A process for the separation and purification of Ginkgo biloba compound GK-A

Abstract

This invention relates to the field of separation and purification of monomers from traditional Chinese medicine, specifically to a process for separating and purifying the compound GK-A from Ginkgo biloba, comprising the following steps: crushing Ginkgo biloba kernels, mixing with water and stirring, separating solids and liquids, adjusting the pH to acidic to obtain a macroporous resin loading solution; then loading the solution onto a macroporous resin column, washing with water to remove impurities, eluting with 5%-50% ethanol solution, collecting the eluent, adjusting the pH of the eluent to 6-9, concentrating to obtain a concentrated eluent; mixing the concentrated eluent with methanol and adjusting the pH to acidic to obtain a preparative loading solution, preparing and separating the GK-A fraction using reversed-phase preparative liquid chromatography; removing acid from the GK-A fraction using reversed-phase preparative liquid chromatography, flushing the column, and collecting the flushing solution; adding sodium hydroxide solution to the flushing solution to obtain a GK-A solution; using medicinal charcoal for adsorption to remove impurities, filtering, concentrating, and drying to obtain a high-yield and high-purity pure GK-A product.

Description

Technical Field

[0001] This invention relates to the field of separation and purification of monomers from traditional Chinese medicine, specifically to a process for separating and purifying Ginkgo biloba compounds. Background Technology

[0002] Ginkgo biloba L., the dried, mature seed of the Ginkgoaceae plant, is a traditional Chinese medicine. The Compendium of Materia Medica states that cooked ginkgo biloba warms the lungs, benefits qi, and relieves asthma and cough. Previous studies screened an N-glycoside indole component, GK-A, from ginkgo biloba, which significantly reduced the frequency of coughs induced by ammonia in mice and by capsaicin in guinea pigs, as well as reduced the frequency of coughs and lung inflammation in cough-variant asthmatic guinea pigs.

[0003] However, ginkgo kernels contain a large amount of protein, oil, and polysaccharides, and the content of its active ingredient GK-A in the medicinal material is only two to three parts per thousand. Current technologies for separating and purifying GK-A from ginkgo cannot guarantee a high yield and high purity, which limits the clinical application of ginkgo to some extent. Therefore, it is necessary to study how to efficiently extract the effective antitussive and antiasthmatic component GK-A from ginkgo for the development and utilization of ginkgo. Summary of the Invention

[0004] Therefore, the present invention provides a process for separating and purifying the Ginkgo biloba compound GK-A, and the obtained pure GK-A has the advantages of high yield and high purity.

[0005] Therefore, the present invention provides the following technical solution:

[0006] This invention provides a process for separating and purifying the Ginkgo biloba compound GK-A, comprising the following steps:

[0007] S1. Crush the ginkgo nuts to obtain ginkgo nut fragments;

[0008] S2. Ginkgo kernel powder is mixed with water, stirred, and separated into solid and liquid components to obtain a supernatant. The pH of the supernatant is then adjusted to acidity to obtain a macroporous resin loading solution.

[0009] S3. Load the macroporous resin loading solution obtained in S2 onto a macroporous resin column, wash with water to remove impurities, then elute with an ethanol solution with a volume concentration of 5%-50%, collect the eluent, adjust the pH of the eluent to 6-9, concentrate it to obtain the concentrated eluent.

[0010] S4. Add methanol solution to the eluent concentrate obtained in S3 to prepare a solution containing 4-6% methanol by volume, adjust the pH to acidic, and obtain the sample loading solution. The sample loading solution is prepared and separated by reversed-phase preparative liquid chromatography, and the GK-A fraction is collected.

[0011] S5. The GK-A fraction obtained in S4 was deacidified by reversed-phase preparative liquid chromatography, then the column was flushed and the flushing solution was collected.

[0012] S6. Add sodium hydroxide solution to the flushing solution in S5 to obtain GK-A solution;

[0013] S7. The GK-A solution obtained in S6 is subjected to adsorption and impurity removal using medicinal charcoal, then filtered, concentrated, and dried to obtain pure GK-A.

[0014] In one alternative embodiment, the ginkgo compound is GK-A, having the structure shown in formula (1).

[0015] Equation (1).

[0016] In one alternative implementation, the crushing method of S1 includes chopping or crushing.

[0017] In one optional embodiment, step S2 includes: mixing crushed ginkgo kernels with water, performing a first stirring, and separating the solid and liquid to obtain a first supernatant and ginkgo kernel residue; adjusting the pH of the first supernatant to 2-4 to obtain a first extract; mixing the ginkgo kernel residue with water, performing a second stirring, and separating the solid and liquid to obtain a second supernatant; adjusting the pH of the second supernatant to 2-4 to obtain a second extract; and combining the first extract and the second extract to obtain a macroporous resin loading solution.

[0018] In one optional embodiment, the mass-to-volume ratio of the liquid in the first stirring refers to the ratio of the mass of the ginkgo kernel fragments to the volume of water, and the mass-to-volume ratio of the liquid in the second stirring refers to the ratio of the mass of the ginkgo kernel residue to the volume of water.

[0019] In one optional embodiment, the mass-to-volume ratio of the liquid to the material in the first stirring step of S2 is 1 kg: (4-10) L, the stirring time is 4-10 h, and the stirring temperature is 20-50 °C.

[0020] In one optional embodiment, the mass-to-volume ratio of the liquid to the material during the second stirring in S2 is 1 kg: (2-6) L, the stirring time is 1-4 h, and the stirring temperature is 20-50 °C.

[0021] In one alternative embodiment, the pH is adjusted in S2 using hydrochloric acid with a mass fraction of 36%-38%.

[0022] In one alternative implementation, in step S4, the pH is adjusted to 2-4.

[0023] In an optional embodiment, in step S4, the reversed-phase preparative liquid chromatography separation uses a methanol solution to elute the preparative loading solution; the methanol solution contains methanol and formic acid, with a methanol volume concentration of 10%-40% and a formic acid volume concentration of 0.01%-0.1%; the methanol solution is an aqueous methanol solution.

[0024] In an optional embodiment, in step S5, the chromatographic column used in the reversed-phase preparative liquid chromatography is an ODS column or a small-pore resin column.

[0025] Optionally, the ODS column includes a Flash column, a medium-pressure ODS column, and a high-pressure ODS column; the ODS column is a reverse-phase chromatography column stationary phase with ODS (octadecylsilane-bonded silica gel) as the packing material; the Flash column is a low-pressure ODS column with a pressure range of <5 bar; the pressure ranges of the medium-pressure ODS column and the high-pressure ODS column are 5-20 bar and 20-60 bar, respectively.

[0026] In one optional embodiment, the adsorption temperature for adsorption and impurity removal in step S7 is 20-60℃, and the adsorption time is 1-5h.

[0027] And / or,

[0028] The mass of the medicinal charcoal is 60%-150% of the mass of GK-A in the GK-A solution.

[0029] In one optional embodiment, in step S6, the amount of sodium hydroxide added is 1.9-2.1 times the amount of GK-A in the flushing solution, and the mass fraction of sodium hydroxide in the sodium hydroxide solution is 8-12%.

[0030] In one alternative embodiment, the mass content of GK-A in the pure GK-A product is >95%.

[0031] In one optional embodiment, the volume concentration of methanol in the methanol solution in S4 is 94%-96%.

[0032] In one optional embodiment, the macroporous resin in S3 is SP700.

[0033] In step S3, with a fixed amount of resin, using SP700 macroporous resin as the resin column can significantly improve the adsorption rate of GK-A.

[0034] In one optional embodiment, the mass percentage of GK-A in the elution concentrate of S3 is above 25%.

[0035] In one optional embodiment, the column flushing step in S5 uses either methanol with a volume concentration of 94%-96% or ethanol with a volume concentration of 94%-96%.

[0036] The technical solution of this invention has the following advantages:

[0037] 1. This invention provides a process for separating and purifying the ginkgo compound GK-A, comprising the following steps: S1. Crushing ginkgo kernels to obtain ginkgo kernel fragments; S2. Mixing the ginkgo kernel fragments with water, stirring, and separating the solid and liquid components to obtain a supernatant, then adjusting the pH of the supernatant to acidic to obtain a macroporous resin loading solution; S3. Loading the macroporous resin loading solution obtained in S2 onto a macroporous resin column, washing with water to remove impurities, then eluting with a 5%-50% (v / v) ethanol solution, collecting the eluent, then adjusting the pH of the eluent to 6-9, and concentrating to obtain a concentrated eluent; S4. Adding methanol solution to the concentrated eluent obtained in S3 to prepare a solution containing 4-6% (v / v) methanol, adjusting the pH to acidic to obtain a preparative loading solution, and separating the preparative loading solution by reversed-phase preparative liquid chromatography, collecting the GK-A fraction; S5. The GK-A fraction obtained in S4 was deacidified by reversed-phase preparative liquid chromatography, then the column was flushed and the flushing solution was collected; S6. Sodium hydroxide solution was added to the flushing solution in S5 to obtain GK-A solution; S7. The GK-A solution obtained in S6 was subjected to adsorption and impurity removal using pharmaceutical charcoal, filtered, concentrated and dried to obtain pure GK-A product.

[0038] Existing methods for extracting traditional Chinese medicine often involve heating and reflux with organic solvents, while this process uses water-stirring extraction, which has advantages in terms of economy, environmental protection, and safety. This process simplifies the process route: some impurities in ginkgo are difficult to completely remove in a single preparation and require a second preparation. This application adopts a single preparation followed by adsorption and impurity removal with medicinal charcoal, which can yield high-yield and high-purity GK-A. The process is simple, the production cost is low, and the toxic components are completely removed.

[0039] Step S2 involves mixing crushed ginkgo kernels with water and then stirring for extraction. This is a low-energy extraction method that avoids the high viscosity of the extract caused by starch gelatinization due to heating and reflux, which could clog the resin column during sample loading. This extraction method ensures the extraction rate of GK-A while avoiding the extraction of impurities such as proteins and polysaccharides caused by heating, thus effectively improving the yield and purity of GK-A extracted from ginkgo kernels. Furthermore, the use of an aqueous extract allows for direct loading onto a macroporous resin column without the need for concentration, reducing energy consumption and lowering production costs.

[0040] In step S4, the reversed-phase preparative liquid chromatography separation method uses methanol solution to elute the preparative loading solution; the volume concentration of methanol in the methanol solution is 10-40%, and the volume concentration of formic acid is 0.01-0.1%. Formic acid is added to the elution solution to improve the separation degree between GK-A and impurities, thereby increasing the efficiency and yield of GK-A preparation and separation.

[0041] 2. This application efficiently separates and purifies GK-A. The GK-A separation and purification process provided by this invention yields pure GK-A with a GK-A content of >95% and a yield of >50%. Attached Figure Description

[0042] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0043] Figure 1 This is a liquid chromatogram of the supernatant in Example 1 of the present invention;

[0044] Figure 2 This is a liquid chromatogram of the macroporous resin eluent in Example 1 of the present invention;

[0045] Figure 3 This is a liquid chromatogram of the GK-A elution fraction prepared and separated in Example 1 of the present invention;

[0046] Figure 4 This is a liquid chromatogram of pure GK-A product after adsorption with medicinal charcoal in Example 1 of the present invention. Detailed Implementation

[0047] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0048] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0049] Reagent source:

[0050] Hydrochloric acid (37% by mass) was purchased from Beijing Tongguang Fine Chemical Co., Ltd.

[0051] Ethanol (95% by volume) was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.

[0052] Sodium hydroxide was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0053] SP700 macroporous resin was purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.

[0054] Small-pore resin, purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.

[0055] ODS packing was purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.

[0056] The medicinal charcoal was purchased from Yushan County Sanqing Activated Carbon Co., Ltd.

[0057] Example 1

[0058] This embodiment provides a process for separating and purifying the Ginkgo biloba compound GK-A, including the following steps:

[0059] Source of medicinal materials: Ginkgo kernels were purchased from Anhui Shengantang Pharmaceutical Co., Ltd., originating from Jiangsu Province. The GK-A content was determined to be 0.251 wt.%.

[0060] S1. Take 5kg of ginkgo kernels and chop them into ginkgo kernel pieces.

[0061] S2. Add 40L of purified water to the crushed ginkgo kernels, stir and extract at 25℃ for 6h, let stand, separate, and obtain the first supernatant and ginkgo kernel residue. Add hydrochloric acid to the first supernatant to adjust the pH to 3.5 to obtain the first extract. Add 15L of purified water to the ginkgo kernel residue, stir and extract at 25℃ for 3h, let stand, separate, and obtain the second supernatant. Add hydrochloric acid to adjust the pH to 3.5 to obtain the second extract. Combine the first and second extracts, let stand for 12h, transfer the third supernatant from the combined extracts, filter the lower sediment in the combined extracts to remove the filter residue, and obtain the filtrate. Combine the third supernatant and the filtrate to obtain the macroporous resin loading solution. The mass of GK-A in the macroporous resin loading solution was found to be 11.5 g, and the extraction rate of GK-A was 92 wt.%.

[0062] S3. Take 1 L of SP700 macroporous resin, soak it in 95% (v / v) ethanol solution for 24 h, and then pack it into a column (diameter-to-height ratio approximately 1:4). Rinse the column with purified water to remove ethanol. Load the macroporous resin sample solution at a flow rate of 3 BV / h. After loading, rinse the resin column with 3 BV of purified water to remove impurities, and then elute with 8 BV of 15% (v / v) ethanol solution at a flow rate of 3 BV / h. Adjust the pH of the eluent collected in S3 to 9 using sodium hydroxide solution, concentrate under reduced pressure, and collect the macroporous resin eluent concentrate. The mass of GK-A in the macroporous resin eluent concentrate is 9.9 g, and the elution rate of GK-A is 86% (v / v).

[0063] S4. A solution containing 5% (v / v) methanol was prepared by adding 95% (v / v) methanol solution to the macroporous resin eluent concentrate. The pH was adjusted to 3.5, and the solution was filtered to obtain the sample loading solution. The solution was loaded onto a medium-pressure ODS column and eluted with 25% (v / v) methanol solution (containing 0.05% (v / v) formic acid). The GK-A fraction was collected, and the mass of GK-A in the fraction was 9.0 g, indicating a GK-A yield of 91%.

[0064] S5. After all samples were prepared, the GK-A fractions were combined, and an equal volume of formic acid aqueous solution (0.05% (v / v)) was added. After mixing, the sample was loaded onto a medium-pressure ODS column (the mass of the packing material was 500 g, and the sample mass accounted for 0.6 wt.% of the mass of the packing material). Then, the column was washed with 5% (v / v) methanol solution for 2 BV. Finally, the column was flushed with 95% (v / v) methanol solution. The flushing solution was collected, and the mass of GK-A in the flushing solution was found to be 8.9 g, and the acid removal yield of GK-A was 99%.

[0065] S6. Add sodium hydroxide solution (the mass fraction of sodium hydroxide in the sodium hydroxide solution is 10 wt.%, and the amount of sodium hydroxide added is twice the amount of GK-A in the rinsing solution) to the rinsing solution, concentrate and dry to obtain crude GK-A, and redissolve the dried product with purified water to obtain a solution of Ginkgo biloba compound GK-A.

[0066] S7. 80% of the mass of medicinal charcoal (GK-A) was added to a solution of Ginkgo biloba compound GK-A for adsorption and impurity removal. The adsorption was carried out at 35°C with stirring for 2 hours. The charcoal was then removed by filtration. The yield of GK-A after adsorption with medicinal charcoal was 92%. The solution after adsorption with medicinal charcoal was concentrated and dried to obtain approximately 8.2 g of pure GK-A. The yield of GK-A was 65%, and the purity was 98.5% as determined by HPLC. The mass content of GK-A on a dried basis was 97.4%.

[0067] Liquid chromatography detection results as follows Figure 1-4 As shown, Figure 1This is a liquid chromatogram of the supernatant in Example 1 of the present invention; Figure 2 This is a liquid chromatogram of the macroporous resin eluent in Example 1 of the present invention; Figure 3 This is a liquid chromatogram of the GK-A elution fraction prepared and separated in Example 1 of the present invention; Figure 4 This is a liquid chromatogram of pure GK-A product after adsorption with medicinal charcoal in Example 1 of the present invention.

[0068] Example 2

[0069] This embodiment provides a process for separating and purifying the Ginkgo biloba compound GK-A, including the following steps:

[0070] Source of medicinal materials: Anguo Ruifeng Beileng Chinese Medicinal Materials Co., Ltd., produced in Jiangsu Province. The GK-A content was determined to be 0.298 wt.%.

[0071] S1. Take 5 kg of ginkgo kernels and chop them into ginkgo kernel pieces.

[0072] S2. Add 20L of purified water to the crushed ginkgo kernels, stir and extract at 40℃ for 8 hours, let stand, separate, and obtain the first supernatant and ginkgo kernel residue. Add hydrochloric acid to the first supernatant to adjust the pH to 4.0 to obtain the first extract. Add 20L of purified water to the ginkgo kernel residue, stir and extract at 40℃ for 4 hours, let stand, separate, and obtain the second supernatant. Add hydrochloric acid to adjust the pH to 4.0 to obtain the second extract. Combine the first and second extracts, let stand for 12 hours, transfer the third supernatant from the combined extracts, filter the lower sediment in the combined extracts to remove the filter residue, and obtain the filtrate. Combine the third supernatant and the filtrate to obtain the macroporous resin loading solution. The mass of GK-A in the macroporous resin loading solution was 13.4 g, and the extraction rate of GK-A was 90%.

[0073] S3. Take 1 L of SP700 macroporous resin, soak it in 95% (v / v) ethanol solution for 24 h, and then pack it into a column (diameter-to-height ratio approximately 1:5). Rinse the column with purified water to remove ethanol. Load the macroporous resin sample solution at a flow rate of 3 BV / h. After loading, rinse the resin column with 3 BV of purified water to remove impurities, and then elute with 3 BV of 40% (v / v) ethanol solution at a flow rate of 3 BV / h. Adjust the pH of the eluent collected in S3 to 7.0 with sodium hydroxide solution, concentrate under reduced pressure, and collect the macroporous resin eluent concentrate. The mass of GK-A in the macroporous resin eluent concentrate is 11.7 g, and the elution rate of GK-A is 87%.

[0074] S4. Add 95% (v / v) methanol solution to the macroporous resin eluent to prepare a solution with a methanol volume concentration of 5% (v / v), adjust the pH to 3, filter, and obtain the sample loading solution; load the sample onto a Flash column and elute with 20% (v / v) methanol solution (in which the volume concentration of formic acid is 0.01% (v / v)), collect the GK-A fraction, and the mass of GK-A in the GK-A fraction is 10.76 g, and the yield of GK-A is 92%.

[0075] S5. After all samples were prepared, the GK-A fractions were combined, and an equal volume of formic acid aqueous solution (0.01% (v / v)) was added. After mixing, the sample was loaded onto a Flash column to remove formic acid (the mass of the packing material was 500 g, and the mass of the sample loaded accounted for 0.8 wt.% of the mass of the packing material). Then, the column was washed with 5% (v / v) methanol solution for 2 BV. Finally, the column was flushed with 95% (v / v) methanol solution, and the flushing solution was collected. The mass of GK-A in the flushing solution was 10.6 g, and the acid removal yield of GK-A was 99%.

[0076] S6. Add sodium hydroxide solution (the mass fraction of sodium hydroxide in the sodium hydroxide solution is 10 wt.%, and the amount of sodium hydroxide added is twice the amount of GK-A in the rinsing solution), concentrate and dry to obtain crude GK-A. Redissolve the dried product with purified water to obtain a solution of Ginkgo biloba compound GK-A.

[0077] S7. 70% of the mass of medicinal charcoal was added to the solution of Ginkgo biloba compound GK-A for adsorption and impurity removal. The adsorption was carried out by stirring at 25°C for 4 hours. The medicinal charcoal was removed by filtration. The yield of GK-A after adsorption by medicinal charcoal was 90%. After concentration and drying, 9.5g of pure GK-A was obtained. The yield of GK-A was 64%. The purity was 98.5% by HPLC. The mass content of GK-A on the dried product was 97.9 wt.%.

[0078] Example 3

[0079] This embodiment provides a process for separating and purifying the Ginkgo biloba compound GK-A, including the following steps:

[0080] Source of medicinal materials: Yuzhongtang Chinese Herbal Medicine Co., Ltd. of Zhangshu City, Shandong Province. The content of GK-A was determined to be 0.202 wt.%.

[0081] S1. Take 5 kg of ginkgo kernels and chop them into ginkgo kernel pieces.

[0082] S2. Add 50L of purified water to the crushed ginkgo kernels, stir and extract at 50℃ for 4h, let stand, separate, and obtain the first supernatant and ginkgo kernel residue. Add hydrochloric acid to the first supernatant to adjust the pH to 2.5 to obtain the first extract. Add 15L of purified water to the ginkgo kernel residue, stir and extract at 50℃ for 4h, let stand, separate, and obtain the second supernatant. Add hydrochloric acid to adjust the pH to 2.5 to obtain the second extract. Combine the first and second extracts, let stand for 12h, transfer the third supernatant from the combined extracts, filter the lower sediment in the combined extracts to remove the filter residue, and obtain the filtrate. Combine the third supernatant and the filtrate to obtain the macroporous resin loading solution. The mass of GK-A in the macroporous resin loading solution was found to be 9.4 g, and the extraction rate of GK-A was 93%.

[0083] S3. Take 1 L of SP700 macroporous resin, soak it in 95% (v / v) ethanol solution for 24 h, and then pack it into a column (diameter-to-height ratio approximately 1:3). Rinse the column with purified water to remove ethanol. Load the macroporous resin sample solution at a flow rate of 3 BV / h. After loading, rinse the resin column with 3 BV of purified water to remove impurities, and then elute with 20 BV of 5% (v / v) ethanol solution at a flow rate of 3 BV / h. Adjust the pH of the eluent collected in S3 to 6.5 with sodium hydroxide solution, concentrate under reduced pressure, and collect the macroporous resin eluent concentrate. The mass of GK-A in the macroporous resin eluent concentrate is 8 g, and the elution rate of GK-A is 85%.

[0084] S4. Add 95% (v / v) methanol to the macroporous resin eluent to prepare a solution with a methanol volume concentration of 5% (v / v), adjust the pH to 3.5, filter, and obtain the sample loading solution; load the sample onto an ODS column under medium pressure, and elute with a 20% (v / v) methanol solution (in which the volume concentration of formic acid is 0.05% (v / v)), collect the GK-A fraction, and the mass of GK-A in the GK-A fraction is 7.18 g, and the preparation yield of GK-A is 90%.

[0085] S5. After all samples have been prepared, combine the GK-A fractions and add an equal volume of formic acid aqueous solution (0.05% (v / v)). Mix well and remove formic acid using a small-pore resin column (packing mass is 350 g, and the sample mass accounts for 1 wt.% of the packing mass). Then rinse with pure water for 2.5 BV, and finally rinse the column with 95% (v / v) ethanol. Collect the rinsing solution. The mass of GK-A in the rinsing solution is 7.04 g, and the acid removal rate of GK-A is 98%.

[0086] S6. Add sodium hydroxide solution (the mass fraction of sodium hydroxide in the sodium hydroxide solution is 10 wt.%, and the amount of sodium hydroxide added is twice the amount of GK-A in the flushing solution), concentrate and dry to obtain crude GK-A, and redissolve the dried product with purified water to obtain a solution of Ginkgo biloba compound GK-A.

[0087] S7. 140% (by weight of GK-A) of medicinal charcoal was added to a solution of Ginkgo biloba compound GK-A for adsorption and impurity removal. The mixture was stirred and adsorbed at 40°C for 4 hours. The charcoal was then removed by filtration, resulting in a 90% yield of GK-A after adsorption. After concentration and drying, 6.3 g of pure GK-A was obtained, with a yield of 62%. HPLC analysis showed a purity of 99.4%, and the mass content of GK-A on a dried basis was 98.3%.

[0088] Comparative Example 1

[0089] This comparative example provides a process for separating and purifying the Ginkgo biloba compound GK-A, including the following steps:

[0090] Source of medicinal materials: Ginkgo kernels were purchased from Anhui Shengantang Pharmaceutical Co., Ltd., originating from Jiangsu Province. The GK-A content was determined to be 0.251%.

[0091] S1. Take 5kg of ginkgo kernels and chop them into ginkgo kernel pieces.

[0092] S2. Add 25L of purified water to the crushed ginkgo kernels, reflux for 2 hours, let stand, separate, and obtain the first supernatant and ginkgo kernel residue. Add hydrochloric acid to the first supernatant to adjust the pH to 4.5 to obtain the first extract. Add 15L of purified water to the ginkgo kernel residue, reflux and stir for 2 hours, let stand, separate, and obtain the second supernatant. Add hydrochloric acid to adjust the pH to 4.5 to obtain the second extract. Combine the first and second extracts, let stand for 12 hours, transfer the third supernatant from the combined extracts, filter the lower sediment in the combined extracts to remove the filter residue, and obtain the filtrate. Combine the third supernatant and the filtrate to obtain the macroporous resin loading solution. The mass of GK-A in the macroporous resin loading solution was 10.0 g, and the extraction rate of GK-A was 80%.

[0093] S3. Take 1 L of SP700 macroporous resin, soak it in 95% ethanol solution for 24 h, and then pack it into a column (diameter-to-height ratio approximately 1:4). Use purified water to flush the column to replace the ethanol. Load the macroporous resin sample solution at a flow rate of 3 BV / h. During the loading process, GK-A leakage occurred, with a leakage rate of 15%. After loading, first flush the resin column with 3 BV of purified water to remove impurities, then elute with 8 BV of 15% ethanol solution at a flow rate of 3 BV / h. Adjust the pH of the eluent collected in S3 to 6.5 using sodium hydroxide solution, concentrate under reduced pressure, and collect the macroporous resin eluent concentrate. The mass of GK-A in the macroporous resin eluent concentrate was found to be 8 g, and the elution rate of GK-A was 80%.

[0094] S4. A solution containing 5% methanol (v / v) was prepared by adding 95% methanol to the macroporous resin eluent concentrate. The pH was adjusted to 4.0, and the solution was filtered to obtain the sample loading solution. The solution was loaded onto a medium-pressure ODS column and eluted with a 35% methanol solution (containing 0.06% formic acid). The GK-A fraction was collected, and the mass of GK-A in the fraction was determined to be 6.0 g, indicating a GK-A yield of 75%.

[0095] S5. After all samples were prepared, the GK-A fractions were combined, and an equal volume of formic acid aqueous solution (0.06%) was added. After mixing, the sample was loaded onto a medium-pressure ODS column (the mass of the packing material was 500 g, and the sample mass accounted for 0.6 wt.% of the mass of the packing material). Then, the column was washed with 5% methanol solution for 2.5 BV. Finally, the column was flushed with 95% methanol, and the flushing solution was collected. The mass of GK-A in the flushing solution was found to be 5.9 g, and the acid removal yield of GK-A was 98%.

[0096] S6. Add sodium hydroxide solution (the mass fraction of sodium hydroxide in the sodium hydroxide solution is 15 wt.%, and the amount of sodium hydroxide added is twice the amount of GK-A in the column flushing solution), concentrate and dry to obtain 5.9 g of GK-A, with a yield of 47%. The purity was 91.5% as determined by HPLC, and the mass content of GK-A on a dried basis was 90%.

[0097] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A process for separating and purifying GK-A, a compound from Ginkgo biloba, characterized in that, Includes the following steps: S1. Crush the ginkgo nuts to obtain ginkgo nut fragments; S2. Ginkgo kernel powder is mixed with water, stirred, and separated into solid and liquid components to obtain a supernatant. The pH of the supernatant is then adjusted to acidity to obtain a macroporous resin loading solution. S3. Load the macroporous resin loading solution obtained in S2 onto a macroporous resin column, wash with water to remove impurities, then elute with an ethanol solution with a volume concentration of 5%-50%, collect the eluent, adjust the pH of the eluent to 6-9, concentrate it to obtain the concentrated eluent. S4. Add methanol solution to the eluent concentrate obtained in S3 to prepare a solution containing 4-6% methanol by volume, adjust the pH to acidic, and obtain the sample loading solution. The sample loading solution is prepared and separated by reversed-phase preparative liquid chromatography, and the GK-A fraction is collected. S5. The GK-A fraction obtained in S4 was deacidified by reversed-phase preparative liquid chromatography, then the column was flushed and the flushing solution was collected. S6. Add sodium hydroxide solution to the flushing solution in S5 to obtain GK-A solution; S7. The GK-A solution obtained in S6 was adsorbed and purified using medicinal charcoal, then filtered, concentrated and dried to obtain pure GK-A. The adsorption temperature for impurity removal in S7 is 20-60℃, and the adsorption time is 1-5h. The mass of the medicinal charcoal is 60%-150% of the mass of GK-A in the GK-A solution.

2. The separation and purification process of Ginkgo biloba compound GK-A according to claim 1, characterized in that, S2 includes: mixing crushed ginkgo kernels with water, stirring for the first time, separating the solid and liquid to obtain a first supernatant and ginkgo kernel residue, adjusting the pH of the first supernatant to 2-4 to obtain a first extract; mixing the ginkgo kernel residue with water, stirring for the second time, separating the solid and liquid to obtain a second supernatant, adjusting the pH of the second supernatant to 2-4 to obtain a second extract, and combining the first extract and the second extract to obtain a macroporous resin loading solution.

3. The separation and purification process of Ginkgo biloba compound GK-A according to claim 2, characterized in that, In S2, the mass-to-volume ratio of the liquid to the material during the first stirring is 1 kg: (4-10) L, the stirring time is 4-10 h, and the stirring temperature is 20-50 °C.

4. The separation and purification process of Ginkgo biloba compound GK-A according to claim 2, characterized in that, In S2, the mass-to-volume ratio of the liquid to the material during the second stirring is 1 kg: (2-6) L, the stirring time is 1-4 h, and the stirring temperature is 20-50 °C.

5. The separation and purification process of Ginkgo biloba compound GK-A according to claim 1, characterized in that, In step S4, the pH is adjusted to 2-4.

6. The separation and purification process of Ginkgo biloba compound GK-A according to claim 1, characterized in that, In the reversed-phase preparative liquid chromatography (RPLC) separation in step S4, a methanol solution is used to elute the sample loading solution. The methanol solution contains methanol and formic acid, with a methanol volume concentration of 10%-40% and a formic acid volume concentration of 0.01%-0.1%.

7. The separation and purification process of Ginkgo biloba compound GK-A according to claim 1, characterized in that, In step S5, the chromatographic column used in the reversed-phase preparative liquid chromatography is an ODS column or a small-pore resin column.

8. The separation and purification process of Ginkgo biloba compound GK-A according to claim 1, characterized in that, In step S6, the amount of sodium hydroxide added is 1.9-2.1 times the amount of GK-A in the flushing solution, and the mass fraction of sodium hydroxide in the sodium hydroxide solution is 8-12%.

9. The separation and purification process for Ginkgo biloba compound GK-A according to any one of claims 1-8, characterized in that, The pure GK-A product contains >95% GK-A by mass.

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