CN103524526B - A kind of isolation and purification method of Bilobalide - Google Patents

Abstract

The invention discloses a method for separating and purifying bilobalide. The method of the present invention comprises the following steps: (1) preparing the ginkgolide enriched product; (2) obtaining the crude product of bilobalide by reverse-phase adsorption resin column chromatography; (3) obtaining the fine product of bilobalide after recrystallization of the crude product of bilobalide . The content of the bilobalide prepared by the method of the invention can reach 98%-99%. The filler used in the method of the invention is MCI-GEL or NM100 reverse-phase adsorption resin, which has good selectivity, can greatly improve the separation efficiency of bilobalide, can be recycled, has low toxicity and low use cost. The solvent of the invention is simple and easy to obtain, the equipment used is conventional equipment, the operation is simple, the energy consumption is small, the production is safe, no pollution, the obtained product has high purity, is suitable for industrial production, and has great application value.

Description

A kind of separation and purification method of bilobalide

technical field

The invention relates to a method for separating and purifying medicines, in particular to a method for separating and purifying bilobalide.

Background technique

The composition of Ginkgo biloba is complex, containing a variety of active ingredients, mainly flavonoids, terpene lactones, organic acids, alkylphenols and alkylphenolic acids, steroidal compounds, trace elements, etc. The medicinal ingredients in Ginkgo biloba are mainly ginkgo flavonoids and ginkgolides. At present, diterpene lactones and sesquiterpene lactones are mainly isolated from ginkgo leaves, namely ginkgolides A, B, C, J, M and bilobalide. Bilobalide is a sesquiterpene lactone with only one pentane in its molecular structure. Bilobalide has strong biological activity and has various neuroprotective effects, including promoting nerve growth, improving memory function in the elderly, and preventing the occurrence of senile dementia. Because the content of terpene lactones in Ginkgo biloba is extremely low, in order to obtain ginkgolides and bilobalides with high medicinal value, Ginkgo biloba or its crude extract must be re-extracted and refined. At present, the extraction and separation methods of ginkgolides reported in the literature mainly include: solvent extraction, solvent extraction-chromatographic column separation, supercritical extraction and separation, high-speed countercurrent chromatography and simulated moving bed chromatography, among which the most widely used is the chromatographic column. In the separation method, silica gel, alumina and reversed-phase C18 silica gel column packing are mostly used as fillers. Chinese invention patent "Method for Extracting and Separating Ginkgolide A, B, C, J and Bilobalide Monomer from Ginkgo Leaf" (Patent Application No. 200710050242.1) and "Method for Extracting and Separating Bilobalide from Ginkgo Leaf" ( Patent Application No. 200710050244.0) uses silica gel column chromatography and n-hexane-ethyl acetate solvent system elution method to separate bilobalide. However, the silica gel used in this method cannot be recycled and reused, and a large amount of organic solvents must be used for elution, which is not conducive to industrial production. Chinese invention patent "Efficient Separation and Purification Method of Ginkgolide A, B, C, J and Bilobalide Monomer" (Patent Application No. 200810046162.3) and "Preparation Method of Separation and Purification of Bilobalide" (Patent Application No. 201210457118.8) Preparative high-performance liquid chromatography column and reversed-phase silica gel column chromatography were used to prepare bilobalide respectively. The disadvantages of this method are that the reversed-phase silica gel has a small sample load, a large amount of methanol or acetonitrile is used, and the process cost is high, which is not conducive to mass production. . Therefore, there are still various defects in the above-mentioned purification and refining method, and high-purity bilobalide cannot be separated efficiently, economically and environmentally friendly, and it is urgent to further improve the method for extracting and separating bilobalide.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the above disadvantages, research and design the separation and purification method of bilobalide which is simple, low in cost, beneficial to environmental protection and suitable for industrial production.

The invention provides a method for separating and purifying bilobalide.

Method of the present invention comprises the following steps:

(1) Preparation of ginkgolide-enriched substance: take ginkgo biloba extract (EGB), extract with an organic solvent, and evaporate the extract to dryness under reduced pressure to obtain ginkgolide-enriched substance;

(2) Reverse-phase adsorption resin column chromatography: Ginkgolide enrichment is passed through reverse-phase adsorption resin, firstly eluted with 0-20% ethanol to remove 1-4BV impurities, and then eluted with 20%-50% ethanol from the resin 1-8BV, collecting the eluate containing bilobalide, recovering the solvent to dryness, and obtaining the crude product of bilobalide;

(3) Recrystallization: The crude product of bilobalide is heated, dissolved and filtered with 10%-60% ethanol, then placed in a refrigerator at 2°C-10°C for 12-48h, crystals are precipitated, crystallized repeatedly for 1-3 times, and crystals are washed with purified water for 1-5 Second, vacuum drying at 60°C-120°C to obtain high-purity bilobalide.

The BV is an elution column volume unit recognized in the art.

In the separation and purification method of bilobalide of the present invention, the step (1) Ginkgo biloba extract (EGB) is commercially available, or prepared by the following method: Ginkgo dry leaves are used as raw materials, crushed, and 40%-80% ethanol Aqueous solution extraction, the amount of extraction solvent is 4-8 times the amount of medicinal materials W/V, the number of extractions is 3-4 times, and the extraction time is 1-2 hours each time, the extracts are combined, concentrated under reduced pressure to recover ethanol until there is no ethanol smell , filter, put the filtrate on a macroporous adsorption resin D101 or HP20 column, the column volume is 1-1.5 times the amount of medicinal material W/V, wash with water for 2-3BV to remove impurities, and elute with 60%-80% ethanol until the color of the eluent changes The eluate was concentrated under reduced pressure to obtain Ginkgo biloba extract.

The organic solvent in step (1) is selected from ethyl acetate, methyl acetate, propyl acetate, butyl acetate, acetone, methyl ethyl ketone, n-butanol or isobutanol or two mixed solvents for extraction , preferably ethyl acetate or acetone, the number of extractions is 1-6 times, preferably 3 times, and the amount of solvent used for each extraction is 5-15 times the amount of Ginkgo biloba extract W/V, preferably 10 times the amount of W/V.

In the step (2), the bilobalide-containing fraction obtained by reverse-phase adsorption resin column chromatography is rotated to dryness under reduced pressure at 55°C-65°C and -0.09--0.1MPa until there is no solvent, and at 40°C-70°C Heat to dissolve in 10%-60% ethanol, filter while it is hot, place the filtrate at 2°C-10°C for 12-48 hours, precipitate crystals, and filter to obtain crude bilobalide.

The reverse-phase adsorption resin filler in step (2) is MCI-GELCHP series reverse-phase adsorption resin or NM100 series reverse-phase adsorption resin.

The MCI-GELCHP series reversed-phase adsorption resin is produced by Mitsubishi Chemical Corporation, the model is CHP20/P120, and its technical parameters are: the particle size is 75-150 μm, the average particle size is 120 μm, the pH range is 0-14, and the pore size is 450 Angstroms, the matrix is polystyrene and divinyl copolymer.

The NM100 series reversed-phase adsorption resin is produced by Suzhou Nawei Technology Co., Ltd., the model is NM100, and its technical parameters are: particle size 50-150 μm, pore size 300 angstroms, and matrix polystyrene and divinylbenzene.

The impurity removal solvent is 0-20% ethanol solution, preferably 10% ethanol solution, and the elution volume is 1-4BV, preferably 1-2BV;

The elution solvent is 20-50% ethanol, preferably 30-40% ethanol, and the elution volume is 3-10BV, preferably 6-8BV.

The crystallization solvent in the step (3) is 10%-60% ethanol; the recrystallization step is repeated recrystallization 1-3 times; the crystallization washing step is washing the crystallization 1-5 times with purified water.

The preferred crystallization solvent is 20%-50% ethanol; the recrystallization step is to repeat recrystallization twice; the crystallization step is to wash the crystallization with purified water 3 times.

The detected content of the bilobalide prepared by the method of the invention is 98%-99%.

Compared with the prior art, the inventive method has achieved good results:

The filler used in the method of the present invention is MCI-GEL or NM100 reversed-phase adsorption resin, and the filler has good selectivity to ginkgolide, can greatly improve the separation efficiency of bilobalide, and the filler can be recycled, which is better than silica gel, The organic solvents used in column chromatography such as alumina have low toxicity and low cost of use.

In addition, because the solvent for separating and purifying bilobalactone is simple and easy to obtain, the equipment used is conventional equipment, so the operation is simple, the energy consumption is small, the non-toxic adsorbent is used for adsorption, and ethanol is used for desorption, so the production is safe and pollution-free, and the obtained product It has high purity, is suitable for industrial production, and has great application value.

detailed description

The present invention will be further described below by way of examples, but not in order to limit the scope of the present invention.

Example 1

Suspend 4 g of Ginkgo biloba extract (EGB) (commercially available, with total ginkgo lactone content ≥ 6%) in 40 mL of water, extract three times with ethyl acetate, 50 mL each time, and store the extraction solution at 60 °C and -0.09--0.1 MPa The solvent was recovered under reduced pressure under certain conditions, the extract was ginkgolide enrichment, the total ginkgolide content was 34.5% as detected by HPLC-ELSD, the extract was heated and dissolved in 100mL water at 60oC, and adsorbed with 100mL MCI-GEL resin. The time is 0.5h, first elute 3BV with 10% ethanol to remove impurities, then elute resin 8BV with 30% ethanol, collect the eluate containing bilobalide after HPLC detection, and store it at 65°C and -0.09--0.1MPa Under reduced pressure, evaporate to dryness under reduced pressure until there is no solvent to obtain the crude product of bilobalide, add 40% ethanol (2.8mL) to the crude product of bilobalide, heat and dissolve at 60°C, put it in the refrigerator (4°C) for 24h, precipitate crystals, and recrystallize repeatedly for 2 Wash the crystal three times with purified water (1 mL each time), heat and dry in vacuum at 65°C to obtain 83 mg of bilobalide, and the HPLC-ELSD detection content is 98.4%.

The detection method of ginkgolide and bilobalide adopts HPLC-ELSD detection method (the reference is Medical Herald, March 2010, 29 (3): 377-378).

Example 2

Suspend 4 g of Ginkgo biloba extract (EGB) (commercially available, total ginkgo lactone content ≥ 6%) in 40 mL of water, extract three times with ethyl acetate, 50 mL each time, extract the solution at 60oC to recover the solvent under reduced pressure, and the extract is Ginkgolide enrichment, the total ginkgolide content detected by HPLC-ELSD is 31.8%, the extract is heated and dissolved in 100mL water at 60°C, and adsorbed with 100mL NM100 resin for 0.5h, first with 10% ethanol Elute 3BV to remove impurities, then elute resin 8BV with 30% ethanol, collect bilobalide-containing eluate after HPLC detection, and rotate under reduced pressure at 65oC and -0.09--0.1MPa until there is no solvent to obtain ginkgo For crude lactone, recover solvent, add 40% ethanol (2.6mL) to crude bilobalide, heat to dissolve at 60°C, place in refrigerator (4°C) for 24h, crystallize, recrystallize twice, wash with purified water (1mL) crystallized for 3 times, heated and dried under vacuum at 65°C to obtain 71 mg of bilobalide, and the content detected by HPLC-ELSD was 98.8%.

Example 3

Take 300g of dried ginkgo leaves (commercially available), crush them, and extract three times under reflux with 80% ethanol. The amount of solvent used is 2.4L, 1.5L and 1.5L, and each extraction is 2h. Under the conditions, concentrate under reduced pressure until there is no ethanol smell, filter, put the filtrate on a macroporous adsorption resin D101 column, wash the column with purified water (1000mL) to remove impurities, and elute with 70% ethanol (2000mL) until the color of the eluent becomes light (thin layer Ginkgo biloba extract can be detected by chromatography), and the eluate is evaporated under reduced pressure at 60°C and -0.09--0.1MPa until there is no solvent to obtain Ginkgo biloba extract (6g). Ginkgo biloba is detected by HPLC-ELSD Total lactone content is 6.5%

Ginkgo biloba extract 6g was extracted three times with ethyl acetate, 150mL each time, the extraction solution was evaporated to dryness under reduced pressure at 60°C to recover the solvent, the extract was ginkgolide enrichment, and the total ginkgolide content was 29.3% as detected by HPLC-ELSD , the extract was heated and dissolved in 20mL of water at 60°C, and adsorbed with 70mL of MCI-GEL resin for 1h. First, 3BV was eluted with 10% ethanol to remove impurities, and then the resin 6BV was eluted with 30% ethanol. After HPLC detection Collect the eluate containing bilobalide, and under the condition of 65°C and -0.09--0.1MPa, rotary evaporate to dryness under reduced pressure until there is no solvent. 3.8mL, after heating and dissolving at 60°C, put it in the refrigerator (4°C) for 24 hours, precipitate crystals, repeat recrystallization 3 times, wash the crystals with purified water (1.5mL) 3 times, heat and dry under vacuum at 80°C to obtain bilobalide 102mg, the content detected by HPLC-ELSD is 98.5%.

Example 4

Take 500g of dried Ginkgo biloba leaves (commercially available), crush them, and extract three times under reflux with 80% ethanol. The amount of solvent used is 4L, 2.5L and 2.5L, and each extraction is 2h. Concentrate under reduced pressure until there is no ethanol smell, filter, put the filtrate on a macroporous adsorption resin D101 column, wash the column with purified water (1500mL) to remove impurities, and elute with 70% ethanol (3000mL) until the color of the eluent becomes light (thin layer Ginkgo biloba extract can be detected without Ginkgo biloba extract), and the eluate was concentrated and dried under reduced pressure at 60°C and -0.09--0.1MPa to obtain Ginkgo biloba extract (7.2g), and the total ginkgo lactone content was detected by HPLC-ELSD was 9.9%.

Ginkgo biloba extract 7.2g was extracted three times with ethyl acetate, 250mL each time, the extraction solution was evaporated to dryness under reduced pressure at 60oC to recover the solvent, the extract was ginkgolide enrichment, and the total ginkgolide content was 31.8% as detected by HPLC-ELSD , the extract was dissolved in 40mL of water by heating at 60°C, and adsorbed with 100mL of NM100 resin for 1h. First, 10% ethanol was used to elute 3BV to remove impurities, and then 30% ethanol was used to elute resin 6BV. After HPLC detection, the collection containing The eluate of bilobalide was evaporated under reduced pressure at 65°C and -0.09--0.1MPa until there was no solvent to obtain the crude product of bilobalide, and the solvent was recovered. Add 4.2mL of 50% ethanol to bilobalide and heat at 60°C After dissolving, put it in the refrigerator (4°C) for 24h to precipitate crystals, repeat recrystallization 3 times, wash the crystals with purified water (1.5mL) 3 times, heat and dry under vacuum at 80°C to obtain bilobalide (153mg, HPLC-ELSD The detected content is 98.4%).

Example 5

Ginkgolide enrichment (1.8g) was prepared according to the method of Example 3, the total ginkgolide content was 37.6% as detected by HPLC-ELSD, the extract was heated and dissolved in 20mL of water at 60oC, and adsorbed with 70mL of MCI-GEL resin, The adsorption time is 1.5h, first elute 4BV with 10% ethanol to remove impurities, then elute resin 8BV with 25% ethanol, collect the eluate containing bilobalide after HPLC detection, and store it at 60°C and -0.09--0.1MPa Under reduced pressure, spin evaporate to dryness under reduced pressure until there is no solvent and concentrate under reduced pressure to obtain the crude product of bilobalide. Recover the solvent, add 6.8mL of 25% ethanol to the bilobalide, heat and dissolve at 50°C, and place it in the refrigerator (4°C) for 36h to precipitate crystals. Repeated recrystallization twice, washed the crystal with purified water (1.5mL) three times, heated and dried under vacuum at 60°C to obtain 205mg of bilobalide, the HPLC-ELSD detection content was 98.7%.

Example 6

Ginkgolide enrichment (3.3g) was prepared according to the method of Example 4. The total ginkgolide content detected by HPLC-ELSD was 18.6%. The extract was heated and dissolved in 40mL of water at 60oC, and adsorbed with 100mL of NM100 resin. The adsorption time For 1.5h, first use 10% ethanol to elute 3BV to remove impurities, then use 35% ethanol to elute resin 6BV, after HPLC detection, collect the eluate containing bilobalide, under the conditions of 55°C and -0.09--0.1MPa, The crude product of bilobalide was obtained by rotary evaporation under reduced pressure until there was no solvent, and the solvent was recovered. Add 6.2mL of 45% ethanol to bilobalide, heat and dissolve at 60°C, place in the refrigerator (4°C) for 24h, crystallize out, and recrystallize repeatedly 3 times. Wash the crystal with purified water (1.5 mL) for 3 times, heat and dry under vacuum at 70°C to obtain bilobalide (241 mg, HPLC-ELSD detection content: 98.8%).

Claims (10)

1. a method for separation and purification of bilobalide, characterized in that the method may further comprise the steps: (1) Preparation of ginkgolide enrichment: take ginkgo biloba extract, extract with ethyl acetate, and evaporate to dryness under reduced pressure to obtain ginkgolide enrichment; (2) Reversed-phase adsorption resin column chromatography: the ginkgolide enrichment is passed through MCI-GELCHP series reverse-phase adsorption resin or NM100 series reverse-phase adsorption resin, first eluted with 0-20% ethanol 1-4BV to remove impurities, Then use 20%-50% ethanol to elute the resin 1-8BV, collect the eluate containing bilobalide, recover the solvent until dry, and obtain the crude product of bilobalide; (3) Recrystallization: The crude product of bilobalide is heated, dissolved and filtered with 10%-60% ethanol, then refrigerated at 2°C-10°C for 12-48h, crystals are precipitated, crystallization is repeated 1-3 times, and the crystals are washed with purified water for 1-5 Second, vacuum-dry at 60°C-120°C to obtain high-purity bilobalide; the content of bilobalide is 98%-99%. 2. the separation and purification method of a kind of bilobalide according to claim 1, is characterized in that, described step (1) ginkgo leaf extract is commercially available, or prepares by following method: take ginkgo dry leaf as raw material, Pulverize and extract with 40%-80% ethanol aqueous solution, the amount of extraction solvent is 4-8 times W/V of the amount of medicinal materials, the number of extractions is 3-4 times, and each extraction time is 1-2 hours, the combined extracts are reduced Concentrate under pressure to recover ethanol until there is no ethanol smell, filter, put the filtrate on a macroporous adsorption resin D101 or HP20 column, the column volume is 1-1.5 times the amount of medicinal materials W/V, wash 2-3BV to remove impurities, 60%-80% Elute with ethanol until the color of the eluate becomes light, and concentrate the eluate under reduced pressure to obtain Ginkgo biloba extract. 3. the separation and purification method of a kind of bilobalide according to claim 1, is characterized in that, described step (1) ethyl acetate extraction number of times is 1-6 time, and the used solvent consumption of each extraction is Ginkgo biloba extract 5-15 times the amount of W/V. 4. the separation and purification method of a kind of bilobalide according to claim 3, is characterized in that, described step (1) ethyl acetate extraction number of times is 3 times, and the used solvent consumption of each extraction is Ginkgo biloba extract 10 Multiple W/V. 5. the separation and purification method of a kind of bilobalide according to claim 1, is characterized in that, described step (2) MCI-GELCHP series reverse phase adsorption resin is produced by Mitsubishi Chemical Corporation, and model is CHP20/P120, Its technical parameters are: the particle size is 75-150 μm, the average particle size is 120 μm, the pH range is 0-14, the pore size is 450 angstroms, and the matrix is polystyrene and divinyl copolymer; the NM100 series reversed-phase adsorption resin Produced by Suzhou Nawei Technology Co., Ltd., the model is NM100, and its technical parameters are: particle size of 50-150 μm, pore size of 300 angstroms, and matrix of polystyrene and divinylbenzene. 6. the separation and purification method of a kind of bilobalide according to claim 1, is characterized in that, described step (2) impurity removal solvent is 0-20% ethanol solution, and elution volume is 1-4BV; The solvent is 20%-50% ethanol, and the elution volume is 3-10BV. 7. the separation and purification method of a kind of bilobalide according to claim 6, is characterized in that, described step (2) impurity removal solvent is 10% ethanol solution, and elution volume is 1-2BV; Elution solvent is 30%-40% ethanol, the elution volume is 6-8BV. 8. the separation and purification method of a kind of bilobalide according to claim 1, is characterized in that, described step (2) with MCI-GELCHP series reverse-phase adsorption resin or NM100 series reverse-phase adsorption resin column chromatography gained containing The fraction of bilobalide was rotary-evaporated under reduced pressure at 55°C-65°C and -0.09--0.1MPa until solvent-free, heated and dissolved in 10%-60% ethanol at 40°C-70°C, filtered while hot, and the filtrate Place it under the condition of 2°C-10°C for 12-48 hours, crystallize out, and filter to obtain the crude product of bilobalide. 9. according to the separation and purification method of a kind of bilobalide described in claim 1, it is characterized in that, the solvent of described step (3) crystallization is 10%-60% ethanol; Recrystallization is repeated recrystallization 1-3 time; Wash the crystals with purified water 1-5 times. 10. according to the separation and purification method of a kind of bilobalide described in claim 9, it is characterized in that, described step (3) crystallization solvent is 20%-50% ethanol; Recrystallization is repeated recrystallization 2 times; Wash with purified water Crystallized 3 times.