Preparation method and purified product of Ginkgolide B

Abstract

The present invention relates to the technical field of natural product purification, and more specifically, to a method for preparing ginkgolide B and a purified product. The method comprises: mixing a ginkgolide extract with water, boiling the mixture, cooling and filtering the mixture to form a precipitate, then mixing and dissolving the precipitate with a hot alcohol solvent, and then cooling and filtering the mixture. The ginkgolide B prepared by this method has higher purity and yield, is simpler, pollution-free, and more suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product purification, and in particular to a preparation method of ginkgolide B and a purified product. Background Art

[0002] Ginkgo biloba extract is listed in the Chinese Pharmacopoeia and is primarily used as a raw material for preparations containing it. Numerous studies have examined the medicinal effects of ginkgo biloba extract. Ginkgo biloba extract primarily contains two components: ginkgo flavonoids and ginkgolides. Ginkgolides are potent antagonists of platelet-activating factor (PAF), exhibiting antiplatelet aggregation, antithrombotic, anti-atherosclerotic, anti-inflammatory, and anti-allergic effects. Among the ginkgolides, Ginkgolide B possesses the strongest physiological activity, being the strongest PAF antagonist discovered to date and used to treat phlegm and blood stasis syndrome in ischemic stroke. Therefore, Ginkgolide B must be isolated and purified from the Ginkgolides. Existing purification methods for Ginkgolide B typically involve column chromatography and recrystallization. Column chromatography is costly and uses organic solvents, which can be environmentally polluting. Existing recrystallization methods, however, require numerous steps, consume large amounts of solvent, and result in significant product loss, resulting in low yields and purity of Ginkgolide B.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The object of the present invention is to provide a preparation method and purified product of Ginkgolide B. The preparation method provided in the embodiment of the present invention has higher purity and yield of Ginkgolide B, is simpler, pollution-free, and more suitable for large-scale production.

[0005] The present invention is achieved in that:

[0006] In a first aspect, the present invention provides a method for preparing ginkgolide B, comprising: mixing a ginkgolide extract with water and boiling the mixture, followed by cooling and filtering to form a filtered precipitate, then mixing and dissolving the filtered precipitate with a hot alcohol solvent, and then cooling and filtering.

[0007] In an optional embodiment, the method comprises: mixing the ginkgo lactone extract with water and boiling for 30-45 minutes, then cooling to 10-30° C., filtering to form the filtered precipitate, then mixing and dissolving the filtered precipitate with 80-95% hot ethanol, cooling to 10-30° C., standing for more than 48 hours, and then filtering.

[0008] In an alternative embodiment, the temperature of the hot alcohol solvent is 30-50°C.

[0009] In an optional embodiment, the preparation method of the ginkgolide extract comprises: concentrating the alcohol eluate of the macroporous adsorption resin adsorbed with the ginkgo leaf extract until there is no alcohol taste;

[0010] Then, water is added to dilute the mixture to form a diluted medicinal solution, wherein each liter of the diluted medicinal solution corresponds to 3-4 kg of Ginkgo biloba original medicine;

[0011] Next, the diluted drug solution is refrigerated at a temperature below 10° C. for more than 24 hours;

[0012] Then, the supernatant is taken and concentrated to form a concentrated solution, wherein each liter of the concentrated solution corresponds to 5.5-8.5 kg of Ginkgo biloba original medicine;

[0013] Next, the mixture is heated to boiling at normal pressure for 1-3 hours, cooled, and refrigerated at a temperature below 10° C. for more than 48 hours, and then filtered, and the filtered solid is dried.

[0014] In an optional embodiment, the preparation steps of the ginkgo leaf extract include: extracting the ginkgo leaf extract with an alcohol solvent by reflux, collecting the refluxed ethanol solution, and then concentrating it until it has no alcohol taste, then diluting it with water and letting it stand to collect the supernatant.

[0015] In an optional embodiment, the step of preparing the alcohol eluent comprises: passing the ginkgo leaf extract through a macroporous adsorption resin, followed by pre-washing and then re-eluting with an alcohol solvent.

[0016] In an optional embodiment, the step of preparing the alcohol eluent comprises: passing the ginkgo leaf extract through a macroporous adsorption resin, followed by pre-washing with 12-15% ethanol, and then eluting again with 50-80% ethanol to form an alcohol eluent.

[0017] In an optional embodiment, the macroporous adsorption resin is a D101 macroporous adsorption resin column.

[0018] In an optional embodiment, the pre-wash and the re-elution are respectively isocratic elutions.

[0019] In a second aspect, the present invention provides a purified product, which is prepared by the method for preparing ginkgolide B according to any one of the aforementioned embodiments.

[0020] The present invention has the following beneficial effects: The present invention can obtain high-purity ginkgolide B through only two crystallizations, namely, water crystallization and alcohol crystallization, with a high yield of ginkgolide B. Furthermore, the preparation method provided by the present invention significantly reduces the number of crystallizations, making it more suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 The present invention provides a process flow chart of the preparation method of apricot lactone B. DETAILED DESCRIPTION

[0023] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0024] The present invention provides a method for preparing Ginkgolide B. Figure 1 , specifically including:

[0025] Ginkgo biloba leaf extract is prepared, crushed, mixed with ethanol (e.g., 70% ethanol, 75% ethanol, 80% ethanol, etc., 70-80% ethanol) and subjected to reflux extraction 3-4 times, each time for 1-2 hours. The refluxed ethanol solution is then collected and mixed, concentrated until there is no alcohol taste, diluted with water to form a dilution, and allowed to stand for 24-36 hours. The supernatant is taken, i.e., the ginkgo biloba leaf extract, wherein the mass ratio of ginkgo biloba leaf to ethanol is 1:3-5, e.g., 1:3, 1:3.5, 1:4, 1:4.5, 1:5, or any other value between 1:3-5; and each kg of the ginkgo biloba leaf extract corresponds to 30-60 liters of the dilution, e.g., each kg corresponds to 30 liters, 35 liters, 40 liters, 45 liters, 50 liters, 55 liters, 60 liters, or any other value between 30-60 liters.

[0026] The supernatant is passed through a macroporous adsorption resin column (200 kg of resin, diameter-to-height ratio of 1:5-1:10) for adsorption. The aqueous solution flowing out of the resin column is discarded, and the macroporous adsorption resin column is then eluted with ethanol (e.g., 10-15% ethanol, such as 12% ethanol, 13% ethanol, 14% ethanol, and 15% ethanol) and discarded. This pre-washing of the macroporous adsorption resin adsorbed with ginkgo flavonoids and ginkgo lactones removes highly polar tanning components and increases the flavonoid content. The use of the aforementioned ethanol concentration can further increase the content of the resulting ginkgo flavonoids.

[0027] Elution is then performed again with 50-80% ethanol (e.g., 50% ethanol, 55% ethanol, 60% ethanol, 65% ethanol, 70% ethanol, 75% ethanol, 80% ethanol, or any other ethanol between 50-80%). The above elutions are all isocratic elutions.

[0028] The alcohol eluate is collected and concentrated until it is free of alcohol, and then diluted with water to form a diluted solution, wherein each liter of the diluted solution corresponds to 3-4 kg of Ginkgo biloba leaf stock. The diluted solution is then refrigerated at below 10°C for at least 24 hours. This step allows some impurities to precipitate, while lactones and flavonoids do not, thereby increasing the purity and yield of ginkgo lactones and ginkgo flavonoids. Furthermore, controlling the concentration of the diluted solution in the above step helps increase the content of the prepared ginkgo lactones; changing this ratio may result in a decrease in ginkgo lactone yield.

[0029] The supernatant is collected and concentrated to form a concentrated solution, wherein each liter of the concentrated solution corresponds to 5.5-8.5 kg of Ginkgo biloba leaf raw material. The concentration of the concentrated solution is controlled to ensure the concentration of the ginkgolide component in the concentrated solution, which is conducive to the precipitation of the ginkgolide component, but the flavonoid component will not precipitate at this concentration, thereby ensuring the yield of ginkgolide and ginkgo flavonoids.

[0030] The solution is then heated to boiling at normal pressure for 1-3 hours, cooled, and refrigerated at a temperature below 10°C for at least 48 hours, followed by filtration to form a solid. After heating at normal pressure for 1-3 hours and boiling at this concentration, the ginkgolide components readily form a uniform colloidal solution, accelerating the crystallization of the ginkgolide components by utilizing the principle of colloid thermal instability. Refrigeration at a temperature below 10°C for at least 48 hours provides sufficient time for the ginkgolide to crystallize, thereby increasing the yield of the ginkgolide.

[0031] It should be noted that although the embodiments of the present invention provide a method for preparing a ginkgolide extract, it is understandable that the following purification can also be performed using an existing purchased ginkgolide extract.

[0032] The filtered solid, i.e., the ginkgolide extract, is mixed and dissolved with water and boiled for 30-45 minutes (e.g., any value between 30-45 minutes, such as 30 minutes, 35 minutes, 40 minutes, and 45 minutes), and then the liquid is cooled to 10-30°C (e.g., any value between 10-30°C, such as 10°C, 15°C, 20°C, 25°C, and 30°C), and then filtered to form a filtered precipitate. The use of boiling water to dissolve all components in the ginkgolide extract is described herein; upon cooling to room temperature, ginkgolide B will precipitate in large quantities, thereby obtaining a crude product of ginkgolide B, while ginkgolides A and C will precipitate in smaller amounts, thereby achieving crude recrystallization.

[0033] The filtered precipitate is then mixed with a hot alcohol solvent (e.g., 80% hot ethanol, 85% hot ethanol, 90% hot ethanol, 95% hot ethanol, or any other concentration between 80-95%), cooled to 10-30°C, allowed to stand for more than 48 hours, and then filtered. The hot alcohol solvent is used to dissolve the crude recrystallized ginkgolide B in a relatively small amount of solution, and then cooled to a supersaturated state in the alcohol solution, followed by precipitation of highly pure ginkgolide B. The hot ethanol temperature is 30-50°C, for example, any value between 30°C, 35°C, 40°C, 45°C, and 50°C.

[0034] The purity determination method of Ginkgolide B is as follows:

[0035] The chromatographic conditions and system suitability test used octadecylsilane bonded silica gel as the filler; methanol-tetrahydrofuran-water (1:15:84) as the mobile phase; and an evaporative light scattering detector for determination.

[0036] Preparation of reference solution: Accurately weigh an appropriate amount of ginkgolide B reference substance, dissolve and dilute with acetone to make a solution containing 0.5 mg per 1 ml.

[0037] Preparation of test solution: Take about 25 mg of the product, accurately weigh it, place it in a 50 ml volumetric flask, and dilute it to the scale with acetone.

[0038] Determination method: Accurately aspirate 5μl and 10μl of the reference solution and 10μl of the test solution, inject them into the liquid chromatograph, and determine them. Calculate the content of ginkgolide B by the external standard two-point logarithmic equation, and then calculate the purity of this product (excluding the drying loss).

[0039] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0040] Example 1

[0041] This embodiment provides a method for preparing a ginkgolide extract, comprising:

[0042] This embodiment provides a method for simultaneously extracting ginkgo brass and ginkgolides, comprising:

[0043] 100 kg of ginkgo leaves were crushed and refluxed with 500 kg of 80% ethanol for 3 times, each time for 1 hour. The refluxed ethanol solution was collected and concentrated to a state without alcohol taste, diluted with water to 6000 L, and allowed to stand for 24 hours. The supernatant was taken and adsorbed on a D101 macroporous adsorption resin column (resin 200 kg, diameter-to-height ratio 1:5). The water flowing out of the resin column was discarded, and the macroporous adsorption resin column was eluted with 15% ethanol (600 L) and discarded. The macroporous adsorption resin column was eluted with 80% ethanol (1500 L), and the solution was collected and concentrated to a state without alcohol taste. The solution was diluted with water to make the diluted solution 25 L, and refrigerated at 4°C for 24 hours. The supernatant was concentrated to 12.5 L, heated to boiling at normal pressure for 3 hours, cooled, and refrigerated at 4°C for 48 hours. The precipitate was filtered and collected and dried to obtain a ginkgo lactone extract (about 267 grams).

[0044] Example 2

[0045] This embodiment provides a method for preparing ginkgolide B, comprising:

[0046] 5 g of the ginkgolide extract prepared in Example 1 was mixed with 500 mL of water, heated to boiling, and kept boiling for 30 minutes. The mixture was then cooled to 25° C. and filtered. The filtrate was discarded and the filtered precipitate was retained. 200 mL of 80% hot ethanol (50° C.) was added to dissolve the precipitate. The mixture was then allowed to stand at 25° C. for 48 hours and filtered again. The filtered crystals were washed with 80% ethanol and dried to obtain 0.479 g of ginkgolide B.

[0047] Example 3

[0048] This embodiment provides a method for preparing ginkgolide B, comprising:

[0049] 5 g of the ginkgolide extract prepared in Example 1 was mixed with 250 mL of water, heated to boiling, and kept boiling for 60 minutes. The mixture was then cooled to 25° C. and filtered. The filtrate was discarded and the filtered precipitate was retained. 400 mL of 95% hot ethanol (50° C.) was added for dissolution, and the mixture was allowed to stand at 25° C. for 48 hours. The mixture was filtered again, and the crystals obtained by filtration were washed with 95% ethanol and dried to obtain 0.541 g of ginkgolide B.

[0050] Example 4

[0051] This embodiment provides a method for preparing ginkgolide B, comprising:

[0052] 5 g of the ginkgolide extract prepared in Example 1 was mixed with 500 mL of water, heated to boiling, and kept boiling for 60 minutes. The mixture was then cooled to 25° C. and filtered. The filtrate was discarded and the filtered precipitate was retained. 200 L of 80% hot ethanol (50° C.) was added to dissolve the precipitate. The mixture was then allowed to stand at 25° C. for 48 hours and filtered again. The filtered crystals were washed with 80% ethanol and dried to obtain 0.421 g of ginkgolide B.

[0053] The ginkgolide B purified in Example 2-4 was tested, and the results are shown in the following table:

[0054]

[0055] Process optimization

[0056] (1) Boil and dissolve

[0057] The effects of boiling on the content of Ginkgolide B were studied in the laboratory stage, as follows:

[0058] Take two portions of 10 g of ginkgo lactone extract, add water to 50 ml, and dissolve the ginkgo lactone extract under two conditions: keeping it at 50°C for 30 minutes and boiling it for 30 minutes (reflux), respectively. After cooling to room temperature, make up for the lost weight, filter it with a microporous filter membrane, and detect the content of each lactone in the filtrate. The results are shown in Table 1.

[0059] Table 1 Content of each ginkgolide in the filtrate

[0060]

[0061] It can be seen that the contents of ginkgolide A and ginkgolide C in the filtrate after boiling at normal pressure are higher than those after being kept warm at 50℃, while the content of ginkgolide B is very low. This shows that purifying the ginkgolide extract by boiling can effectively remove ginkgolide A and C while retaining ginkgolide B, thereby improving the purity of the crude ginkgolide B.

[0062] (2) Dissolved in ethanol at 30-50℃

[0063] The effect of temperature on purification during ethanol crystallization was studied in the laboratory stage, as follows:

[0064] 3g of crude Ginkgolide B was divided into three portions, each 1g. The crude Ginkgolide B was dissolved in ethanol at different temperatures. The volume of ethanol used, the crystallization rate, and the purity of Ginkgolide B were recorded. 0.5g of crude Ginkgolide B (mixed with water, boiled, cooled, and filtered to obtain a solid) was dissolved in ethanol at different conditions. The solids were then cooled to the same temperature and allowed to stand for 48 hours before the mother liquor was removed to form Ginkgolide B. The resulting products were then washed with a small amount of 80% ethanol. The results are shown in the table below.

[0065]

[0066] As can be seen from the table, dissolution using hot ethanol can save a significant amount of ethanol solvent and achieve crystallization of Ginkgolide B in a shorter time. However, dissolution using 70°C hot ethanol results in a faster crystallization rate due to the reduced amount of ethanol added, resulting in a decrease in the purity of Ginkgolide B. Therefore, 50°C hot ethanol is the optimal ethanol concentration for dissolving and crystallizing Ginkgolide B.

[0067] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing ginkgolide B, characterized in that: include: The ginkgolide extract is mixed with water and boiled for 30-45 minutes, then cooled to 10-30°C and filtered to form a filtered precipitate. The filtered precipitate is then mixed and dissolved with 80-95% hot ethanol, cooled to 10-30°C, allowed to stand for more than 48 hours, and then filtered; the temperature of the hot alcohol solvent is 30-50°C; The preparation method of the ginkgolide extract comprises: concentrating the alcohol eluate of the macroporous adsorption resin adsorbed with the ginkgo leaf extract until there is no alcohol taste; Then, water is added to dilute the mixture to form a diluted medicinal solution, wherein each liter of the diluted medicinal solution corresponds to 3-4 kg of Ginkgo biloba original medicine; Next, the diluted drug solution is refrigerated at a temperature below 10° C. for more than 24 hours; Then, the supernatant is collected and concentrated to form a concentrated solution, wherein each liter of the concentrated solution corresponds to 5.5-8.5 kg of Ginkgo biloba original drug; Next, the mixture is heated to boiling point for 1-3 hours at normal pressure, cooled, and refrigerated at a temperature below 10° C. for more than 48 hours, and then filtered to form the ginkgolide extract.

2. The method for preparing ginkgolide B according to claim 1, wherein The preparation steps of the ginkgo leaf extract include: extracting the ginkgo leaf original medicine by refluxing with an alcohol solvent, collecting the refluxed ethanol solution, and then concentrating it until there is no alcohol taste, then diluting it with water and letting it stand before collecting the supernatant.

3. The method for preparing ginkgolide B according to claim 1, wherein The preparation step of the alcohol eluent comprises: passing the ginkgo leaf extract through a macroporous adsorption resin, followed by pre-washing and then re-eluting with an alcohol solvent.

4. The method for preparing ginkgolide B according to claim 1 or 2, characterized in that: The preparation steps of the alcohol eluent include: passing the ginkgo leaf extract through a macroporous adsorption resin, then pre-washing with 12-15% ethanol, and then eluting again with 50-80% ethanol to form an alcohol eluent.

5. The method for preparing ginkgolide B according to claim 4, wherein: The macroporous adsorption resin is selected from D101 macroporous adsorption resin column.

6. The method for preparing ginkgolide B according to claim 4, characterized in that: The pre-wash and re-elution were isocratic elution.

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