Preparation method of ginkgo total lactone and bilobalide monomer contained in ginkgo total lactone

Abstract

The invention discloses a preparation method of ginkgo total lactone and bilobalide monomers contained in the ginkgo total lactone, which comprises the following steps: (1) dissolving a ginkgo leaf extract, extracting with a mixed solvent, taking a mixed solvent layer, and concentrating under reduced pressure until the mixed solvent layer is dry to obtain a ginkgo total lactone crude product; (2) adding dilute ethanol into the crude product of the ginkgo total lactone, heating, pulping, crystallizing, filtering and drying to obtain the ginkgo total lactone; (3) adding a high-concentration ethanol water solution into the ginkgo total lactone, heating, dissolving, crystallizing, filtering, concentrating mother liquor to be dry, repeating the step, and concentrating secondary mother liquor to be dry to obtain a bilobalide monomer crude product; and (4) dissolving the crude bilobalide product with dilute ethanol, adsorbing through macroporous adsorption resin, eluting, concentrating to be dry, and recrystallizing once to obtain the bilobalide monomer. The method has the advantages of few process steps, simplicity in operation, low cost and high product content and yield, and is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to a preparation method of ginkgo biloba lactones and bilobalide monomers contained therein, and belongs to the technical field of extraction and purification of plant active ingredients. Background Art

[0002] Ginkgo biloba extract, a traditional Chinese medicine extracted and separated from Ginkgo biloba leaves, contains multiple active ingredients and possesses unique therapeutic effects in combating Alzheimer's disease and preventing and treating cerebrovascular and cardiovascular system dysfunction. The main active ingredients in Ginkgo biloba extract are flavonoids and ginkgolides, the latter of which are unique to Ginkgo biloba.

[0003] Ginkgolide compounds belong to the terpenoid family and include bilobalide, bilobalide A, bilobalide B, bilobalide C, bilobalide J, bilobalide K, and bilobalide M. The main terpenoid lactone components in ginkgo biloba leaves and ginkgo leaf extracts are bilobalide, bilobalide A, bilobalide B, and bilobalide C. Existing research indicates that bilobalide compounds are highly effective platelet-activating factor antagonists with unique pharmacological effects and therapeutic value. They can be used to treat conditions such as stroke, shock, transplant rejection, hemodialysis, and asthma, and have protective effects against ischemic damage and the central nervous system. However, due to the complex structure of ginkgolides and the extreme difficulty of artificial synthesis, the preparation of total ginkgolides and ginkgolide monomers through ginkgo leaf extraction has become the primary source of ginkgolides.

[0004] As terpenoids, bilobalide belongs to the sesquiterpenoid class, while the other lactones are diterpenoids. Their molecular structures are relatively similar, with minimal polarity differences. In aqueous alcohol solutions, the solubility of terpenoid lactones follows the order: bilobalide > ginkgolide C > ginkgolide A > ginkgolide B. The solubilities of ginkgolide A and ginkgolide B are similar, as are those of bilobalide and ginkgolide C. This makes the extraction and separation of individual monomers within the bilobalide class more difficult. Because bilobalide belongs to the sesquiterpenoid class, its solubility is somewhat similar to that of the brass and colloids found in ginkgo extracts, often blending together in a sticky paste. This makes its separation and purification more challenging than that of the other lactones.

[0005] In recent years, ginkgo extract has been used as raw material for the extraction and separation of bilobalide in China. The existing methods for extracting and purifying bilobalide are mainly extraction-column adsorption separation and column adsorption separation (including polyamide column, alumina column, reverse adsorption resin column, high-performance chromatography column, silica gel column, etc.). The extraction-column adsorption separation method mainly uses ethyl acetate, dichloromethane and other fat-soluble solvents to perform preliminary separation of ginkgo total lactones and other impurities, and then combines the column separation method to prepare bilobalide.

[0006] Patent CN201310482318.3 discloses a method for extracting ginkgo leaf extract using a single ethyl acetate solution. The content of ginkgo lactones in the crude ginkgo lactones obtained by extraction is 29.3% to 34.5%, which is too low. Subsequently, it is necessary to pass it through a reverse adsorption resin to prepare the crude bilobalide product, and then recrystallize it three times to obtain a bilobalide content of more than 98%.

[0007] Patent CN00111986.9 discloses a method for extracting ginkgo leaf extract using a single fat-soluble solvent to obtain a crude product of total ginkgo lactones, which is then purified by column chromatography to obtain total ginkgo lactones, the main components of which are ginkgo lactone A and ginkgo lactone B, without bilobalide.

[0008] This shows that the problem with the single solvent extraction method is that the ginkgo lactone content in the crude ginkgo lactone product is too low and contains too many impurities. It can only be regarded as a preliminary separation and purification and requires further purification.

[0009] In the column adsorption separation method, the results of the polyamide column and the extraction method are equivalent, and the problems are basically the same. At the same time, the processing capacity of the polyamide column is too small, and the amount of water or dilute ethanol used for elution is too large; the fillers used in other patents and literature are mostly silica gel, alumina and reverse adsorption resin fillers, etc. Generally, ordinary silica gel and alumina cannot be reused many times, the amount of solid waste generated is large, environmental protection management is difficult, production costs increase, and they are not suitable for industrial production. Summary of the Invention

[0010] To address at least one problem existing in the above-mentioned prior art, the present invention provides a method for preparing total ginkgo lactones and bilobalide monomers contained therein, which has few process steps, simple operation, low cost, high product content and yield, and is suitable for industrial production.

[0011] In order to achieve the above object, the present invention adopts the following technical solution: a method for preparing total ginkgo lactones and bilobalide monomers contained therein, comprising the following steps:

[0012] (1) Ginkgo biloba extract is obtained according to the preparation method of the Chinese Pharmacopoeia, dissolved in water or dilute ethanol by stirring, then extracted 2-3 times with a mixed solvent, allowed to stand for stratification, the mixed solvent layer is collected, combined, the combined mixed solvent layer is washed with water, allowed to stand for stratification, the mixed solvent layer is collected, and concentrated to dryness under reduced pressure to obtain a crude product of total ginkgo lactones with a content of more than 70%;

[0013] Wherein, the mixed solvent is a mixture of solvent A and solvent B, solvent A is a fat-soluble solvent, and solvent B is a low molecular weight alkane solvent;

[0014] (2) adding dilute ethanol to the crude ginkgo lactones obtained in step (1), heating and beating the mixture, cooling the mixture to room temperature, standing the mixture for crystallization, filtering the mixture, collecting the filter cake, and drying the mixture to obtain a ginkgo lactone content of more than 95%;

[0015] (3) dissolving the total ginkgo lactones obtained in step (2) by heating with a high-concentration ethanol solution, crystallizing at room temperature, filtering, removing the filter cake, collecting the mother liquor, and concentrating the mother liquor to dryness, repeating this step, and concentrating the secondary mother liquor to dryness to obtain a crude product of bilobalide;

[0016] (4) dissolving the crude bilobalide monomer obtained in step (3) in dilute ethanol, adsorbing it on a macroporous adsorption resin, and then eluting it with dilute ethanol. The eluate is collected, concentrated to dryness under reduced pressure, and recrystallized once to obtain a bilobalide monomer with a content of more than 99%.

[0017] In step (1), the mixed solvent is a mixture of solvent A and solvent B, solvent A is a fat-soluble solvent, not limited to dichloromethane, ethyl acetate, methyl ethyl ketone, ethyl formate, chloroform, dichloroethane, 1-butanol, etc., preferably solvent A is ethyl acetate or dichloromethane; solvent B is a low molecular weight alkane solvent, not limited to n-hexane, n-pentane, n-heptane, petroleum ether, etc., preferably solvent B is n-hexane.

[0018] Through experiments, we found two benefits of using a mixed solvent in step 1: First, the mixed solvent has the properties of both solvent A and solvent B, which significantly reduces the solubility of impurities in the mixed solvent, thereby increasing the total lactone content of the crude ginkgo lactones obtained by extraction; taking dichloromethane as an example, the total lactone content of the crude ginkgo lactones obtained by washing with a single dichloromethane extraction is 30-40%, while the total lactone content of the crude ginkgo lactones obtained by washing with a mixed solvent consisting of dichloromethane and n-hexane (the volume ratio of dichloromethane to n-hexane is 3:1) is greater than 70%; second, it is significantly The solubility of Ginkgolide C was reduced, while the solubility of Ginkgolide A, Ginkgolide B and Bilobalide was not significantly reduced. The mixed solvent composed of dichloromethane and solvent B was particularly effective. Experiments showed that the extraction rate of Ginkgolide C in Ginkgo biloba extract by dichloromethane was significantly lower than that of Ginkgolide A, Ginkgolide B and Bilobalide. By controlling the ratio of the mixed solvent components in a specific ratio of dichloromethane and solvent B, the extraction rate of Ginkgolide C can be controlled to about 20%, while Ginkgolide A, Ginkgolide B and Bilobalide can still be maintained at more than 90%. We were very surprised by this. This may be because there are too many hydroxyl groups in the structure of Ginkgolide C, which causes it to be repelled by the alkane solvent in the mixed solvent. After step (2), the content of Ginkgolide C in the total Ginkgolide prepared was ≤5%, which is of great significance for our subsequent preparation of high-content Bilobalide. Because the solubility and resin adsorption properties of Ginkgolide C and bilobalide are very similar, subsequent recrystallization and macroporous resin separation are very ineffective. Ultimately, after passing through the macroporous adsorption resin, the effluent contains too high a concentration of Ginkgolide C, requiring multiple purification steps to obtain more than 99% bilobalide. Therefore, controlling the Ginkgolide C content to a minimum in advance is also a key to this technology.

[0019] Preferably, in the mixed solvent in step (1), solvent A is dichloromethane, solvent B is n-hexane, and the volume ratio of dichloromethane to n-hexane is 1 to 10:1, preferably 3:1.

[0020] Preferably, in the mixed solvent in step (1), solvent A is ethyl acetate, solvent B is n-hexane, and the volume ratio of ethyl acetate to n-hexane is 1 to 6:1, preferably 2:1.

[0021] Preferably, in step (1), the concentration of dilute ethanol is 10-30%, preferably 20%.

[0022] In step (2), the concentration of dilute ethanol is 20-40%, preferably 30%. This concentration of ethanol can take into account both the crystallization yield and the content of total ginkgo lactones.

[0023] Through experiments, it was also found that the prerequisite for the precipitation of bilobalide in step (2) is that mixed solvent extraction is used in step (1), so that the total lactone content of the crude ginkgo lactones obtained by extraction and washing reaches more than 70%, while the total lactone content of the crude ginkgo lactones obtained by single solvent extraction is 30-40%, and bilobalide will not precipitate preferentially. After the crude ginkgo lactones with a content of more than 70% is refined by pulping with dilute ethanol, bilobalide will precipitate in large quantities. The prepared ginkgo lactones with a content of more than 95% has a ginkgo lactone A content of 20-25%, a ginkgo lactone B content of 15-20%, a bilobalide content of ≥50%, and a ginkgo lactone C content of ≤5%.

[0024] In step (3), the ethanol concentration of the high-concentration ethanol aqueous solution is 60 to 95%, preferably 60 to 75%.

[0025] Through experiments, it was further found that two crystallizations were used in step 3, and the total crystallization yield of ginkgolide A and ginkgolide B was over 75%, the total crystallization yield of ginkgolide C was about 30%, and basically no bilobalide was precipitated, and the rest was all in the two mother liquors. In the crude bilobalide obtained by concentrated drying, the bilobalide content was about 80%, and the sum of the contents of ginkgolide A, ginkgolide B and ginkgolide C was about 20%. If subsequent recrystallization was performed, the effect was very poor. At least three recrystallizations were required to prepare bilobalide with a content of more than 99%, and the yield was about 30%.

[0026] In step (4) of the present invention, the concentration of dilute ethanol is 10-30%, preferably 20%. If the alcohol concentration is too low, the amount of dissolution will be too large; if the alcohol concentration is too high, the amount loaded onto the column will be reduced. At the same time, during the elution process with low-concentration dilute ethanol, ginkgolide A will be eluted prematurely.

[0027] In step (4) of the present invention, the macroporous adsorption resin is a conventional macroporous adsorption resin, not limited to one or more of DM131, HPD417, HPD-BJQH, LX-5, LX-32, LX-60, etc.; preferably LX-32.

[0028] Through experiments, step (4) utilizes the difference in adsorption of macroporous adsorption resin for terpenoid lactones to achieve the separation and purification of bilobalide. Under the condition of non-excessive loading, all four terpenoid lactones are adsorbed onto the resin column during the dilute ethanol dissolution and loading process. After elution to a certain volume with dilute ethanol, bilobalide and a small amount of ginkgolide C are preferentially eluted, while ginkgolide A and ginkgolide B are not eluted at this time. By collecting the dilute ethanol eluate and concentrating it to dryness under reduced pressure, bilobalide with a content of about 95% is obtained, and the column yield reaches more than 98%, with basically no loss; after another recrystallization, a small amount of ginkgolide C is removed, and bilobalide monomer with a content of more than 99% can be obtained.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The present invention uses ginkgo leaf extract as raw material and adopts mixed solvent extraction to prepare a crude product of ginkgo lactones with a total lactone content of more than 70%. In contrast, the crude product of ginkgo lactones prepared by the traditional single solvent extraction method has a total lactone content of 30%-40%, which requires further purification.

[0031] (2) The present invention uses ginkgo leaf extract as raw material and adopts a mixed solvent with a specific ratio for extraction. It is unexpectedly found that by controlling the ratio of the mixed solvent components, the extraction rate of ginkgolide C can be controlled to about 20%. The content of ginkgolide C in the prepared total ginkgolide is ≤5%, which is beneficial to subsequent purification, saves time and cost, and improves the refining efficiency of bilobalide.

[0032] (3) In the present invention, the crude product of total ginkgo lactones can be prepared with a high content of total ginkgo lactones, which is ≥95%, by only one-step beating operation. At the same time, the content of bilobalide in the total ginkgo lactones is ≥50%. The traditional crystallization method is step-by-step crystallization, and bilobalide is precipitated from the mother liquor prepared from ginkgo lactone B, ginkgo lactone A, and ginkgo lactone C. The mother liquor after step-by-step crystallization not only retains ginkgo lactone A, ginkgo lactone B, and ginkgo lactone C, but also contains many impurities and is dark in color. The subsequent preparation of bilobalide requires multiple recrystallizations to obtain a high content of bilobalide, which has many steps, slow efficiency, low yield, and unstable product quality.

[0033] (4) The crude bilobalide of the present invention is separated and purified by a resin method; however, the traditional recrystallization method has a very poor effect and requires at least three recrystallizations to prepare a bilobalide monomer with a content of more than 99%, with a yield of about 30%.

[0034] (5) The column chromatography material used in the preparation process of the present invention is a macroporous adsorption resin, which is easy to regenerate and reuse, saves costs, generates less solid waste, and is beneficial to environmental protection.

[0035] (6) In all the resin methods of the present invention, the yield of bilobalide before and after column treatment reaches more than 98%. In the subsequent recrystallization step, ginkgolide C is removed, and bilobalide monomer with a content of more than 99.0% can be prepared, which can fully meet the requirements of pharmaceutical preparations.

[0036] (7) The present invention has few process steps, simple operation, low cost, high product content and yield, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 1 is a high performance liquid chromatogram of bilobalide monomer in Example 1 of the present invention.

[0038] Figure 2 The figure is a high performance liquid chromatogram of total ginkgo lactones in Example 4 of the present invention. DETAILED DESCRIPTION

[0039] The following is a clear and complete description of the technical solutions used in the implementation of the present invention. The embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0040] The ginkgo leaf extract of the present invention is obtained according to the ginkgo leaf extract preparation method specified in the Chinese Pharmacopoeia. The ginkgo leaf extract contains ≥24.0% total flavonol glycosides and ≥6.0% total terpenoid lactones. While the total content of ginkgo terpenoid lactones in ginkgo leaves varies with tree age, season, and region, the ratio of ginkgolides A, B, and C to bilobalide among the ginkgo terpenoid lactones is relatively stable, and the ratio of ginkgolides A, B, and C to bilobalide in the prepared ginkgo leaf extract is also relatively stable.

[0041] Example 1

[0042] A method for preparing ginkgo biloba lactones and bilobalide monomers contained therein, comprising the following specific steps:

[0043] Step 1: 2 kg of Ginkgo biloba extract (Ginkgo biloba total lactone content 6.78%, including Ginkgolide A content 1.35%, Ginkgolide B content 0.92%, Ginkgolide C content 0.91%, bilobalide content 3.6%) was added to 20 L of 20% ethanol and stirred to dissolve. The mixture was then extracted three times with a mixed solvent consisting of dichloromethane and n-hexane in a volume ratio of 3:1, each time using 30 L of the mixed solvent layer. The mixed solvent layer extracts were combined and washed with 10 L of water. The extract was washed with pure water, allowed to stand and separate into layers, and the mixed solvent layer was collected and concentrated under reduced pressure to dryness to obtain 142.62 g of crude ginkgo lactones. Testing showed that the crude ginkgo lactones had a total lactone content of 80.72%, of which the ginkgo lactone A content was 18.45% with a yield of 97.46%, the ginkgo lactone B content was 12.62% with a yield of 97.82%, the ginkgo lactone C content was 1.98%, and the bilobalide content was 47.67% with a yield of 94.43%.

[0044] Step 2: 140 g of the crude ginkgo lactones obtained in step 1 (total lactone content: 80.72%) was taken, 700 mL of 30% ethanol was added, and the mixture was heated to 50-60° C. for 1 hour, then cooled to room temperature and allowed to stand for crystallization for more than 4 hours, filtered, the filter cake was collected, and dried to obtain 106.65 g of ginkgo lactones; after testing, the total lactone content of the ginkgo lactones was 98.06%, of which the content of ginkgo lactone A was 23.11%, the content of ginkgo lactone B was 16.11%, the content of ginkgo lactone C was 2.35%, the content of bilobalide was 56.49%, and the yield was 90.27%;

[0045] Step 3, taking 105g of the total ginkgo lactones obtained in step 2 (total lactone content 98.05%), adding 525mL of 90% ethanol solution and stirring and heating at a temperature of 60°C to dissolve, then standing at room temperature for crystallization for more than 4h, filtering with suction, collecting 1 mother liquor, recycling 1 mother liquor and evaporating to dryness, adding 700mL of 65% ethanol solution and stirring and heating at a temperature of 60°C to dissolve, then standing at room temperature for crystallization for more than 4h, filtering with suction, collecting 2 mother liquors, recycling 2 mother liquors and evaporating to dryness to obtain 70.0g of crude bilobalide, which was tested to have a total lactone content of 97.34%, of which ginkgolide A content was 8.74%, ginkgolide B content was 4.66%, ginkgolide C content was 2.45%, and bilobalide content was 81.49%, with a yield of 96.71%;

[0046] Step 4: Take 10 g of the crude bilobalide obtained in step 3, stir and dissolve it with 20% ethanol 2L, pass it through 200 ml of LX-32 resin, elute with 20% ethanol, monitor the elution effluent by liquid phase, and start collecting when bilobalide flows out of the elution effluent; stop collecting when there is basically no bilobalide in the elution effluent, combine the elution effluent, concentrate under reduced pressure to dryness, dissolve it by heating with 90% ethanol 25 ml, filter it while hot, add 50 ml of water to the filtrate, let it stand at room temperature for crystallization for more than 4 hours, filter it with suction, and dry the filter cake to obtain 7.81 g of bilobalide monomer with a yield of 95.84%, and a total yield of bilobalide of 78.57%. After testing (such as Figure 1 Shown): bilobalide content 99.9%.

[0047] Example 2

[0048] A method for preparing ginkgo biloba lactones and bilobalide monomers contained therein, comprising the following specific steps:

[0049] Step 1: 2 kg of Ginkgo biloba extract (6.78% total ginkgolide content, including 1.35% ginkgolide A, 0.92% ginkgolide B, 0.91% ginkgolide C, and 3.6% bilobalide content) was added to 20 L of 20% ethanol and stirred to dissolve. The mixture was then extracted three times with a mixed solvent of dichloromethane and n-hexane in a volume ratio of 5:1, each time using 20 L of the mixed solvent layer. The extracts were combined and washed with 6 L of The extract was washed with pure water, allowed to stand and separate into layers, and the mixed solvent layer was collected and concentrated under reduced pressure to recover the mixed solvent to dryness to obtain 159.13 g of crude ginkgo lactones. Testing showed that the crude ginkgo lactones had a total lactone content of 73.81%, of which the content of ginkgo lactone A was 16.63% with a yield of 98.01%, the content of ginkgo lactone B was 11.37% with a yield of 98.33%, the content of ginkgo lactone C was 2.9%, and the content of bilobalide was 42.91% with a yield of 94.83%.

[0050] Step 2: 155 g of the crude ginkgo lactones obtained in step 1 (total lactone content: 73.81%) was taken, 750 mL of 30% ethanol was added, and the mixture was heated to 50-60° C. and beaten for 1 hour. The mixture was then cooled to room temperature and allowed to stand for crystallization for more than 4 hours. The mixture was filtered, the filter cake was collected, and dried to obtain 100.05 g of ginkgo lactones. After testing, the total lactone content of the ginkgo lactones was 98.84%, of which the content of ginkgo lactone A was 23.84%, the content of ginkgo lactone B was 16.42%, the content of ginkgo lactone C was 3.83%, the content of bilobalide was 54.75%, and the yield was 82.36%.

[0051] Step 3, taking 99.5g of the total ginkgo lactones obtained in step 2 (total lactone content 98.84%), adding 500mL of 85% ethanol solution and stirring and heating at a temperature of 60°C to dissolve, then standing at room temperature for crystallization for more than 4h, filtering with suction, collecting 1 mother liquor, recycling 1 mother liquor and evaporating to dryness, adding 700mL of 60% ethanol solution and stirring and heating at a temperature of 60°C to dissolve, then standing at room temperature for crystallization for more than 4h, filtering with suction, collecting 2 mother liquors, recycling 2 mother liquors and evaporating to dryness to obtain 65.71g of crude bilobalide, which was tested to have a total lactone content of 98.29%, including 8.6% ginkgolide A, 4.84% ginkgolide B, 4.03% ginkgolide C, 80.82% bilobalide and a yield of 97.49%;

[0052] Step 4. Take 10g of the crude bilobalide obtained in step 3, stir and dissolve it with 2L of 20% ethanol, pass it through 200ml of LX-32 resin, elute with 20% ethanol, monitor the elution effluent in the liquid phase, and start collecting when bilobalide flows out of the elution effluent; stop collecting when there is basically no bilobalide in the elution effluent, combine the elution effluent, concentrate under reduced pressure to dryness, heat and dissolve it with 25ml of 90% ethanol, filter it while hot, add 50ml of water to the filtrate, let it stand at room temperature for crystallization for more than 4h, filter it with suction, and dry the filter cake to obtain 7.56g of bilobalide monomer with a yield of 93.54%, a total yield of bilobalide of 71.23%, and a bilobalide content of 99.6% after testing.

[0053] Example 3

[0054] A method for preparing ginkgo biloba lactones and bilobalide monomers contained therein, comprising the following specific steps:

[0055] Step 1: 2 kg of ginkgo leaf extract (ginkgo lactone content 6.71%, including ginkgolide A content 1.31%, ginkgolide B content 0.94%, ginkgolide C content 1.04%, bilobalide content 3.42%) was added to 20 L of 20% ethanol and stirred to dissolve, and then extracted with a mixed solvent consisting of ethyl acetate and n-hexane in a volume ratio of 2:1 for 3 times, each time using 30 L, and the mixed solvent layer extracts were combined and washed with 10 L of The extract was washed with pure water, allowed to stand and separate into layers, and the mixed solvent layer was collected and concentrated under reduced pressure to recover the mixed solvent to dryness to obtain 147.77 g of crude ginkgo lactones. Testing showed that the crude ginkgo lactones had a total lactone content of 76.71%, including 17.32% ginkgolide A with a yield of 97.69%, 12.50% ginkgolide B with a yield of 98.25%, 2.86% ginkgolide C, and 44.03% bilobalide with a yield of 95.12%.

[0056] Step 2: 146 g of the crude ginkgo lactones obtained in step 1 (total lactone content: 76.72%) was taken, 870 mL of 30% ethanol was added, and the mixture was heated to 50-60° C. and beaten for 1 hour. The mixture was then cooled to room temperature and allowed to stand for crystallization for more than 4 hours. The mixture was filtered, the filter cake was collected, and dried to obtain 103.48 g of ginkgo lactones. After testing, the total lactone content of the ginkgo lactones was 98.41%, of which the content of ginkgo lactone A, ginkgo lactone B, ginkgo lactone C, and bilobalide was 23.01%, 16.95%, 3.5%, and 54.95% respectively, with a yield of 88.46%.

[0057] Step 3, taking 102g of total ginkgo lactones obtained in step 2 (total lactone content 98.41%), adding 600mL of 80% ethanol solution and stirring and heating at a temperature of 60°C to dissolve, then standing at room temperature for crystallization for more than 4h, filtering with suction, collecting 1 mother liquor, recycling 1 mother liquor and evaporating to dryness, adding 600mL of 65% ethanol solution and stirring and heating at a temperature of 60°C to dissolve, then standing at room temperature for crystallization for more than 4h, filtering with suction, collecting 2 mother liquors, recycling 2 mother liquors and evaporating to dryness to obtain 66.64g of crude bilobalide, which was tested to have a total lactone content of 97.81%, including 8.23% of ginkgolide A, 5.00% of ginkgolide B, 3.69% of ginkgolide C, 80.89% of bilobalide and a yield of 96.18%;

[0058] Step 4. Take 10g of the crude bilobalide obtained in step 3, stir and dissolve it with 2L of 20% ethanol, pass it through 200ml of LX-32 resin, elute with 20% ethanol, monitor the elution effluent in the liquid phase, and start collecting when bilobalide flows out of the elution effluent; stop collecting when there is basically no bilobalide in the elution effluent, combine the elution effluent, concentrate under reduced pressure to dryness, heat and dissolve it with 25ml of 90% ethanol, filter it while hot, add 50ml of water to the filtrate, let it stand at room temperature for crystallization for more than 4h, filter it with suction, and dry the filter cake to obtain 7.74g of bilobalide monomer with a yield of 96.77%, a total yield of bilobalide of 78.31%, and a bilobalide content of 99.7%.

[0059] Example 4

[0060] A method for preparing ginkgo biloba lactones and bilobalide monomers contained therein, comprising the following specific steps:

[0061] Step 1: 2 kg of Ginkgo biloba extract (Ginkgo biloba total lactone content 6.71%, including ginkgolide A content 1.31%, ginkgolide B content 0.94%, ginkgolide C content 1.04%, bilobalide content 3.42%) was added to 20 L of 20% ethanol and stirred to dissolve, and then extracted with a mixed solvent consisting of ethyl acetate and n-hexane in a volume ratio of 3:1 for 3 times, each time using 20 L, and the mixed solvent layer extracts were combined and washed with 6 L of The extract was washed with pure water, allowed to stand and separate into layers, and the mixed solvent layer was collected and concentrated under reduced pressure to recover the mixed solvent to dryness to obtain 163.96 g of crude ginkgo lactones. Testing showed that the crude ginkgo lactones had a total lactone content of 70.14%, of which the content of ginkgo lactone A was 15.76% with a yield of 98.63%, the content of ginkgo lactone B was 11.35% with a yield of 99.0%, the content of ginkgo lactone C was 3.22%, and the content of bilobalide was 39.81% with a yield of 95.43%.

[0062] Step 2: Take 162 g of crude ginkgo lactones obtained in step 1 (total lactone content 70.14%), add 750 mL of 30% ethanol, heat to 50-60 ° C and beat for 1 hour, then cool to room temperature and stand for crystallization for more than 4 hours, filter, collect the filter cake, and dry to obtain 100.0 g of ginkgo lactones; after testing (such as Figure 2 The total lactone content of Ginkgo biloba lactones is 97.5%, of which Ginkgolide A content is 23.53%, Ginkgolide B content is 17.06%, Ginkgolide C content is 4.35%, and bilobalide content is 52.56%, and the yield is 74.1%;

[0063] Step 3, taking 98g of the total ginkgo lactones obtained in step 2 (total lactone content 97.5%), adding 500mL of 85% ethanol solution and stirring and heating at a temperature of 60°C to dissolve, then standing at room temperature for crystallization for more than 4h, filtering with suction, collecting 1 mother liquor, recycling 1 mother liquor and evaporating to dryness, adding 700ml of 65% ethanol solution and stirring and heating at a temperature of 60°C to dissolve, then standing at room temperature for crystallization for more than 4h, filtering with suction, collecting 2 mother liquors, recycling 2 mother liquors and evaporating to dryness to obtain 67.49g of crude bilobalide, which was tested to have a total lactone content of 96.88%, including 11.75% ginkgolide A content, 5.94% ginkgolide B content, 4.45% ginkgolide C content, 74.74% bilobalide content and a yield of 81.50%;

[0064] Step 4. Take 10g of the crude bilobalide obtained in step 3, stir and dissolve it with 2L of 20% ethanol, pass it through 200ml of LX-32 resin, elute with 20% ethanol, monitor the elution effluent in the liquid phase, and start collecting when bilobalide flows out of the elution effluent; stop collecting when there is basically no bilobalide in the elution effluent, combine the elution effluent, concentrate under reduced pressure to dryness, add 25ml of 90% ethanol and heat to dissolve, filter while hot, add 50ml of water to the filtrate, let it stand at room temperature for crystallization for more than 4h, filter with suction, and dry the filter cake to obtain 6.9g of bilobalide monomer with a yield of 92.32%, a total yield of bilobalide of 70.31%, and a bilobalide content of 99.55% after testing.

[0065] Comparative Example 1

[0066] Step 1. Take 10 g of the crude bilobalide obtained in step 3 of Example 1, heat and stir to dissolve with 75 ml of 60% ethanol, let stand at room temperature for more than 6 hours to crystallize, filter, and dry the filter cake to obtain 4.83 g. After testing, the total lactone content is 99.96%, of which the ginkgolide A content is 2.36%, the ginkgolide B content is 3.71%, the ginkgolide C content is 0.38%, and the bilobalide content is 93.52%.

[0067] Step 2: The mother liquor in step 1 of this comparative example was concentrated to dryness, 52 ml of 60% ethanol was added, heated with stirring to dissolve, and allowed to stand at room temperature for more than 6 hours for crystallization. The mixture was filtered and the mother liquor was collected. The mother liquor was concentrated to dryness, 32 ml of 60% ethanol was added, heated with stirring to dissolve, and allowed to stand at room temperature for more than 6 hours for crystallization. The mixture was filtered and the filter cake was dried to obtain 1.72 g. The total lactone content was 99.74%, of which the ginkgolide A content was 2.23%, the ginkgolide B content was 3.42%, the ginkgolide C content was 0.61%, and the bilobalide content was 93.48%.

[0068] Step 3. Combine the filter cakes obtained in steps 1 and 2 of this comparative example, add 32 ml of 70% ethanol, heat with stirring to dissolve, let stand at room temperature for crystallization for more than 6 h, filter, collect the filter cake, heat the filter cake with 20 ml of 70% ethanol, stir and dissolve, let stand at room temperature for crystallization for more than 6 h, filter, collect the filter cake, and dry to obtain 2.67 g. After testing, the total lactone content was 99.95%, of which the ginkgolide A content was 0.07%, the ginkgolide B content was 0.55%, the ginkgolide C was not detected, the bilobalide content was 99.33%, and the total yield of bilobalide was 26.70%.

[0069] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be embodied in other specific forms without departing from the spirit and essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0070] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for preparing ginkgo biloba lactones and bilobalide monomers contained therein, characterized in that: The following steps are involved: (1) Ginkgo biloba extract is obtained according to the preparation method of the Chinese Pharmacopoeia, dissolved in water or dilute ethanol by stirring, then extracted 2-3 times with a mixed solvent, allowed to stand for stratification, the mixed solvent layer is collected, combined, the combined mixed solvent layer is washed with water, allowed to stand for stratification, the mixed solvent layer is collected, and concentrated to dryness under reduced pressure to obtain a crude product of total ginkgo lactones with a content of more than 70%; Wherein, the mixed solvent is a mixture of solvent A and solvent B, solvent A is a fat-soluble solvent, and solvent B is a low molecular weight alkane solvent; (2) adding dilute ethanol to the crude ginkgo lactones obtained in step (1), heating and beating the mixture, cooling the mixture to room temperature, standing the mixture for crystallization, filtering the mixture, collecting the filter cake, and drying the mixture to obtain a ginkgo lactone content of more than 95%; (3) dissolving the total ginkgo lactones obtained in step (2) by heating with a high-concentration ethanol solution, crystallizing at room temperature, filtering, collecting the mother liquor, and concentrating the mother liquor to dryness, repeating this step, and concentrating the second mother liquor to dryness to obtain a crude product of bilobalide; (4) dissolving the crude bilobalide monomer obtained in step (3) in dilute ethanol, adsorbing it on a macroporous adsorption resin, and then eluting it with dilute ethanol. The eluate is collected, concentrated to dryness under reduced pressure, and recrystallized once to obtain a bilobalide monomer with a content of more than 99%.

2. A method for preparing ginkgo biloba lactones and bilobalide monomers contained therein according to claim 1, characterized in that: In step (1), the solvent A is one of dichloromethane, ethyl acetate, methyl ethyl ketone, ethyl formate, chloroform, dichloroethane or 1-butanol; and the solvent B is one of n-hexane, n-pentane, n-heptane or petroleum ether.

3. A method for preparing ginkgo biloba lactones and bilobalide monomers contained therein according to claim 2, characterized in that: In step (1), the solvent A is dichloromethane or ethyl acetate; and the solvent B is n-hexane.

4. The method for preparing a total ginkgo lactone and bilobalide monomer contained therein according to claim 3, wherein: In step (1), in the mixed solvent, solvent A is dichloromethane, solvent B is n-hexane, and the volume ratio of the dichloromethane to n-hexane is 1 to 10:

1.

5. The method for preparing a ginkgolide and bilobalide monomers contained therein according to claim 4, wherein: The volume ratio of the dichloromethane to n-hexane is 3:

1.

6. The method for preparing the ginkgolides and bilobalide monomers contained therein according to claim 3, wherein: In step (1), in the mixed solvent, solvent A is ethyl acetate, solvent B is n-hexane, and the volume ratio of the ethyl acetate to n-hexane is 1 to 6:

1.

7. The method for preparing the ginkgo biloba lactones and the bilobalide monomers contained therein according to claim 6, wherein: The volume ratio of ethyl acetate to n-hexane is 2:

1.

8. The method for preparing a total ginkgo lactone and bilobalide monomer contained therein according to claim 1, wherein: In step (1) and step (4), the concentration of dilute ethanol is 10-30%; In step (2), the concentration of dilute ethanol is 20-40%; In step (3), the ethanol concentration of the high-concentration ethanol aqueous solution is 60-95%.

9. The method for preparing a total ginkgo lactone and bilobalide monomer contained therein according to claim 1, wherein: In step (4), the macroporous adsorption resin is one or more of DM131, HPD417, HPD-BJQH, LX-5, LX-32 or LX-60.

10. The method for preparing the ginkgo bilobalide and the bilobalide monomers contained therein according to claim 1, wherein: In the total ginkgo lactones with a content of more than 95% obtained in step (2), the content of ginkgo lactone A is 20-25%, the content of ginkgo lactone B is 15-20%, the content of bilobalide is ≥50%, and the content of ginkgo lactone C is ≤5%.

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