CN113801133B - Preparation method of ginkgolide and monomer contained in ginkgolide - Google Patents
Preparation method of ginkgolide and monomer contained in ginkgolide Download PDFInfo
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- CN113801133B CN113801133B CN202111259321.XA CN202111259321A CN113801133B CN 113801133 B CN113801133 B CN 113801133B CN 202111259321 A CN202111259321 A CN 202111259321A CN 113801133 B CN113801133 B CN 113801133B
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Abstract
The invention discloses a preparation method of ginkgolides and monomers contained in the ginkgolides, which comprises the steps of obtaining ginkgo biloba extract eluent according to a preparation method of Chinese pharmacopoeia, concentrating the ginkgo biloba extract eluent to a column, eluting with ethanol with different concentrations in sequence, collecting the ethanol eluents with different concentrations, treating the ethanol eluents respectively to obtain crystallization liquid with certain ethanol concentration, and crystallizing at low temperature to obtain crude ginkgolides; recrystallizing the crude product of bilobalide in ethanol water solution to obtain bilobalide with content of more than 99%, which comprises bilobalide A, bilobalide B and bilobalide C; the method comprises the steps of respectively obtaining ginkgolide A, ginkgolide B and ginkgolide C monomers with the content of more than 99.5% by utilizing the dissolution difference of each component of ginkgolide in ethanol with different concentrations. The method only relates to a solvent ethanol, has simple operation and high product purity, and is suitable for industrial production.
Description
Technical Field
The invention relates to a preparation method of ginkgolides and monomers contained in the ginkgolides, and belongs to the field of extraction and purification of plant active ingredients.
Background
Currently, the most studied ginkgo biloba is ginkgo biloba extract, the main ingredients of which include flavonoids and ginkgolide compounds. The bilobalide compound belongs to terpenoids, and is bilobalide, bilobalide A, bilobalide B, bilobalide C, bilobalide J, and bilobalide M. The ginkgo leaf contains 0.2-0.5% of total terpene lactones, and the existing research shows that the ginkgo leaf lactones are a high-efficiency platelet activating factor antagonist, have unique pharmacological action and therapeutic value, can be used for treating diseases such as apoplexy, shock, transplant rejection, hemodialysis, asthma and the like, and have protective action on ischemic injury and central nervous system. However, the structure of bilobalide is complex, and the artificial synthesis difficulty is very high, so that the bilobalide compound obtained by extracting ginkgo leaves and the bilobalide monomer become the main sources of bilobalide.
The preparation method of bilobalide or each bilobalide monomer provided by the prior art only considers the extraction and separation of bilobalide components, but can only obtain the bilobalide components from ginkgo leaves or ginkgo leaf extracts (the mass of the total bilobalide of the finished ginkgo leaf extracts accounts for 70-75 percent of the total bilobalide in the ginkgo leaves), while the other main component flavonoid compounds in the ginkgo leaves or the ginkgo leaf extracts can not be used as the ginkgo leaf extracts due to the large quantity loss of the bilobalide, namely, the cross part of the bilobalide compounds contained in the chromatographic column adsorption generated in the refining process of the produced ginkgo leaf extracts (namely, the impurity part generated in the refining process of the produced ginkgo leaf extracts, the mass of the total bilobalide accounts for 15-20 percent of the total bilobalide in the ginkgo leaves) does not fully consider the comprehensive utilization of resources, resulting in a large waste of resources. And the content of the ginkgolide compound and each lactone monomer prepared by the prior art is not very high, multiple solvents are involved in the process, the industrial production is not easy, the production cost is high, or the used chromatographic materials are alumina and silica gel, so that the chromatographic materials cannot be repeatedly used, the solid waste generation amount is large, and the environmental protection treatment difficulty is large.
Therefore, the invention provides a preparation method of ginkgolides and monomers contained in the ginkgolides based on the economic consideration of industrial production of comprehensive utilization of resources.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a preparation method of ginkgolides and monomers contained in the ginkgolides, which makes full use of resources, is simple to operate, has high purity of the obtained product, and is suitable for industrial production; the preparation method comprises the steps of taking the cross part containing the ginkgolide compound adsorbed by a chromatographic column generated in the refining process of producing the ginkgo biloba extract as a raw material source, adopting ethanol water solutions with different concentrations for elution, dilution and crystallization, and obtaining the high-purity ginkgolide compound and the contained ginkgolide monomer by matching with resin enrichment, separation and recrystallization.
In order to achieve the purpose, the invention adopts the following technical scheme: a method for preparing bilobalide and monomer contained therein comprises the following steps:
step 1: according to the method for preparing the ginkgo leaf extract in Chinese pharmacopoeia, ethanol eluent containing the ginkgo leaf extract is obtained, the ethanol eluent is concentrated until the dry matter proportion is 10-15 percent of elution concentrated solution, the cooled ethanol eluent is put into a processed macroporous absorption resin a, the ethanol eluent with the concentration of 20-40 percent (v/v) is used for elution, the effluent liquid of the macroporous absorption resin a and the ethanol eluent with the concentration of 20-40 percent are collected and merged and concentrated to be dried, the ginkgo leaf extract is obtained, the content of total flavonol glycosides in the ginkgo leaf extract is more than or equal to 24 percent, the content of total terpene lactones is more than or equal to 6 percent, in order to obtain the cross part which is generated in a chromatographic column of the macroporous absorption resin a in the refining process of producing the ginkgo leaf extract, namely the cross part which contains the ginkgolide compounds and is remained in the macroporous absorption resin a, the cross part is sequentially eluted in the macroporous absorption resin a by the ethanol with the concentration of 50-70 percent (v/v) and the ethanol with the concentration of 90-95 percent (v/v) concentration of 90-95 percent, collecting 50-70% ethanol eluate and 90-95% ethanol eluate, wherein the 50-70% ethanol eluate is rich in bilobalide, and the 90-95% ethanol eluate contains impurities and bilobalide;
step 2: taking 90-95% ethanol eluent in the step 1, recovering ethanol to be complete, adding pure water to one fourth to one third of the volume of the stock solution, adding a pH regulator to regulate the pH value of the solution to be below 2, cooling to 15-30 ℃, carrying out centrifugal separation to remove insoluble matters to obtain clear water solution, putting the clear water solution on a treated macroporous adsorption resin b, eluting by using 10-20% (v/v) ethanol, removing effluent of the macroporous adsorption resin b and 10-20% (v/v) ethanol eluent, namely removing impurities at the cross part of a ginkgolide compound, in order to obtain ginkgolides enriched in the macroporous adsorption resin b, eluting by using 50-70% (v/v) ethanol, and collecting 50-70% ethanol eluent;
and 3, step 3: mixing the 50-70% ethanol eluate in step 1 and the 50-70% (v/v) ethanol eluate in step 2, recovering ethanol until the ethanol concentration is 10-30% (v/v), cooling for crystallization, filtering for separation to obtain crude product of ginkgolides, heating and pulping with 10-30% (v/v) ethanol solution with 10-20 times volume (ml) of the mass (g) value, cooling for crystallization, filtering for separation, and drying to obtain refined ginkgolides, wherein the refined ginkgolides are ginkgolide A, ginkgolide B and ginkgolide C, and the total lactone content is not less than 99%;
and 4, step 4: taking a refined product of ginkgolide, adding 90-95% (v/v) concentration ethanol with the mass (g) value of 20-25 times volume (ml), heating for dissolving, cooling for crystallization, filtering and separating to obtain a crude product of ginkgolide B, repeating the step to recrystallize the crude product of ginkgolide B for 2-3 times, drying the filtered solid in vacuum to obtain a ginkgolide B monomer with the content being more than or equal to 99.5%, and collecting mother liquor filtered in each step, wherein the mother liquor contains ginkgolide A and ginkgolide C;
and 5: combining all the mother liquor in the step 4, adding pure water to reduce the ethanol concentration to 50-70% (v/v), filtering and separating to obtain filtrate, concentrating to dryness, adding 90-95% (v/v) ethanol with the volume (ml) which is 20-25 times of the mass (g) of the filtrate, heating to dissolve the filtrate, adding pure water to reduce the ethanol concentration to 50-70% (v/v), cooling to crystallize, filtering and separating to obtain filtered mother liquor rich in ginkgolide A and ginkgolide C, and applying the rest filtered solids obtained in the step to the refined ginkgolide product in the step 4;
step 6: taking the filtered mother liquor rich in the ginkgolide A and the ginkgolide C in the step 5, adding pure water to reduce the concentration of ethanol in the mother liquor to 20-30% (v/v), and filtering and separating to obtain a solid rich in the ginkgolide A and a mother liquor rich in the ginkgolide C;
and 7: taking the solid rich in ginkgolide A in the step 6, adding 90-95% (v/v) ethanol with the mass (g) value of 15-20 times and the volume (ml) of the solid, heating for dissolving, cooling for crystallization, filtering and separating to obtain a crude product of ginkgolide A, then repeating the step to recrystallize the crude product of ginkgolide A for 1-2 times, carrying out vacuum drying on the filtered solid to obtain a ginkgolide A monomer, wherein the content of the ginkgolide A monomer is more than or equal to 99.5%, carrying out vacuum concentration on the filtered mother liquor obtained in the step to recycle ethanol until the filtered solid is dried, and applying the solid to the refined ginkgolide product in the step 4;
and 8: taking mother liquor rich in ginkgolide C in the step 6, adding the mother liquor into a macroporous adsorption resin C which is processed and balanced by 20% ethanol, adding effluent liquid of the macroporous adsorption resin C into a macroporous adsorption resin d which is processed and balanced by 20% ethanol, circularly eluting ginkgolide C in the macroporous adsorption resin C by using the effluent liquid of the macroporous adsorption resin d, circularly adding the effluent liquid containing ginkgolide C eluted by the macroporous adsorption resin C into the resin d to be adsorbed, repeating the operation until the ginkgolide C in the macroporous adsorption resin C is completely eluted, then respectively eluting the macroporous adsorption resin C and the macroporous adsorption resin d by using 50-70% (v/v) ethanol, collecting 50-70% ethanol eluent of the macroporous adsorption resin d, concentrating to be dry, adding 90-95% (v/v) ethanol with the volume (ml) which is 5-10 times of the mass (g) value of the ethanol, heating, dissolving, cooling and crystallizing, filtering and separating, vacuum drying the filtered solid to obtain a ginkgolide C monomer with the content of more than or equal to 99.5%, then vacuum concentrating the filtered mother liquor, recovering ethanol until the filtered mother liquor is dried to obtain a solid which can be applied to the refined ginkgolide product in the step 4, and applying 50-70% (v/v) ethanol eluent of the macroporous adsorption resin C to the 50-70% ethanol eluent in the step 3.
Preferably, in step 1, the macroporous adsorbent resin a is one or more of HPD5000, LX200, DA201 resins.
Wherein, the column loading amount of the macroporous adsorption resin a is as follows: the amount of the concentrated eluate with dry matter ratio of 10-15% (v/v) is 5-8BV, and the amounts of ethanol with concentration of 20-40% (v/v), 50-70% (v/v) and 90-95% (v/v) are 3-6BV respectively.
Preferably, in step 2, the macroporous adsorbent resin b is one or more of DM131, HPD417 and HPD-BJQH resin.
Wherein, the column loading amount of the macroporous adsorption resin b is as follows: the amount of the clear aqueous solution is 20-25BV, 10-20 (v/v)%, 50-70% (v/v) ethanol is 2-3 BV.
Preferably, in the step 8, the macroporous adsorption resin c is one or more of LX-5, LX-32 and LX-60 resins, and the macroporous adsorption resin d is one or more of HC100A, HC700 and HC80B resins.
Wherein the bed volume of the macroporous adsorption resin d is the same as that of the macroporous adsorption resin c, and the column loading amount of the macroporous adsorption resin c is as follows: the amount of mother liquor rich in ginkgolide C and the effluent liquid of the macroporous adsorption resin d are respectively 4-8 (v/v) BV, the amount of 50-70% (v/v) ethanol used is 2-3BV, and the column loading amount of the macroporous adsorption resin d is as follows: the amount of effluent liquid of the macroporous adsorbent resin c is 4-8 (v/v) BV respectively, and the amount of 50-70% (v/v) ethanol is 2-3 BV.
Preferably, in step 3, the refined product of ginkgolides contains ginkgolide A10-20%, ginkgolide B70-85%, and ginkgolide C5-15%.
Preferably, the pH regulator in the step 2 is one or more of sulfuric acid, hydrochloric acid and phosphoric acid; the pH value is adjusted to 1-2.
Preferably, the cooling crystallization temperature is 0-15 ℃, and the crystallization time is 8-24 h; the vacuum degree of the concentration and the vacuum drying in the steps 2 to 8 is less than-0.09 MPa, the evaporation temperature is less than 60 ℃, and the vacuum drying time is 6 to 8 hours; the heating pulping and heating dissolution are carried out under normal pressure to reach a reflux state.
The invention has the beneficial effects that:
(1) the invention utilizes the cross part behind the chromatographic column in the refining process of producing the ginkgo biloba extract as the raw material for extracting and purifying the ginkgolide compound and the monomer contained in the ginkgolide compound, ensures the quality of the ginkgo biloba extract product, obtains the target product of the invention, realizes the comprehensive utilization of resources to the maximum extent, has important significance and also has good economic benefit;
(2) the purity of the target product prepared by the invention is high, the total lactone content of the ginkgolide compound is more than or equal to 99 percent, the purity of the monomer ginkgolide A is more than or equal to 99.5 percent, and the purity of the monomer ginkgolide B is more than or equal to 99.5 percent; the purity of the monomer ginkgolide C is more than or equal to 99.5 percent, and the requirement of a pharmaceutical preparation can be completely met;
(3) in the preparation process, toxic and harmful solvents or a plurality of solvents are not used, only one ethanol water solution is involved, and the ethanol is repeatedly recycled, so that the residual variety of the solvents in the product is reduced, and the quality safety of the product is improved;
(4) the chromatographic materials used in the preparation process are macroporous adsorption resin, so that the preparation process is easy to regenerate and recycle, and less in solid waste;
(5) the preparation process of the invention is relatively friendly to the environment, and can effectively improve the health guarantee of operators;
(6) the invention has simple process operation and small equipment investment and is suitable for industrial production.
Drawings
FIG. 1: liquid chromatogram of refined product of bilobalide;
FIG. 2: a ginkgolide A liquid chromatogram map;
FIG. 3: a ginkgolide B liquid chromatogram map;
FIG. 4 is a schematic view of: a liquid chromatogram of ginkgolide C.
Detailed Description
The technical solution in the implementation of the present invention is described in detail below with reference to embodiments, which are only a part of embodiments of the present invention, but not all embodiments. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.
The dry matter mentioned in the examples refers to the residue of the solution after removal of the solvent, and the dry matter ratio refers to the percentage of the mass of the residue relative to the volume of the solution. Unless otherwise specified, all are conventional methods.
The ethanol concentrations mentioned in the examples are volume concentrations.
Example 1
Step 1: taking 100kg of dried folium Ginkgo (total flavonol glycoside content is 0.84%, total ginkgolide content is 0.26%), pulverizing, extracting with 70% ethanol under heating, according to the method for preparing folium Ginkgo extract of Chinese pharmacopoeia, obtaining ethanol eluate containing folium Ginkgo extract part 210L, concentrating the eluate to 25L, cooling to 20 deg.C, adding into 5L HPD5000 macroporous adsorbent resin, eluting with 20% ethanol of 30L, mixing the effluent of HPD5000 macroporous adsorbent resin with 20% ethanol eluate 54.5L, concentrating and recovering ethanol under-0.09 MPa vacuum degree for 6 hr, drying under-0.09 MPa vacuum degree and 80 deg.C, pulverizing to obtain 2.9kg of folium Ginkgo extract (total flavonol glycoside content is 25.3%, total terpene lactone content is 6.5%, total ginkgolic acid content is 0.4 ppm), producing macroporous part of HPD5000 adsorbent resin chromatographic column after refining process of folium Ginkgo extract production, continuously eluting with 20L of 50% ethanol to elute part of the enriched bilobalide in the cross section of the HPD5000 macroporous adsorbent resin chromatographic column, continuously eluting with 30L of 90% ethanol to elute impurities and the rest bilobalide in the cross section of the HPD5000 macroporous adsorbent resin chromatographic column, and respectively collecting 20.3L of 50% ethanol eluate and 30.6L of 90% ethanol eluate;
step 2: taking 30.6L of 90% ethanol eluent in the step 1, recovering ethanol to be complete under vacuum of-0.09 MPa, adding pure water to 8L, dropwise adding 98% sulfuric acid to adjust the pH value to 2, cooling to 30 ℃, centrifugally separating to remove insoluble solids to obtain 7.7L of clear water liquid, feeding the clear water liquid into 350ml of DM131 macroporous adsorption resin to adsorb ginkgolides, firstly eluting with 700ml of 10% ethanol, removing effluent of the DM131 macroporous adsorption resin and 10% ethanol eluent, namely removing impurities at the cross part of a ginkgolide compound, continuously eluting with 1050ml of 50% ethanol to obtain ginkgolides enriched in the DM131 macroporous adsorption resin, and collecting 1100ml of 50% ethanol eluent;
and step 3: combining 20.3L of the 50% ethanol eluate in step 1 with 1100ml of the 50% ethanol eluate in step 2, 21.4L, concentrating and recovering ethanol to 6.8L in volume under-0.09 MPa vacuum degree, with the concentration of ethanol in the feed liquid being about 10%, cooling at 15 deg.C, crystallizing for 24 hr, filtering, separating, measuring the components with liquid chromatograph (shown in figure 1), which is bilobalide containing bilobalide A, bilobalide B and bilobalide C, to obtain crude product of bilobalide 33.5g, adding the crude product into 670ml of 10% ethanol, heating and pulping under normal pressure until the solution boils and refluxes, cooling at 15 deg.C, crystallizing for 24 hr, filtering, separating, drying the filtered solid at-0.09 MPa vacuum degree and 60 deg.C for 6 hr to obtain refined product of bilobalide 22.7g, measuring with liquid chromatograph, and calculating the content of each component, the content of ginkgolide A is 16.6%, the content of ginkgolide B is 71.3%, the content of ginkgolide C is 11.5%, and the content of total lactones is 99.4%;
and 4, step 4: taking 22g of refined bilobalide, adding 440ml of 90% ethanol, heating under normal pressure for reflux dissolution, cooling, placing at 15 ℃ for cooling crystallization for 24 hours, filtering and separating, measuring by a liquid chromatograph, as shown in figure 3, obtaining 14.5g of crude bilobalide B, adding 290ml of 90% ethanol into the crude bilobalide B, heating under normal pressure for reflux dissolution, cooling, placing at 15 ℃ for cooling crystallization for 24 hours, filtering and separating, obtaining 10.2g of crude bilobalide B, adding 190ml of 90% ethanol into the crude bilobalide B, heating under normal pressure for reflux dissolution, cooling, placing at 15 ℃ for cooling crystallization for 24 hours, filtering and separating, placing the filtered solid under the conditions of-0.09 MPa vacuum degree and 60 ℃ for drying for 6 hours, obtaining 6.7g of bilobalide B monomer, measuring by a liquid chromatograph and calculating, the content of the bilobalide B monomer is 99.8%, collecting the mother liquor filtered in each step;
and 5: combining 890ml of all the mother liquor obtained in the step 4, adding 710ml of pure water to reduce the ethanol concentration in the mother liquor to 50%, filtering and separating to obtain 1590ml of filtrate, concentrating and recovering ethanol to dryness under-0.09 MPa of vacuum degree to obtain 6.2g of solid, measuring and calculating the solid by a liquid chromatograph, adding 125ml of 90% ethanol into the solid to perform heating reflux dissolution under normal pressure, adding 100ml of water to reduce the ethanol concentration to 50%, cooling and then placing at 15 ℃ for cooling crystallization for 24 hours, filtering and separating to obtain 220ml of filtered mother liquor rich in ginkgolides A and C, and combining the filtered solid obtained in the step and applying the solid to the refined ginkgolides in the step 4;
step 6: taking 220ml of the filtered mother liquor rich in the ginkgolide A and the ginkgolide C in the step 5, adding 330ml of pure water to reduce the ethanol concentration in the mother liquor to 20%, and filtering and separating to obtain 2.8g of solid rich in the ginkgolide A and 540ml of mother liquor rich in the ginkgolide C;
and 7: taking 2.8g of the solid rich in ginkgolide A in step 6, adding 40ml of 90% ethanol, heating, refluxing for dissolving, cooling, placing at 15 ℃, cooling, crystallizing for 24 hours, filtering, separating, measuring by a liquid chromatograph, as shown in figure 2, obtaining 1.9g crude product of bilobalide A, adding 30ml 90% ethanol into the crude product of bilobalide A, heating under reflux at normal pressure for dissolving, cooling at 15 deg.C for crystallization for 24 hr, filtering for separation, drying the filtered solid at-0.09 MPa vacuum degree and 60 deg.C for 6 hr to obtain 1.3g ginkgolide A monomer, measuring with liquid chromatograph, calculating, the content of the ginkgolide A monomer is 99.9 percent, and the filtered mother liquor obtained in the step is merged, concentrating under-0.09 MPa vacuum degree, recovering ethanol to obtain dry solid 1.5g, and mechanically applying to the refined product of bilobalide obtained in step 4;
and 8: taking 540ml of mother liquor rich in ginkgolide C in the step 6, adding 100ml of mother liquor into LX-5 macroporous adsorption resin balanced by 20% ethanol, collecting 550ml of LX-5 macroporous adsorption resin effluent, adding 100ml of HC100A macroporous adsorption resin balanced by 20% ethanol, collecting 550ml of HC100A macroporous adsorption resin effluent, circularly eluting LX-5 macroporous adsorption resin by using HC100A macroporous adsorption resin effluent, eluting ginkgolide C in LX-5 macroporous adsorption resin into HC100A macroporous adsorption resin for adsorption, performing operation for 2 times, respectively eluting LX-5 macroporous adsorption resin and HC100A macroporous adsorption resin by using 200ml of 50% ethanol, mixing 50% ethanol in LX-5 macroporous adsorption resin with 50% ethanol in the step 3, collecting 50% ethanol eluent in HC100A macroporous adsorption resin, and keeping the vacuum degree of-0.09 MPa, concentrating and recovering ethanol to dryness, determining by a liquid chromatograph, as shown in figure 4, obtaining 1.5g of crude ginkgolide C product, adding 10ml of 90% ethanol, heating under normal pressure, refluxing and dissolving, cooling, placing at 15 ℃, cooling, crystallizing for 24 hours, filtering, separating, drying the filtered solid for 6 hours at 60 ℃ under the vacuum degree of-0.09 MPa to obtain 1.2g of ginkgolide C monomer, determining by the liquid chromatograph, calculating to obtain the content of the ginkgolide C monomer of 99.7%, concentrating the filtered mother liquor under the vacuum degree of-0.09 MPa, recovering ethanol to dryness to obtain solid, and applying the solid to the refined ginkgolide product in the step 4.
Example 2
Step 1: taking 100kg of dried folium Ginkgo (total flavonol glycoside content is 0.95%, total ginkgolide content is 0.35%), pulverizing, extracting with 75% ethanol under heating, obtaining 220L of ethanol eluent containing folium Ginkgo extract part according to Chinese pharmacopoeia, concentrating the eluent to 26L, cooling to 20 deg.C, adding into LX200 macroporous adsorbent resin with volume of 5L, eluting with 20L of 30% ethanol, mixing the effluent of LX200 macroporous adsorbent resin with 46.2L of 20% ethanol eluent, concentrating and recovering ethanol to completion under vacuum degree of-0.095 MPa, drying under vacuum degree of-0.095 MPa and temperature of 80 deg.C for 8 hr, pulverizing to obtain 3.2kg of folium Ginkgo extract (total flavonol glycoside content is 26.7%, total terpene lactone content is 7.6%, total ginkgolic acid content is 0.2 ppm), producing macroporous adsorption resin chromatographic column of LX200 in the refining process of folium Ginkgo extract, continuously eluting with 30L of 60% ethanol to elute the ginkgolides partially enriched in the cross part of the LX200 macroporous adsorbent resin chromatographic column, continuously eluting with 20L of 92% ethanol to elute the impurities and the residual ginkgolides in the cross part of the LX200 macroporous adsorbent resin chromatographic column, and respectively collecting 29.6L of 60% ethanol eluate and 20.7L of 92% ethanol eluate;
and 2, step: taking 20.7L of 92% ethanol eluent in the step 1, recovering ethanol to be complete under vacuum of-0.095 MPa, adding pure water to 7L, dropwise adding 35% hydrochloric acid to adjust the pH value to 1, cooling to 20 ℃, centrifugally separating to remove insoluble solids to obtain 6.8L of clear water liquid, adding the clear water liquid into 300ml of HPD417 macroporous adsorption resin to adsorb ginkgolides, firstly eluting with 750ml of 12% ethanol, removing the effluent of the HPD417 macroporous adsorption resin and 10% ethanol eluent, namely removing impurities at the cross part of ginkgolides compounds, in order to obtain ginkgolides enriched in the HPD417 macroporous adsorption resin, then continuously eluting with 900ml of 60% ethanol, and collecting 950ml of 60% ethanol eluent;
and 3, step 3: combining 29.6L of the 60% ethanol eluate in the step 1 with 950ml of the 60% ethanol eluate in the step 2, concentrating and recovering ethanol to 7.9L in volume under-0.095 MPa of vacuum degree, wherein the ethanol concentration of the feed liquid is about 20%, cooling, placing at 10 ℃ for cooling and crystallizing for 20 hours, filtering and separating, measuring by a liquid chromatograph (shown in figure 1), obtaining 56.2g of a crude product of ginkgolides for ginkgolides containing ginkgolides A, ginkgolides B and ginkgolides C, adding the crude product into 850ml of 20% ethanol, heating and pulping under normal pressure until the solution boils and refluxes, cooling, placing at 10 ℃ for cooling and crystallizing for 20 hours, filtering and separating, placing the filtered solid under-0.095 MPa of vacuum degree and drying at 55 ℃ for 7 hours to obtain 35.8g of a refined product of ginkgolides, measuring and calculating by the liquid chromatograph, wherein the content of ginkgolides A in the refined product of ginkgolides is 12.4%, The content of ginkgolide B is 80.5%, the content of ginkgolide C is 6.7%, and the content of total lactones is 99.6%;
and 4, step 4: adding 870ml of 92% ethanol into 35g of refined ginkgolide, heating under normal pressure, refluxing for dissolving, cooling at 10 ℃, cooling for 20 hours, filtering and separating to obtain 24.2g of crude ginkgolide B, adding 600ml of 92% ethanol into the crude ginkgolide B, heating under normal pressure, refluxing for dissolving, cooling at 10 ℃, cooling for 20 hours, filtering and separating, determining by a liquid chromatograph (shown in figure 3) to obtain 15.2g of crude ginkgolide B, adding 400ml of 92% ethanol into the crude ginkgolide after the first crystallization, heating under normal pressure, refluxing for dissolving, cooling at 10 ℃ for 20 hours, filtering and separating, drying the filtered solid at 55 ℃ under-0.095 MPa vacuum degree to obtain 9.9g of ginkgolide B monomer, determining by the liquid chromatograph, calculating, the content of the filtrate is 100.2 percent, and mother liquor obtained after each filtration in the step is collected;
and 5: merging all the mother liquor 1840ml obtained in the step 4, adding 980ml of pure water to reduce the ethanol concentration in the mother liquor to 60%, filtering and separating to obtain 2800ml of filtrate, concentrating and recovering ethanol to dryness under the vacuum degree of-0.095 MPa to obtain 11.7g of solid, measuring and calculating by a liquid chromatograph, adding 290ml of 92% ethanol into the solid, heating and refluxing to dissolve the solid at normal pressure, adding 155ml of pure water to reduce the ethanol concentration to 60%, cooling and placing at 10 ℃ for cooling and crystallizing for 20 hours, filtering and separating to obtain 430ml of filtered mother liquor rich in ginkgolides A and C, and merging the filtered solid obtained in the step to be applied to the refined ginkgolides in the step 4;
step 6: taking 430ml of the filtered mother liquor rich in the ginkgolide A and the ginkgolide C in the step 5, adding 600ml of pure water to reduce the ethanol concentration in the mother liquor to 25%, and filtering and separating to obtain 3.2g of solid rich in the ginkgolide A and 1020ml of mother liquor rich in the ginkgolide C;
and 7: taking 3.2g of the solid rich in ginkgolide A in the step 6, adding 75ml of 92% ethanol, heating for dissolving, cooling, placing at 10 ℃, cooling for crystallization for 20 hours, filtering, separating, and determining by a liquid chromatograph, wherein the solid is ginkgolide A as shown in figure 2 to obtain 2.1g of crude ginkgolide A; adding 50ml of 92% ethanol into the crude product, heating under reflux for dissolving at normal pressure, cooling, placing at 10 ℃ for cooling and crystallizing for 20 hours, filtering and separating to obtain a solid, placing at-0.095 MPa vacuum degree, drying at 55 ℃ for 7 hours to obtain 1.2g of a ginkgolide A monomer, measuring and calculating by using a liquid chromatograph to obtain 99.7% of the ginkgolide A monomer, combining the filtered mother liquor obtained in the step, concentrating and recovering the ethanol until the dry solid is 2.0g under-0.09 MPa vacuum degree, and then applying the dry solid to the refined ginkgolide product in the step 4;
and step 8: and (4) taking 1020ml of the mother liquor rich in ginkgolide C in the step (6), adding 150ml of the mother liquor into LX-32 macroporous adsorption resin balanced by 20% ethanol, collecting 1000ml of LX-32 resin effluent, adding 150ml of the mother liquor into HC700 macroporous adsorption resin balanced by 20% ethanol, and collecting 980ml of HC700 macroporous adsorption resin effluent. Circularly eluting LX-32 resin with effluent of HC700 macroporous adsorbent resin, eluting ginkgolide C in LX-32 macroporous adsorbent resin into HC700 macroporous adsorbent resin, and adsorbing, performing such operation for 3 times, respectively eluting LX-32 macroporous adsorbent resin and HC700 macroporous adsorbent resin with 370ml of 60% ethanol, mixing 60% ethanol eluate of LX-32 macroporous adsorbent resin with 60% ethanol eluate of step 3, collecting 60% ethanol eluate of HC700 macroporous adsorbent resin, concentrating and recovering ethanol to dryness under-0.095 MPa vacuum, measuring with liquid chromatograph as shown in FIG. 4 to obtain crude product of ginkgolide C1.7 g, adding 12ml of 92% ethanol, heating under normal pressure, refluxing for dissolving, cooling at 10 deg.C, cooling for 20 hr, filtering, separating, and placing the filtered solid under-0.095 MPa vacuum degree, Drying at 55 deg.C for 7 hr to obtain 1.3g of ginkgolide C monomer, determining by liquid chromatograph, calculating to obtain 99.8% content, concentrating the filtered mother liquor under-0.095 MPa, recovering ethanol to dry to obtain solid, and mechanically applying to the refined ginkgolide product of step 4.
Example 3
Step 1: taking 100kg of dried folium Ginkgo (total flavonol glycoside content is 1.13%, total ginkgolide content is 0.42%), pulverizing, heating and extracting with 75% ethanol, obtaining 200L of ethanol eluate containing folium Ginkgo extract part according to the method for preparing folium Ginkgo extract of Chinese pharmacopoeia, concentrating the eluate to 24L, cooling to 20 deg.C, adding into 5L DA201 macroporous adsorbent resin, eluting with 30L of 40% ethanol, mixing the eluate of DA201 macroporous adsorbent resin with 20% ethanol eluate 46.2L, concentrating and recovering ethanol to complete under-0.097 MPa vacuum degree, drying under-0.097 MPa vacuum degree and 80 deg.C for 8 hr, pulverizing to obtain 3.3kg of folium Ginkgo extract (total flavonol glycoside content is 30.5%, total flavonoid lactone content is 8.9%, total ginkgolic acid content is 0.5 ppm), refining to produce macroporous part of DA201 adsorbent resin chromatographic column, continuously eluting with 15L of 70% ethanol to elute the ginkgolides partially enriched in the cross part of the DA201 macroporous adsorbent resin chromatographic column, continuously eluting with 25L of 95% ethanol to elute the impurities and the residual ginkgolides in the cross part of the DA201 macroporous adsorbent resin chromatographic column, and respectively collecting 14.8L of 70% ethanol eluate and 25.3L of 95% ethanol eluate;
step 2: taking 25.3L of 95% ethanol eluent in the step 1, recovering ethanol to be complete under vacuum of-0.097 MPa, adding pure water to 6.5L, dropwise adding 85% phosphoric acid to adjust the pH value to 2, cooling to 15 ℃, centrifugally separating to remove insoluble solids to obtain 6.2L of clear water liquid, adding the clear water liquid into 250ml of HPD-BJQH macroporous adsorption resin to adsorb ginkgolides, firstly eluting with 750ml of 15% ethanol, removing effluent of the HPD-BJQH macroporous adsorption resin and 10% ethanol eluent, namely removing impurities of a crossed part containing ginkgolides, continuously eluting with 500ml of 70% ethanol to obtain ginkgolides enriched in the HPD-BJQH macroporous adsorption resin, and collecting 520ml of 70% ethanol eluent;
and 3, step 3: combining 14.8L of the 70% ethanol eluate in the step 1 with 520ml of the 70% ethanol eluate in the step 2, collecting 15.32L, concentrating and recovering ethanol under-0.097 MPa vacuum degree to 2.5L, wherein the ethanol concentration of the feed liquid is about 30%, cooling, crystallizing at 0 deg.C for 8 hr, filtering, separating, measuring with liquid chromatograph (shown in figure 1), to obtain bilobalide crude product 63.5g, adding the crude product into 650ml of 30% ethanol, heating and pulping under normal pressure to boil and reflux, cooling, crystallizing at 0 deg.C for 8 hr, filtering, separating, drying the filtered solid at-0.097 MPa vacuum degree and 50 deg.C for 8 hr to obtain refined bilobalide product 41.3g, measuring with liquid chromatograph, and calculating to obtain refined bilobalide product containing bilobalide A12.5%, bilobalide B76.3%, The content of ginkgolide C is 10.5%, and the content of total lactones is 99.3%;
and 4, step 4: taking 40g of refined product of ginkgolide, adding 800ml of 95% ethanol, heating under reflux at normal pressure for dissolving, cooling, placing at 0 ℃ for cooling and crystallizing for 8 hours, filtering and separating to obtain 27.5g of crude product of ginkgolide B; adding 550ml of 95% ethanol into the crude product of ginkgolide B, heating, refluxing and dissolving under normal pressure, cooling, placing at 0 ℃ for cooling and crystallizing for 8 hours, filtering and separating, measuring by a liquid chromatograph, as shown in figure 3, obtaining ginkgolide B crude product 19.7g, adding 400ml of 95% ethanol into the crude product of ginkgolide B after the first crystallization, heating, refluxing and dissolving under normal pressure, cooling, placing at 0 ℃ for cooling and crystallizing for 8 hours, filtering and separating, drying the filtered solid for 8 hours at 50 ℃ under the vacuum degree of-0.097 MPa to obtain ginkgolide B monomer 14.2g, measuring and calculating by the liquid chromatograph, wherein the content of the ginkgolide B monomer is 99.7%, and collecting the mother liquor filtered in each step;
and 5: combining 1730ml of all the mother liquor obtained in the step 4, adding 460ml of pure water to reduce the ethanol concentration in the mother liquor to 70%, filtering and separating to obtain 2150ml of filtrate, concentrating and recovering ethanol until the ethanol concentration in the mother liquor is reduced to 12.1g under the vacuum degree of-0.097 MPa, measuring and calculating by a liquid chromatograph, wherein the solid contains 37.2% of ginkgolide A, 28.9% of ginkgolide B and 33.3% of ginkgolide C, adding 240ml of 95% ethanol, heating, refluxing and dissolving under normal pressure, adding 85ml of pure water to reduce the ethanol concentration to 70%, cooling, placing at 0 ℃ for cooling and crystallizing for 8 hours, filtering and separating to obtain filtered mother liquor 310m rich in ginkgolides A and C, and combining the filtered solids obtained in the step and mechanically applying the filtered solids to the refined ginkgolides in the step 4;
and 6: taking 310ml of the filtered mother liquor rich in the ginkgolide A and the ginkgolide C in the step 5, adding 400ml of pure water to reduce the ethanol concentration in the mother liquor to 30%, and filtering and separating to obtain 3.3g of solid rich in the ginkgolide A and 700ml of mother liquor rich in the ginkgolide C;
and 7: taking 3.3g of the solid rich in ginkgolide A in the step 6, adding 65ml of 95% ethanol, heating, refluxing for dissolving, cooling, placing at 0 ℃ for cooling and crystallizing for 8 hours, filtering, separating, and determining by a liquid chromatograph (shown in figure 2) to obtain crude ginkgolide A2.5 g; adding 40ml of 95% ethanol into the crude product of the ginkgolide A, heating, refluxing and dissolving under normal pressure, cooling, placing at 0 ℃ for cooling and crystallizing for 8 hours, filtering and separating, placing the filtered solid at-0.097 MPa vacuum degree and 50 ℃ for drying for 8 hours to obtain 1.7g of ginkgolide A monomer, measuring and calculating by using a liquid chromatograph, wherein the content of the ginkgolide A monomer is 100.1%, combining the filtered mother liquor obtained in the step, concentrating and recovering ethanol until the dry solid is 1.5g under-0.097 MPa vacuum degree, and then applying the concentrated solid to the refined product of the ginkgolide in the step 4;
and 8: taking 700ml of the mother liquor rich in ginkgolide C in the step 6, adding 100ml of the mother liquor into LX-60 macroporous adsorption resin balanced by 20% ethanol, collecting 700ml of LX-60 resin effluent, adding 100ml of HC80B macroporous adsorption resin balanced by 20% ethanol, collecting 980ml of HC80B macroporous adsorption resin effluent, circularly eluting LX-60 macroporous adsorption resin by using HC80B macroporous adsorption resin effluent, eluting ginkgolide C in LX-60 macroporous adsorption resin into HC80B macroporous adsorption resin, performing the operation for 4 times, respectively eluting LX-60 macroporous adsorption resin and HC80B macroporous adsorption resin by using 200ml of 70% ethanol, merging the LX-60 macroporous adsorption resin 70% ethanol eluate with the 70% ethanol eluate in the step 3, collecting HC80B macroporous adsorption resin 70% ethanol eluate, and performing vacuum evaporation under-0.097 MPa, concentrating and recovering ethanol to dryness, determining by a liquid chromatograph, as shown in figure 4, obtaining 2.3g of crude ginkgolide C product, adding 20ml of 95% ethanol, heating under reflux and dissolving at normal pressure, cooling, placing at 0 ℃ for cooling and crystallizing for 8 hours, filtering and separating, placing the filtered solid at-0.097 MPa vacuum degree and 50 ℃ for drying for 8 hours to obtain 1.5g of ginkgolide C monomer, determining by the liquid chromatograph, calculating to obtain the content of the ginkgolide C monomer of 99.9%, concentrating and recovering ethanol to dryness to obtain solid by placing the filtered mother liquor at-0.097 MPa vacuum degree, and applying to the refined ginkgolide product in the step 4.
According to the embodiments, the invention provides a preparation method of ginkgolides and monomers contained in the ginkgolides, on the basis of not influencing the quality index of the ginkgo leaf extract, the invention utilizes the cross part adsorbed in a chromatographic column for refining the ginkgo leaf extract as a raw material, the total content of the ginkgolides accounts for about 15-20% of the total ginkgolides in the ginkgo leaf, through the preparation process, the comprehensive utilization of resources is realized to the maximum extent, the quality of the ginkgo leaf extract product is ensured, and simultaneously the target product is obtained, the purity of the target product is high, the total lactone content of a ginkgolide compound is more than or equal to 99%, the purity of a monomer ginkgolide A is more than or equal to 99.5%, and the purity of a monomer ginkgolide B is more than or equal to 99.5%; the purity of the monomer ginkgolide C is more than or equal to 99.5 percent, the requirement of a pharmaceutical preparation can be completely met, the method has important significance and good economic benefit, the product quality of the ginkgo biloba extract is ensured, and the ginkgolide lost in the refining process is fully utilized; the difference of the content of the ginkgolides in the ginkgo leaves is large, so that the yield difference of a refined product of the ginkgolides relative to the ginkgo leaves is large, but in the separation process of the ginkgolides monomers, the generated filtered mother liquor or filtered solid or concentrated and dried solid can be applied to the refined product of the ginkgolides, so that the purified ginkgolides monomers of the refined product of the ginkgolides are basically not lost, the preparation process is relatively environment-friendly, the health guarantee of operators can be effectively improved, the process operation is simple, the equipment investment is small, and the method is suitable for industrial production.
Finally, it should be noted that the above embodiments are only used for illustrating and not limiting the technical solutions of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the present invention without departing from the spirit and scope of the present invention, and all modifications or partial substitutions should be covered by the scope of the claims of the present invention.
Claims (7)
1. A preparation method of ginkgolides and monomers contained in the ginkgolides is characterized by comprising the following steps:
step 1: according to a preparation method of a ginkgo leaf extract in Chinese pharmacopoeia, obtaining an ethanol eluent containing a ginkgo leaf extract part, concentrating the ethanol eluent until the dry matter proportion is 10-15% of an elution concentrated solution, cooling the elution concentrated solution, putting the cooled elution concentrated solution into a processed macroporous adsorption resin a, eluting the macroporous adsorption resin a with 20-40% (v/v) ethanol, collecting and combining an effluent liquid of the macroporous adsorption resin a and a 20-40% ethanol eluent, concentrating the elution concentrated solution until the elution solution is dry to obtain the ginkgo leaf extract, sequentially eluting the macroporous adsorption resin a with 50-70% (v/v) ethanol and 90-95% (v/v) ethanol, and collecting 50-70% ethanol eluent and 90-95% ethanol eluent;
and 2, step: taking the 90-95% ethanol eluate in step 1, recovering ethanol to full, adding pure water to one fourth to one third of the volume of the stock solution, adding a pH regulator to adjust the pH value of the solution to be below 2, cooling to 15-30 ℃, centrifuging to remove insoluble substances to obtain a clear water solution, putting the clear water solution on a treated macroporous adsorption resin b, sequentially eluting with 10-20% (v/v) and 50-70% (v/v) ethanol, and collecting 50-70% ethanol eluate;
and step 3: mixing the 50-70% ethanol eluate in step 1 and the 50-70% (v/v) ethanol eluate in step 2, recovering ethanol until the ethanol concentration is 10-30% (v/v), cooling for crystallization, filtering for separation to obtain crude product of ginkgolides, heating and pulping with 10-30% (v/v) ethanol solution with 10-20 times volume (ml) of the mass (g) value, cooling for crystallization, filtering for separation, and drying to obtain refined ginkgolides, wherein the refined ginkgolides are ginkgolide A, ginkgolide B and ginkgolide C, and the total lactone content is not less than 99%;
and 4, step 4: taking a refined product of ginkgolide, adding 90-95% (v/v) concentration ethanol with the mass (g) value of 20-25 times volume (ml), heating for dissolving, cooling for crystallization, filtering and separating to obtain a crude product of ginkgolide B, repeating the step to recrystallize the crude product of ginkgolide B for 2-3 times, vacuum drying the filtered solid to obtain a ginkgolide B monomer, wherein the content of the ginkgolide B monomer is more than or equal to 99.5%, and collecting the mother liquor filtered in each step;
and 5: combining all the mother liquor obtained in the step (4), adding pure water to reduce the ethanol concentration to 50-70% (v/v), filtering and separating to obtain filtrate, concentrating to dryness, adding 90-95% (v/v) ethanol with the volume (ml) which is 20-25 times of the mass (g) value of the filtrate, heating to dissolve the filtrate, adding pure water to reduce the ethanol concentration to 50-70% (v/v), cooling to crystallize, filtering and separating to obtain filtered mother liquor rich in ginkgolide A and ginkgolide C, and applying the rest filtered solids obtained in the step to the refined ginkgolide product obtained in the step (4);
step 6: taking the filtered mother liquor rich in the ginkgolide A and the ginkgolide C in the step 5, adding pure water to reduce the concentration of ethanol in the mother liquor to 20-30% (v/v), and filtering and separating to obtain a solid rich in the ginkgolide A and a mother liquor rich in the ginkgolide C;
and 7: taking the solid rich in ginkgolide A in the step 6, adding 90-95% (v/v) ethanol with the mass (g) value of 15-20 times and the volume (ml) of the solid, heating for dissolving, cooling for crystallization, filtering and separating to obtain a crude product of ginkgolide A, then repeating the step to recrystallize the crude product of ginkgolide A for 1-2 times, carrying out vacuum drying on the filtered solid to obtain a ginkgolide A monomer, wherein the content of the ginkgolide A monomer is more than or equal to 99.5%, carrying out vacuum concentration on the filtered mother liquor obtained in the step to recycle ethanol until the filtered solid is dried, and applying the solid to the refined ginkgolide product in the step 4;
and 8: taking mother liquor rich in ginkgolide C in the step 6, adding the mother liquor into a macroporous adsorption resin C which is processed and balanced by 20% ethanol, adding effluent liquid of the macroporous adsorption resin C into a macroporous adsorption resin d which is processed and balanced by 20% ethanol, circularly eluting ginkgolide C in the macroporous adsorption resin C by using the effluent liquid of the macroporous adsorption resin d, circularly adding the effluent liquid containing ginkgolide C eluted by the macroporous adsorption resin C into the resin d to be adsorbed, repeating the operation until the ginkgolide C in the macroporous adsorption resin C is completely eluted, then respectively eluting the macroporous adsorption resin C and the macroporous adsorption resin d by using 50-70% (v/v) ethanol, collecting 50-70% ethanol eluent of the macroporous adsorption resin d, concentrating to be dry, adding 90-95% (v/v) ethanol with the volume (ml) which is 5-10 times of the mass (g) value of the ethanol, heating, dissolving, cooling and crystallizing, filtering and separating, vacuum drying the filtered solid to obtain a ginkgolide C monomer with the content of more than or equal to 99.5%, then vacuum concentrating the filtered mother liquor and recovering ethanol until the obtained solid is dried, wherein the solid can be applied to the refined ginkgolide product in the step (4), and applying 50-70% (v/v) ethanol eluent of the macroporous adsorption resin C to the 50-70% ethanol eluent in the step (3);
the macroporous adsorption resin a is one or more of HPD5000, LX200 and DA201 resins;
the macroporous adsorption resin b is one or more of DM131, HPD417 and HPD-BJQH resin;
the macroporous adsorbent resin c is one or more of LX-5, LX-32 and LX-60 resins, and the macroporous adsorbent resin d is one or more of HC100A, HC700 and HC80B resins.
2. The method for preparing ginkgolides and the monomers contained in ginkgolides according to claim 1, wherein the column loading of macroporous adsorbent resin a is as follows: the amount of the concentrated eluate with dry matter ratio of 10-15% (v/v) is 5-8BV, and the amounts of ethanol with concentration of 20-40% (v/v), 50-70% (v/v) and 90-95% (v/v) are 3-6BV respectively.
3. The method for preparing ginkgolides and the monomers contained in ginkgolides according to claim 1, wherein the column loading of macroporous adsorbent resin b is as follows: the amount of the clear aqueous solution is 20-25BV, 10-20 (v/v)%, 50-70% (v/v) ethanol is 2-3 BV.
4. The method for preparing ginkgolides and monomers contained in claim 1, wherein the bed volume of macroporous adsorption resin d is the same as that of macroporous adsorption resin c, and the column loading amount of macroporous adsorption resin c is as follows: the amount of mother liquor rich in ginkgolide C and the effluent liquid of the macroporous adsorption resin d are respectively 4-8 (v/v) BV, the amount of 50-70% (v/v) ethanol used is 2-3BV, and the column loading amount of the macroporous adsorption resin d is as follows: the effluent amount of the macroporous adsorbent resin c is 4-8 (v/v) BV, and the ethanol with the concentration of 50-70% (v/v) is 2-3 BV.
5. The method for preparing ginkgolides and the monomers contained in ginkgolides according to claim 1, wherein in step 3, the content of ginkgolide A, ginkgolide B and ginkgolide C in the refined ginkgolides product is 10-20%, 70-85% and 5-15%.
6. The method for preparing ginkgolides and the monomers contained in the ginkgolides according to claim 1, wherein in the step 2, the pH regulator is one or more of sulfuric acid, hydrochloric acid and phosphoric acid; the pH value is 1-2.
7. The method for preparing ginkgolides and monomers contained in claim 1, wherein the temperature for cooling crystallization is 0-15 ℃, and the crystallization time is 8-24 h; the vacuum degree of the concentration and the vacuum drying in the steps 2 to 8 is less than-0.09 MPa, the evaporation temperature is less than 60 ℃, and the vacuum drying time is 6 to 8 hours; the heating pulping and heating dissolution are carried out under normal pressure to reach a reflux state.
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| DE3940094A1 (en) * | 1989-12-04 | 1991-06-06 | Montana Ltd | ACTIVE CONCENTRATES AND NEW ACTIVE COMBINATIONS FROM BLACKERS OF GINKGO BILOBA, A METHOD FOR THE PRODUCTION THEREOF, AND THE ACTIVE CONCENTRATES THE DRUG COMBINATIONS CONTAINING DRUG COMPOUNDS |
| EP1033994A4 (en) * | 1997-11-25 | 2002-04-03 | Pharmanex Inc | Methods of preparation of bioginkgo |
| CN100497344C (en) * | 2005-01-07 | 2009-06-10 | 上海同田生物技术有限公司 | Preparation of high-purity ginkgolide |
| CN101134758B (en) * | 2007-10-15 | 2010-04-14 | 桂林市振达生物科技有限责任公司 | Method for extracting and separating bilobalide A, B, C, J and bilobalide monomer from ginkgo leaf |
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| CN102464666B (en) * | 2010-11-09 | 2015-04-08 | 上海医药工业研究院 | Preparation method for ginkgolide C |
| CN102464665B (en) * | 2010-11-09 | 2015-04-08 | 上海医药工业研究院 | Method for preparing ginkgolide A |
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| CN102070647B (en) * | 2010-12-28 | 2012-11-07 | 浙江大学 | Method for separating ginkgolide B from ginkgolide mixture |
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