Ginkgo biloba extract, preparation method and application of ginkgo biloba extract in improvement of cerebral ischemia

Abstract

The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a ginkgo biloba extract, a preparation method and application of the ginkgo biloba extract in improvement of cerebral ischemia. The preparation method of the ginkgo biloba extract comprises the following steps: (1) crushing dried ginkgo biloba, and adding a compound enzyme aqueous solution for soaking to obtain a mixture 1; (2) adding a surfactant and absolute ethyl alcohol into the mixture 1, performing ultrasonic extraction, and filtering to obtain a ginkgo leaf extracting solution; and (3) filtering the ginkgo leaf extracting solution through a ceramic membrane, concentrating until the ginkgo leaf extracting solution has no ethanol smell, adding the ginkgo leaf extracting solution into a polyamide resin and macroporous resin combined resin column for elution, collecting eluent, concentrating and drying to obtain the ginkgo leaf extract. According to the ginkgo biloba extract, the specific compound enzyme, the surfactant and the purified resin are selected, and the ginkgo biloba extract obtained through extraction is high in effective component content and remarkable in cerebral ischemia improving effect.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to a ginkgo leaf extract, a preparation method and application thereof in improving cerebral ischemia. Background Art

[0002] Cerebral ischemia is a common pathological state of the nervous system. Ischemic cerebrovascular disease belongs to the scope of "ischemic stroke" in traditional Chinese medicine. It is a focal brain dysfunction caused by insufficient or interrupted local cerebral blood supply and oxygen supply. Common ones include transient ischemic attack, cerebral embolism, and cerebral thrombosis. It is a common and frequently-occurring disease that seriously threatens human life and health, and is the main disease leading to death and loss of ability. Ischemia-reperfusion injury (IRI) refers to the pathological phenomenon that the degree of tissue damage worsens after the blood supply is restored after the tissue suffers from ischemia for a certain period of time. At present, drug intervention treatment of ischemic brain injury has become an important research topic of concern to the society and the medical community, and the research on finding effective therapeutic drugs to prevent and treat cerebral ischemia has received more and more attention.

[0003] Modern medical research on the mechanism of cerebral ischemia includes energy metabolism disorder, platelet aggregation, vasoconstriction and microcirculatory disorder, free radical damage, excitatory amino acid toxicity, intracellular calcium ion overload, inflammatory damage, the role of NO and NOS, cell apoptosis, etc. Cerebral ischemic injury is the result of a cascade of damage caused by cerebral blood circulation disorder, which causes cell energy metabolism failure and initiates the damage cascade. This damage cascade involves different stages such as excitatory amino acid (EAA) toxicity, peri-infarct depolarization, inflammation and cell apoptosis. Modern medicine mainly uses drug intervention to prevent and treat cerebral ischemia. The drugs that can be used clinically to prevent and treat cerebral ischemia mainly include antiplatelet drugs such as aspirin, clopidogrel, ticlopidine and aspirin-dipyridamole, anticoagulants such as heparin and low molecular weight heparin, oral anticoagulants such as warfarin and dicoumarol injection, thrombolytics such as urokinase (UK) and streptokinase (SK), vasodilators such as ergoline, free radical scavengers such as superoxide dismutase, dexamethasone, mannitol, vitamin E, calcium antagonists such as nimodipine, glutamate receptor antagonists, NOS inhibitors and statins, among which aspirin is the most widely used antiplatelet aggregation. Many literatures report that these drugs have the effect of preventing and treating cerebral ischemia, but the existing drugs are not suitable for repeated use due to their high price, limited administration methods, and many side effects, or their application is limited due to long medication time and large dosage.

[0004] The main active ingredients of Ginkgo biloba are flavonoids and terpene lactones. Ginkgo biloba contains a high content of flavonoids, mainly flavonoid glycosides, flavonol glycosides, biflavonoid aglycones, flavonoid aglycones and other flavonoid components, among which flavonoid glycosides and flavonol glycosides include quercetin, kaempferol, isorhamnetin and glucose or rhamnose combinations. Terpene lactones in Ginkgo biloba are terpenes with lactone structures, including ginkgolide A, B, C, J, M and bilobalide. Ginkgo biloba extract can act on the heart, brain and peripheral vascular system, improve peripheral blood circulation and promote blood circulation, and has the effects of promoting blood circulation, removing blood stasis, unblocking collaterals and relaxing pulses. It is clinically used to treat dizziness, coronary heart disease, angina pectoris, etc. caused by blood stasis type cerebral arteriosclerosis.

[0005] In addition, ginkgo leaves also contain alkylphenolic acid compounds, which are composed of ginkgolic acid, ginkgo phenol and ginkgo biloba diphenol. Ginkgo acid has cytotoxic, embryotoxic, sensitizing and mutagenic effects. When the concentration of ginkgolic acid reaches a certain level, it will greatly reduce the survival rate of rat liver cells. Therefore, the European and American pharmacopoeia standards are that the content of ginkgolic acid in ginkgo leaf extract is less than 5ppm, and the Chinese pharmacopoeia standard is that the content of ginkgolic acid in ginkgo leaf extract is less than 10ppm.

[0006] Chinese invention patent application CN107693552A discloses a ginkgo leaf preparation for treating cerebral ischemia, its preparation method and use. The invention administers ginkgo leaf extract and NADPH in combination, which has a significant therapeutic effect on cerebral ischemia; the combined administration of ginkgo leaf extract and NADPH in a specific ratio has a significantly better therapeutic effect on cerebral ischemia than the therapeutic effect of either alone, and has a synergistic effect.

[0007] Chinese invention patent application CN101099758A discloses a compound ginkgo leaf preparation, which is composed of ginkgo leaf extract, ginseng extract and borneol. It can not only be used to prevent and treat coronary heart disease and angina pectoris, but also has a good protective effect on cerebral ischemic damage.

[0008] However, the therapeutic effect of the above-mentioned simple ginkgo leaf extract is not ideal. The present invention proposes a specific extraction process to prepare the ginkgo leaf extract, which is used to improve the effect of improving cerebral ischemia. Summary of the invention

[0009] In view of the deficiencies in the prior art, the present invention provides a ginkgo leaf extract, a preparation method and an application thereof in improving cerebral ischemia.

[0010] The present invention is achieved through the following technical solutions:

[0011] A method for preparing a ginkgo leaf extract comprises the following steps:

[0012] (1) crushing dried ginkgo leaves, adding them into a complex enzyme aqueous solution and soaking them to obtain a mixture 1;

[0013] (2) adding a surfactant and anhydrous ethanol to the mixture 1, performing ultrasonic extraction, and filtering to obtain a ginkgo leaf extract;

[0014] (3) filtering the ginkgo leaf extract through a ceramic membrane, concentrating until there is no ethanol smell, adding it to a combined resin column of polyamide resin and macroporous resin for elution, collecting the eluate, concentrating and drying it to obtain the ginkgo leaf extract.

[0015] Preferably, in step (1), the ginkgo leaves are crushed to 40-80 mesh, and the mass ratio of the ginkgo leaves to the complex enzyme aqueous solution is 1:2-3.

[0016] Preferably, the complex enzyme in step (1) is cellulase, pectinase and glucosidase in a mass ratio of 1-2:1-2:0.5, the soaking time is 2-4 hours, and the added amount of the complex enzyme is 6%-8% of the mass of the ginkgo leaves.

[0017] Preferably, the surfactant in step (2) is a mixture of sodium dodecyl sulfate and rhamnolipid, and the mass ratio of sodium dodecyl sulfate to rhamnolipid is 4-5:1.

[0018] Preferably, in step (2), the amount of the surfactant added is 5%-10% of the mass of the mixture 1, and the amount of the anhydrous ethanol added is 1-2 times the mass of the mixture 1.

[0019] Preferably, the frequency of the ultrasonic extraction in step (2) is 50-70 KHz, and the extraction time is 20-30 min.

[0020] Preferably, the pore size of the ceramic membrane in step (3) is 100-200 nm, and the elution method is: firstly washing with 2-3 column volumes of water to remove impurities, and then eluting with 3-4 column volumes of 50-60% ethanol.

[0021] Preferably, in step (3), the weight ratio of the polyamide resin to the macroporous resin is 1:0.5-5, and the loading method is: the polyamide resin is in the upper layer and the macroporous resin is in the lower layer. The polyamide is nylon-6 and the macroporous resin model is D-101.

[0022] The present invention also relates to the ginkgo leaf extract obtained by the preparation method. The ginkgo leaf extract comprises: a total flavonol glycoside content of ≥24% and a total lactone content of ≥9.5%.

[0023] The present invention also relates to the application of the ginkgo leaf extract prepared by the preparation method in the preparation of health products or medicines for improving cerebral ischemia.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention uses a specific complex enzyme, surfactant and purification resin to extract the ginkgo leaf extract, which has a high content of effective ingredients and a significant effect in improving cerebral ischemia;

[0026] (2) The present invention studies the therapeutic effect of Ginkgo biloba extract on rats with cerebral ischemia-reperfusion injury, and proves that Ginkgo biloba extract can improve the neurological function of ischemic rats, reduce the area of ​​cerebral infarction and brain edema in rats, which strongly proves that the Ginkgo biloba extract of the present invention has a significant antagonistic effect on cerebral ischemia injury and can be used to prepare anti-cerebral ischemia drugs. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0028] Example 1

[0029] A method for preparing ginkgo leaf extract, comprising the following steps:

[0030] (1) Grinding dried ginkgo leaves into 60 mesh, adding a composite enzyme aqueous solution 2.5 times the mass of the ginkgo leaves and soaking for 3 hours, wherein the composite enzyme is cellulase, pectinase and glucosidase in a mass ratio of 2:1:0.5, and the amount of the composite enzyme added is 7% of the mass of the ginkgo leaves, to obtain a mixture 1;

[0031] (2) adding 8% of a surfactant (sodium dodecyl sulfate and rhamnolipid in a mass ratio of 4:1) and 1.5 times the mass of anhydrous ethanol to the mixture 1, performing ultrasonic extraction at 60 kHz for 25 min, and filtering to obtain a ginkgo leaf extract;

[0032] (3) The ginkgo leaf extract is filtered through a 200 nm ceramic membrane, concentrated until there is no ethanol smell, and added to a resin column composed of polyamide resin (nylon-6) and macroporous resin (D-101) for elution, with the polyamide resin in the upper layer and the macroporous resin in the lower layer, and the weight ratio of nylon-6 to D-101 macroporous resin is 1:3; during elution, it is first washed with water for 2 times the column volume to remove impurities, and then eluted with 55% ethanol solution for 4 times the column volume, the eluate is collected, concentrated and dried to obtain the ginkgo leaf extract.

[0033] The test was carried out according to the content determination method of Ginkgo leaf extract in the 2020 edition of the "Chinese Pharmacopoeia": total flavonol glycoside content was 36.9%, total lactone content was 12.7% and total ginkgo phenolic acid content was 0.3ppm.

[0034] Example 2

[0035] A method for preparing ginkgo leaf extract, comprising the following steps:

[0036] (1) Grinding dried ginkgo leaves into 80 meshes, adding a composite enzyme aqueous solution twice the mass of the ginkgo leaves and soaking for 2 hours, wherein the composite enzyme is cellulase, pectinase and glucosidase in a mass ratio of 1:1:0.5, and the amount of the composite enzyme added is 6% of the mass of the ginkgo leaves, to obtain a mixture 1;

[0037] (2) adding 5% of a surfactant (sodium dodecyl sulfate and rhamnolipid in a mass ratio of 5:1) and 1 times the mass of anhydrous ethanol to the mixture 1, performing ultrasonic extraction at 50 kHz for 30 min, and filtering to obtain a ginkgo leaf extract;

[0038] (3) The ginkgo leaf extract is filtered through a 100 nm ceramic membrane, concentrated until there is no ethanol smell, and added to a resin column combined with a polyamide resin (nylon-6) and a macroporous resin (D-101) for elution, with the polyamide resin in the upper layer and the macroporous resin in the lower layer, and the weight ratio of nylon-6 to D-101 macroporous resin is 1:0.5; during elution, it is first washed with water for 3 times the column volume to remove impurities, and then eluted with a 50% ethanol solution for 3 times the column volume, the eluate is collected, concentrated and dried to obtain the ginkgo leaf extract.

[0039] The test was carried out according to the content determination method of Ginkgo leaf extract in the 2020 edition of the "Chinese Pharmacopoeia": total flavonol glycoside content was 35.8%, total lactone content was 12.1% and total ginkgo phenolic acid content was 0.5ppm.

[0040] Example 3

[0041] A method for preparing ginkgo leaf extract, comprising the following steps:

[0042] (1) Grinding dried ginkgo leaves into 40 meshes, adding a composite enzyme aqueous solution 3 times the mass of the ginkgo leaves and soaking for 3 hours, wherein the composite enzyme is cellulase, pectinase and glucosidase in a mass ratio of 2:2:0.5, and the amount of the composite enzyme added is 8% of the mass of the ginkgo leaves, to obtain a mixture 1;

[0043] (2) adding 10% of a surfactant (sodium dodecyl sulfate and rhamnolipid in a mass ratio of 4.5:1) and 2 times the mass of anhydrous ethanol to the mixture 1, performing ultrasonic extraction at 70 kHz for 20 min, filtering, and obtaining a ginkgo leaf extract;

[0044] (3) The ginkgo leaf extract is filtered through a 150 nm ceramic membrane, concentrated until there is no ethanol smell, and added to a resin column combined with a polyamide resin (nylon-6) and a macroporous resin (D-101) for elution, with the polyamide resin in the upper layer and the macroporous resin in the lower layer, and the weight ratio of nylon-6 to D-101 macroporous resin is 1:5; during elution, it is first washed with water for 2.5 times the column volume to remove impurities, and then eluted with 60% ethanol for 3.5 times the column volume, the eluate is collected, concentrated and dried to obtain the ginkgo leaf extract.

[0045] The test was carried out according to the content determination method of Ginkgo leaf extract in the 2020 edition of the "Chinese Pharmacopoeia": total flavonol glycoside content was 36.2%, total lactone content was 12.3% and total ginkgo phenolic acid content was 0.4ppm.

[0046] Comparative Example 1

[0047] The difference between this comparative example and Example 1 is that, step (1): the dried ginkgo leaves are crushed into 60 meshes, and a complex enzyme aqueous solution 2.5 times the mass of the ginkgo leaves is added and soaked for 3 hours, wherein the complex enzyme is a 2:1 ratio of cellulase and pectinase, and the amount of the complex enzyme added is 7% of the mass of the ginkgo leaves, to obtain a mixture 1.

[0048] Comparative Example 2

[0049] The difference between this comparative example and Example 1 is that, step (1): the dried ginkgo leaves are crushed into 60 meshes, an aqueous solution of glucosidase 2.5 times the mass of the ginkgo leaves is added and soaked for 3 hours, the amount of glucosidase added is 7% of the mass of the ginkgo leaves, and a mixture 1 is obtained.

[0050] Comparative Example 3

[0051] The difference between this comparative example and Example 1 is that the surfactant in step (2) is only sodium dodecyl sulfate.

[0052] Comparative Example 4

[0053] The only difference between this comparative example and Example 1 is that the surfactant in step (2) is only rhamnolipid.

[0054] Comparative Example 5

[0055] The only difference between this comparative example and Example 1 is that in the resin column of the polyamide resin (nylon-6) and D-101 macroporous resin combined in step (3), the macroporous resin is in the upper layer and the polyamide resin is in the lower layer.

[0056] Comparative Example 6

[0057] The only difference between this comparative example and Example 1 is that the macroporous resin in step (3) is AB-8 macroporous resin.

[0058] Effect test

[0059] In order to verify the anti-cerebral ischemic injury effect of the ginkgo leaf extract of the present invention, the following experiments were conducted:

[0060] Treatment method: 120 male SD rats weighing (280±20) g were taken, and focal cerebral ischemia / reperfusion model was established by thread embolization method. The successfully modeled SD rats were randomly divided into 12 groups: sham operation group, model group, positive drug administration group (nimodipine 9 mg / kg), Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group, Comparative Example 5 group and Comparative Example 6 group, wherein the dosage of Example 1-3 group and Comparative Example 1-6 group was 6 g / kg. The corresponding drugs were given by gavage for 12 days.

[0061] Statistical methods: All data were expressed as mean ± standard deviation (mean ± SD), and SPSS software (version 19.0) was used for statistical analysis. Nonparametric tests were used to analyze the neurobehavioral scores among the experimental groups, and one-way ANOVA was used for the rest. When the variance was homogeneous, LSD test was selected, and when the variance was unequal, Games Howell test was selected. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significant.

[0062] Detect the following indicators:

[0063] (1) Neurological deficit score: After the last administration, the behavioral deficits of SD rats were graded and scored according to the Bederson method. The criteria are as follows:

[0064] 0 points: no neurological symptoms were observed;

[0065] 1 point: When the animal's tail is lifted and suspended in the air, the forelimb on the contralateral side of the operation shows wrist and elbow flexion, shoulder internal rotation, elbow abduction, and close to the chest wall;

[0066] 2 points: The animal is placed on a smooth surface, and the resistance decreases when the operated shoulder is pushed toward the opposite side;

[0067] 3 points: the animal circles or rotates toward the side opposite to the surgery when walking freely;

[0068] 4 points: flaccid paralysis, no spontaneous movement of the limbs.

[0069] The test results are shown in Table 1.

[0070] Table 1 Neurological deficit scores

[0071] Group Neurological function score Sham operation group <![CDATA[0 a ]]> Model Group <![CDATA[2.7 b ]]> Positive drug group 0.8c Example 1 Group 1.1d Example 2 Group 1.5d Example 3 Group 1.3d Comparative Example 1 2.3e Comparative Example 2 2.5b Comparative Example 3 1.9f Comparative Example 4 2.2e Comparative Example 5 1.7f Comparative Example 6 2.0e

[0072] Note: Different letters in the same column indicate significant statistical differences between the corresponding groups, p < 0.05.

[0073] (2) Detection of cerebral infarction area and brain water content in rats

[0074] After sodium pentobarbital anesthesia, the whole brain was removed. The whole brain was placed in a -20℃ refrigerator and frozen for 20 minutes. When the rat brain became hard, a sharp blade was used to make coronal sections at the optic chiasm and 2 mm before and after it. The brain tissue was cut into five slices. The sliced ​​brain slices were quickly placed in a PBS buffer solution containing 1% TTC and incubated at 37℃ in the dark for 10 minutes. After 10 minutes, the brain slices were gently removed, arranged in order, the moisture around the brain slices was wiped dry, and photos were taken with a digital camera. The digital photos were analyzed using Image J image analysis software, and the formula for calculating the infarction rate is as follows:

[0075] Infarction rate (%) = (brain area on the ischemic side opposite to the ischemic side - brain area of ​​the non-infarcted area on the ischemic side) / brain area on the ischemic side opposite to the ischemic side × 100%.

[0076] The stained brain tissue was weighed, and the weight at this time was the wet weight of the brain tissue. It was then placed in a 110°C oven for 24 hours and weighed again, and the weight at this time was the dry weight of the brain tissue. The formula for calculating the water content of the brain tissue is as follows:

[0077] Water content of brain tissue (%) = (wet weight - dry weight) / wet weight × 100%.

[0078] The test results are shown in Table 2.

[0079] Table 2 Detection results of cerebral infarction area and brain water content

[0080] Group Infarction rate (%) Brain tissue water content (%) Sham operation group <![CDATA[0 a ]]> 76.2 Model Group <![CDATA[62.5 b ]]> 82.5 Positive drug group <![CDATA[20.6 c ]]> 78.4 Example 1 Group <![CDATA[22.4 c ]]> 79.6 Example 2 Group <![CDATA[26.7 c ]]> 80.1 Example 3 Group <![CDATA[24.1 c ]]> 79.5 Comparative Example 1 <![CDATA[30.2 d ]]> 81.2 Comparative Example 2 <![CDATA[31.4 d ]]> 80.9 Comparative Example 3 <![CDATA[29.1 d ]]> 81.6 Comparative Example 4 <![CDATA[31.0 d ]]> 80.8 Comparative Example 5 <![CDATA[35.7 d ]]> 81.0 Comparative Example 6 <![CDATA[38.6 d ]]> 81.7

[0081] Note: Different letters in the same column indicate significant statistical differences between the corresponding groups, p < 0.05.

[0082] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A method for preparing a ginkgo leaf extract, characterized in that: The following steps are involved: (1) crushing dried ginkgo leaves, adding them into a complex enzyme aqueous solution and soaking them to obtain a mixture 1; (2) adding a surfactant and anhydrous ethanol to the mixture 1, performing ultrasonic extraction, and filtering to obtain a ginkgo leaf extract; (3) filtering the ginkgo leaf extract through a ceramic membrane, concentrating until there is no ethanol smell, adding it to a combined resin column of polyamide resin and macroporous resin for elution, collecting the eluate, concentrating and drying it to obtain the ginkgo leaf extract.

2. The preparation method according to claim 1, characterized in that: In step (1), the ginkgo leaves are crushed to 40-80 meshes, and the mass ratio of the ginkgo leaves to the complex enzyme aqueous solution is 1:2-3.

3. The preparation method according to claim 1, characterized in that: The compound enzyme in step (1) is cellulase, pectinase and glucosidase in a mass ratio of 1-2:1-2:0.5, the soaking time is 2-4 hours, and the added amount of the compound enzyme is 6%-8% of the mass of the ginkgo leaves.

4. The preparation method according to claim 1, characterized in that: The surfactant in step (2) is a mixture of sodium dodecyl sulfate and rhamnolipid, and the mass ratio of sodium dodecyl sulfate to rhamnolipid is 4-5:

1.

5. The preparation method according to claim 1, characterized in that: In step (2), the amount of the surfactant added is 5%-10% of the mass of the mixture 1, and the amount of the anhydrous ethanol added is 1-2 times the mass of the mixture 1.

6. The preparation method according to claim 1, characterized in that: The frequency of the ultrasonic extraction in step (2) is 50-70 KHz, and the extraction time is 20-30 min.

7. The preparation method according to claim 1, characterized in that: The pore size of the ceramic membrane in step (3) is 100-200 nm, and the elution method is: firstly wash with 2-3 column volumes of water to remove impurities, and then elute with 3-4 column volumes of 50-60% ethanol.

8. The preparation method according to claim 1, characterized in that: The weight ratio of the polyamide resin to the macroporous resin in step (3) is 1:0.5-5, and the loading method is: the polyamide resin is in the upper layer and the macroporous resin is in the lower layer. The polyamide is nylon-6 and the macroporous resin model is D-101.

9. A ginkgo leaf extract prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The ginkgo leaf extract comprises: a total flavonol glycoside content of ≥24% and a total lactone content of ≥9.5%.

10. Use of the ginkgo leaf extract prepared by the preparation method according to any one of claims 1 to 8 in preparing a health product or medicine for improving cerebral ischemia.