CN115594654A

CN115594654A - Extraction method and application of 7-demethyl ginkgetin and ginkgetin ester

Info

Publication number: CN115594654A
Application number: CN202211338979.4A
Authority: CN
Inventors: 巴卫松; 张爱龙; 王永利; 张兰桐
Original assignee: Hebei Quanmei Biotechnology Co ltd
Current assignee: Hebei Quanmei Biotechnology Co ltd
Priority date: 2022-10-28
Filing date: 2022-10-28
Publication date: 2023-01-13
Anticipated expiration: 2042-10-28
Also published as: CN115594654B

Abstract

The invention relates to the technical field of extraction of natural medicines, and provides an extraction method and application of 7-demethyl ginkgetin and ginkgetin ester. The extraction method of the 7-demethylation ginkgetin and the ginkgetin ester comprises the following steps: s1, mixing a ginkgo leaf extract with a disodium hydrogen phosphate solution, performing ultrasonic treatment, and centrifuging to obtain a centrifugate I and filter residues; s2, mixing the filter residue with a disodium hydrogen phosphate solution, performing ultrasonic treatment, centrifuging, and collecting a centrifugate to obtain a centrifugate II; s3, combining the centrifugate I and the centrifugate II and then extracting to obtain an ester phase I and a water phase; s4, performing secondary extraction on the water phase obtained in the S3, and collecting an ester phase to obtain an ester phase II; s5, combining the ester phase I and the ester phase II, evaporating and concentrating, washing with water, and freeze-drying to obtain the 7-demethyl ginkgetin/bilobanone ester. By the technical scheme, the problem that 7-demethyl ginkgetin, total flavonol glycoside and terpene lactone cannot be simultaneously extracted from the ginkgo leaf extract in the prior art is solved.

Description

Extraction method and application of 7-demethyl ginkgetin and ginkgetin ester

Technical Field

The invention relates to the technical field of extraction of natural medicines, in particular to an extraction method and application of 7-demethyl ginkgetin and ginkgetin ester.

Background

Ginkgo leaves are used by Chinese medicine for treating memory loss, stomach pain, dysentery, hypertension, mental stress and respiratory problems such as asthma, bronchitis and poor circulation and anxiety caused by them. The ginkgo leaf extract mainly contains 6 kinds of biflavones (ginkgo biflavone, isoginkgetin, 7-demethyl ginkgo biflavone, amentoflavone, sciadonna biflavone and 1-5' -methoxy demethyl ginkgo biflavone) and 21 kinds of flavonol glycosides (8 kinds of quercetin glycoside +9 kinds of kaempferide glycoside +4 kinds of isorhamnetin glycoside) and 4 kinds of terpene lactones (bilobalide + ginkgolide A + ginkgolide B + ginkgolide C), has wide medicinal value and application, can play medicinal effects in the aspects of resisting inflammation, resisting allergy, expanding blood vessels, protecting heart and cerebral vessels, improving peripheral blood circulation, reducing serum cholesterol, assisting in anticancer and the like besides having obvious antagonistic PAF receptors, and can be widely applied to the prevention and the health care of systemic diseases such as heart and cerebral vessels, nerves and the like.

Chinese patent CN106109510A discloses a process for extracting ginkgetin from ginkgo biloba extract, the content of the obtained total flavone is 41.56-46.14%, but the extracted total flavone only includes 6 biflavones and 21 total flavonol glycosides, and terpene lactones as one of the effective components of ginkgo biloba are not extracted.

Disclosure of Invention

The invention provides an extraction method and application of 7-demethyl ginkgetin and ginkgetin ester, which solves the problem that 7-demethyl ginkgetin, total flavonol glycoside and terpene lactone can not be extracted from a ginkgo leaf extract simultaneously in the related technology.

The technical scheme of the invention is as follows:

the extraction method of 7-demethyl ginkgetin and ginkgetin ester comprises the following steps:

s1, mixing a ginkgo leaf extract with a disodium hydrogen phosphate solution, performing ultrasonic treatment, and centrifuging to obtain a centrifugate I and filter residues;

s2, mixing the filter residue with a disodium hydrogen phosphate solution, performing ultrasonic treatment, centrifuging, and collecting a centrifugate to obtain a centrifugate II;

s3, combining the centrifugate I and the centrifugate II and then extracting to obtain an ester phase I and a water phase;

s4, performing secondary extraction on the water phase obtained in the S3, and collecting an ester phase to obtain an ester phase II;

s5, combining the ester phase I and the ester phase II, evaporating and concentrating, washing with water, and freeze-drying to obtain the 7-demethyl ginkgetin/bilobanone ester.

As a further technical scheme, the ginkgo biloba extract in the S1 has the ginkgo flavonol glycoside content of not less than 24 percent, the ginkgo terpene lactone (calculated by the total amount of the ginkgolides A, B, C and the bilobalide) content of not less than 6 percent, and the 7-demethyl ginkgo biflavone content of not less than 0.1 thousandth.

As a further technical scheme, the disodium hydrogen phosphate solution in the S1 and the S2 is 0.15 percent of disodium hydrogen phosphate solution.

As a further technical scheme, the mass-volume ratio of the ginkgo biloba extract to the disodium hydrogen phosphate solution in the S1 is 1kg (10-15) L.

As a further technical scheme, the temperature of the ultrasound in the S1 and the S2 is 55-70 ℃.

As a further technical scheme, in the S3, the pH is adjusted to 4.74-4.84 before extraction.

As a further technical scheme, ethyl acetate and n-heptane in a volume ratio of 6.

As a further technical scheme, ethyl acetate and n-butanol in a volume ratio of 4.5.

As a further technical scheme, in the S4, the pH value is adjusted to 3.74-3.84 before the second extraction.

As a further technical scheme, the extract obtained by the extraction method of the 7-demethyl ginkgetin and the ginkgetin ester is applied to the preparation of the medicine for treating eye diseases.

As a further technical solution, the application includes: the method has the advantages of removing free radicals, prolonging blood coagulation time, reducing fluorescence leakage area of the rhesus CNV model, reducing retina thickening, and inhibiting leakage and bleeding of rhesus retinas in NPDR period.

The working principle and the beneficial effects of the invention are as follows:

1. the invention discloses a method for extracting 7-demethyl ginkgetin/bilobanone ester from ginkgo leaf extract, which has simple process and high yield of extracted 7-demethyl ginkgetin/bilobanone ester, wherein the content of 7-demethyl ginkgetin in the obtained 7-demethyl ginkgetin/bilobanone ester reaches 0.037%, the content of total flavonol glycosides reaches 45.70%, and the content of terpene lactone reaches 31.20%.

2. The 7-demethyl ginkgetin/ginkgetin ester extracted by the invention has the effect of removing DPPH free radicals, can obviously prolong the blood coagulation time, has obvious improvement effect on the fluorescence leakage and retina thickening of a rhesus CNV model, and has obvious pharmaceutical activity of inhibiting the leakage, bleeding and the like of rhesus retinas in the NPDR period.

Drawings

The invention is described in further detail below with reference to the drawings and the detailed description.

FIG. 1 is a HPLC test result chart of 7-demethyl ginkgetin control solution provided by the present invention;

FIG. 2 is a HPLC detection result chart of 7-demethylginkgetin in 7-demethylginkgetin/ginkgetin ester provided in example 1 of the present invention;

FIG. 3 is a HPLC detection result chart of the quercetin, kaempferide and isorhamnetin reference substance solution provided by the invention;

FIG. 4 is a chart showing the HPLC detection results of total flavonol glycosides in 7-demethyl ginkgetin/bilobanone ester provided in example 1 of the present invention;

FIG. 5 is a diagram of the HPLC detection result of the ginkgo biloba leaf total lactone reference solution provided by the present invention;

FIG. 6 is a HPLC detection result of terpene lactones in 7-demethyl ginkgo biflavone/ginkgo ketoester provided by example 1 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any inventive step, are intended to be within the scope of the present invention.

The ginkgo biloba extract powder in the following examples and comparative examples was obtained from Linyi Rui kang Liang biology Ltd.

Example 1

S1, weighing 1.8kg of raw material ginkgo leaf extract powder, adding 22L of 0.15% disodium hydrogen phosphate solution, performing ultrasonic extraction for 30min at 55 ℃, and centrifuging at 4000r/min to obtain a centrifugal liquid I and filter residue;

s2, performing ultrasonic extraction on filter residues for 20min at 60 ℃ by using 18L of 0.15% disodium hydrogen phosphate solution, centrifuging at 4000r/min, and collecting a centrifugate to obtain a centrifugate II;

s3, combining the centrifugate I and the centrifugate II, adjusting the pH to 4.74 by using a 25% (g/v) sodium hydroxide solution, extracting for 3 times by using an equal volume of solvent, and combining ester phases to obtain an ester phase I and a water phase, wherein the solvent consists of ethyl acetate and n-heptane with the volume ratio of 6;

s4, adjusting the pH of the water phase obtained in the S3 to 3.74 by using a 10% phosphoric acid solution, and extracting by using an equal-volume solvent to obtain an ester phase II, wherein the solvent consists of ethyl acetate and n-butyl alcohol in a volume ratio of 4.5;

s5, combining the ester phase I and the ester phase II, evaporating and concentrating to dryness, adding 4.5L of water to elute solvent residues, collecting extract, freeze-drying, pulverizing and sieving to obtain 0.4kg of 7-demethyl ginkgetin/bilobanone ester.

The 7-demethyl ginkgetin/bilobanone ester contains 7-demethyl ginkgetin 0.037%, total flavonol glycoside 45.70%, and terpene lactone 31.20%.

Example 2

S1, weighing 1.8kg of raw material ginkgo leaf extract powder, adding 18L of 0.15% disodium hydrogen phosphate solution, performing ultrasonic extraction for 30min at 60 ℃, and centrifuging at 4000r/min to obtain a centrifugal liquid I and filter residue;

s2, performing ultrasonic extraction on filter residues for 20min at 55 ℃ by using a 0.15% disodium hydrogen phosphate solution 18L, centrifuging at 4000r/min, and collecting a centrifugal liquid to obtain a centrifugal liquid II;

s3, combining the centrifugate I and the centrifugate II, adjusting the pH to 4.8 by using a 25% (g/v) sodium hydroxide solution, extracting for 3 times by using an equal volume of solvent, and combining ester phases to obtain an ester phase I and a water phase, wherein the solvent consists of ethyl acetate and n-heptane with the volume ratio of 6;

s4, adjusting the pH of the water phase obtained in the S3 to 3.8 by using a 10% phosphoric acid solution, and extracting by using an equal-volume solvent to obtain an ester phase II, wherein the solvent consists of ethyl acetate and n-butanol with the volume ratio of 4.5;

s5, combining the ester phase I and the ester phase II, evaporating and concentrating, adding 4.8L of water to elute solvent residues, collecting extract, freeze-drying, crushing and sieving to obtain 0.39kg of 7-demethyl ginkgetin/ginkgetin ester.

The 7-demethyl ginkgetin/bilobanone ester contains 7-demethyl ginkgetin 0.028%, total flavonol glycoside 42.80% and terpene lactone 27.70%.

Example 3

S1, weighing 1.8kg of ginkgo leaf extract powder, adding 27L of 0.15% disodium hydrogen phosphate solution, performing ultrasonic extraction for 30min at 70 ℃, and centrifuging at 4000r/min to obtain a centrifugal liquid I and filter residue;

s2, performing ultrasonic extraction on filter residues for 20min at 70 ℃ by using 18L of 0.15% disodium hydrogen phosphate solution, centrifuging at 4000r/min, and collecting a centrifugal liquid to obtain a centrifugal liquid II;

s3, combining the centrifugate I and the centrifugate II, adjusting the pH to 4.84 by using a 25% (g/v) sodium hydroxide solution, extracting for 3 times by using an equal volume of solvent, and combining ester phases to obtain an ester phase I and a water phase, wherein the solvent consists of ethyl acetate and n-heptane with the volume ratio of 6;

s4, adjusting the pH of the water phase obtained in the S3 to 3.84 by using a 10% phosphoric acid solution, and extracting by using an equal-volume solvent to obtain an ester phase II, wherein the solvent consists of ethyl acetate and n-butyl alcohol in a volume ratio of 4.5;

s5, combining the ester phase I and the ester phase II, evaporating and concentrating, adding 5L of water to elute solvent residues, collecting extract, freeze-drying, crushing and sieving to obtain 0.41kg of 7-demethyl ginkgetin/bilobanone ester.

The 7-demethyl ginkgetin/ginkgetin ester contains 7-demethyl ginkgetin 0.029%, total flavonol glycoside 43.30%, and terpene lactone 27.00%.

Example 4

S1, weighing 1.8kg of raw material ginkgo leaf extract powder, adding 27L of 0.15% disodium hydrogen phosphate solution, performing ultrasonic extraction for 30min at 70 ℃, and centrifuging at 4000r/min to obtain a centrifugal liquid I and filter residue;

s4, adjusting the pH of the water phase obtained in the S3 to 6.5 by using a 25% (g/v) sodium hydroxide solution, and extracting by using an equal-volume solvent to obtain an ester phase II, wherein the solvent consists of ethyl acetate and n-butanol in a volume ratio of 4.5;

s5, combining the ester phase I and the ester phase II, evaporating and concentrating, adding 5L of water to elute solvent residues, freeze-drying, crushing and sieving to obtain 0.32kg of 7-demethyl ginkgetin/bilobanone ester.

The 7-demethyl ginkgetin/bilobanone ester contains 7-demethyl ginkgetin 0.024%, total flavonol glycoside 37.80%, and terpene lactone 25.90%.

Comparative example 1

S1, weighing 1.8kg of raw material ginkgo leaf extract powder, adding 27L of water at 70 ℃, dissolving, and centrifuging at 4000r/min to obtain a centrifugate I and filter residues;

s5, combining the ester phase I and the ester phase II, evaporating and concentrating, adding 5L of water to elute solvent residues, freeze-drying, crushing and sieving to obtain 0.37kg of 7-demethyl ginkgetin/ginkgo ketonic ester.

The 7-demethyl ginkgetin/bilobanone ester contains 7-demethyl ginkgetin 0.026%, total flavonol glycoside 41.20%, and terpene lactone 26.60%.

1. Product index analysis:

(1) 7-Demethyl Ginkgo Bilobanone assay (HPLC method)

Chromatographic conditions are as follows: and (3) chromatographic column: c18 column (4.6 mm. Times.250mm, 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 to 30% of B (30 to 31 min); 30% B (31-35 min); flow rate: 1.0mL/min; column temperature: at 40 ℃; sample injection amount: 10 mu L of the solution; detection wavelength: 330nm.

Preparation of a reference solution: standard stock solutions: taking 0.0300g of 7-demethyl ginkgetin, adding 2.0mL of dimethyl sulfoxide for ultrasonic dissolution, adding 80mL of 75% methanol, ultrasonic mixing uniformly, and fixing the volume with 75% methanol; standard working solution: the standard stock solution was diluted with 75% methanol solution to a standard working solution with a concentration of 10. Mu.g/mL.

Preparing a test solution: taking 0.1000g of sample, adding 45mL of 75% methanol, performing ultrasonic-assisted extraction for 10min, cooling to room temperature, diluting to constant volume with 75% methanol, filtering with a 0.45 μm filter membrane, and collecting filtrate, and measuring with a computer.

And (3) sample determination: respectively sucking 10 μ L of each of the reference solution and the sample solution, performing separation and measurement, and performing qualitative determination and quantitative determination by external standard method based on the retention time of the reference solution.

And (4) calculating a result: the content of the 7-demethylginkgetin in the sample is expressed by a 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; units are 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.

The determination results of 7-demethylginkgetin content in 7-demethylginkgetin/ginkgetin ester obtained in example 1 are shown in fig. 1 and fig. 2, and the product has 7-demethylginkgetin content of 0.37mg/g (0.037%) calculated on dried product.

(2) Total flavonol glycoside determination (HPLC method)

Chromatographic conditions are as follows: filling agent: octadecylsilane chemically bonded silica; mobile phase: methanol 0.4% phosphoric acid solution = 48; detection wavelength: 360nm; the number of theoretical plates should not be lower than 2500 calculated from the peak of quercetin.

Preparation of control solutions: taking a proper amount of quercetin, kaempferide and isorhamnetin reference substances, precisely weighing, and adding methanol to prepare a mixed reference solution containing 30 mu g of quercetin, kaempferide and 3 mu g of isorhamnetin per 1mL to obtain the final product.

Preparing a test solution: accurately weighing 35mg of a sample, adding 25mL of a mixed solution of methanol and 25% hydrochloric acid solution =4, placing the mixed solution in a water bath at 80 ℃ for heating reflux for 30min, quickly cooling to room temperature, transferring the mixed solution to a 50mL measuring flask, diluting the mixed solution to a scale with methanol, shaking uniformly, filtering, and taking a filtrate to obtain the compound preparation.

And (3) sample determination: respectively sucking 10 μ L of reference solution and test solution, measuring, calculating the contents of quercetin, kaempferide and isorhamnetin according to the corresponding correction factors in the following table by taking the peak area of quercetin reference as reference, and determining the peak positions of quercetin, kaempferide and isorhamnetin according to the relative retention time of the chromatographic peak of the component to be measured and the chromatographic peak of quercetin, wherein the relative retention time is within +/-5% of a specified value (if the relative retention time deviates more than 5%, the corresponding replaced reference is confirmed as reference), thus obtaining the final product. The relative retention times and correction factors (F) are given in the table below:

total flavonol glycoside content = (quercetin content + kaempferide content + isorhamnetin content) × 2.51.

The determination results of the total flavonol glycosides content in the 7-demethyl ginkgetin/ginkgetin ester obtained in example 1 are shown in fig. 3 and 4, and the total flavonol glycosides content of the product is 45.7% by dry product.

(3) Terpene lactone assay

Chromatographic conditions are as follows: filling agent: octadecylsilane chemically bonded silica; mobile phase: n-propanol tetrahydrofuran: water = 1; detecting with an evaporative light scattering detector; the number of theoretical plates is not less than 2500 calculated according to the peak of bilobalide.

Preparation of control extract solution: taking appropriate amount of folium Ginkgo total lactone control extract, precisely weighing, and adding methanol to obtain solution containing 2.5mg per lmL.

Preparing a test solution: accurately weighing 0.15g of the product, adding 10mL of water, heating in a water bath to dissolve, adding 2% hydrochloric acid solution for 2 drops, shaking and extracting with ethyl acetate for 4 times (15 mL, 10mL and 10 mL), combining the extracting solutions, washing with 5% sodium acetate solution for 20mL, separating the sodium acetate solution, washing with ethyl acetate 10mL, combining the ethyl acetate extracting solution and the washing solution, washing with water for 2 times and 20mL each time, separating the water solution, washing with ethyl acetate 10mL, combining the ethyl acetate solutions, recovering the solvent till dryness, dissolving the residual liquid with methanol, transferring to a 5mL volumetric flask, adding methanol to the scale, shaking uniformly, filtering, and taking the filtrate to obtain the product.

And (3) sample determination: respectively sucking 5 μ L and 10 μ L of the reference extract solution and 5-L0 μ L of the sample solution, measuring, and calculating the contents of bilobalide, bilobalide A, bilobalide B and bilobalide C by using an external standard two-point method logarithmic equation.

The determination results of terpene lactone content in 7-demethyl ginkgetin/bilobanone ester obtained in example 1 are shown in fig. 5 and fig. 6, and the terpene lactone content of the product is 31.2% calculated on dried product.

According to the method, the content of 7-demethyl ginkgetin, total flavonol glycoside and terpene lactone in the 7-demethyl ginkgetin/ginkgetin ester obtained in the examples 2 to 4 and the comparative example 1 is tested, and the yield is calculated, wherein the calculation formula is as follows:

yield = 7-mass of demethylated ginkgetin/ginkgo ketoester actually obtained/mass of ginkgo biloba extract powder 100%

The results of the content and yield tests are reported in table 1:

TABLE 1 yield of 7-demethylginkgetin/bilobanone ester and content of 7-demethylginkgetin, total flavonol glycosides, terpene lactones

As can be seen from table 1, the yields of 7-demethylginkgetin/bilobanone ester obtained in examples 1 to 3 of the present invention reached more than 21.67%, wherein the content of 7-demethylginkgetin was more than 0.028%, the content of total flavonol glycosides was more than 42.8%, and the content of terpene lactones was more than 27%, and example 4 differs from example 3 only in that the pH of the aqueous phase obtained in S3 was adjusted to 6.5 with 25% (g/v) sodium hydroxide solution in S4, and the yields of 7-demethylginkgetin/bilobanone ester obtained and the contents of 7-demethylginkgetin, total flavonol glycosides, and terpene lactones therein were all lower than in example 3, so that the yields of 7-demethylginkgetin/bilobanone ester and the yields of 7-demethylginkgetin, total flavonol glycosides, and terpene lactones obtained when the pH of the aqueous phase obtained in S3 was adjusted to 3.74 to 3.84 with phosphoric acid solution in S4 were the highest.

Comparative example 1 differs from example 3 only in that comparative example 1 is that ginkgo biloba extract powder is directly dissolved in water, and after separation, the filter residue is extracted with disodium hydrogen phosphate solution; and in the embodiment 3, the extract powder of the ginkgo leaves is directly extracted by using a disodium hydrogen phosphate solution, the filter residue is extracted by using the disodium hydrogen phosphate solution, and the yield of the 7-demethyl ginkgetin/ginkgo ketone ester obtained in the comparative example 1 and the contents of the 7-demethyl ginkgetin, total flavonol glycoside and terpene lactone in the 7-demethyl ginkgetin/ginkgo ketone ester are lower than those in the embodiment 3.

2. Application study: the application study of the 7-demethyl ginkgetin/ginkgetin ester obtained in example 1 on the aspects of scavenging free radicals, prolonging blood coagulation time, reducing fluorescence leakage area of rhesus CNV model, reducing retina thickening, and inhibiting leakage and hemorrhage of rhesus retina in NPDR period.

(1) Test research on in-vitro DPPH free radical elimination of 7-demethyl ginkgetin/ginkgetin ester

The purpose is as follows: the test is intended to measure the in vitro scavenging capacity of 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH free radical) by adopting an enzyme labeling method.

The method comprises the following steps: preparing a DPPH ethanol solution with the concentration of 0.2mmol/L, and refrigerating for later use; preparing the test substance into the test substance absolute ethyl alcohol solution with the total flavonol glycoside concentration of 0.01mg/mL, 0.05mg/mL, 0.1mg/mL, 0.2mg/mL, 0.4mg/mL and 0.8mg/mL, discarding the peripheral holes from 2B holes in order to eliminate the edge effect of a 96-hole plate,

test groups: add 20. Mu.L of test substance and 200. Mu.L of DPPH solution to each well, repeat 3 wells, and record the measurement as A _i ；

Control group: add 20. Mu.L of absolute ethanol and 200. Mu.L of DPPH solution to each well, repeat 3 wells, and record the measurement as A _j ；

Blank group: add 20. Mu.L of test substance and 200. Mu.L of absolute ethanol, repeat 3 wells, and record the measurement as A ₀ ；

Storing in dark place, reacting at 40min at room temperature, and measuring light absorption value at 517nm with enzyme labeling instrument. From the measured data, the DPPH clearance of the test substance was calculated.

DPPH radical clearance (%) = [1- (a) _i -A _j )/A ₀ ]×100％

As a result: EC for eliminating DPPH free radical by 7-demethyl ginkgetin/bilobanone ester based on total drug concentration in test substance ₅₀ The value was 300. Mu.g/mL.

And (4) conclusion: under the test conditions, the 7-demethyl ginkgetin/ginkgetin ester provided by the invention has the effect of eliminating DPPH free radical, and EC ₅₀ The value was 300. Mu.g/mL.

(2) Study on influence of 7-demethylation ginkgetin/ginkgo ketone ester intragastric administration on blood coagulation time of mice

The purpose is as follows: the test is intended to observe the anticoagulation of 7-demethylginkgetin/bilobanone ester.

The method comprises the following steps: 30 KM mice with qualified quarantine, 19.5-23.1 g and male and female halves are taken and randomly divided into 3 groups according to weight and sex, namely a solvent control group, a gold multi-reference control group and a test object group, wherein each group comprises 10 mice and the male and female halves. The test group is given with 25.6mg/kg of 7-demethyl ginkgetin/ginkgo ketone ester suspension, the reference control group is given with 60.8mg/kg of gold multi-suspension, the solvent control group is given with pure water, and the two groups are both administered by intragastric administration, the administration volume is 0.4mL/10 g/time, 1 day 2 times, and 5 days of continuous administration. Blood is taken from the inner canthus 0.5h after the last administration, 1 drop of blood is dropped on a glass slide, a stopwatch is used for timing immediately, a pin is used for gently picking from the edge of the blood drop to the middle 1 time every 30s, the timing is stopped when a blood streak is picked up, and the time is recorded, namely the blood coagulation time.

As a result: the mean blood coagulation time of the mice in the vehicle control group is 141 +/-65(s), the average blood coagulation time of the gold multi-reference control group is 215 +/-85(s), and the average blood coagulation time of the test object group is 287 +/-51(s); compared with the vehicle control group, the blood coagulation time of the test object group and the blood coagulation time of the reference control group are obviously prolonged (P is less than 0.05 or 0.01). Compared with a reference control group, the blood coagulation time of the test group is obviously prolonged (P is less than 0.05).

And (4) conclusion: under the condition of the test, under the clinical equivalent dose, the 7-demethyl ginkgetin/ginkgetin ester provided by the invention is 25.6mg/kg, 2 times per day, and the continuous gavage administration is carried out for 5 days, so that the blood coagulation time of a mouse can be obviously prolonged, and the anticoagulation effect is stronger than that of a reference control gold under the same dose.

(3) Pharmacodynamic study of 7-demethyl ginkgetin/ginkgetin ester on laser-induced Choroidal Neovascularization (CNV) rhesus model

The purpose is as follows: in the research, 7-demethyl ginkgetin/ginkgetin ester is orally taken for 4 weeks every day continuously, and the inhibition effect of the 7-demethyl ginkgetin/ginkgetin ester on generation of Choroidal Neovascularization (CNV) caused by rhesus laser is observed, so that a design basis is provided for subsequent experiments.

The method comprises the following steps: in the experiment, 33 healthy rhesus monkeys were used for binocular fundus laser photocoagulation to form a CNV molding, and 9 spots were photocoagulated around the macular fovea in each eye. On day 19 of molding (D-2, before administration), 33 monkeys were subjected to FP, FFA, and OCT examination, and 22 monkeys (female 8, male 14) were screened for animals having at least 1 class IV spot in one eye, and 22 animals (36 eyes in total meet the selection criteria) were divided into 6 groups according to the fluorescence leakage area of class IV spots, namely, a placebo group (6, 10 eyes), a positive drug combretastatin group (0.5 mg/eye group (3, 5 eyes), a positive drug extract 6mg/kg group (3, 5 eyes), a test sample 7-demethylginkgetin/ginkgetin 3mg/kg group (3, 5 eyes), a test sample 7-demethylginkgetin/ginkgetin 6mg/kg group (4, 6 eyes), and a 7-demethylginkgetin/ginkgetin 12mg/kg group (3, 5 eyes).

On the 21 st day (D0) of molding, 6 groups of animals were administered with a single intravitreal injection of 50. Mu.L of combaici cypress injection per eye of 0.5mg combicap per eye group; the placebo group orally takes the empty capsules 2 times a day; the folium Ginkgo extract 6mg/kg, 7-demethyl ginkgetin/bilobanone ester 3mg/kg, 6mg/kg and 12mg/kg groups are administered orally 2 times per day.

FP, FFA, OCT examinations were performed on day 14 (D14) and day 28 (D28) before and after dosing; cage-side observations and food intake recordings were made on animals daily; before administration and D28 administration, weighing and blood biochemical and blood routine index examination are carried out. The main curative effect indexes comprise the number of IV-level light spots before and after administration, the change rate of the fluorescence leakage area of the IV-level light spots and the change rate of the retinal thickness of the IV-level light spots.

As a result: on day 19 of molding (D-2, before administration), FP showed abnormal pigment in the fundus oculi photocoagulation region, scar formation, and grey white; FFA is seen with a typical fluorescein leak at the early stage and a strong fluorophore at the late stage; OCT can be seen that the retina is in a shape of a block, a fusiform shape or irregular enlargement and has a liquid dark area under the neural epithelium of the retina, and the retina is obviously thickened.

(1) Placebo group: with D14 and D28, the number of IV spots increased and the area of fluorescein leakage expanded, and the retinal thickness of the IV spots increased somewhat. The rhesus monkey laser photocoagulation induced CNV is similar to clinical CNV in expression, and the disease condition continues to progress without drug intervention.

(2) Positive drug combretastatin 0.5 mg/eye group: after single intravitreal injection administration, the number of IV-level light spots is obviously reduced, the fluorescence leakage area is obviously reduced, and the thickness of the IV-level light spot retina membrane is also obviously reduced; statistically significant differences (p < 0.01) compared to placebo group.

(3) Positive drug ginkgo leaf extract 6mg/kg group: after daily oral administration, the number of IV-grade light spots is not obviously increased, the leakage area of the IV-grade light spots is slightly increased but the increase amplitude is smaller than that of a placebo group, and the thickness of the IV-grade light spot retina is not obviously developed, which shows that the 6mg/kg dose of the ginkgo biloba extract has a certain inhibition effect on the development of CNV, but each pharmacodynamic index is not statistically significantly different (p is more than 0.05) compared with the placebo group.

(4) Test article 7-demethylation ginkgetin/ginkgetin ester 3mg/kg group: after daily oral administration, the number of IV-grade light spots is not obviously increased, the leakage area of the IV-grade light spots is slightly increased but is less than that of a placebo group, and the thickness of the IV-grade light spot retina is not obviously developed, which indicates that the 3mg/kg dose of 7-demethyl ginkgetin/bilobanone ester has a certain inhibition effect on the development of CNV, but each pharmacodynamic index has no statistically significant difference (p is more than 0.05) compared with the placebo group.

(5) Test article 7-demethylation ginkgetin/ginkgetin ester 6mg/kg group: after daily oral administration, the number of IV spots was significantly reduced, the fluorescence leakage area was significantly reduced, and the retinal thickness was also significantly reduced at IV spots, with statistically significant differences (p <0.01 or p < 0.05) compared to placebo.

(6) The test sample 7-demethylation ginkgo biflavone/ginkgo ketoester 12mg/kg group: after daily oral administration, the number of IV spots was significantly reduced, the fluorescence leakage area was significantly reduced, and the retinal thickness was also significantly reduced at IV spots, with statistically significant differences (p <0.01 or p < 0.05) compared to placebo.

During the test period, the animals in each group did not have obvious abnormalities related to administration, such as food intake, drinking, behavior and activity, mental state, body surface hair color, biochemical index of blood, routine index of blood, and the like.

And (4) conclusion: after the test is modeled, the rhesus monkey has the characteristics similar to clinical CNV, the drug effect result of the positive control group Corbai Xipu is similar to that reported in the literature, and the test system is proved to be stable and reliable.

Under the test system, the test sample 7-demethylginkgetin/ginkgetin ester with different doses has good safety, wherein the 7-demethylginkgetin/ginkgetin ester with 6mg/kg dose and 12mg/kg dose has obvious improvement effect on fluorescence leakage and retina thickening of a rhesus monkey CNV model.

(4) Study on effectiveness and safety of 7-demethyl-apricotin/ginkgo ketoester against spontaneous non-proliferative diabetic retinopathy (NPDR)

The purpose is as follows: the study plan evaluates the effectiveness and safety of the test substance on rhesus monkey spontaneous non-proliferative diabetic retinopathy (NPDR) by continuously observing the oral administration of the test substance (7-demethyl-apricot biflavone/ginkgo ketoester) for 60 days.

The method comprises the following steps: 15 male NPDR/DME rhesus monkeys aged 15-25 years and having a diabetic disease course of more than 3 years were selected for this study, and Fundus Photography (FP) or Fundus Fluorescence Angiography (FFA) examinations were performed with Kowa VX-20Fundus Camera, and the diagnosis criteria for NPDR were met: retinal hemorrhage, capillary leakage, hard or cystic leakage, microangiomas, venous beading changes, intraretinal microvascular abnormalities (IRMA) changes, retinal nonperfusion area formation, and the like; or a diagnostic standard for DME: the CFT of a single eye or double eyes is more than or equal to 214 mu m or the average thickness of the EDTRS area of the retina is more than or equal to 319 mu m. The group was divided into 4 groups, eylea 2 mg/eye group 5/4, test article 6mg/kg group 6/3, test article 12mg/kg group 8/4, and placebo group 7/4. Eylea 2 mg/eye group, each animal selected for single or double eye for intravitreal injection. The test samples of 6mg/kg and 12mg/kg groups were administered by nasal gavage once a day for 60 days.

The main efficacy indexes are as follows: 1) The effect of dynamic FP/FFA examination on retinal capillary leakage and/or hemorrhage in spontaneous NPDR rhesus monkeys; 2) The effect of OCT examination on foveal thickness and mean thickness of the EDTRS area. Other observation indexes are as follows: blood biochemistry, body weight, food intake, clinical observation and the like. Throughout the experimental period, FP/FFA/OCT examinations were performed before dosing and on day D60, body weight examinations were performed every 30 days, glycolipid metabolism index measurements were performed every 15 days, and food intake and clinical observations were performed 1 time per day.

As a result: effect on retinal capillary leakage and/or hemorrhage \ edema in spontaneous NPDR rhesus monkeys

Placebo group (n =4,7 eyes): the baseline FFA examination showed that 4 rhesus monkeys all had NPDR stage performance, which were: #251 eyes had IRMA; #1659 Small non-perfused areas of retina were visible in both eyes, and OD infratemporal venous branches were beaded; #4841OD branch of the superior temporal vein visible as venous beading, with IRMA around it, small non-perfused area visible beside the OS macula; #6709OS one vein branch above and one vein branch below the OS was beaded and surrounded by IRMA. There were NPDR-related changes in a total of 7 eyes. Compared to baseline, no significant changes were seen in these abnormalities and the disease appeared stable upon administration of placebo D60.

Mean thickness of the EDTRS area significantly increased by 2 ± 2 μm (p < 0.05) and foveal thickness of the macula increased by 1 ± 3 μm on D60 days of dosing compared to baseline.

Eylene 2 mg/eye group (n =4, 5 eyes were ivt injected): the baseline FFA examination showed that 4 rhesus monkeys all had NPDR stage performance, which were: #1543 multiple eyes small non-perfusion area and mottled fluorescence, the late period of the optic disc of both eyes has obvious fluorescence leakage, the branch of the inferior temporal vein of OS is changed like a bead string; #1841 the superior venous branch of OS has a beaded change and is surrounded by IRMA; #4713 eyes with small perfused areas and capillary leaks; #12465OD branches of the superior temporal vein were bead-like altered and surrounded by IRMA. All injected eyes had NPDR-associated changes.

Compared with the baseline period, #1543 OCT examination 30-60 days after a single injection showed a significant reduction in both ocular edema and hard exudation, with a decrease in the mean thickness of the nine-grid plot. No significant progress was seen during treatment of 3 eyes/3D 60 of the remaining animals. In conclusion, 2/5 eyes showed efficacy under the conditions of this trial, with significant reduction in cystic leakage and retinal edema, similar to clinical patient benefit.

Test article 6mg/kg group (n =3,6 eyes): the baseline FFA examination showed that 3 rhesus monkeys all had NPDR stage performance, which were: #6189OD the first branch of the nasal vein is in the form of a loop and has 2 IRMA around it, small perfusion areas and capillary leakage around the macula of both eyes; #12465OD supranasal venous branches were beaded with IRMA around. Capillary leakage in both eyes; #4831 both eyes had small perfusion areas in the posterior segment and local capillary leakage. There were NPDR-related changes in a total of 6 eyes. Compared to the baseline period, #4831 reduced local capillary leakage in the posterior pole of both eyes on day D60 of dosing compared to pre-dosing. The disease manifestations in the remaining animals were not significantly altered at day D60.

Compared with baseline values, the mean thickness change in the EDTRS region was 0 + -2 μm and the foveal thickness of the macula increased by 2 + -4 μm on day D60; no statistical difference was observed between the change values and the placebo group.

Test article 12mg/kg group (n =4,8 eyes): the baseline FFA examination showed that 4 rhesus monkeys all had NPDR stage performance, which were: #1221 local capillary leakage in the retina was seen in both eyes; local capillary leakage of retina can be seen in #1361 eyes; #1569 binocular disks showed IRMA-like or neovascularization-like changes; there are changes in the beading of the retinal vein branches, significant leakage of the optic discs of both eyes, and local leakage of the retinal capillaries of both eyes. OD like beaded changes with retinal vein branches; the macula part of #1863 eyes was observed to have small non-perfusion area and multiple bleeding spots. There were NPDR-related changes in 8 eyes altogether.

Compared with the baseline period, #1569 had significant leakage relief from both ocular discs at D60 days of dosing, local leakage from both ocular retinal capillaries was similarly relieved, OCT examination showed that both retinal bleeds were visible before and after dosing, but OS had increased retinal bleeds and fluid appeared after dosing; #1863 at D60 administration, there was a significant reduction in the point of hemorrhage in both eyes. The disease manifestations in the remaining animals were not significantly altered at day D60.

Mean thickness in the EDTRS area was significantly reduced by 1 ± 2 μm at day D60 compared to baseline values, with a significant reduction in variance compared to placebo (p < 0.01); the fovea thickness decreased by 2 ± 2 μm, and no statistical difference was observed in the change values compared to placebo.

And (4) conclusion: the product 7-demethyl ginkgetin/ginkgetin ester has activity on NPDR stage rhesus monkey retina leakage and hemorrhage, and shows dose correlation. The 7-demethyl ginkgetin/bilobanone ester was well tolerated throughout the experiment.

In conclusion, the 7-demethyl ginkgetin/ginkgetin ester provided by the invention has obvious pharmaceutical activities on scavenging free radicals, prolonging blood coagulation time, reducing fluorescence leakage area of a rhesus CNV model, reducing retina thickening, inhibiting leakage and bleeding of rhesus retina in NPDR period, and the like, wherein the anti-platelet aggregation activity for prolonging blood coagulation time and reducing retina thickening activity is stronger than that of a ginkgo leaf extract in international medicinal standard, and the ginkgo leaf extract in the international medicinal standard has no effect of inhibiting retina thickening.

The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1.7-extraction method of demethylation ginkgo biflavone and ginkgo ketoester, which is characterized by comprising the following steps:

2. The method of claim 1, wherein the ginkgo biloba extract in S1 is ginkgo biloba extract having a ginkgo flavonol glycoside content of not less than 24%, a ginkgo terpene lactone (calculated on the total of ginkgolides a, B, C and bilobalide) content of not less than 6%, and a 7-demethyl ginkgo biloba extract content of not less than 0.1 ‰.

3. The method of extracting 7-demethylginkgetin and bilobanone ester according to claim 1, wherein the disodium hydrogen phosphate solution in S1 and S2 is 0.15% disodium hydrogen phosphate solution.

4. The method for extracting 7-demethyl ginkgetin and bilobanone ester according to claim 1, wherein the mass-to-volume ratio of the ginkgo biloba extract to the disodium hydrogen phosphate solution in S1 is 1kg (10-15) L.

5. The method for extracting 7-demethylginkgetin and ginkgetin ester according to claim 1, wherein the temperature of the ultrasound in S1 and S2 is 55-70 ℃.

6. The method of claim 1, wherein the pH of the solution of S3 is adjusted to 4.74-4.84 before extraction.

7. The method of claim 1, wherein the extraction in S3 comprises using ethyl acetate and n-heptane as extraction agents in a volume ratio of 6.

8. The method for extracting 7-demethylginkgetin and bilobanone ester according to claim 1, wherein the second extraction in S4 uses ethyl acetate and n-butanol at a volume ratio of 4.5.

9. The method of claim 1, wherein the pH of the solution of S4 is adjusted to 3.74-3.84 before the second extraction.

10. Use of the extract obtained by the extraction method of 7-demethylginkgetin and ginkgetin ester according to any one of claims 1 to 9 in the preparation of a medicament for treating eye diseases.