to provide the use of the above extract for the preparation of a medicament for the prevention and/or treatment of cerebral ischemic injury including chronic ischemic stroke.
The invention has the technical advantages that:
(1) The ginkgo leaf organic acid extract provided by the invention has definite components and has a synergistic effect in treating cerebral ischemia injury, especially chronic ischemic cerebral apoplexy. The test results of the monomer compounds show that the drug effect of the 6-HKA is superior to that of other 3 organic acids. As will be appreciated by those of ordinary skill in the art, increasing the proportion of other 3 organic acid monomers at the same concentration will decrease the proportion of 6-HKA to result in a mixture having a neuroprotective effect less than or equal to the corresponding effect of 6-HKA monomers.
The test result of the mouse neuroma blast N2a oxygen sugar deprivation reoxygenation (OGD/R) injury model shows that the cell survival rate of the samples 1-3 provided by the invention is 73.6% -77.6%, which is obviously higher than that of the samples 6-HKA (64.2%), and is shown in the table 1. The total organic acid content of samples 1-3 was similar to sample 5, eliminating the hypothesis that the total organic acid content affects neuroprotection. It can be deduced from this that the neuroprotection of samples 1 to 3 is advantageous in determining the content and content ratio of protocatechuic acid,6-HKA, gallic acid and parahydroxybenzoic acid. The specific organic acid content range corresponding to the samples 1-3 has the technical effect of neuroprotection and synergy, wherein the content ratio of protocatechuic acid/6-HKA is 4-6, the content ratio of protocatechuic acid/gallic acid is 9-12, and the content ratio of protocatechuic acid/parahydroxybenzoic acid is 8-14. Table 2 shows the organic acid extract components protected by the present invention.
TABLE 1 results of the test of the organic acid extract protected by the present invention on the oxygen glucose deprivation and reoxygenation model of mouse neuroma cells
Grouping | Name of the name | N | Survival rate% (mean+ -SD) |
Blank space | Blank | 4 | 98.9%±6.8% |
OGD/R model | OGD/R | 5 | 51.8%±7.7% △△△ |
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-hydroxykynuroquinolinic acid | OGD/R+6-HKA | 4 | 64.2%±7.3% |
OGD/R+ gallic acid | OGD/R+GA | 4 | 59.1%±12.1% |
OGD/R+p-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%* |
Note that: △△△ representation in contrast to blank, p<0.01; * Representation is compared to the OGD/R model group, p<0.05。
TABLE 2 organic acid extract composition of the invention
(2) The invention adopts the combination of raw material control and separation extraction to realize the preparation process protection of the ginkgo leaf organic acid extract. The ginkgo leaf extract is prepared by adopting ginkgo leaf extract raw material control, adopting boiling water or ethanol with the concentration of less than 10 percent to dissolve, adopting polyamide column chromatography, taking 5 to 10 percent ethanol eluent, and then carrying out macroporous resin column chromatography, purifying and concentrating.
Comparing the preparation process of the organic acid of samples 1-5, it is found that sample 2 and sample 5 adopt the same preparation process, and only the raw materials are different. Wherein the content of 6-HKA in the ginkgo leaf extract raw material of sample 5 is 1.24mg/g, and the content of 6-HKA in sample 5 is 6.62%, which are 1/2 of the corresponding value of sample 2. It can be seen that the control of the components of the ginkgo leaf extract as raw material has an effect on the organic acid content of the finished product.
The invention limits the content of organic acid in the raw material ginkgo leaf extract: the content of 6-HKA is more than 2mg/g, and the content of gallic acid and p-hydroxybenzoic acid is more than 1mg/g, so that the quality control of raw materials can be realized.
Sample 4 and sample 2 are prepared from the raw materials in the quality control range, and the preparation process is different from that of a polyamide column and macroporous resin column eluent; the corresponding obtained sample 4 was lower in total organic acid content, protocatechuic acid and 6-HKA content than sample 2. From this, it can be seen that samples 1-3 only achieve synergy under specific raw material control and specific separation extraction conditions. The raw materials, preparation process and finished product pairs of samples 2, 4 and 5 are shown in Table 3, and the drug effect results are shown in Table 4.
Table 3 raw materials, preparation process and finished product comparison of samples 2, 4 and 5
Table 4 samples 2, 4 and 5 comparison of results of the model test of oxygen glucose deprivation and reoxygenation in mouse neuroma blast
Note that: △△△ representation ofIn contrast to the blank, p<0.01; * Representation is compared to the OGD/R model group, p<0.05。
(3) The ginkgo leaf extract meeting national pharmacopoeia standards is taken as a raw material, boiled water or ethanol with the concentration of less than 10% is adopted for dissolution, polyamide column chromatography is adopted, 5% -10% ethanol eluent is taken, macroporous resin column chromatography is further carried out, and purification and concentration are carried out, thus obtaining the ginkgo leaf extract.
The invention adopts the preparation method of step 1) and step 2) in the prior patent application CN202010732010.X 'a ginkgo leaf flavone extract and a preparation method and a characteristic spectrum construction method' of a research and development team, and the ginkgo leaf flavone extract is obtained after macroporous resin column chromatography and concentration. Wherein, the sample liquid in the step 2) is eluted by polyamide column purified water, and organic acid and lactone components are collected by eluting with ethanol with concentration less than 10 percent, and the operation does not interfere the collection of flavonoid components by eluting with 0.1 to 0.5 percent sodium carbonate solution in the subsequent step. The technical scheme of the invention realizes the reutilization of the production waste liquid and reduces the production cost. In addition, the preparation process adopts low-concentration ethanol as a solvent and an eluting reagent, thereby realizing green production.
(4) The ginkgolic acid is not detected by the organic acid extract of the ginkgo leaves, so that the requirement that the ginkgolic acid in the ginkgo leaf extract required by Chinese pharmacopoeia is less than 1ppm is met, and the application is safe.
Drawings
FIG. 1 shows a liquid phase diagram of ginkgo leaf extract, wherein GA is gallic acid, PA is protocatechuic acid, HBA is p-hydroxybenzoic acid, and 6-HKA is 6-hydroxykynurenic acid.
Fig. 2 shows cytotoxicity test results of ginkgo leaf organic acid extract in mouse neuroma blast, wherein Blank is Blank, NBP is positive control butylphthalide, EDA is positive control edaravone, GBE is preparation raw material ginkgo leaf extract, PA is protocatechuic acid, GA is gallic acid, HBA is p-hydroxybenzoic acid, EOA1 is sample 1, EOA2 is sample 2, EOA3 is sample 3, EOA4 is sample 4, and EOA5 is sample 5. The higher the cell viability, the less cytotoxic.
FIG. 3 shows the results of a test of the organic acid extract of ginkgo leaf in a mouse neuroma blast glucose deprivation reoxygenation model, wherein Blank is a Blank control group, OGD/R is a model group, OGD/R+NBP is a butylphthalide positive control group, OGD/R+EDA is an edaravone positive control group, OGD/R+GBE is a prepared raw material ginkgo leaf extract group, OGD/R+PA is a protocatechuic acid group, OGD/R+GA is a gallic acid group, OGD/R+HBA is a parahydroxybenzoic acid group, OGD/R+EOA1 is a sample 1 group, OGD/R+EOA2 is a sample 2 group, OGD/R+EOA3 is a sample 3 group, OGD/R+EOA4 is a sample 4 group, and OGD/R+EOA5 is a sample 5 group. The higher the cell viability, the better the neuroprotection.
FIG. 4 shows the results of the test of organic acid extract in mice with chronic ischemic cerebral apoplexy, and FIG. 4-1 shows brain slices of the results of the test of arterial embolism in the brains of mice, wherein Model is a Model group, EOA2 is a sample 2 group, and the greater the light area on the right side of the slices, the higher the damage degree of brain tissues on the right side; FIG. 4-2 is a graph showing cerebral infarction volume rate of middle cerebral artery embolism in mice; figures 4-3 are neurological symptom scoring profiles in the brain of mice. The lower the neurological symptom score, the better the neuroprotective effect.
FIG. 5 shows the results of a photochemical embolism experiment in mice, wherein the Model group is a Model group, and EOA2 is a sample 2 group; the lower the step-by-step rate is, the better the photochemical embolism inhibition effect of the mice is.
Detailed Description
The invention will be further illustrated with reference to specific examples, but the scope of the invention as claimed is not limited to the following embodiments.
The ginkgo leaf extract used in the following examples is prepared according to the 2020 edition of Chinese pharmacopoeia, manufacturer: zhejiang Kang Enbei pharmaceutical Co., ltd., production lot: 191106;190301;190109;180307;180807;180912.
example 1 organic acid component detection method
The method for detecting the organic acid components and the content in the ginkgo leaf extract and the ginkgo leaf organic acid extract is not limited to HPLC, UPLC, LC-MS and other methods.
The UPLC detection method provided by the invention is optimized on the basis of the existing patent ZL201510826946.8 of the research team, namely a method for measuring the content of organic acid in ginkgo leaf extract. Adopting UPLC to replace an HPLC detection method; the pretreatment step of removing procyanidine from bovine serum albumin is omitted, and the quantitative detection of 5 organic acids is realized within 14 min. The specific detection method comprises the following steps:
1. preparation of test solution
Taking appropriate amount of folium Ginkgo extract sample, precisely weighing, placing in 10mL measuring flask, adding small amount of purified water, ultrasound for 20min, cooling, adding purified water to volume to scale, shaking, filtering, and collecting filtrate.
2. Preparation of control solution
And respectively taking appropriate amounts of the protocatechuic acid reference substance, the gallic acid reference substance and the p-hydroxybenzoic acid reference substance in respective 50mL measuring flasks, precisely weighing, adding 40% methanol for dissolving, fixing the volume to a scale, and shaking uniformly to obtain protocatechuic acid reference substance stock solution, gallic acid reference substance stock solution and p-hydroxybenzoic acid reference substance stock solution.
Taking a proper amount of the 6-hydroxykynurenic acid reference substance, accurately weighing the 6-hydroxykynurenic acid reference substance in a 100mL measuring flask, dissolving the 6-hydroxykynurenic acid reference substance in methanol, fixing the volume to a scale, and shaking the solution uniformly to obtain the 6-HKA reference substance stock solution.
3. UPLC detection conditions
Chromatographic conditions and system adaptation test: octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.1% trifluoroacetic acid solution is used as a mobile phase; the flow rate is 0.4mL/min; the detection wavelength is 254nm; the column temperature was 30 ℃. The flow ratios are shown in table 5, for example:
table 5 flow comparative example
Time (min) | 0.1% trifluoroacetic acid (%) | 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 |
Precisely sucking 5 μl of reference solution and sample solution, respectively, and injecting into ultra-high performance liquid chromatograph for measurement.
4. Experimental results
4.1 linearity and Range
The protocatechuic acid reference substance, the 6-HKA reference substance, the gallic acid reference substance and the p-hydroxybenzoic acid reference substance are precisely weighed, reference substance stock solutions with different concentrations are prepared, the analysis is carried out according to the chromatographic conditions, the peak area is taken as an ordinate (y), the reference substance concentration (mug/mL) is taken as an abscissa, a standard curve is drawn, and the results are shown in Table 6.
TABLE 6 Linear regression equation for UPLC detection of organic acids
Organic acid | Regression equation | Linear range (μg/mL) |
Protocatechuic acid | y=44730.9338x+4525.3107,R2=1.0000 | 40.04~200.2 |
6-HKA | y=122148.1405x-9249.5026,R2=1.0000 | 7.776~38.88 |
Gallic acid | y=116010.113x-17159.2753,R2=1.0000 | 5.896~29.48 |
Para-hydroxybenzoic acid | y=76795.6281x+10988.6000,R2=0.9997 | 5.896~29.48 |
4.2 organic acid content in Ginkgo biloba leaf extract
The sample solution of ginkgo leaf extract was analyzed according to the above chromatographic conditions, and the experimental results are shown in table 7.
TABLE 7 organic acid content (unit: mg/g) in ginkgo leaf extract raw materials
Batch 1 | Batch 2 | Batch 3 | Batch 4 | Batch 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 |
Para-hydroxybenzoic acid | 1.75 | 1.17 | 1.26 | 1.41 | 0.65 |
Totals to | 19.95 | 19.54 | 21.74 | 20.33 | 23.04 |
Example 2 preparation of organic acid extract sample 1
(1) Taking 500g of batch 1 ginkgo leaf extract as a raw material;
(2) Dissolving the raw materials in the step (1) by boiling water to prepare a sample loading liquid 1;
(3) Passing the sample loading liquid 1 through a 10-30-mesh polyamide column, eluting with 10% ethanol at a flow rate of 1BV/h, collecting effluent and ethanol eluent, concentrating to 2 column volumes, and filtering to obtain filtrate as sample loading liquid 2;
(4) Loading liquid 2 is passed through a D101 macroporous resin column, eluting with 10% ethanol for 5BV at an eluting flow rate of 1BV/h, collecting ethanol eluent, concentrating and drying at 50-70deg.C to obtain folium Ginkgo organic acid extract sample 1.
Example 3 preparation of organic acid extract sample 2
(1) Taking 2kg of batch 2 ginkgo leaf extract as a raw material;
(2) Dissolving the raw materials in the step (1) by 8% ethanol to prepare a sample loading liquid 1;
(3) Passing the sample loading liquid 1 through a 30-60-mesh polyamide column, eluting with 5% ethanol at a flow rate of 3BV/h, collecting effluent and ethanol eluent, concentrating to 1 column volume, and filtering to obtain filtrate as sample loading liquid 2;
(4) Loading the sample liquid 2 through a D101 macroporous resin column, eluting with 15% ethanol at a flow rate of 3BV/h, collecting ethanol eluate, concentrating at 50-70deg.C, and drying to obtain folium Ginkgo organic acid extract sample 2.
Example 4 preparation of organic acid extract sample 3
(1) Taking 500g of batch 3 ginkgo leaf extract as a raw material;
(2) Dissolving the raw materials in the step (1) by using 10% ethanol to prepare a sample loading liquid 1;
(3) Passing the sample loading liquid 1 through a 30-60-mesh polyamide column, eluting with 5% ethanol at a flow rate of 2BV/h, collecting effluent and ethanol eluent, concentrating to 2 column volumes, and filtering to obtain filtrate as sample loading liquid 2;
(4) Loading liquid 2 passes through an AB-8 macroporous resin column, eluting with 15% ethanol for 3BV at an eluting flow rate of 2BV/h, collecting ethanol eluent, concentrating and drying at 50-70 ℃ to obtain ginkgo leaf organic acid extract sample 3.
Example 5 preparation of organic acid extract sample 4
(1) Taking 500g of batch 4 ginkgo leaf extract as a raw material;
(2) Dissolving the raw materials in the step (1) by boiling water to prepare a sample loading liquid 1;
(3) Passing the sample loading liquid 1 through a 10-30-mesh polyamide column, eluting with 15% ethanol at a flow rate of 2BV/h, collecting effluent and ethanol eluent, concentrating to 2 column volumes, and filtering to obtain filtrate as sample loading liquid 2;
(4) Loading liquid 2 passes through a D101 macroporous resin column, 5BV is eluted by 5% ethanol, the eluting flow rate is 3BV/h, the ethanol eluent is collected, and the concentration and drying are carried out at 50-70 ℃ to obtain a ginkgo leaf organic acid extract sample 4.
Example 6 preparation of organic acid extract sample 5
(1) Taking 500g of batch 5 ginkgo leaf extract as a raw material;
(2) Dissolving the raw materials in the step (1) by 8% ethanol to prepare a sample loading liquid 1;
(3) Passing the sample loading liquid 1 through a 30-60-mesh polyamide column, eluting with 5% ethanol at a flow rate of 3BV/h, collecting effluent and ethanol eluent, concentrating to 1 column volume, and filtering to obtain filtrate as sample loading liquid 2;
(4) Loading liquid 2 passes through a D101 macroporous resin column, eluting with 15% ethanol for 5BV, eluting with flow rate of 3BV/h, collecting ethanol eluate, concentrating and drying at 50-70deg.C to obtain folium Ginkgo organic acid extract sample 5.
The organic acid extracts of examples 2 to 6 described above had a transfer rate of 55% to 65%.
Example 7 determination of organic acid component content
The ginkgo leaf extracts prepared in examples 2 to 7 were subjected to content measurement in the same manner as in example 1. The experimental results are shown in table 8:
TABLE 8 organic acid component content of samples 1 to 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% |
Para-hydroxybenzoic acid | 7.68% | 6.65% | 5.80% | 6.92% | 5.17% |
Totals to | 86.46% | 91.67% | 91.59% | 79.42% | 86.07% |
Example 8 Effect of different organic acid samples on the survival rate of oxygen glucose deprived reoxygenated mouse neuroma blasts (N2 a cells)
1. Pharmaceutical formulation
Commercial ginkgo leaf extract, 5 organic acid compounds, organic acid extract samples 1-5, positive control butylphthalide and edaravone are prepared into 100mg/mL stock solution by using DMSO solution, and stored at-20 ℃. The medium was diluted to 1. Mu.g/mL for use at the time.
2. Culture of mouse neuroma blast
Mouse neuroma cells (N2 a) were routinely cultured in DMEM medium containing 10% fetal calf serum, 100U/mL penicillin and 0.1mg/mL streptomycin at 37℃with 5% CO 2 Culturing in a +95% air incubator. When a confluence of about 75% is reached, cell passaging is performed.
3. Cytotoxicity evaluation of drugs
Selecting a mouse neuroma blast N2a cell, treating the cell with N-2 (1% of a DMEM culture solution without bovine serum) for 24 hours, differentiating the cell into a neuron-like cell, changing the cell into a normal culture medium, pre-dosing, incubating for 2 hours, and respectively adding ginkgo leaf extract, 5 organic acid compounds, organic acid extract samples 1-5, positive control butylphthalide and edaravone for detection, wherein each group is provided with 3 holes. And (3) measuring an OD value at 490nm by using an enzyme-labeled instrument by using a CCK-8 method, and detecting toxicity evaluation of the drug on cells.
4. Study of N2a cytoprotective action of drugs on OGD/R injury
The primary neurons of mice were treated with an oxygen-glucose deprivation/reperfusion (OGD/R) model and tested for neuroprotection by organic acids in an in vitro simulated ischemic environment. N2a cells (3000 cells/well, 100 μl of conventional DMEM per well, 96-well plate) were seeded and allowed to adhere overnight, and then the cells were differentiated into neuron-like cells after 24h treatment with N-2 (1%, 100 μl of serum-free DMEM per well). The normal medium (100. Mu.L of conventional DMEM per well) was changed and pre-treated with ginkgo leaf extract, 5 organic acid compounds, organic acid extract samples 1-5, positive control butylphthalide and edaravone each 1. Mu.g/mL for 2h, respectively. After washing with PBS (1 time, 100. Mu.L/well), the medium was replaced with glucose-free and serum-free DMEM (100. Mu.L/well). Placing a cell plate on CO 2 In incubator (5% CO) 2 +95%N 2 25L/min,6 min) and incubated (37 ℃) for 4h. Treated with conventional DMEM (100. Mu.L/well) followed by conventional incubation (5% CO) 2 +95% air, 37 ℃) for 4h of cells was used as OGD/R model group. After the OGD/R model was established, cells were treated with different drugs for 24h. The protection effect of the drug on cells was detected by measuring OD value at 490nm with an enzyme-labeled instrument by CCK-8 method.
5. Test results
5.1 cytotoxicity evaluation
The experimental results are shown in table 9.
TABLE 9 cytotoxicity test results of organic acid extracts
The test results show that the ginkgo leaf extract, the organic acid compounds and the samples 1-5 have better safety at the administration concentration of 1 mug/mL.
5.2 results of N2a cytoprotective action of drugs on OGD/R injury
Under the condition that the oxygen deprivation model is successful, the N2a cell survival rate of the 4 organic acid compounds is greater than that of the OGD/R model group, which indicates that the 4 monomer compounds have N2a cell protection effect. Wherein, the efficacy of the 6-HKA is better than that of other 3 organic acids and positive control butylphthalide. As shown in table 10.
TABLE 10N 2a cytoprotective effect results of organic acid extracts
Grouping | Name of the name | N | Survival rate% (mean+ -SD) |
Blank space | Blank | 4 | 98.9%±6.8% |
OGD/R model | OGD/R | 5 | 51.8%±7.7% △△△ |
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-hydroxykynuroquinolinic acid | OGD/R+6-HKA | 4 | 64.2%±7.3% |
OGD/R+ gallic acid | OGD/R+GA | 4 | 59.1%±12.1% |
OGD/R+p-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 that: △△△ representation in contrast to blank, p<0.01; * Representation is compared to the OGD/R model group, p<0.05。
The N2a cell viability of samples 1-3 was 73.6% to 77.6%, significantly higher than 6-HKA (64.2%). Moreover, the total organic acid content of samples 1-3 is 86.46% -91.67%, which is similar to the total organic acid content of sample 5 by 87.61%, and the hypothesis that the total organic acid content influences the neuroprotection is eliminated. From this, it can be inferred that the specific organic acid content ranges corresponding to samples 1 to 3 have the technical effect of neuroprotection synergy. Wherein the content ratio of protocatechuic acid/6-HKA is 4-6, the content ratio of protocatechuic acid/gallic acid is 9-12, and the content ratio of protocatechuic acid/p-hydroxybenzoic acid is 8-14.
The invention adopts the preparation method of step 1) and step 2) in the prior patent application CN202010732010.X 'a ginkgo leaf flavone extract and a preparation method and a characteristic spectrum construction method' of a research and development team, and the ginkgo leaf flavone extract is obtained after macroporous resin column chromatography. Wherein, the sample liquid in the step 2) is eluted by polyamide column purified water, and the organic acid and lactone components are collected by eluting with ethanol with concentration less than 10 percent, and the operation does not interfere the collection of flavonoid components by eluting with 0.1 to 0.5 percent (v/v) sodium carbonate solution in the subsequent step. Therefore, the invention fully enriches the effective components of the neuroprotection in the ginkgo leaf extract, and realizes the reutilization of the production waste liquid; the ginkgo leaf organic acid extract has the advantages of being clear in component and obvious in neuroprotection while reducing production cost.
The preparation process of analysis samples 1-5 found that sample 5 and sample 2 used the same preparation process, with only a large difference in raw materials. Wherein the content of 6-HKA in sample 5 was 1.24mg/g, and the content of 6-HKA in sample 5 was 6.62%, which were 1/2 of that in sample 2. Under the condition that the transfer rate of the organic acid extract meets 55% -65%, the control of the components of the raw material ginkgo leaf extract has an influence on the organic acid content of the finished product. The content of organic acid in the ginkgo leaf extract of the raw materials is limited by combining the component range of the ginkgo leaf extract of the raw materials 1-3 and excluding the data with larger difference in the raw materials of the sample 5, the content of 6-HKA is more than 2mg/g, and the content of gallic acid and p-hydroxybenzoic acid is more than 1mg/g, so that the quality control of the raw materials is realized.
Sample 4 and sample 2 are prepared from the raw materials within the quality control range, and the preparation process is different in the concentration of ethanol in the eluent of the polyamide column and the macroporous resin column, and the content of total organic acid, protocatechuic acid and 6-HKA of the corresponding obtained sample 4 is lower than that of sample 2. From this, samples 1 to 3 were obtained under specific raw material control and specific separation and extraction conditions. The preparation process parameters of the samples 1 to 3 are combined, the individual process parameters in the sample 4 are eliminated, and the protection scope of the preparation process is limited as follows:
(1) Taking ginkgo leaf extract meeting national pharmacopoeia standards as a raw material, wherein the content of 6-hydroxykynurenic acid is more than 2mg/g, the content of parahydroxybenzoic acid is more than 1mg/g, and the content of gallic acid is more than 1mg/g;
(2) Dissolving the raw materials in the step (1) with boiling water or ethanol with concentration of less than 10% to prepare a sample loading liquid 1;
(3) Passing the sample loading liquid 1 through a polyamide column, eluting with 5% -10% ethanol at a flow rate of 1 BV/h-3 BV/h and 3 BV-5 BV, collecting effluent and ethanol eluent, concentrating to 1-2 times of column volume, and filtering to obtain filtrate as sample loading liquid 2;
(4) The sample liquid 2 passes through a macroporous resin column, 3 BV-5 BV is eluted by 10% -15% ethanol, the flow rate is 1 BV/h-3 BV/h, the ethanol eluent is collected, concentrated and dried, and the ginkgo leaf organic acid extract is obtained.
Example 9 Effect of organic acid extracts on chronic ischemic cerebral apoplexy mice
According to the cell experiment result of example 8, the drug effect of the sample 2 was further verified by chronic ischemic stroke mice.
1. Experimental animal
C57BL/6 male mice for 8-10 weeks, supplied by Shanghai Laetto laboratory animal Co., ltd., animal eligibility number SCXK (Shanghai) 2017-0005. In the experimental process, animal feeding, drug administration, surgery and material drawing are all in compliance with the experimental animal management regulations.
2. Drug formulation and administration mode
Organic acid sample 2: the preparation is prepared by dissolving the medicine with physiological saline before test to obtain clarified liquid medicine with low concentration (1 mg/mL), medium concentration (3 mg/mL) and high concentration (10 mg/mL), and adding 1M NaHCO 3 Regulating pH of the medicinal liquid to neutrality. The mode of administration of sample 2 in this study was intragastric, with doses of 10mg/kg,30mg/kg and 100mg/kg, respectively.
3. Establishment of middle cerebral artery embolism model of mouse
Male C57BL/6 mice of 8-10 weeks old were adaptively bred for 3 days, randomly divided into model groups (normal saline, 10mL/kg, i.g.) and administration groups according to body weight, fasted without water-deprivation for 12h, pre-administered for 5 days, and each group was given normal saline solvent without water-deprivation before pMCAO surgeryThe medicines with the same dosage and the medicinal group solution are prepared at present, the medicines are dissolved by normal saline before experiments, and are prepared into clear liquid medicine, and 1M NaHCO is used 3 Regulating pH of the medicinal liquid to neutrality. Mice were anesthetized with isoflurane. Cortical cerebral blood flow of middle cerebral artery (middle cerebral artery, MCA) was monitored by doppler laser flow cytometry. The skin of the mouse head was separated, the bregma was exposed, and the front end of the laser Doppler blood flow meter optical fiber was fixed to the animal skull at a position 2mm behind the bregma and 6mm to the right of the midline. The mice were supine, the right common carotid artery was isolated, the distal end was ligated, and the proximal end was closed with hemostat jaws. A sharp blunted 6-0 nylon monofilament suture was inserted approximately 10mm in length into the right internal carotid artery using microsurgery to block the origin of MCA, resulting in a Middle Cerebral Artery Occlusion (MCAO) model. Observing cerebral blood flow meter data, and if the blood flow value reduction is more than 80%, establishing a model successfully; if the blood flow value decreases by less than 80%, the animal is excluded. Animals in the model group were anesthetized, blood flow recorded and common carotid artery isolated using the same procedure, but were embolized without the insertion of a tether.
4. Experimental operation
Mice were randomly divided into model groups (saline, 10mL/kg, i.g.), organic acid samples 2 groups (10 mg/kg,30mg/kg,100mg/kg, i.g.), each group of saline, different doses of organic acid samples 2 were administered prior to surgery, and each group of mice was evaluated for neurological symptoms score and cerebral infarction volume statistics 24h after MCAO surgery. Brains were taken from each group of mice 24h after surgery, and infarct volume statistics were performed using 2,3, 5-triphenyltetrazolium chloride (TTC) staining technique.
Neurological symptoms were scored 24 hours after ischemia, with specific scores: score 0, no symptoms; 1, after the rat tail is caught and lifted, the mouse rotates leftwards; 2 minutes, the mouse cannot walk linearly after being placed on a plane and rotates leftwards; 3 minutes, the mice cannot walk or rotate and roll to one side; 4 minutes, no spontaneous activities; 5 minutes, death. Mice were euthanized 24 hours post-surgery, brain tissue was taken at 1mm intervals, and coronally sectioned and stained with 0.25% ttc (Sigma, T8877) to determine cerebral infarct volume. Cerebral edema can be corrected by calculating the infarct area by an indirect method using Image-Pro Plus 7.0. The cerebral infarction volume rate of mice= (sum of left-side non-infarcted areas-sum of right-side non-infarcted areas)/sum of left-side non-infarcted areas x 100%.
5. Test results
The test results are shown in Table 11 and FIG. 4. Compared with the model group, the administration of 100mg/kg of the organic acid sample 2 can significantly reduce the cerebral infarction volume rate (p < 0.01). Mice were scored according to symptoms, and it was found that different doses of organic acid sample 2 had a tendency to reduce the neurological symptom score, and that 30mg/kg of organic acid sample 2 significantly reduced the neurological symptom score in mice compared to the model group. The results show that the ginkgo leaf organic acid extract sample 2 shows potential anti-chronic ischemic stroke effect in the MCAO model.
Table 11 mouse cerebral arterial embolism test results (mean±sd, n=6)
Note that: * Represents p <0.05 compared to the model group; * Represents p <0.01 compared to the model group.
Example 10 Effect of organic acid extracts on photochemical embolic mice
1. Experimental animal
Reference is made to the experimental animals of example 9.
2. Drug formulation and administration mode
Reference is made to the formulation and mode of administration of the drug of example 9.
3. Establishment of photochemical embolism model for mice
Male C57BL/6 mice of 8-10 weeks old were adaptively bred for 3 days, randomly divided into model groups (normal saline, 10mL/kg, i.g.) and administration groups according to body weight, fasted without water inhibition for 12h, and pre-administered and intragastric administration for 7 days, and subjected to a light thrombus operation. The mice are anesthetized with isoflurane, and after the mice are anesthetized and unconscious for 3-5 min, the heads of the mice are fixed by using a small animal stereotactic instrument. The rose bengal of 11.875mg/mL is injected intraperitoneally according to the dosage of 0.1mL/10g, and the rose bengal is waited to be distributed in the cerebral vessels (more than 5 min) through the circulatory system.The scalp of the mice was cut off and the light source was placed 1.5mm to the right of the front halogen of the mice. The mice were covered with sanitary napkin during 15min of irradiation with a cold light source of 16000 lux. After the irradiation is completed, the scalp is sutured and sterilized with iodophor. The mice were placed on an electric blanket and irradiated with a heating lamp. 24h post-surgery, mice were weighed and given a model group, mice in the dosing group were given 14 consecutive days, and behavioural tests were performed on alternate days (1, 3, …,15 days). The medicinal solutions are prepared by dissolving the medicinal materials with normal saline before experiment, preparing into clear medicinal liquid, and mixing with 1M NaHCO 3 Regulating pH of the medicinal liquid to neutrality.
4. Experimental operation
Mice were randomly divided into model groups (normal saline, 10mL/kg, i.g.), organic acid samples 2 groups (10 mg/kg,30mg/kg, i.g.), and the mice of each group were given their corresponding drugs 14 consecutive days after PT surgery, and the effect of organic acid on PT-induced focal cerebral ischemic injury of mice was detected by analyzing the motor function index of the grid step-out rate mice on every other day (1, 3, …,15 days).
According to the method reported in literature, the test mice are placed in steel grids (length and width are respectively 32cm and 20cm, and the side length of the upper grid is 1.2cm multiplied by 1.2 cm), so that the mice can freely walk on the grids for more than 3min, and cameras are placed under the grids to shoot the walking process. Video was played at 0.5-fold speed, and the number of steps taken by the forelimb of the mouse in 100 steps was recorded, with step rate = number of feet lost/100 x 100%.
5. Test results
The test results are shown in Table 12 and FIG. 5. The administration of both 10mg/kg and 30mg/kg of the organic acid sample 2 reduced the step rate of mice compared to the model group, wherein 10mg/kg significantly reduced the step rate of mice on days 9, 13 (p < 0.05), and 30mg/kg of the organic acid sample 2 significantly reduced the step rate of mice on day 13 (p < 0.05), which suggested that ginkgo leaf organic acid sample 2 could partially alleviate motor sensory functions of mice after cerebral ischemia in PT model.
Table 12 results of step rate in photochemical embolism test in mice (mean±sd, n=9)
Note that: * The representation compares p <0.05 to the model set.
Claims (8)
1. A ginkgo leaf organic acid extract, the preparation method of the extract comprising the following steps:
(1) Taking ginkgo leaf extract meeting national pharmacopoeia standards as a raw material;
(2) Dissolving the raw materials in the step (1) with boiling water or ethanol below 10% (v/v) to prepare a sample loading liquid 1;
(3) Loading liquid 1 passes through a polyamide column, 3 BV-5 BV is eluted by 5% -10% (v/v) ethanol, the flow rate is 1 BV/h-3 BV/h, effluent liquid and ethanol eluent are collected, concentrated to 1-2 times of column volume, and filtered, and filtrate is taken as loading liquid 2;
(4) Loading the sample liquid 2 through a macroporous resin column, eluting 3 BV-5 BV with 10% -15% (v/v) ethanol at a flow rate of 1 BV/h-3 BV/h, collecting ethanol eluent, concentrating and drying to obtain ginkgo leaf organic acid extract;
the content of 6-hydroxykynurenic acid in the ginkgo leaf extract in the step (1) is more than 2mg/g, the content of parahydroxybenzoic acid is more than 1mg/g, and the content of gallic acid is more than 1mg/g.
2. The extract according to claim 1, wherein the active ingredients of the extract comprise, by weight, 60% -68% of protocatechuic acid, 10% -14% of 6-hydroxykynurenic acid, 5% -8% of gallic acid, 3% -8% of p-hydroxybenzoic acid, and the total organic acid content of the extract is more than 80%.
3. The extract according to claim 2, wherein the protocatechuic acid, 6-hydroxykynurenic acid, gallic acid and parahydroxybenzoic acid content satisfy the following conditions: the content ratio of protocatechuic acid/6-hydroxykynurenine is 4-6, or the content ratio of protocatechuic acid/gallic acid is 9-12, or the content ratio of protocatechuic acid/p-hydroxybenzoic acid is 8-14.
4. The extract of claim 1, wherein the ginkgo leaf extract in the step (1) is selected by an organic acid content detection method according to an organic acid component index.
5. The extract according to claim 1, wherein the polyamide column in the step (3) is 10 to 30 mesh or 30 to 60 mesh.
6. The extract according to claim 1, wherein the macroporous resin column type in the step (4) is D101 or AB-8, and the concentration temperature is 50-70 ℃.
7. Use of the extract according to claim 1 for the preparation of a medicament for the prevention and/or treatment of cerebral ischemic injury.
8. The use according to claim 7, wherein the cerebral ischemic injury comprises chronic ischemic stroke.
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