Preparation method of ginkgo leaf extract with high content of ginkgolide and flavone

Abstract

The invention discloses a ginkgo biloba extract preparation method, which belongs to the technical field of medicine, and comprises the following steps: S1, ginkgo biloba is subjected to wall breaking, crushing and sieving, ethanol is added for extraction, and an obtained extracting solution is filtered, centrifuged and concentrated to obtain a primary concentrated solution; s2, diluting and centrifuging the primary concentrated solution, adsorbing and purifying the primary concentrated solution with macroporous resin, and washing with water to obtain a desorption solution; s3, the desorption liquid is subjected to resin deacidification and secondary concentration and deodorization processes, and a ginkgo leaf primary extract is obtained; and S4, adding ethyl acetate and an enzyme compound into the concentrated solution for extraction, performing liquid-liquid separation, performing centrifugal separation, concentration, cooling, filtration and drying on the supernatant to obtain high-content ginkgolide, and performing concentration and drying on the subnatant to obtain ginkgetin. According to the method, the content of ginkgolic acid is low, and high-content ginkgo brass and ginkgolide are obtained at the same time.

Description

Technical Field

[0001] The invention relates to the technical field of extracting active ingredients of natural products, in particular to a method for preparing a ginkgo leaf extract. Background Art

[0002] Ginkgo flavonoids and ginkgolides contained in ginkgo leaf extract have many biological activities, such as anti-oxidation, free radical scavenging, vasodilation, blood circulation and blood stasis removal, tendon activating and collateral activating, etc. It is a good medicine for the treatment of cardiovascular and cerebrovascular diseases. Its products are widely used in medical treatment, health care products, food, cosmetics and other fields. However, ginkgo leaf extract also contains a small amount of ginkgolic acid compounds with sensitization and mutagenicity. It has been confirmed that when the content of ginkgolic acid is less than 10 ppm, it is harmless to the human body; with the improvement of living standards, the relevant international standards now require the content of ginkgolic acid to be less than 5 ppm. At present, the main methods for removing ginkgolic acid from ginkgo leaf extract are: supercritical carbon dioxide method, macroporous resin method and organic solvent extraction method, or a combination of two or more of these methods. Among them, organic solvent extraction method is more widely used, and the commonly used solvents are hydrocarbon compounds such as petroleum ether, n-hexane, and cyclohexane.

[0003] For example, patent CN116115656A discloses a method of using solvent extraction and resin adsorption to concentrate the alcohol eluent of the macroporous adsorption resin adsorbing the ginkgo leaf extract until there is no alcohol taste; dilute with water to form a diluted solution, and concentrate to form a concentrated solution, cool it, and filter it to obtain ginkgo brass and ginkgolide. However, the process used in the above patent has the following disadvantages: the ginkgo leaf extraction method described in the patent does not involve the removal process of impurities such as ginkgo phenolic acid and chlorophyll, and cannot achieve the separation of high-content ginkgo flavonoids and ginkgolide. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing a ginkgo leaf extract with high content of ginkgolide and flavonoids, which ensures that the content of ginkgolic acid is less than 5ppm and can simultaneously obtain high content of ginkgo brass and ginkgolide.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: A method for preparing a ginkgo leaf extract with high content of ginkgolide and flavonoids comprises the following steps: S1 Ginkgo biloba leaves are crushed and passed through a 60-80 mesh sieve, then added with ethanol for extraction, and the obtained extract is filtered, centrifuged, and concentrated to obtain a primary concentrate; S2 The concentrated solution is diluted, centrifuged, subjected to adsorption and purification treatment with a macroporous resin, and washed with water to obtain a desorbed solution; S3 performs resin deacidification and secondary concentration and deodorization processes on the desorbed liquid to obtain a concentrated solution of the primary extract of ginkgo leaves; S4 adds ethyl acetate and enzyme complex to the concentrated liquid of ginkgo leaf primary extract for extraction and liquid-liquid separation. The upper liquid is centrifuged, concentrated, cooled, filtered and dried to obtain high-content ginkgolide, and the lower liquid is concentrated and dried to obtain ginkgo flavonoids.

[0006] The technical solution of the present invention is further improved in that: in step S1, the ginkgo leaf powder is sieved through a 60-80 mesh sieve, the mass ratio of the ginkgo leaf powder to the alcohol is 1:2.2-3.5, and the extract obtained by extraction with 70%-75% ethanol is concentrated at a temperature of 45-55° C. to an ethanol content of 20%-30%.

[0007] A further improvement of the technical solution of the present invention is that: in step S2, the concentrated liquid is diluted into a diluted liquid with a solid content of 30-45%.

[0008] A further improvement of the technical solution of the present invention is that: in step S2, the dilution liquid is centrifuged in a centrifuge with a centrifugal speed of 6000-10000 rpm.

[0009] The technical solution of the present invention is further improved in that: in step S2, the macroporous resin adsorption adopts any one of D101, HPD100, LKY134 or DM2 macroporous adsorption resin columns; the resin is treated with 0.05 mol / L hydrochloric acid or sodium hydroxide.

[0010] The technical solution of the present invention is further improved in that: the concentration temperature of the secondary concentration and deodorization in step S3 is 50-55°C, the time is 1-2.5h, the viscosity of the concentrate is controlled at 80-200mPa•s, and the residual n-hexane is controlled below 500ppm.

[0011] The technical solution of the present invention is further improved in that: during the extraction process of step S4, the ratio of the concentrate to ethyl acetate is 1.3-3.5:1, the enzyme complex is cellulase and polygalacturonase, the mass ratio of cellulase and polygalacturonase is 2:1, the extraction is stirred for 10 to 30 minutes and then allowed to stand for stratification, and liquid-liquid extraction is separated 3 times.

[0012] The technical solution of the present invention is further improved in that: in step S4, the upper liquid is concentrated, cooled, and filtered to obtain ginkgo lactone, the solid content of the concentrated liquid is 40%-42%, the temperature is controlled at 4-10°C, and the standing time is 10-12h; ethanol is added to the lower liquid, concentrated, and dried to obtain ginkgo flavonoids, the ethanol content is 80%-90%, and the solid content of the concentrated liquid is 40-44%.

[0013] A further improvement of the technical solution of the present invention is that step S4 adopts any one of radiation drying, convection drying, spray drying or freeze drying.

[0014] The further improvement of the technical solution of the present invention is that the content of ginkgo lactone after extraction is 90% to 96%, and the content of ginkgo flavonoids is 79% to 91%.

[0015] Due to the adoption of the above technical solution, the technical progress achieved by the present invention is: The method for preparing ginkgo leaf extract of the present invention can obtain high-content ginkgolide and ginkgoflavonoids through extraction with ethyl acetate, and a series of centrifugal separation, concentration, cooling, concentration, drying, etc., innovative process and application of ratio, time and temperature. The high-content ginkgolide and ginkgoflavonoids can be used as additives for health products, and can also be directly used as drugs for treating hyperlipidemia and hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work. Figure 1 It is a flowchart of the present invention. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with embodiments: A method for preparing a ginkgo leaf extract having high content of ginkgolide and flavonoids, such as Figure 1 As shown, the following steps are included: S1, drying the ginkgo leaves, grinding them into powder, passing through a 60-80 mesh sieve, taking 300-650 g of the ginkgo leaf powder and adding 70%-75% ethanol according to weight, the mass ratio of the ginkgo leaf powder to the alcohol being 1:2.2-3.5, filtering the obtained extract, centrifuging, and concentrating at a temperature of 45-55° C. to obtain a primary concentrate after concentrating to an ethanol content of 20%-30%, preferably 22%-25%; S2, the concentrated solution is diluted, centrifuged, and purified by macroporous resin adsorption to obtain a desorbed solution, wherein the macroporous resin adsorption adopts any one of D101, HPD100, LKY134 or DM2 macroporous adsorption resin columns; the resin is treated with 0.05 mol / L hydrochloric acid or sodium hydroxide.

[0018] S3, subjecting the desorbed liquid to resin deacidification and secondary concentration and deodorization processes, the concentration temperature is 50-55°C, the time is 1-2.5 hours, and the viscosity of the concentrated liquid is controlled at 80-200 mPa•s to obtain a concentrated liquid of ginkgo leaf extract with a ginkgo phenolic acid content of less than 5 PPM.

[0019] S4, adding ethyl acetate to the concentrated solution of the primary extract of ginkgo leaves for extraction to achieve liquid-liquid extraction and separation, wherein the ratio of the concentrated solution to ethyl acetate is 1.3-3.5:1, the enzyme complex is cellulase and polygalacturonase, and the mass ratio of cellulase and polygalacturonase is 2:1. After stirring and extracting for 10 to 30 minutes, let it stand and separate the layers, and repeat the liquid-liquid extraction and separation steps 3 times.

[0020] The upper liquid is centrifuged, concentrated, cooled, filtered and dried to obtain high-content ginkgolide, wherein the temperature of cooling, crystallizing and concentrating the upper liquid is controlled at 0-5°C, and the standing time is 6-10h. The concentrated liquid with a solid content of 40%-42% is controlled at 4-10°C, and the standing time is 10-12h. It is dissolved to an ethanol content of 70%-80%, and then crystallized and dried to obtain high-content ginkgolide. The lower liquid is dissolved in ethanol, concentrated and dried to obtain ginkgo flavonoids. The drying method is any one of radiation drying, convection drying, spray drying or freeze drying.

[0021] Example 1 Take a certain amount of ginkgo leaf powder 500g, add 1500g of 70% ethanol to dissolve, extract, filter and centrifuge the obtained extract, concentrate at 45-55℃, and concentrate to 25% ethanol content; dilute the concentrate, centrifuge, and adsorb and purify it with D101 macroporous resin column to obtain a desorption solution; then perform resin deacidification and secondary concentration and deodorization at a temperature of 50-55℃ for 1h, and the viscosity of the obtained concentrate is 92mPa·s. A ginkgo leaf extract concentrate with a ginkgo phenolic acid content of 4.7PPM is obtained. The concentrate is added in a ratio of 3.5:1 ethyl acetate, the enzyme complex is cellulase and polygalacturonase, and the mass ratio of cellulase and polygalacturonase is 2:1. After stirring and extracting for 10-30min, stand and separate, and extract 3 times in the same way. The upper layer liquid was centrifuged, cooled at 4°C, and allowed to stand for 8 hours for crystallization. The liquid was concentrated to a solid content of 40%, the temperature was controlled at 6°C, and the standing time was 12 hours. After crystallization and spray drying, the ginkgolide content was 91.3%. The lower layer liquid was dissolved in 83% ethanol, concentrated, and spray dried to obtain a ginkgo flavonoid content of 85.6%. Example 2 Take a certain amount of ginkgo leaf powder 500g, add 1250g of 70% ethanol to dissolve, extract, filter and centrifuge the obtained extract, concentrate at 45-55℃, and concentrate to 23% ethanol content; dilute the concentrate, centrifuge, and adsorb and purify it with D101 macroporous resin column to obtain a desorption solution; then perform resin deacidification and secondary concentration and deodorization at a temperature of 50-55℃ for 2.5h, and the viscosity of the obtained concentrate is 169mPa·s. A ginkgo leaf extract concentrate with a ginkgo phenolic acid content of 3.6PPM is obtained. The concentrate is added in a ratio of 1.5:1 ethyl acetate, the enzyme complex is cellulase and polygalacturonase, and the mass ratio of cellulase and polygalacturonase is 2:1. After stirring and extracting for 10-30min, stand and separate, and extract 3 times in the same way. The upper layer liquid was centrifuged, cooled at 4°C, and allowed to stand for 8 hours for crystallization. The liquid was concentrated to a solid content of 42%, the temperature was controlled at 6°C, and the standing time was 12 hours. After crystallization and spray drying, the ginkgolide content was 94%. The lower layer liquid was dissolved in 84% ethanol, concentrated, and spray dried to obtain a ginkgo flavonoid content of 81.2%. Example 3 Take a certain amount of ginkgo leaf powder 500g, add 1300g of 70% ethanol to dissolve, extract, filter and centrifuge the obtained extract, concentrate at 45-55℃, and concentrate to 22% ethanol content; dilute and centrifuge the concentrate, and adsorb and purify it with D101 macroporous resin column to obtain a desorption solution; then perform resin deacidification and secondary concentration and deodorization at a temperature of 50-55℃ for 2h, and the viscosity of the obtained concentrate is 172mPa·s. A ginkgo leaf extract concentrate with a ginkgo phenolic acid content of 4.5PPM is obtained. The concentrate is added in a ratio of 1.3:1 ethyl acetate, the enzyme complex is cellulase and polygalacturonase, and the mass ratio of cellulase and polygalacturonase is 2:1. After stirring and extracting for 10-30min, stand and separate, and extract 3 times in the same way. The upper layer liquid was centrifuged, cooled at 4°C, and allowed to stand for 8 hours for crystallization. The liquid was concentrated to a solid content of 41%, the temperature was controlled at 6°C, and the standing time was 12 hours. After crystallization and spray drying, the ginkgo lactone content was 93%. The lower layer liquid was dissolved in 85% ethanol, concentrated, and spray dried to obtain a ginkgo flavonoid content of 85%. Example 4 Take a certain amount of ginkgo leaf powder 500g, add 1300g of 70% ethanol to dissolve, extract, filter and centrifuge the obtained extract, concentrate at 45-55℃, and concentrate to 25% ethanol content; dilute the concentrate, centrifuge, and adsorb and purify it with D101 macroporous resin column to obtain a desorption solution; then perform resin deacidification and secondary concentration and deodorization at a temperature of 50-55℃ for 1.5h, and the viscosity of the obtained concentrate is 182mPa·s. A ginkgo leaf extract concentrate with a ginkgo phenolic acid content of 2.1PPM is obtained. The concentrate is added in a ratio of 2:1 ethyl acetate, the enzyme complex is cellulase and polygalacturonase, and the mass ratio of cellulase and polygalacturonase is 2:1. After stirring and extracting for 10-30min, stand and separate, and extract 3 times in the same way. The upper layer liquid was centrifuged, cooled at 4°C, and allowed to stand for 8 hours for crystallization. The liquid was concentrated to a solid content of 42%, the temperature was controlled at 6°C, and the standing time was 12 hours. After crystallization and spray drying, the ginkgo lactone content was 92.5%. The lower layer liquid was dissolved in 81% ethanol, concentrated, and spray dried to obtain a ginkgo flavonoid content of 89%.

[0022] Example 5 Take a certain amount of ginkgo leaf powder 500g, add 1300g of 70% ethanol to dissolve, extract, filter and centrifuge the obtained extract, concentrate at 45-55℃, and concentrate to 24% ethanol content; dilute the concentrate, centrifuge, HPD100 macroporous resin column adsorption purification treatment to obtain desorption liquid; then resin deacidification, three times of concentration and deodorization, temperature is 50-55℃, time is 2h, and the viscosity of the obtained concentrate is 85mPa·s. Ginkgo leaf extract concentrate with a ginkgo phenolic acid content of 4.9PPM is obtained. The concentrate is added in a ratio of 3.5:1 ethyl acetate, the enzyme complex is cellulase and polygalacturonase, the mass ratio of cellulase and polygalacturonase is 2:1, and the extraction is stirred for 10-30min and then allowed to stand for stratification, and the same method is used for extraction 3 times. The upper layer liquid was centrifuged, cooled at 4°C, and allowed to stand for 8 hours for crystallization. The liquid was concentrated to a solid content of 40%, the temperature was controlled at 6°C, and the standing time was 12 hours. After crystallization and spray drying, the ginkgo lactone content was 93%. The lower layer liquid was dissolved in 89% ethanol, concentrated, and spray dried to obtain a ginkgo flavonoid content of 84%.

[0023] Comparative Example 1 Take a certain amount of ginkgo leaf powder 500g, add 1500g of 70% ethanol to dissolve, extract, filter and centrifuge the obtained extract, concentrate at 45-55℃, and concentrate to 24% ethanol content; dilute and centrifuge the concentrate, and purify it by adsorption on HPD100 macroporous resin column to obtain a desorption solution; then deacidify the resin, concentrate and deodorize it twice, the temperature is 60-75℃, the time is 2h, and the viscosity of the obtained concentrate is 50mPa·s. A ginkgo leaf extract concentrate with a ginkgo phenolic acid content of 4.9PPM is obtained. The concentrate is added with ethyl acetate in a ratio of 3.5:1, stirred and extracted for 10-30min, and then allowed to stand for stratification, and the same method is used for extraction 3 times. The upper layer liquid is centrifuged, cooled at 4℃, allowed to stand for 8h to crystallize, the temperature is controlled at 8℃, the standing time is 10h, and after spray drying, the ginkgo lactone content is 79%. The lower layer liquid is dissolved in 85% ethanol, concentrated, and spray dried to obtain a ginkgo flavonoid content of 54%.

[0024] The embodiments described above are merely descriptions of preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a ginkgo leaf extract having a high content of ginkgolides and flavonoids, characterized in that The following steps are involved: S1 Ginkgo biloba leaves are crushed and passed through a 60-80 mesh sieve, then added with ethanol for extraction, and the obtained extract is filtered, centrifuged, and concentrated to obtain a primary concentrate; S2 The concentrated solution is diluted, centrifuged, subjected to adsorption and purification treatment with a macroporous resin, and washed with water to obtain a desorbed solution; S3 performs resin deacidification and secondary concentration and deodorization processes on the desorbed liquid to obtain a concentrated solution of the primary extract of ginkgo leaves; S4 adds ethyl acetate and enzyme complex to the concentrated liquid of ginkgo leaf primary extract for extraction and liquid-liquid separation. The upper liquid is centrifuged, concentrated, cooled, filtered and dried to obtain high-content ginkgolide, and the lower liquid is concentrated and dried to obtain ginkgo flavonoids.

2. The method for preparing a ginkgo leaf extract with high content of ginkgolides and flavonoids according to claim 1, characterized in that: In step S1, the ginkgo leaf powder is sieved through a 60-80 mesh sieve, the mass ratio of the ginkgo leaf powder to the alcohol is 1:2.2-3.5, and the extract obtained by extraction with 70%-75% ethanol is concentrated at a temperature of 45-55° C. to an ethanol content of 20%-30%.

3. The method for preparing a ginkgo leaf extract with high content of ginkgolides and flavonoids according to claim 1, characterized in that: Step S2: diluting the concentrated solution into a dilution solution with a solid content of 30-45%.

4. The method for preparing a ginkgo leaf extract with high content of ginkgolides and flavonoids according to claim 3, characterized in that: Step S2: The diluted solution is centrifuged in a centrifuge at a speed of 6000-10000 rpm.

5. The method for preparing a ginkgo leaf extract with high content of ginkgolides and flavonoids according to claim 1, characterized in that: In step S2, the macroporous resin adsorption adopts any one of D101, HPD100, LKY134 or DM2 macroporous adsorption resin columns; the resin is treated with 0.05 mol / L hydrochloric acid or sodium hydroxide.

6. The method for preparing a ginkgo leaf extract with high content of ginkgolides and flavonoids according to claim 1, characterized in that: The concentration temperature of the secondary concentration and deodorization in step S3 is 50-55°C, the time is 1-2.5h, the viscosity of the concentrate is controlled at 80-200mPa•s, and the residual n-hexane is controlled below 500ppm.

7. The method for preparing a ginkgo leaf extract with high content of ginkgolides and flavonoids according to claim 1, characterized in that: In step S4, the ratio of the concentrate to ethyl acetate is 1.3-3.5:1, the enzyme complex is cellulase and polygalacturonase, the mass ratio of cellulase to polygalacturonase is 2:1, the extraction is stirred for 10-30 minutes, then the mixture is allowed to stand and separate, and the liquid-liquid extraction is performed 3 times.

8. The method for preparing a ginkgo leaf extract with high content of ginkgolides and flavonoids according to claim 1, characterized in that: Step S4: the upper liquid is concentrated, cooled, and filtered to obtain ginkgo lactones, the solid content of the concentrated liquid is 40%-42%, the temperature is controlled at 4-10°C, and the standing time is 10-12h; ethanol is added to the lower liquid, concentrated, and dried to obtain ginkgo flavonoids, the ethanol content is 80%-90%, and the solid content of the concentrated liquid is 40-44%.

9. The method for preparing a ginkgo leaf extract with high content of ginkgolides and flavonoids according to claim 1, characterized in that: Step S4 adopts any drying method including radiation drying, convection drying, spray drying or freeze drying.

10. The method for preparing a ginkgo leaf extract with high content of ginkgolides and flavonoids according to any one of claims 1 to 9, characterized in that: The content of ginkgo lactone after extraction is 90% to 96%, and the content of ginkgo flavonoids is 79% to 91%.

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