CN111166775A

CN111166775A - Ginkgo leaf extract, ginkgo leaf extract medicament and application thereof

Info

Publication number: CN111166775A
Application number: CN202010110791.9A
Authority: CN
Inventors: 巴卫松; 王永利; 张兰桐
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-02-24
Filing date: 2020-02-24
Publication date: 2020-05-19

Abstract

The invention relates to the field of drug research and development, and particularly relates to a ginkgo leaf extract, a ginkgo leaf extract medicament and application thereof. The ginkgo biloba extract comprises 7-demethyl ginkgetin, ginkgo total flavonol glycoside and terpene lactone, wherein the total mass of the ginkgo total flavonol glycoside and the terpene lactone accounts for more than 50 percent of the mass of the ginkgo biloba extract, and the mass of the 7-demethyl ginkgetin accounts for more than five ten-thousandths of the mass of the ginkgo biloba extract. The folium Ginkgo extract has good therapeutic effect on eye diseases, especially macular degeneration and diabetic retinopathy, and can be used for treating macular degeneration diseases, diabetic retinopathy and other eye diseases caused by the macular degeneration diseases and the diabetic retinopathy.

Description

Ginkgo leaf extract, ginkgo leaf extract medicament and application thereof

Technical Field

The invention relates to the field of drug research and development, and particularly relates to a ginkgo leaf extract, a ginkgo leaf extract medicament and application thereof.

Background

In the last 60 years, many studies on ginkgo biloba extracts have been made, and it has been found that ginkgo biloba extracts have many physiological and biochemical functions, such as expanding blood vessels, improving microcirculation, resisting oxidation, resisting platelet activating factors, protecting cell mitochondria and the like, so that ginkgo biloba extracts have various clinical applications, such as treating coronary heart disease, angina pectoris, hypertension, hyperlipidemia, asthma, diabetic nephropathy, cerebral infarction and other diseases, but many other applications of ginkgo biloba extracts have not been found.

In view of this, the present invention is proposed.

Disclosure of Invention

The invention provides a ginkgo leaf extract, a ginkgo leaf extract medicament and application thereof. The folium Ginkgo extract has good therapeutic effect on eye diseases, especially macular degeneration and diabetic retinopathy, and can be used for treating macular degeneration diseases, diabetic retinopathy and other eye diseases caused by the macular degeneration diseases and the diabetic retinopathy.

The invention is realized by the following steps:

in a first aspect, the embodiments of the present invention provide a ginkgo biloba extract, which includes 7-demethyl ginkgetin, ginkgo total flavonol glycosides and terpene lactones, wherein the total mass of the ginkgo total flavonol glycosides and the terpene lactones accounts for more than 50% of the mass of the ginkgo biloba extract, and the mass of the 7-demethyl ginkgetin accounts for more than five ten-thousandths of the mass of the ginkgo biloba extract; wherein the mass percentages of the above substances are on a dry basis.

Further, in a preferred embodiment of the invention, the mass of the ginkgo total flavonol glycosides accounts for 18.5-37.7% of the mass of the ginkgo biloba extract, the mass of the terpene lactones accounts for 40.4-53.7% of the mass of the ginkgo biloba extract, and the mass of the 7-demethyl ginkgo biflavone accounts for five-thousandths of the mass of the ginkgo biloba extract;

preferably, the ginkgo biloba extract further comprises ginkgo biloba total acids;

preferably, the content of total acids of ginkgo biloba is less than 1 ppm.

Further, in preferred embodiments of the present invention, the ginkgo total flavonol glycosides include quercetin glycoside, kaempferol glycoside and isorhamnetin glycoside;

preferably, the quercetin glycoside comprises the following 8 compounds;

preferably, the kaempferol glycoside comprises the following 9 compounds;

preferably, the isorhamnetin glycoside comprises the following 4 compounds:

further, in preferred embodiments of the present invention, the terpene lactones include bilobalide, ginkgolide a, ginkgolide B, and ginkgolide C.

In a second aspect, an embodiment of the present invention provides a pharmaceutical preparation comprising the above-mentioned ginkgo biloba extract.

Further, in a preferred embodiment of the present invention, the dosage form of the ginkgo biloba extract medicament is a solid preparation or a liquid preparation;

preferably, the solid preparation includes any one of tablets, granules and powders;

preferably, the liquid formulation includes any one of oral liquid, injection and eye drop.

Further, in a preferred embodiment of the present invention, when the ginkgo biloba extract medicament is a tablet, the ginkgo biloba extract medicament includes an auxiliary material and the ginkgo biloba extract;

preferably, the mass of the ginkgo biloba extract in each tablet accounts for 19.75-20.29% of the mass of the ginkgo biloba extract medicament.

Further, in a preferred embodiment of the present invention, the auxiliary materials include diluents, excipients, binders, disintegrating and foaming agents, flavoring agents, and lubricants;

preferably, the diluent comprises lactose, the excipient comprises mannitol, the binder comprises sodium carboxymethyl starch, the disintegrating and foaming agent comprises fumaric acid and sodium bicarbonate, the flavoring agent comprises aspartame and berry essence, and the lubricant comprises magnesium stearate;

preferably, the ginkgo biloba extract medicament comprises 31.25 to 44.13 parts of ginkgo biloba extract, 32.5 to 44.37 parts of lactose, 32.5 to 44.37 parts of mannitol, 13.5 to 18.43 parts of sodium carboxymethyl starch, 25 to 34.13 parts of fumaric acid, 10 to 13.65 parts of aspartame, 2.5 to 3.4 parts of berry essence, 10 to 13.65 parts of sodium bicarbonate and 1 to 1.37 parts of magnesium stearate in parts by weight.

In a third aspect, the embodiments of the present invention further provide an application of the above ginkgo biloba extract or ginkgo biloba extract medicament in preparing a medicament for treating an eye disease;

preferably, the drug is a drug that inhibits the formation of neovascular membrane cells in the macular region;

preferably, the drug is a drug capable of scavenging free radicals and inhibiting platelet aggregation;

preferably, the ocular disease comprises macular degeneration;

preferably, the ocular disease comprises diabetic retinopathy;

preferably, the ocular disease comprises retinal thickening.

Further, in a preferred embodiment of the present invention, in the rhesus monkey animal experiment, the effective dose of the ginkgo biloba extract is 1.5-3.0 mg/Kg.

The invention has the beneficial effects that: the ginkgo biloba extract disclosed by the invention has an excellent treatment effect on eye diseases, particularly macular degeneration and diabetic retinopathy, and simultaneously has a good treatment effect on other eye diseases caused by macular degeneration or diabetic retinopathy by adopting 7-demethyl ginkgetin, ginkgo total flavonol glycosides and terpene lactones and limiting the content of the 7-demethyl ginkgetin, the ginkgo total flavonol glycosides and the terpene lactones in the ginkgo biloba extract.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a diagram showing HPLC test results of three mixed standards of quercetin, kaempferol and isorhamnetin according to the embodiment of the present invention;

FIG. 2 is a diagram showing HPLC detection results of three aglycones, quercetin, kaempferide and isorhamnetin, of ginkgo biloba extract of example 1 according to the present invention;

FIG. 3 is a diagram showing HPLC detection results of four mixed standards of bilobalide, bilobalide A, bilobalide B and bilobalide C provided by the embodiment of the present invention;

FIG. 4 is a HPLC detection result chart of four terpene lactones of Ginkgo biloba extract of example 1, bilobalide A, bilobalide B and bilobalide C;

FIG. 5 is a HPLC detection result chart of a 7-nor-ginkgetin standard provided by an embodiment of the present invention;

FIG. 6 is a HPLC check result chart of 7-norginkgetin of Ginkgo biloba leaf extract of example 1 according to an embodiment of the present invention;

FIG. 7 is a diagram of the HPLC detection result of the standard ginkgo total acid provided by the embodiment of the present invention;

fig. 8 is a HPLC detection result chart of total ginkgolic acids of ginkgo biloba extract of example 1 according to the embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The embodiments of the present invention provide a ginkgo biloba extract, a pharmaceutical preparation containing the same, and applications thereof.

Firstly, the embodiment of the invention provides a ginkgo biloba leaf extract, which comprises 7-demethyl ginkgetin, ginkgo total flavonol glycoside and terpene lactone, wherein the total mass of the ginkgo total flavonol glycoside and the terpene lactone accounts for more than 50% of the mass of the ginkgo biloba leaf extract, and the mass of the 7-demethyl ginkgetin accounts for more than five ten-thousandths of the mass of the ginkgo biloba leaf extract. By adopting the substances and controlling the content of the substances, the ginkgo leaf extract has excellent treatment effect on macular degeneration and diabetic retinopathy, and meanwhile, the medicament has low toxic and side effects.

Specifically, 7-demethylginkgetin has a certain cytotoxic effect and can inhibit choroidal neovascularization, but if its content is too high, it is liable to damage normal cells, so that it can inhibit neovascularization while avoiding damage to normal cells in the above range. The ginkgo biloba total flavonol glycosides have the functions of super oxidation resistance and partial platelet aggregation resistance, and the terpene lactones have the functions of super platelet aggregation resistance and partial free radical removal, so that the combination of the three can effectively treat macular degeneration and diabetic retinopathy, and the ginkgo biloba extract has low toxic and side effects.

Specifically, the total ginkgo flavonol glycosides account for 18.5-37.7% of the mass of the ginkgo biloba extract (calculated on a dry basis), the terpene lactones account for 40.4-53.7% of the mass of the ginkgo biloba extract (calculated on a dry basis), and the 7-demethyl ginkgo biflavone accounts for five-thousandths to one thousandth of the mass of the ginkgo biloba extract. Further respectively controlling the contents of the ginkgo total flavonol glycoside, the terpene lactone and the 7-demethyl ginkgo biflavone, and further ensuring the treatment effect and the toxic and side effect of the ginkgo leaf extract.

Further, the ginkgo total flavonol glycosides include quercetin glycoside, kaempferol glycoside and isorhamnetin glycoside; wherein the 8 kinds of quercetin glycosides comprise the following 8 kinds of compounds;

the kaempferol glucoside comprises the following 9 compounds;

the isorhamnetin glycoside comprises the following 4 compounds:

the terpene lactones include bilobalide, bilobalide A, bilobalide B and bilobalide C.

Further, the ginkgo biloba extract also comprises ginkgo biloba total acid; and the content of the total ginkgolic acids is less than 1 ppm. The safety of the ginkgo biloba extract can be further ensured by controlling the content of the ginkgo biloba total acid in the ginkgo biloba extract.

Furthermore, the ginkgo biloba extract also comprises free quercetin, free kaempferol, free isorhamnetin and the like, and the content of the substances meets the regulation of the report (No. 66 in 2015) of the test method for releasing ginkgo biloba supplement by the State food and drug administration for the supplement test of the ginkgo biloba drug, wherein the regulation of the supplement test method for the detection items of the ginkgo biloba extract, the ginkgo biloba leaves and the ginkgo biloba capsules comprises the free quercetin, the free kaempferol and the free isorhamnetin.

Further, in rhesus monkey animal experiments, the effective dose of the ginkgo biloba extract is 1.5-3.0 mg/Kg.

The preparation method of the ginkgo biloba extract can refer to the methods in the prior art, and only needs to ensure that the classification and the content of the active ingredients in the prepared ginkgo biloba extract meet the requirements of the embodiments of the invention.

Furthermore, the embodiment of the invention also provides a ginkgo biloba extract medicament, which comprises the ginkgo biloba extract. Namely, the ginkgo biloba extract can be compounded with auxiliary materials in the prior art to obtain medicaments of different formulations. Specifically, the dosage form of the ginkgo biloba extract medicament is a solid preparation or a liquid preparation; wherein the solid preparation comprises any one of tablets, granules and powder; the liquid preparation includes any one of oral liquid, injection and eye drop.

The ginkgo biloba extract may be prepared into other dosage forms such as cream, gel and the like according to requirements, and the preparation method of the medicament is a conventional preparation method of the corresponding dosage form.

Although the ginkgo biloba extract and the auxiliary materials can be mixed to prepare the corresponding preparation according to the existing preparation, the selection of the auxiliary materials of different preparation forms is different, and even if the same preparation form is adopted, the selection of the different auxiliary materials can also cause certain influence on the release, disintegration, stability and the like of the medicament, so that the appropriate auxiliary materials need to be selected to ensure that the prepared ginkgo biloba extract medicament can be practically applied to clinic.

For example, when the ginkgo biloba extract preparation is a tablet, the selected auxiliary materials include a diluent, an excipient, a binder, a disintegrating and foaming agent, a flavoring agent and a lubricant; specifically, the diluent comprises lactose, the excipient comprises mannitol, the binder comprises sodium carboxymethyl starch, the disintegrating and foaming agent comprises fumaric acid and sodium bicarbonate, the flavoring agent comprises aspartame and berry essence, and the lubricant comprises magnesium stearate.

Further, when the ginkgo biloba extract medicament is a tablet, the ginkgo biloba extract medicament comprises 31.25-44.13 parts of the ginkgo biloba extract, 32.5-44.37 parts of lactose, 32.5-44.37 parts of mannitol, 13.5-18.43 parts of sodium carboxymethyl starch, 25-34.13 parts of fumaric acid, 10-13.65 parts of aspartame, 2.5-3.4 parts of berry essence, 10-13.65 parts of sodium bicarbonate and 1-1.37 parts of magnesium stearate in parts by weight. The proportion of each auxiliary material and the ginkgo biloba extract is controlled, the prepared tablet is ensured to have good disintegration degree, release degree, stability and the like, and the drug effect of the tablet is further ensured.

Furthermore, the weight of each tablet of the prepared tablets is 158.25-217.50 mg generally, the content of the ginkgo biloba extract is 31.25-44.13 mg generally, and the mass of the ginkgo biloba extract in each tablet is 19.75-20.29% of the mass of the ginkgo biloba extract medicament after conversion. The content of the ginkgo leaf extract in each tablet is controlled, which is beneficial to ensuring the drug effect of the tablets.

Therefore, the embodiment of the invention also provides an application of the ginkgo biloba extract or the ginkgo biloba extract medicament in preparing a medicament for treating eye diseases, wherein the medicament is a medicament for inhibiting formation of neovascular membrane cells in a macular region; or a drug capable of scavenging free radicals and inhibiting platelet aggregation, and ocular diseases including macular degeneration and diabetic retinopathy and retinal thickening.

The ginkgo leaf extract or the ginkgo leaf extract medicament provided by the embodiment of the invention can also be applied to ocular blood flow and nerve disorders, such as retinopathy and nerve disorders caused by senile and diabetes mellitus, blurred vision, chronic glaucoma and cataract; acute and chronic cardiac and cerebral insufficiency and its sequelae, such as myocardial ischemia, stroke, attention deficit, memory deterioration, senile dementia; disorders of the blood flow and nerves of the ear, such as tinnitus, vertigo, hearing loss, and labyrinthine symptoms of the ear; peripheral circulatory disorders such as various arterial occlusions, intermittent claudication, cold hands and feet paralysis, soreness of the extremities; bronchial smooth muscle spasm, e.g., bronchospastic asthma caused by pollen, dust, and cold air-induced allergies; eliminating or relieving cardiotoxicity caused by chemotherapy drugs, especially adriamycin.

The following embodiments are provided to describe the ginkgo biloba extract, the pharmaceutical preparation of the ginkgo biloba extract and the application thereof.

Example 1

This example provides a ginkgo biloba extract, wherein the total ginkgo flavonol glycosides account for 37.7% of the mass of the ginkgo biloba extract, the terpene lactones account for 37% of the mass of the ginkgo biloba extract, the 7-demethyl ginkgetin accounts for 0.521 ‰ of the mass of the ginkgo biloba extract, the content of total ginkgo acids is less than 1ppm, the content of free quercetin is 1.85 ‰, the content of free kaempferide is 1.04 ‰, and the content of free isorhamnetin is 0.127 ‰.

The preparation method of the ginkgo leaf extract comprises the following steps: the product is an extract purified product prepared by extracting and processing ginkgo leaf extract which meets the medicinal standard of Chinese pharmacopoeia 2015 edition. Taking a standard ginkgo leaf extract, dissolving in hot water, performing ultrasonic treatment and centrifugal separation on the extract to precipitate, and sequentially adopting ethyl acetate: n-heptane (8:1), ethyl acetate: respectively extracting with butanol (6:1) and two extractants, concentrating under reduced pressure, eluting residual solvent, lyophilizing, pulverizing, measuring the content of total flavonol glycoside and terpene lactone in the two purified products, compounding, and sieving.

Example 2

This example provides a ginkgo biloba extract, wherein the total ginkgo flavonol glycosides account for 36.10% of the mass of the ginkgo biloba extract, the terpene lactones account for 36.00% of the mass of the ginkgo biloba extract, the 7-demethyl ginkgetin accounts for 0.51% of the mass of the ginkgo biloba extract, the total ginkgo acids content is less than 1ppm, the free quercetin accounts for 1.96%, the free kaempferide accounts for 1.24%, and the free isorhamnetin accounts for 0.14%.

The preparation method of the ginkgo biloba extract is shown in example 1.

Example 3

This example provides a ginkgo biloba extract, wherein the total ginkgo flavonol glycosides account for 18.50% of the mass of the ginkgo biloba extract, the terpene lactones account for 53.70% of the mass of the ginkgo biloba extract, the 7-demethyl ginkgetin accounts for 1.03% of the mass of the ginkgo biloba extract, the total ginkgo acids content is less than 1ppm, the free quercetin accounts for 2.58%, the free kaempferide accounts for 0.55%, and the free isorhamnetin accounts for 0.22%.

The preparation method of the ginkgo leaf extract comprises the following steps: the product is an extract purified product prepared by extracting and processing ginkgo leaf extract which meets the medicinal standard of Chinese pharmacopoeia 2015 edition. Taking a standard ginkgo leaf extract, dissolving in hot water, performing ultrasonic treatment, performing centrifugal separation and precipitation, and adopting ethyl acetate: extracting with n-heptane (8:1) extractant, concentrating under reduced pressure, eluting residual solvent, lyophilizing, pulverizing, measuring total flavonol glycoside and terpene lactone content in purified product, and sieving.

Example 4

This example provides a ginkgo biloba extract, wherein the total ginkgo flavonol glycosides account for 20.80% of the mass of the ginkgo biloba extract, the terpene lactones account for 53.40% of the mass of the ginkgo biloba extract, the 7-demethyl ginkgetin accounts for 0.89% of the mass of the ginkgo biloba extract, the total ginkgo acids content is less than 1ppm, the free quercetin accounts for 2.58%, the free kaempferide accounts for 0.55%, and the free isorhamnetin accounts for 0.22%.

The preparation of the ginkgo biloba extract is described in example 3.

Example 5

This example provides a pharmaceutical preparation of ginkgo biloba extract, which is a tablet, comprising 25.86g of the ginkgo biloba extract of example 1, 26 g of lactose, 26 g of mannitol, 10.8 g of sodium carboxymethyl starch (7.2 g of the above are added, 3.6 g of the above are added), 20 g of fumaric acid, 8 g of aspartame, 2g of berry essence, 8 g of sodium bicarbonate and 0.8 g of magnesium stearate, wherein 800 tablets are prepared in total, each tablet weighs 159.3 mg, and each tablet contains 32.325 mg of ginkgo biloba extract, that is, each tablet contains 20.29% of ginkgo biloba extract.

The preparation of the ginkgo biloba extract medicament of the tablet of the embodiment is a preparation method of a conventional tablet, and comprises the steps of uniformly mixing, preparing a soft material, granulating, drying, granulating, mixing and tabletting.

Example 6

This example provides a pharmaceutical preparation of ginkgo biloba extract, which is a tablet, comprising 25g of the ginkgo biloba extract of example 2, 26 g of lactose, 26 g of mannitol, 10.8 g of sodium carboxymethyl starch (7.2 g of the above are added, 3.6 g of the above are added), 20 g of fumaric acid, 8 g of aspartame, 2g of berry essence, 8 g of sodium bicarbonate and 0.8 g of magnesium stearate, and 800 tablets are prepared in total, each tablet weighs 158.3 mg, and the content of the ginkgo biloba extract in each tablet is 31.25 mg, that is, the content of the ginkgo biloba extract in each pharmaceutical preparation is 19.74%.

Example 7

This example provides a pharmaceutical preparation of ginkgo biloba extract, which is a tablet, comprising 25.86g of the ginkgo biloba extract of example 3, 26 g of lactose, 26 g of mannitol, 10.8 g of sodium carboxymethyl starch (7.2 g of the above are added, 3.6 g of the above are added), 20 g of fumaric acid, 8 g of aspartame, 2g of berry essence, 8 g of sodium bicarbonate and 0.8 g of magnesium stearate, and 586 tablets were prepared in total, each tablet weighs 217.5 mg, and the ginkgo biloba extract content in each tablet is 44.13 mg, that is, the ginkgo biloba extract content in each pharmaceutical preparation of ginkgo biloba extract is 20.29%.

Detection of

Content detection is carried out on each component of the ginkgo biloba extract prepared in the example 1, a sample is the ginkgo biloba extract provided in the example 1, and the specific detection is as follows:

(1) total flavonol glycosides

The instrument is a high performance liquid chromatograph (provided with an ultraviolet detector); electronic balance (parts per million); an ultrasonic cleaner; a constant temperature water bath kettle.

The reagent comprises quercetin, kaempferide and isorhamnetin reference substance; acetonitrile (chromatographically pure); water (deionized water); methanol, hydrochloric acid, phosphoric acid (analytical grade).

Preparing a reference substance solution: taking a proper amount of quercetin, kaempferide and isorhamnetin reference substances, precisely weighing, and adding methanol to prepare a mixed solution containing 30 mu g of quercetin, kaempferide and isorhamnetin per 1 mL.

Sample treatment, about 35mg of a sample is taken and put in a 250mL flat-bottomed flask, 50mL of methanol-25% hydrochloric acid (4:1, fresh formula) mixed solution is added, the mixture is refluxed in a water bath at 80 ℃ for 30min, the mixture is rapidly cooled to room temperature, the mixture is transferred to a 100mL volumetric flask, the mixture is diluted to a scale mark by methanol, the mixture is shaken up and filtered by a 0.45 mu m filter membrane, and the filtrate is taken and put on a machine for measurement.

Chromatographic conditions of chromatographic column: c18 column (4.6 mm. times.250 mm, 5.0 μm); mobile phase: acetonitrile: water: phosphoric acid 40:60: 0.25; the flow rate is 1.0 mL/min; column temperature: 35 ℃; sample introduction amount: 10 mu L of the solution; detection wavelength: 360 nm.

And (3) sample determination: accurately weighing 10 μ L (or same volume) of each of the sample solution and the reference solution, injecting into a high performance liquid chromatograph for separation and determination, and performing qualitative determination and quantitative determination by external standard method according to retention time of the reference solution.

And (4) calculating a result:

in the formula:

X_i-the content of quercetin, kaempferide and isorhamnetin in the sample,%;

A_s-peak area of the sample solution;

A_std-peak area of control solution;

C_std-the concentration of the control solution; units are micrograms per milliliter (μ g/mL);

v-sample volume to volume in milliliters (mL);

m is the sample weighing in grams (g);

10000-unit conversion factor.

X＝(X₁+X₂+X₃)×2.51

In the formula:

x-total flavonol glycoside content of the sample,%.

X₁、X₂、X₃-the contents of quercetin, kaempferide and isorhamnetin in the sample are respectively percent. The calculation results are expressed as the arithmetic mean of the replicates, with 3 significant digits remaining.

The detection results are shown in figure 1 and figure 2, and the calculated content of the ginkgo total flavonol glycosides is 37.70% (calculated on a dry basis).

(2) Terpene lactones

The instrument comprises the following steps: high performance liquid chromatography (equipped with evaporative light scattering detector); electronic balance (parts per million); an ultrasonic cleaner.

Reagent: bilobalide A, bilobalide B, bilobalide C and bilobalide reference substance; methanol (chromatographically pure); ethyl acetate, tetrahydrofuran, hydrochloric acid, sodium acetate (analytical purity); water (deionized water).

Preparing a reference substance solution: accurately weighing appropriate amount of bilobalide A, bilobalide B, bilobalide C and bilobalide reference substances, and adding 50% methanol to obtain 0.30mg mixed solution containing bilobalide A, bilobalide B, bilobalide C and bilobalide per 1 mL.

Sample treatment: accurately weighing 40mg of a sample in a 50mL polypropylene graduated centrifuge tube, adding 10mL of water, placing the sample in ultrasonic waves for ultrasonic dissolution, adding 2 drops of 37% hydrochloric acid, shaking and extracting the solution for 4 times (15mL, 10mL and 10mL) by using ethyl acetate, combining extracting solutions, washing the extracting solutions by using 20mL of 5% sodium acetate solution, separating the sodium acetate solution, and then washing the solution by using 10mL of ethyl acetate. Mixing the ethyl acetate extractive solution and the washing solution, washing with water for 2 times, each time 20mL, separating the water solution, washing with ethyl acetate 10mL, mixing the ethyl acetate solutions, concentrating the obtained extractive solution under reduced pressure to dry, transferring into 25mL volumetric flask with methanol, metering to desired volume, shaking, filtering with 0.45 μm filter membrane, collecting the filtrate, and measuring.

Chromatographic conditions are as follows: a chromatographic column: c18 column (4.6 mm. times.250 mm, 5.0 μm); mobile phase: methanol: tetrahydrofuran: water 25:10: 65; the flow rate is 1.0 mL/min; column temperature: 35 ℃ is carried out. Detector parameters: agilent Technologies ELSD;

Evaporator Temperature：35℃；Nebulizer Temperature：35℃；Gas Flow Rate：1.5SLM。

and (3) sample determination: precisely sucking 1 μ L, 2 μ L, 5 μ L and 10 μ L of reference solution and 10-L00 μ L of test solution, respectively, injecting into a liquid chromatograph, measuring, determining the retention time of the reference solution, and calculating the contents of ginkgolide A, ginkgolide B, ginkgolide C and bilobalide by using external standard four-point method logarithmic equation.

And (4) calculating a result:

X_i＝EXP[(LnS_y-A)/B)]/V_j×V_d×F/m/10

in the formula:

X_i-the content of ginkgolide a, ginkgolide B and ginkgolide C and bilobalide in the sample,%;

S_y-sample peak area;

a-intercept;

b-slope;

V_j-sample size in microliters (μ L);

V_d-sample volumetric volume in milliliters (mL);

f is the dilution factor of the sample solution;

m is the sample weighing in grams (g);

10-unit conversion factor.

X＝X₁+X₂+X₃+X₄

In the formula:

x-terpene lactone content,%, in the sample.

X₁、X₂、X₃、X₄-the content of ginkgolide a, ginkgolide B, ginkgolide C and bilobalide in the sample, respectively,%. The calculation results are expressed as the arithmetic mean of the replicates, with 3 significant digits remaining.

The detection results, as shown in fig. 3 and 4, calculated to give a terpene lactone content of 37.00% (on a dry basis).

(3) 7-demethyl ginkgetin

The instrument comprises the following steps: high performance liquid chromatography (with ultraviolet detector); electronic balance (parts per million); an ultrasonic cleaner.

Reagent: 7-demethylation ginkgetin reference substance; methanol, acetonitrile (chromatographically pure); water (deionized water); methanol, dimethyl sulfoxide, phosphoric acid (analytical grade).

Preparing a reference substance solution: standard stock solutions: precisely weighing 0.0300g of 7-demethyl ginkgetin, placing in a volumetric flask with 100mL, adding 2.0mL of dimethyl sulfoxide for ultrasonic dissolution, adding 80mL of 75% methanol, ultrasonically mixing, and fixing the volume with 75% methanol; standard working solution: the standard stock solution was diluted stepwise with 75% methanol solution to a series of standard working solutions with concentrations of 29.4. mu.g/mL, 5.88. mu.g/mL, 1.18. mu.g/mL, 0.236. mu.g/mL, and 0.0588. mu.g/mL, respectively.

Sample treatment: precisely weighing 0.1000g of sample, placing the sample in a 50mL volumetric flask, adding 45mL of 75% methanol, performing ultrasonic-assisted extraction for 10min, cooling to room temperature, metering volume with 75% methanol, filtering with a 0.45 μm filter membrane, and taking the filtrate and measuring on a machine.

Chromatographic conditions are as follows: a chromatographic column: c18 column (4.6 mm. times.250 mm, 5.0 μm); mobile phase: 0.1% phosphoric acid (a) -acetonitrile (B), gradient elution: 30% B (0-4 min); 30-100% B (4-25 min); 100% B (25-30 min); 100-30% B (30-31 min); 30% B (31-35 min); the flow rate is 1.0 mL/min; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution; detection wavelength: 330 nm.

And (4) calculating a result: the content of the 7-demethylginkgo biflavone in the sample is expressed by mass fraction X, the numerical value is expressed by milligram per gram (‰), and the calculation formula is as follows:

in the formula:

A_s-peak area of 7-demethylginkgetin in the sample solution;

A_std-peak area of 7-demethylginkgetin in control solution;

C_std-the concentration of 7-demethylginkgetin in the control solution in micrograms per milliliter (μ g/mL);

v-sample volume to volume in milliliters (mL);

m is the sample weighing in grams (g);

1000-unit conversion factor.

The calculation results are expressed as the arithmetic mean of the replicates, with 3 significant digits remaining.

The detection result is shown in fig. 5 and fig. 6, and the content of the 7-demethylation ginkgetin is calculated to be 0.521 per mill.

(4) Ginkgo total acid

Measuring by high performance liquid chromatography (general rule 0512).

Chromatographic conditions and system applicability test with octadecylsilane chemically bonded silica as filler (column length 150mm, column inner diameter 4.6mm, particle size 5 μm); using acetonitrile containing 0.1% trifluoroacetic acid as mobile phase A and water containing 0.1% trifluoroacetic acid as mobile phase B, and carrying out gradient elution according to the specification in the following table; the detection wavelength was 310 nm. The number of theoretical plates is not less than 4000 according to the peak of neoacidity of ginkgo.

Preparing reference solution by precisely weighing appropriate amount of neoacid ginkgolide reference, and adding methanol to obtain solution containing 1 μ g of neoacid ginkgolide reference per lmL as reference solution; and preparing a proper amount of total ginkgolic acid reference substance into a solution containing 20 mu g of total ginkgolic acid per lmL with methanol to serve as a reference solution for positioning.

Preparing test solution by weighing about 2g of the powder, precisely weighing, placing in a conical flask with a stopper, precisely adding 10mL of methanol, weighing, making it dissolve by Lap-Bee sound, cooling, supplementing the lost weight with methanol, shaking, filtering, and collecting the filtrate.

The determination method comprises precisely sucking sample solution, reference solution and positioning reference solution by 50 μ 1, respectively, injecting into liquid chromatograph, calculating total peak area of chromatogram peak corresponding to total ginkgolic acid reference in the sample solution, and calculating total ginkgolic acid content by semen Ginkgo neo-acid reference external standard method.

The content of total ginkgolic acid in the product is not more than 1.0 mg/kg.

Test results referring to fig. 7 and 8, test results: the total acids of ginkgo biloba were not detected (< 1 ppm).

(5)

Ethyl acetate was measured by gas chromatography (general rule 0521) at 0.5%.

N-heptane should not exceed 0.5% as determined by gas chromatography (general rule 0521).

The n-butanol content should not be measured by gas chromatography (general rule 0521) at 0.5%.

The peak area ratio of the flavone aglycone is calculated according to the total flavonol glycoside chromatogram under the content determination items, and the peak area ratio of the quercetin to the kaempferide is 0.8-1.2.

The ginkgo biloba leaf extracts of examples 2 to 4 were also tested in the same manner as described above, and the results were matched with the contents of the respective substances of the corresponding examples.

Experimental example 1

The ginkgo biloba extract of example 1 (test 1) and the ginkgo biloba extract of example 3 (test 2) were tested for their ability to scavenge DPPH radicals.

The detection method comprises the following steps:

the test was set up with a reference control,

the injection, experimental method and reference substance are compared with the test substance for eliminating DPPH free radical.

Accurately weighing 7.9mg of DPPH reagent, dissolving with absolute ethyl alcohol (analytically pure), diluting to 100mL of volume, preparing a DPPH ethanol solution with the final concentration of 0.2mmol/L, placing in a brown bottle, and refrigerating for later use or at 2-8 ℃.

Preparing the ginkgo biloba extracts of examples 1 and 3 into a test sample, namely an absolute ethyl alcohol solution, by taking the concentration of the total ginkgo flavonol glycosides as a reference; in order to eliminate the edge effect of the 96-well plate, the peripheral wells are discarded without use, starting from the 2B well, 20 mul of the test sample and 200 mul of DPPH solution are added into each well, 3 wells are repeated, and the measured value is recorded as Ai; adding 20 mul of absolute ethyl alcohol and 200 mul of DPPH solution into each hole of the control group, repeating 3 holes, and recording the measured value as Aj; the blank was added with 20. mu.l of the test article and 200. mu.l of absolute ethanol, and 3 wells were repeated, and the measurement was designated A0. Light-proof protectorAnd reacting at room temperature for 40min, and measuring the light absorption value at 517nm by using an enzyme-labeling instrument. Calculating DPPH clearance of the sample according to the measured data, adopting EXCEL and ORIGIN 8.0 to process and analyze the data, drawing a dose-effect relation curve of the sample for eliminating free radicals by taking the logarithmic value of the concentration of the sample as an abscissa and the clearance as an ordinate, and solving the EC of the sample₅₀And (4) concentration.

DPPH radical scavenging ratio (%) - [1- (A)_i-A_j)/A₀]×100％

In order to verify the reliability of the test, a series of solutions of sodium ascorbate are prepared by using a known strong reducing agent vitamin C (sodium ascorbate) as a positive control, the radical clearance rate of the solutions is determined according to the method, and simultaneously, in order to further verify the repeatability of the test, the tests are repeated once again after the test is completed.

DPPH free radical scavenging EC of positive control sodium ascorbate in two experiments₅₀The values are 65.72 mu g/ml and 70.06 mu g/ml respectively, so that the reliability of the test method is verified, and the results of two tests on the DPPH removing capacity of the test products 1-2 and the reference control are summarized as follows:

remarking: EC (EC)₅₀The value is the concentration of ginkgo total flavonol glycosides

Under the present test conditions, the effectiveness of the ginkgo biloba extract of example 1 (test article 1) and the ginkgo biloba extract of example 3 (test article 2) and the reference control for DPPH radical scavenging is established. The test samples (1-2) have equivalent ability of removing DPPH free radicals and are all obviously stronger than gold.

Experimental example 2

The ginkgo biloba extract of example 1 (test 1) and the ginkgo biloba extract of example 3 (test 2), the reference control (gold multiinjection) were examined for anti-platelet aggregation activity.

The ginkgo biloba leaf extract of example 1 was tested for its anticoagulant effect in mice by measuring the Clotting Time (CT) in mice. The specific method comprises the following steps:

30 quarantine-qualified KM mice are taken, and are 4-6 weeks old, half male and half female, and are randomly divided into 5 groups (namely a negative control group, a positive control group, the ginkgo biloba extract (test article 1) group in example 1, the ginkgo biloba extract (test article 2) group in example 3 and a reference control article group) according to gender, and each group comprises 6 mice. The ginkgo biloba extract of example 1 (test article 1) group is administered by single tail vein injection, the ginkgo biloba extract of example 1 (test article 1) group of example 3 (test article 2) group is administered by the ginkgo biloba extract of example 3, the gold multi-injection is administered by the reference control article group, the administration dosage is 2.45mg/kg, the administration volume is 0.07mL/10g, the physiological saline with the same volume is administered by the negative control article group, and the heparin sodium injection with the same volume is administered by the positive control article group (the administration concentration is 1250 units/mL). After 10 minutes of administration, the mice were subjected to intracameral blood sampling using a broken capillary (about 1.5cm long), after the 1 st drop of blood was removed, 1 drop of blood was dropped onto the slide glass, immediately counted using a stopwatch, gently picked up 1 time from the edge of the blood drop to the middle with a pin every 30s, when a blood streak was picked up, the timing was stopped, and the time was recorded, which was the in vitro clotting time.

The results are expressed as mean ± standard deviation (x ± s), statistically processed using EXCEL software, and the difference in clotting time between each group and the negative control group was compared by the paired T test, with a test level P value of 0.05.

The blood coagulation time of all mice in the positive control (heparin sodium) group is more than 600s, and the statistical significance is longer than that of the negative control group, so that the test method is reliable. The detection results are as follows:

remarking: 1: the test article 1-2 and gold multi-group administration dosage is calculated by ginkgo leaf extract, 2: the administration dosage of the heparin sodium pre-test is 12500 units/kg, and the dosage of the formal test is 8750 units/kg;

*：P＜0.05；**：P＜0.01。

under the test conditions, the effectiveness of the ginkgo biloba extract of example 1 (test sample 1), the ginkgo biloba extract of example 3 (test sample 2) and the reference control in prolonging the clotting time of mice was established. Compared with a negative control substance, the blood coagulation time of the mouse can be prolonged by each test substance and each reference substance under the condition of approximate multi-clinical dose of gold, and the effect of the test substances 1-2 is stronger than that of the gold.

Experimental example 3

The ginkgo biloba leaf extract of example 1 was tested for its effect on thickening of the retina.

The detection method comprises the following steps:

fundus laser modeling was performed on 4 rhesus monkeys, specifically, intramuscular injection anesthesia was performed with ketamine 8 mg/kg. Both eyes of the animals were laser-photocoagulated at 8 spots around the fovea in the macular area. The laser parameters are as follows: the wavelength is 532nm, the energy is 0.6-0.7W, and the spot diameter is 50 μm. Fundus angiography and OCT examination were performed 3 weeks after laser photocoagulation. The formation of tertiary or quaternary fluorescence bleed spots by the eye was considered successful in modeling, for a total of 5 successful eyes.

The ginkgo biloba leaf extract of example 1 was orally administered at 1.5mg/kg 21 days after molding. The administration is carried out once daily (1.5mg/kg/d) 16 days before the administration, and twice daily (i.e., 3.0mg/kg/d in total) 17 to 28 days after the administration. The clinical symptoms of the animals were observed every day after the molding, and the examination was performed 14 days and 28 days after the administration, respectively, and the improvement rate of the fluorescein leakage area and the improvement rate of the fundus retinal thickening were calculated.

TABLE 1 improvement of fluorescein leakage area (%)

As can be seen from table 1, the CNV fluorescein leakage area of 1 eye in the ginkgo biloba extract provided by the example of the present invention is reduced, and the CNV fluorescein leakage area of two eyes at D28 is smaller than that of D14.

Table 2 improvement rate of fundus retinal thickening (%)

Eyeball number	Administration of D14	Administration of D28
			1	-64.1	-75.3
2	-142.3	-151.9
			3	113.9	188.9
4	18.2	76.1
			5	-25.9	-21.5

As can be seen from table 2, CNV retinal thickening was reduced in 3 out of 5 eyes after administration, and CNV retinal thickening was lower at D28 than at D14 in 1 eye.

The ginkgo biloba extract provided by the embodiment of the invention has a certain reduction effect trend on the thickening of the retina.

Meanwhile, the change of the characteristics of the animals in the experimental process is detected, and the conditions of ingestion, behavior and activity, body surface hair color, blood biochemistry, body weight and the like of the experimental animals are not abnormal, which indicates that the ginkgo leaf extract provided by the embodiment of the invention has low toxic and side effects.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The ginkgo leaf extract is characterized by comprising 7-demethyl ginkgetin, ginkgo total flavonol glycoside and terpene lactone, wherein the total mass of the ginkgo total flavonol glycoside and the terpene lactone accounts for more than 50% of the mass of the ginkgo leaf extract, and the mass of the 7-demethyl ginkgetin accounts for more than five ten-thousandths of the mass of the ginkgo leaf extract.

2. The ginkgo biloba extract according to claim 1, wherein the total ginkgo flavonol glycosides account for 18.5-37.7% by weight, the terpene lactones account for 40.4-53.7% by weight, and the 7-demethyl ginkgetin accounts for five-thousandths to one-thousandth of the mass of the ginkgo biloba extract;

preferably, the content of total acids of ginkgo biloba is less than 1 ppm.

3. The ginkgo biloba leaf extract according to claim 1 or 2, wherein the ginkgo biloba total flavonol glycosides comprise quercetin glycosides, kaempferol glycosides and isorhamnetin glycosides;

preferably, the quercetin glycoside comprises the following 8 compounds;

preferably, the kaempferol glycoside comprises the following 9 compounds;

preferably, the isorhamnetin glycoside comprises the following 4 compounds:

4. the ginkgo biloba leaf extract according to claim 1 or 2, wherein the terpene lactones comprise bilobalide, ginkgolide a, ginkgolide B and ginkgolide C.

5. A pharmaceutical preparation comprising the extract of Ginkgo biloba leaves as claimed in any one of claims 1 to 4.

6. The pharmaceutical preparation of claim 5, wherein the pharmaceutical preparation is in the form of a solid or liquid preparation;

preferably, the liquid formulation includes any one of injection, oral liquid and eye drop.

7. The ginkgo biloba extract medicament of claim 5, wherein when the ginkgo biloba extract medicament is a tablet, the ginkgo biloba extract medicament comprises an excipient and the ginkgo biloba extract;

8. The pharmaceutical preparation of claim 7, wherein the excipients include diluents, excipients, binders, disintegrating and foaming agents, flavoring agents and lubricants;

9. Use of the ginkgo biloba extract of any one of claims 1-4 or the ginkgo biloba extract medicament of any one of claims 5-8 in the manufacture of a medicament for treating an ocular disease;

preferably, the ocular disease comprises macular degeneration;

preferably, the ocular disease comprises diabetic retinopathy;

preferably, the ocular disease comprises retinal thickening.

10. The use of claim 9, wherein the effective amount of the ginkgo biloba extract is 1.5-3.0mg/Kg in the rhesus monkey animal experiment.