A method for extracting and purifying ginkgolide B

Abstract

The present invention relates to a method for extracting and purifying ginkgolide B. The method comprises: (1) crushing ginkgo leaves, adding a mixed acid solution for soaking and extraction; (2) concentrating ginkgo leaf extract 1, boiling, adding talcum powder, filtering, refrigerating, and filtering; (3) adding ethyl acetate to extract 2, separating the layers, adding water, separating the layers, filtering, and spin-drying; (4) dissolving the crude extract in ethanol, eluting through a superacidic alumina chromatography column; (5) concentrating the eluate, refrigerating, filtering, and drying; (6) adding the crude ginkgolide B to a mixed solution of n-heptane and ethanol for crystallization. This method not only solves the problems of low extraction rate in steam distillation and large amount of solvent loss in organic solvent extraction, but also improves the yield of ginkgolide B while ensuring the purity of ginkgolide B. It has the advantages of simple preparation process, easy operation, low organic solvent consumption, low cost, high yield and purity, and is suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine extraction methods, and particularly relates to a method for extracting and purifying ginkgolide B. Background Art

[0002] my country accounts for 70% of the world's ginkgo biloba resources. Ginkgo biloba extract (GBE), a product extracted from ginkgo biloba, is the preferred raw material for the prevention and treatment of cardiovascular and cerebrovascular diseases in both the domestic and international markets. The active ingredients of ginkgo biloba extract are primarily flavonoid glycosides and terpenes. Flavonoid glycosides account for 24%, primarily quercetin, kaempferol, and isorhamnetin; terpenes account for 6%, primarily ginkgolides (GA, GB, GC) and bilobalide (BB). Ginkgolide B, one of the terpenoid lactones in GBE, is the most potent platelet-activating factor antagonist discovered to date. It is clinically effective in treating thrombosis, acute pancreatitis, and cardiovascular disease, surpassing other similar drugs. Recent studies have shown that the extract can also be used to treat metastatic cancer.

[0003] However, the structure of ginkgolide compounds is complex, and it is difficult to prepare them by chemical synthesis at present, and the cost is high. Therefore, ginkgolide B is mainly obtained by separating from Ginkgo biloba leaves. Chinese patent CN111171043B discloses a method for extracting ginkgolide B. This method adopts organic solvent extraction, organic solvent separation under carrier conditions, water stratification, and crystallization in three stages. The ginkgolide B yield obtained reaches 0.63g / kg. This method has a large amount of organic solvent consumption and a low yield. Chinese patent CN101392000A discloses a method for separating and purifying ginkgolide monomers. This method is prepared by high-performance liquid phase after multi-step organic solvent extraction using ginkgo leaf extract. This method requires a large amount of organic solvent extraction, high-performance liquid phase separation, and macroporous adsorption resin purification. The method has many steps, and the organic solvent consumption is large, and the cost is high, so it is not suitable for industrialized production. Chinese patent CN101747338A discloses a method for preparing ginkgolide compounds, which is a process for preparing a ginkgolide monomer. The method comprises the following steps: using ginkgo leaf or ginkgo leaf extract as raw material, extracting the lactones by water extraction and then subjecting the extract to a silica gel column to organic solvent extraction, collecting each lactone fraction, and then recrystallizing the lactone fraction. The extraction yield of the aqueous solution is low, and the organic solvent consumption is large. Chinese patent CN101134758A discloses a method for preparing a ginkgolide monomer. The method comprises the following steps: using ginkgo leaf as raw material, extracting the lactones by alcohol extraction and ethyl acetate extraction, and subjecting the extract to a selective polar non-porous adsorption resin to obtain total ginkgolides. The total lactones are then subjected to a silica gel column to collect each lactone fraction and recrystallization to obtain each lactone monomer. The steps are complicated and the cost is high. Up to now, there is no method that is particularly suitable for industrialized production, which is simple, easy to operate, and has low cost, and which obtains a high-yield, high-purity ginkgolide B. Summary of the Invention

[0004] The object of the present invention is to provide a method for extracting and purifying ginkgolide B with simple steps, low cost, low organic solvent consumption, and high purity and high yield.

[0005] The present invention provides a method for extracting and purifying ginkgolide B, comprising:

[0006] (1) Ginkgo biloba leaves are crushed, added to a mixed acid solution, soaked and extracted, and filtered to obtain a ginkgo biloba extract 1, wherein the mixed acid solution is a mixed solution of HCl solution and tartaric acid solution in a volume ratio of 1:1 to 3:1, and the mass ratio of ginkgo biloba leaves to the mixed acid solution is 1:20 to 40;

[0007] (2) Concentrating the ginkgo biloba extract 1 in step (1), heating and boiling, adding talcum powder and stirring until the temperature drops to room temperature, continuing stirring and filtering, refrigerating the filtrate, and filtering to obtain extract 2;

[0008] (3) heating the extract 2 in step (2), adding ethyl acetate and stirring at the same temperature, separating the layers to obtain an organic phase 1, adding water and stirring again, separating the layers to obtain an organic phase 2, filtering, and drying the mixture to obtain a crude extract;

[0009] (4) adding ethanol to the crude extract in step (3) to dissolve, filtering, passing through an acidic alumina chromatography column, and eluting with 85-95% ethanol to obtain an eluate;

[0010] (5) concentrating the eluate in step (4) to an alcohol content of 60%-70%, refrigerating at 4-10° C., filtering, and drying to obtain a crude product of ginkgolide B;

[0011] (6) Add the crude product of ginkgolide B in step (5) to a mixed solution of n-heptane and ethanol, heat and stir at 70-80° C. until dissolved, let it stand and cool to 20-30° C., keep warm for crystallization, filter, and dry to obtain ginkgolide B.

[0012] Preferably, the soaking and extraction temperature in step (1) is 90-100° C., and the soaking and extraction time is 3-5 hours.

[0013] Preferably, the pH of the HCl solution in step (1) is 2-3; the pH of the tartaric acid solution is 2-4.

[0014] Preferably, in step (2), the concentration is performed to a relative density of 1.05-1.15; and the heating and boiling time is 20 to 40 minutes.

[0015] Preferably, the talc powder is activated at 110-120° C. for 0.5-1.5 hours before being added in step (2) to increase the adsorption capacity.

[0016] Preferably, the weight of talc in step (2) is 0.1 to 0.2 times the weight of the extracted concentrated solution.

[0017] Preferably, the refrigeration temperature in step (2) is 0-5°C and the refrigeration time is 6-12 hours.

[0018] Preferably, in step (3), the mixture is heated to 40-50° C. and the stirring time is 20-30 min.

[0019] Preferably, the stirring temperature in step (3) is room temperature, and the stirring time is 20-30 minutes.

[0020] Preferably, in step (3), the weight of ethyl acetate is 1-2 times the weight of the extract 2; and the volume of water is 1-2 times the volume of ethyl acetate.

[0021] Preferably, the refrigeration time in step (5) is more than 12 hours.

[0022] Preferably, the weight of the mixed solution of n-heptane and ethanol in step (6) is 10 to 12 times the weight of the crude ginkgolide B.

[0023] Preferably, the mass ratio of n-heptane to ethanol in step (6) is 1:3 to 1:6.

[0024] Preferably, the crystallization time in step (6) is 3-4 hours.

[0025] Beneficial effects

[0026] The invention combines the advantages of a steam distillation method and an organic solvent extraction method, solves the problems of low extraction rate of the steam distillation method and large amount of solvent loss of the organic solvent extraction method, improves the yield of ginkgolide B, and ensures the purity of ginkgolide B. The invention has the advantages of simple preparation process, easy operation, low organic solvent consumption, low cost, high yield and purity, is suitable for industrial production, and has great application value.

[0027] The present invention has a production cost of about 70 to 92 yuan / g (production cost=total production cost divided by the weight of the product ginkgolide B), an organic solvent consumption of about 1.5 to 2.3 L / g (organic solvent consumption=total volume of organic solvent consumption divided by the weight of the product ginkgolide B), a ginkgolide B yield of not less than 0.78 g / kg (ginkgolide B yield=weight of the final product ginkgolide B product in g / weight of ginkgo leaves in kg), a purity determined by HPLC greater than 99%, and a transfer rate of not less than 39% (transfer rate of ginkgolide B=(weight of the final product ginkgolide B / weight of ginkgolide B in ginkgo leaves)×100%). DETAILED DESCRIPTION

[0028] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0029] The content of ginkgolide B in the ginkgo leaves of the embodiment is about 0.2%.

[0030] Example 1

[0031] A method for extracting and purifying ginkgolide B comprises:

[0032] (1) removing impurities from ginkgo leaves, washing with water, and drying, crushing 10 kg of dried ginkgo leaves, adding a mixed acid solution, heating to 90° C., soaking for 5 h, and filtering to obtain a ginkgo leaf extract 1, wherein the mixed acid solution is a mixed solution of HCl solution and tartaric acid solution in a volume ratio of 1:1, the mass ratio of ginkgo leaves to the mixed acid solution is 1:20, the pH of the HCl solution is 2, and the pH of the tartaric acid solution is 2;

[0033] (2) The ginkgo leaf extract 1 in step (1) was concentrated to a relative density of 1.05, heated and boiled for 20 minutes, transferred to a container, talcum powder was added, stirred and cooled to room temperature, and then continued to stir and began to filter. The filtrate was refrigerated at 0°C for 6 hours, filtered, and the filtrate was taken to obtain extract 2, wherein the talcum powder was activated at 115°C for 1 hour before being added, and the weight of the talcum powder was 0.1 times the weight of the extract concentrate;

[0034] (3) heating the extract 2 in step (2) to 40° C., adding ethyl acetate and stirring for 20 min, wherein the weight of ethyl acetate is 1 times the weight of the extract 2, separating the layers to obtain an organic phase 1, and then adding purified water and stirring for 20 min at room temperature, wherein the volume of purified water is 1 times the volume of ethyl acetate, separating the layers to obtain an organic phase 2, filtering, and drying by spin drying to obtain a crude extract;

[0035] (4) adding 95% ethanol to the crude extract in step (3) and stirring and dissolving it for 40 minutes, wherein the ratio of the volume of ethanol (mL) to the weight of the crude extract (g) is 10, filtering it with a filter membrane, adding an acidic alumina chromatography column treated with 95% ethanol, wherein the weight ratio of the acidic alumina to the crude extract is 20:1, the diameter-to-height ratio of the acidic alumina is 1:10, the acidic alumina is 300 mesh, and eluting it with 3 times (the ratio of the volume of ethanol (mL) to the weight of the crude extract (g)) of 95% ethanol to obtain an eluate;

[0036] (5) The eluate obtained in step (4) was concentrated to an alcohol content of 60%, refrigerated at 4° C. for 13 h, filtered, and dried to obtain a crude product of ginkgolide B;

[0037] (6) The crude product of ginkgolide B obtained in step (5) was added to a mixed solution of n-heptane and ethanol (the weight of the mixed solution of n-heptane and ethanol was 10 times the weight of the crude product of ginkgolide B), heated and stirred at 70°C until dissolved, the mass ratio of n-heptane to ethanol was 1:3, and the mixture was allowed to stand and cool to 20°C. The mixture was kept warm for 3 hours for crystallization, filtered, and dried to obtain 7.8 g of ginkgolide B with a purity of 99.4%, a yield of 0.78 g / kg, and a transfer rate of 39%.

[0038] The production cost of this embodiment is about 79 yuan / g, and the organic solvent consumption is about 2.2 L / g.

[0039] Example 2

[0040] A method for extracting and purifying ginkgolide B comprises:

[0041] (1) removing impurities from ginkgo leaves, washing with water, and drying, crushing 28 kg of dried ginkgo leaves, adding a mixed acid solution, heating to 100° C., soaking for 3 h, and filtering to obtain a ginkgo leaf extract 1, wherein the mixed acid solution is a mixed solution of HCl solution and tartaric acid solution in a volume ratio of 3:1, the mass ratio of ginkgo leaves to the mixed acid solution is 1:40, the pH of the HCl solution is 2.8, and the pH of the tartaric acid solution is 3.5;

[0042] (2) The ginkgo leaf extract 1 in step (1) was concentrated to a relative density of 1.15, heated and boiled for 40 minutes, transferred to a container, talcum powder was added, stirred and cooled to room temperature, and then continued to stir and began to filter. The filtrate was refrigerated at 5°C for 12 hours, filtered, and the filtrate was taken to obtain extract 2, wherein the talcum powder was activated at 115°C for 1 hour before being added, and the weight of the talcum powder was 0.2 times the weight of the extract concentrate;

[0043] (3) The extract 2 in step (2) was heated to 50° C., and then ethyl acetate was added and stirred for 30 minutes. The weight of ethyl acetate was twice the weight of the extract 2. The organic phase 1 was obtained by separation. Purified water was added and stirred for 30 minutes at room temperature. The volume of purified water was twice the volume of ethyl acetate. The organic phase 2 was obtained by separation. The organic phase 2 was filtered and dried to obtain a crude extract.

[0044] (4) adding 95% ethanol to the crude extract in step (3) and stirring and dissolving it for 45 minutes, wherein the ratio of the volume of ethanol (mL) to the weight of the crude extract (g) is 12, filtering it with a filter membrane, adding an acidic alumina chromatography column treated with 95% ethanol, wherein the weight ratio of the acidic alumina to the crude extract is 20:1, the diameter-height ratio of the acidic alumina is 1:10, the acidic alumina is 300 mesh, and eluting it with 2.5 times (the ratio of the volume of ethanol (mL) to the weight of the crude extract (g)) of 95% ethanol to obtain an eluate;

[0045] (5) concentrating the eluate obtained in step (4) to an alcohol content of 70%, refrigerating at 10° C. for 20 h, filtering, and drying to obtain a crude product of ginkgolide B;

[0046] (6) The crude product of ginkgolide B obtained in step (5) was added to a mixed solution of n-heptane and ethanol (the weight of the mixed solution of n-heptane and ethanol was 12 times the weight of the crude product of ginkgolide B), heated at 80°C with stirring until dissolved, the mass ratio of n-heptane to ethanol being 1:6, and allowed to stand and cool to 30°C. The mixture was kept warm for 4 hours for crystallization, filtered, and dried to obtain 22.4 g of ginkgolide B with a purity of 99.2%, a yield of 0.80 g / kg, and a transfer rate of 40%.

[0047] The production cost of this embodiment is about 92 yuan / g, and the organic solvent consumption is about 1.9 L / g.

[0048] Example 3

[0049] A method for extracting and purifying ginkgolide B comprises:

[0050] (1) removing impurities from ginkgo leaves, washing with water, and drying, crushing 50 kg of dried ginkgo leaves, adding a mixed acid solution, heating to 95° C., soaking for 4 hours, and filtering to obtain a ginkgo leaf extract 1, wherein the mixed acid solution is a mixed solution of HCl solution and tartaric acid solution in a volume ratio of 2:1, the mass ratio of ginkgo leaves to the mixed acid solution is 1:30, the pH of the HCl solution is 3, and the pH of the tartaric acid solution is 4;

[0051] (2) The ginkgo leaf extract 1 in step (1) was concentrated to a relative density of 1.10, heated and boiled for 30 minutes, transferred to a container, talcum powder was added, stirred, cooled to room temperature, continued to stir and began to filter, the filtrate was refrigerated at 5°C for 10 hours, filtered, and the filtrate was taken to obtain extract 2, wherein the talcum powder was activated at 115°C for 1 hour before being added, and the weight of the talcum powder was 0.16 times the weight of the extract concentrate;

[0052] (3) heating the extract 2 in step (2) to 45° C., adding ethyl acetate and stirring for 25 minutes, wherein the weight of ethyl acetate is 1.5 times the weight of the extract 2, separating the layers to obtain an organic phase 1, adding purified water and stirring for 25 minutes at room temperature, wherein the volume of purified water is 1.5 times the volume of ethyl acetate, separating the layers to obtain an organic phase 2, filtering, and drying by spin drying to obtain a crude extract;

[0053] (4) adding 95% ethanol to the crude extract in step (3) and stirring and dissolving it for 45 minutes, wherein the ratio of the volume of ethanol (mL) to the weight of the crude extract (g) is 11, filtering with a filter membrane, adding an acidic alumina chromatography column treated with 95% ethanol, wherein the weight ratio of the acidic alumina to the crude extract is 22:1, the diameter-height ratio of the acidic alumina is 1:10, the acidic alumina is 300 mesh, and eluting with 2.0 times (the ratio of the volume of ethanol (mL) to the weight of the crude extract (g)) of 95% ethanol to obtain an eluate;

[0054] (5) Concentrating the eluate obtained in step (4) to an alcohol content of 65%, refrigerating at 8° C. for 25 h, filtering, and drying to obtain a crude product of ginkgolide B;

[0055] (6) The crude product of ginkgolide B obtained in step (5) was added to a mixed solution of n-heptane and ethanol (the weight of the mixed solution of n-heptane and ethanol was 12 times the weight of the crude product of ginkgolide B) and heated at 75°C with stirring until dissolved, the mass ratio of n-heptane to ethanol being 1:5. The mixture was allowed to stand and cool to 25°C, kept warm for 4 hours for crystallization, filtered, and dried to obtain 41.5 g of ginkgolide B with a purity of 99.5%, a yield of 0.83 g / kg, and a transfer rate of 41.5%.

[0056] The production cost of this embodiment is about 83 yuan / g, and the organic solvent consumption is about 1.9 L / g.

[0057] Comparative Example 1

[0058] According to Example 2, 28 kg of dried ginkgo leaves were crushed and divided into seven groups, and the following experiments were performed on each group:

[0059] Group 1: HCl solution was added to the crushed ginkgo leaves, the pH of the HCl solution was 2.8, the mass ratio of ginkgo leaves to HCl solution was 1:40, heated to 100°C, soaked for 3 hours, and filtered to obtain ginkgo leaf extract 1.

[0060] Group 2: Add tartaric acid solution to the crushed ginkgo leaves. The pH of the tartaric acid solution is 3.5, and the mass ratio of ginkgo leaves to tartaric acid solution is 1:40. Heat to 100°C, soak for 3 hours, and filter to obtain ginkgo leaf extract 1.

[0061] Group 3: A mixed acid solution was added to the crushed ginkgo leaves. The mixed acid solution was a mixed solution of HCl solution and tartaric acid solution in a volume ratio of 1:2, the mass ratio of ginkgo leaves to the mixed acid solution was 1:40, the pH of the HCl solution was 2.8, and the pH of the tartaric acid solution was 3.5. The mixture was heated to 100°C, soaked for 3 hours, and filtered to obtain ginkgo leaf extract 1.

[0062] Group 4: A mixed acid solution was added to the crushed ginkgo leaves. The mixed acid solution was a mixed solution of HCl solution and tartaric acid solution in a volume ratio of 5:1, the mass ratio of ginkgo leaves to the mixed acid solution was 1:40, the pH of the HCl solution was 2.8, and the pH of the tartaric acid solution was 3.5. The mixture was heated to 100°C, soaked for 3 hours, and filtered to obtain ginkgo leaf extract 1.

[0063] Group 5: A mixed acid solution was added to the crushed ginkgo leaves. The mixed acid solution was a mixed solution of HCl solution and citric acid solution in a volume ratio of 3:1, the mass ratio of ginkgo leaves to the mixed acid solution was 1:40, the pH of the HCl solution was 2.8, and the pH of the citric acid solution was 3.5. The mixture was heated to 100°C, soaked for 3 hours, and filtered to obtain ginkgo leaf extract 1.

[0064] Group 6: Add citric acid solution to the crushed ginkgo leaves. The pH of the citric acid solution is 3.5, and the mass ratio of ginkgo leaves to the citric acid solution is 1:40. Heat to 100°C, soak for 3 hours, and filter to obtain ginkgo leaf extract 1.

[0065] Group 7: A mixed acid solution was added to the crushed ginkgo leaves. The mixed acid solution was a mixed solution of HCl solution and tartaric acid solution in a volume ratio of 3:1, the mass ratio of ginkgo leaves to the mixed acid solution was 1:40, the pH of the HCl solution was 2.8, and the pH of the tartaric acid solution was 3.5. The mixture was heated to 100°C, soaked for 3 hours, and filtered to obtain ginkgo leaf extract 1.

[0066] The ginkgo leaf extracts 1 obtained from the seven groups of experiments were subjected to steps (2) to (6) of Example 2 to obtain ginkgolide B.

[0067] The weight, purity and yield of Ginkgolide B obtained from the above seven experiments are shown in Table 1.

[0068] Table 1

[0069] Ginkgolide B Weight (g) purity(%) Yield (g / kg) Group 1 1.84 98.6 0.46 Group 2 2.2 98.4 0.55 Group 3 2.24 98.5 0.56 Group 4 2.32 98.6 0.58 Group 5 2.12 98.6 0.53 Group 6 2.24 98.8 0.56 Group 7 3.24 99.2 0.81

[0070] From this comparative example, it can be seen that the yield of Ginkgolide B (Group 7) of the present invention is higher than that of the first to sixth groups of the control, and it has the advantages of high yield and high purity.

[0071] Comparative Example 2

[0072] According to Example 2, 20 kg of dried ginkgo leaves were taken and divided into five groups. Step (1) of Example 2 was repeated for each of the five groups to obtain ginkgo leaf extract 1. The ginkgo leaf extract 1 was subjected to the following experiments:

[0073] Group 1: According to step (2) of Example 2, talcum powder was directly added without heating and boiling. The rest was the same as step (2) of Example 2 to obtain extract 2.

[0074] Group 2: According to step (2) of Example 2, talc was replaced with diatomaceous earth, and the rest was the same as step (2) of Example 2 to obtain extract 2.

[0075] The third group: according to step (2) of Example 2, heat and boil for 40 minutes, stir and cool to room temperature, continue stirring and start filtering. Other steps are the same as step (2) of Example 2 to obtain extract 2.

[0076] Group 4: According to step (2) of Example 2, talcum powder was added, stirred, and cooled to room temperature, then continued to stir and began to filter. The filtrate was not refrigerated to obtain Extract 2.

[0077] Group 5: Repeat step (2) of Example 2 to obtain extract 2.

[0078] The extracts 2 obtained from the above five groups of experiments were used to repeat steps (3) to (6) of Example 2 to obtain ginkgolide B.

[0079] The weight, purity and yield of Ginkgolide B in the above five experiments are shown in Table 2.

[0080] Table 2

[0081] Ginkgolide B Weight (g) purity(%) Yield (g / kg) Group 1 2.52 97.5 0.63 Group 2 2.2 97.7 0.55 Group 3 2.0 96.8 0.5 Group 4 2.4 97.0 0.60 Group 5 3.20 99.2 0.80

[0082] It can be seen from this comparative example that the purity and yield of Ginkgolide B (the fifth group) of the present invention are higher than those of the first, second, third and fourth groups of the control, and has the advantages of high yield and high purity.

[0083] Comparative Example 3

[0084] Ginkgolide B was obtained according to the method of Example 3 in Chinese patent CN111171043B, specifically:

[0085] (1) After weighing the ginkgo leaves, wash them with water to remove dust, and then crush them. Add a citric acid solution with a pH value of 5 (solid-liquid ratio of 1 kg:3 L), and soak them under stirring for 20 minutes. Then add an 80% ethanol solution (solid-liquid ratio of 1 kg:8 L) and heat and reflux at 85°C for 2.5 hours. Then filter and remove impurities to obtain a filtrate. The filtrate is reduced pressure and concentrated at a pressure of -0.1 MPa and 55°C until there is no ethanol taste, thereby obtaining a ginkgo leaf extract.

[0086] (2) Activated carbon and α-alumina were mixed in a weight ratio of 12:1 to obtain a carrier, which was then added to a second organic solvent of ethyl acetate and methyl acetate (volume ratio 8:1), and then the ginkgo leaf extract obtained in step (1) was added, and the mixture was heated under reflux at 85° C. for 0.5 h. The amount of the carrier was 0.8 g and the amount of the second organic solvent was 15 ml relative to 1 g of ginkgo leaf extract. The material was then filtered to obtain a crude extract of ginkgolide B, which was then concentrated to a solid content of 0.05 g / ml to obtain a concentrate.

[0087] (3) To 1 part by volume of the concentrate, 0.2 parts by volume of distilled water was added, the mixture was mixed evenly, and the mixture was allowed to stand for separation. The organic solvent layer was separated, and the mixture was then concentrated under reduced pressure at a pressure of -0.1 MPa and 55° C. until no organic solvent smell was observed, thereby obtaining a concentrate containing ginkgolide B.

[0088] (4) The concentrate containing ginkgolide B was dissolved in ethyl acetate, and crystallized at 10°C and a stirring speed of 200 rpm for 1.5 hours. The temperature was then lowered by 8°C and allowed to stand for crystallization for 10 hours to obtain crystals I. After 70% of crystals I were separated, 40% ethanol was added and the system was cooled to 4°C and crystallization was continued for 12 hours to obtain crystals II. Crystals II were separated and combined with crystals I to obtain ginkgolide B crystals. The ginkgolide B content in the ginkgolide B product was detected and analyzed by HPLC. The yield of ginkgolide B = ginkgolide B product weight / ginkgo leaf weight.

[0089] The purity, yield, production cost and organic solvent consumption of this comparative example and Examples 1-3 of the present invention are shown in Table 3.

[0090] Table 3

[0091]

[0092] As can be seen from Table 3, the yield of Ginkgolide B of the present invention is significantly higher than that of Comparative Example 3, and has the advantages of low production cost and low organic solvent consumption.

Claims

1. A method for extracting and purifying ginkgolide B, comprising: (1) Ginkgo biloba leaves are crushed, added to a mixed acid solution, soaked and extracted, and filtered to obtain a ginkgo biloba extract 1, wherein the mixed acid solution is a mixed solution of HCl solution and tartaric acid solution in a volume ratio of 1:1 to 3:1, and the mass ratio of ginkgo biloba leaves to the mixed acid solution is 1:20 to 40; (2) Concentrating the ginkgo leaf extract 1 in step (1), heating and boiling, adding talcum powder and stirring until the temperature drops to room temperature, continuing stirring and filtering, refrigerating the filtrate, and filtering to obtain extract 2, wherein the extract is concentrated to a relative density of 1.05-1.15, the heating and boiling time is 20-40 minutes; the refrigeration temperature is 0-5°C, and the refrigeration time is 6-12 hours; (3) heating the extract 2 in step (2), adding ethyl acetate and stirring at the same temperature, separating the layers to obtain an organic phase 1, adding water and stirring again, separating the layers to obtain an organic phase 2, filtering, and drying the mixture to obtain a crude extract; (4) adding ethanol to the crude extract in step (3) to dissolve, filtering, passing through an acidic alumina chromatography column, and eluting with 85-95% ethanol to obtain an eluate; (5) concentrating the eluate in step (4) to an alcohol content of 60%-70%, refrigerating at 4-10° C., filtering, and drying to obtain a crude product of ginkgolide B; (6) Add the crude product of ginkgolide B in step (5) to a mixed solution of n-heptane and ethanol, heat and stir at 70-80° C. until dissolved, let it stand and cool to 20-30° C., keep warm for crystallization, filter, and dry to obtain ginkgolide B, wherein the mass ratio of n-heptane to ethanol is 1:3-1:6, and the crystallization time is 3-4 h.

2. The extraction and purification method according to claim 1, wherein In the step (1), the soaking and extraction temperature is 90-100° C., and the soaking and extraction time is 3-5 hours.

3. The extraction and purification method according to claim 1, wherein In the step (1), the pH of the HCl solution is 2-3; the pH of the tartaric acid solution is 2-4.

4. The extraction and purification method according to claim 1, wherein In the step (2), the weight of the talc powder is 0.1 to 0.2 times the weight of the extracted concentrated solution.

5. The extraction and purification method according to claim 1, wherein In the step (3), the mixture is heated to 40-50° C. and stirred at this temperature for 20-30 min; the mixture is stirred again at room temperature for 20-30 min.

6. The extraction and purification method according to claim 1, wherein In step (3), the weight of ethyl acetate is 1-2 times the weight of the extract 2; and the volume of water is 1-2 times the volume of ethyl acetate.

7. The extraction and purification method according to claim 1, wherein The refrigeration time in step (5) is more than 12 hours.

8. The extraction and purification method according to claim 1, wherein The weight of the mixed solution of n-heptane and ethanol in step (6) is 10 to 12 times the weight of the crude product of Ginkgolide B.

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