CN109053417B - Preparation method of high-purity ginkgolic acid - Google Patents
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- C07C65/01—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups
- C07C65/03—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups monocyclic and having all hydroxy or O-metal groups bound to the ring
- C07C65/05—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups monocyclic and having all hydroxy or O-metal groups bound to the ring o-Hydroxy carboxylic acids
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Abstract
The invention discloses a preparation method of high-purity ginkgolic acid, which comprises the following steps: (1) separating semen Ginkgo from semen Ginkgo, drying the testa in shade, and extracting the dried testa with petroleum ether for three times to obtain total ginkgolic acid extract; (2) separating and enriching total ginkgolic acid components by pH zone countercurrent chromatography to obtain corresponding components; (3) separating and purifying the enriched component in a circulating countercurrent chromatography mode; the preparation method is lower than the prior art, has simple and convenient operation and high efficiency, and can separate and prepare the ginkgolic acid monomer compound with the purity of more than 95 percent from the ginkgo peels in a large batch.
Description
Technical Field
The invention relates to an efficient preparation method of effective components of traditional Chinese medicines, in particular to a preparation method of high-purity ginkgolic acid.
Technical Field
The ginkgolic acid compounds are another important type of medicinal components in ginkgo midleaves, ginkgo biloba and fruit peels except flavone and lactone, and are derivatives of 6-alkenyl salicylic acid. Modern pharmacological research shows that the ginkgolic acid components have various pharmacological activities of resisting virus, inhibiting bacteria, resisting inflammation, resisting cancer, resisting parasites and the like. The ginkgolic acid component is a low-polarity structural analogue, so that the separation difficulty is high. At present, the traditional separation methods of monomer components include silica gel column chromatography, preparative liquid chromatography, macroporous resins and CO2Critical extraction method, Yangtong Longong, etcResearches show that the separated ginkgolic acid is prepared by adopting a high performance liquid chromatography, and a large amount of fatty acid exists in the separated ginkgolic acid; study on separation technology of ginkgolic acid in Yi Xiu Lian and semen Ginkgo testa, purity of ginkgolic acid separated by macroporous resin S-8 is 85.59%, and CO is used2The purity of the ginkgolic acid separated by the critical extraction method is 77.66 percent, and the separation purity of the ginkgolic acid by the two methods is not high; later, researchers improved the process deficiency, made the product sensitive to the allergy, and extracted the ginkgolic acid by the improved ethanol extraction method, and then purified the ginkgolic acid by the membrane separation technology, and the purity of the obtained ginkgolic acid was improved from 33.9% to 77.8%. Therefore, the method is time-consuming and labor-consuming, pollutes the environment, has low sample purity, has irreversible adsorption effect on the sample by repeated column chromatography, and has low preparation efficiency and higher cost for separating the ginkgolic acid compound monomer; therefore, a new simple and efficient method for preparing high-purity ginkgolic acid is still needed to be continuously searched.
High-speed Counter-current Chromatography (HSCCC) is a continuous High-efficiency and rapid liquid-liquid distribution Chromatography separation technology which is developed in 30 years, does not need any solid support, avoids various problems of easy dead adsorption, loss, denaturation and the like of a sample caused by a solid support or a carrier, and obviously reduces the distribution efficiency and the solvent consumption when other liquid Chromatography is used for preparative separation, ensures higher peak resolution of HSCCC, has large separation amount, no sample loss, High recovery rate, mild separation environment and saves the solvent. The high-speed counter-current chromatography can directly carry out a large amount of crude extraction samples or synthesis mixtures, the separation result can reach quite high purity, and the method is widely applied to the preparation, separation and purification of chemical substances in the fields of biology, medicine, environmental protection and the like. The pH zone countercurrent chromatography is a novel preparation type separation technology developed on the basis of countercurrent chromatography, utilizes the difference of material dissociation constant and hydrophobicity for separation, has the advantages of large separation amount, good separation effect and the like, and is widely applied to separation of alkaloid and organic acid components. At present, the separation and preparation of the ginkgolic acid monomeric compounds by adopting high-speed countercurrent chromatography and pH zone countercurrent chromatography is not disclosed for a while.
Disclosure of Invention
The invention aims to solve the defects in the technical background, and discloses a method for preparing a ginkgolic acid monomer compound, which has the advantages of lower preparation cost than that of the prior art, simple and convenient operation and high efficiency, and can separate and prepare the ginkgolic acid monomer compound with the purity of more than 95% from ginkgo peels in a large batch.
In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:
a method for preparing ginkgolic acid components comprises the following steps:
(1) separating semen Ginkgo from semen Ginkgo, drying the testa in shade, and extracting the dried testa with petroleum ether for three times to obtain total ginkgolic acid extract;
ginkgolic acid is a low-polarity compound, and low-polarity petroleum ether is adopted according to the principle of similar compatibility, so that the extraction efficiency is highest, and other types of effective components, such as flavone and ginkgolide, cannot be extracted.
(2) Separating and enriching total ginkgolic acid components by pH zone countercurrent chromatography to obtain corresponding components;
the pH zone refining countercurrent chromatography is a special countercurrent chromatography separation preparation technology developed on the basis of the application of common preparation type high-speed countercurrent chromatography separation and purification of organic acid components, is eluted in sequence according to the pKa of a compound, and is particularly suitable for the separation of organic acid substances. The pH zone refining countercurrent chromatography has the advantages of high sample introduction amount, high separation efficiency, good separation effect, easy impurity collection, realization of pH monitoring, high concentration of components to be separated and the like, and is widely applied to the research field and practice at present. Because the tail end of the ginkgolic acid compound long-chain fatty acid has a carboxylic acid group, and because the long chain length is different, the pKa of the compound has certain difference, the pH zone refining countercurrent chromatography can be adopted for enrichment and separation.
(3) And (3) separating and purifying the enriched components in a circulating countercurrent chromatography mode to obtain the ginkgolic acid monomers with the purity higher than 95%.
The conventional countercurrent chromatographic separation mode adopts a one-time separation mode, namely, after the separation process of a compound is sample injection, the compound is eluted from the head end of a countercurrent chromatographic instrument to the tail end by a mobile phase, so that the separation is realized. However, ginkgolic acid is a low-polarity structural analogue, and different compounds are only different in the length of an aliphatic chain and the position of a double bond, so that the conventional one-time countercurrent chromatography elution mode cannot realize separation. According to the plate theory of chromatography, the longer the separation column, the higher the number of plates, and the higher the separation efficiency. The circulating countercurrent chromatography mode is just for reference of the chromatography theory, and the number of the tower plates of a single countercurrent chromatograph is increased, so that the separation of the ginkgolic acid compounds is realized.
Preferably, the ether extraction in step (1) has a boiling range of 60 to 90 ℃;
preferably, the specific operation process of pH zone countercurrent chromatography for separation and enrichment in step (2) is as follows:
a) formulating a two-phase solvent system
Preparing a solvent system from n-hexane/ethyl acetate/methanol/water according to a volume ratio of 2: 0.8-1.2: 3: 0.8-1.2, placing the solvent system in a separating funnel, shaking uniformly, standing for layering, and separating into an upper phase and a lower phase;
b) dissolving the sample
Adding 5-30nmol/L hydrochloric acid as reserved acid into the upper phase, adding 5-30nmol/L triethylamine as eluting alkali into the lower phase, and dissolving the total ginkgolic acid extract extracted in the step (1) into 10mL of acidified upper phase and 10mL of non-alkalized lower phase;
c) sample introduction and separation
Placing a sample injection valve of a semi-preparative high-speed countercurrent chromatograph in a sample injection state, filling a chromatographic separation column with a stationary phase at a certain flow velocity by using a pump, and stopping the pump; starting a speed controller, enabling a chromatographic separation column of a high-speed flow chromatograph to rotate forwards, when the rotating speed reaches 800 revolutions per minute, injecting a dissolved sample into a sample injection valve of the counter-current chromatograph by using an injector, rotating the sample injection valve to be in a column connection state, enabling the sample to enter the chromatographic separation column, setting the flow rate of a mobile phase to be 2.0mL/min, starting a pump to pump the mobile phase, and receiving components I, II, III, IV and V according to a detector ultraviolet spectrogram, wherein the component III is a peak 3 monomer in a total sample, the component V is a peak 5 monomer in the total sample, the component I is a mixture of peak 1 and peak 2 monomers in the total sample, and the component IV is a mixture of peak 4 and peak 5 monomers in the total sample.
Further, it is preferable that the total ginkgolic acid extract in the step (b) is 2.0 g.
Preferably, the specific operation process of the separation and purification in the circulating countercurrent chromatography mode in the step (3) is as follows:
a) switching the six-way valve to a conventional separation mode, carrying out fluid dynamic balance on a countercurrent chromatography two-phase solvent system, and after the balance is good, injecting the obtained sample of the component I or IV;
b) switching the six-way valve to perform the circulating countercurrent chromatography of the sample, thereby realizing the internal circulating separation of the sample;
c) and after the separated samples are fully separated, switching the six-way valve, entering a conventional elution mode, sequentially collecting separated peaks in a branch pipe according to an ultraviolet detection result, performing internal circulation on the component I for 10 times to obtain monomers of peaks 1 and 2, and performing internal circulation on the component IV for 7 times to obtain monomers of peaks 4 and 5.
Further, preferably, the two-phase solvent system in step a) is configured as follows: preparing a solvent system from n-heptane/ethyl acetate/methanol/acetic acid in a volume ratio of 5:4: 0.8-1.2, placing the solvent system in a separating funnel, shaking uniformly, standing for layering, and separating an upper phase from a lower phase after balancing for a period of time.
Further, preferably, the step a) of mixing sample feeding is to dissolve the unseparated mixed sample in 5mL of upper phase and 5mL of lower phase.
Further, preferably, the sample injection process in the step a) is as follows: placing a sample injection valve of a semi-preparative high-speed countercurrent chromatograph in a sample injection state, filling a chromatographic separation column with a stationary phase at a certain flow velocity by using a pump, and stopping the pump; starting a speed controller to enable a chromatographic separation column of the high-speed flow chromatograph to rotate forwards at the rotating speed of 800 revolutions per minute, setting the flow rate of a mobile phase to be 2.0mL per minute, starting pumping the mobile phase, injecting a dissolved sample into a sample injection valve of the counter-current chromatograph by using an injector after the hydrodynamic balance is achieved, and rotating the sample injection valve to be in a column connection state to enable the sample to enter the chromatographic separation column.
Preferably, the column volume of the high-speed countercurrent chromatograph is 300mL, the sample loading amount is 500mg, the rotation speed is 800 r/min, the upper phase is a stationary phase, the lower phase is a mobile phase, the flow rate is 2.0mL/min, and the detection wavelength is 254 nm.
The application of the method in the separation and preparation of the ginkgolic acid monomeric compounds is also within the protection scope of the invention.
Has the advantages that:
the preparation cost of the invention is lower than that of the prior art, the operation is simple and convenient, the efficiency is high, and the ginkgolic acid monomer compound with the purity of more than 95 percent can be separated and prepared from the ginkgo nut peel in a large batch.
Drawings
FIG. 1 is a process flow diagram of the present invention;
FIG. 2 is a zonal countercurrent chromatographic separation profile of a ginkgolic acid total sample;
FIG. 3 cycle countercurrent chromatogram of component I;
FIG. 4 a cyclic countercurrent chromatogram of component V;
FIG. 5 is a high performance liquid chromatogram of each part of a countercurrent chromatography of a crude ginkgolic acid extract.
Detailed Description
The present invention will be described with reference to the following embodiments and drawings.
Example 1 sample extraction
Separating testa Ginkgo from semen Ginkgo, drying testa Ginkgo in shade, extracting with 10L petroleum ether (boiling range of 60-90 deg.C) under reflux for 3 times (each time for 2 hr) for 5kg, and filtering the extract with vacuum filter. All extracts were combined and rotary evaporated to dryness at 55 ℃ to give 247g of total ginkgolic acid extract, which was further purified by zonal countercurrent chromatography in a refrigerator at 4 ℃.
Example 2 separation and enrichment of total Ginkgo acid extract Using pH zone countercurrent chromatography
Preparing a solvent system from a two-phase solvent system of n-hexane/ethyl acetate/methanol/water (2:1:3:1, v/v) according to the solvent ratio, placing the solvent system in a separating funnel, shaking uniformly, standing for layering, separating an upper phase and a lower phase after balancing for a period of time, adding 10mmol/L hydrochloric acid into an upper phase as a reserved acid, adding 10mmol/L triethylamine into a lower phase as an eluting alkali, and dissolving 2.0g of total ginkgoic acid extract into 10mL of an acidified upper phase and 10mL of an non-alkalinized lower phase for later use. A semi-preparative high-speed counter-current chromatograph is used, which is composed of plunger pump, sampling valve, ultraviolet detector, recorder and chromatographic separation column (spiral pipe column formed by winding polytetrafluoroethylene tubes in multiple layers with capacity of 300 mL). And starting the speed controller to enable the chromatographic separation column of the high-speed flow chromatograph to rotate forwards, and when the rotating speed reaches 800 revolutions per minute, injecting the dissolved sample into a sample injection valve of the counter-current chromatograph by using an injector, wherein the sample injection valve is rotated to be in a column connection state, so that the sample enters the chromatographic separation column. The flow rate of the mobile phase is set to be 2.0mL/min, the mobile phase is pumped, and then the components I-V in FIG. 2 are received according to the ultraviolet spectrogram of the detector. And detecting that the component III is the peak 3 in the total sample, the component V is the peak 5 in the total sample, the component I is a mixture of the peaks 1 and 2 in the total sample, and the component IV is a mixture of the peaks 4 and 5 in the total sample, finally concentrating each part, and further separating the components I and IV by circulating countercurrent chromatography.
Example 3 separation and purification of ginkgolic acid monomers using a circulating countercurrent chromatography mode
Four ginkgolic acid monomers were separated from fractions I and IV using a separation method of cyclic countercurrent chromatography. Compared with the conventional countercurrent chromatography, the circulation countercurrent method is additionally provided with a six-way valve or two three-way valves, so that the switching between the conventional separation mode and the circulation separation mode can be realized, and the method is specifically divided into 3 steps: 1) switching the six-way valve to a conventional separation mode, carrying out fluid dynamic balance on a countercurrent chromatography two-phase solvent system, and injecting a component I or IV sample in the figure 2 after the balance is good; 2) switching the six-way valve to perform the circulating countercurrent chromatography of the sample, thereby realizing the internal circulating separation of the sample; 3) and after the separated samples are fully separated, switching the six-way valve, entering a conventional elution mode, and sequentially collecting the separated peak branch pipes according to the ultraviolet detection result. The column volume of the high-speed counter-current chromatograph is 300mL, the sample loading amount is 500mg, the rotating speed is 800 r/min, the upper phase is a stationary phase, the lower phase is a mobile phase, the flow rate is 2.0mL/min, and the detection wavelength is 254 nm. N-heptane/ethyl acetate/methanol/acetic acid (5:4:1:1, v/v)
The specific operation steps are as follows: switching the six-way valve to a conventional separation mode, firstly preparing a solvent system from a two-phase solvent system of n-heptane/ethyl acetate/methanol/acetic acid (5:4:1:1, v/v) according to the solvent ratio in the step 1, placing the solvent system into a separating funnel, shaking uniformly, standing for layering, separating an upper phase and a lower phase after balancing for a period of time, taking a crude product of the component I or IV, and dissolving the crude product in 5mL of an upper phase and 5mL of a lower phase for later use. A semi-preparative high-speed counter-current chromatograph is used, which is composed of plunger pump, sampling valve, ultraviolet detector, recorder and chromatographic separation column (spiral pipe column formed by winding polytetrafluoroethylene tubes in multiple layers with capacity of 300 mL). Starting a speed controller to enable a chromatographic separation column of the high-speed flow chromatograph to rotate forwards, setting the flow rate of a mobile phase to be 2.0mL/min when the rotating speed reaches 800 revolutions per minute, starting pumping the mobile phase, injecting a dissolved sample into a sample injection valve of the counter-current chromatograph by using an injector after the hydrodynamic balance is achieved, and rotating the sample injection valve to be in a column connection state to enable the sample to enter the chromatographic separation column. Switching the six-way valve to the circular separation mode, and then determining whether to switch the conventional countercurrent chromatography according to the result of the ultraviolet detector in fig. 3 or fig. 4. And after the peak to be separated is completely separated, switching the six-way valve, collecting chromatographic peaks, sequentially obtaining a component I, obtaining monomers of peaks 1 and 2 through 10 times of internal circulation, and obtaining monomers of peaks 4 and 5 through 7 times of internal circulation. And finally, separating to obtain a peak 1, a peak 2, a peak 3, a peak 4 and a peak 5, wherein five high-purity monomers are obtained, the purity of the monomers is over 95 percent through liquid chromatography detection, and the detection result is shown in a figure 5.
And (3) structural identification: comparing peak time of separated ginkgolic acid component monomer with peak time of mixed standard sample of ginkgolic acid (figure 5). Simultaneously, an Agilent 5973N mass spectrometer is adopted to obtain mass spectrum information, wherein the peak 1: 319.2[ M-H]-1Is ginkgolic acid (GA 13: 0); peak 2: 347.3[ M-H]-1Is ginkgolic acid (GA 15: 1); peak 3: 345.3[ M-H]-1Heptadecadienoic ginkgolic acid (GA 17: 2); peak 4: 373.3[ M-H]-1Is hydroginkgolic acid (GA 15: 0); peak 5: 371.3[ M-H]-1Is heptadecadecene ginkgolic acid (GA 17: 1).
Example 2
The difference from example 1 is that n-hexane/ethyl acetate/methanol/water 2: 1.2:3:0.8, n-heptane/ethyl acetate/methanol/acetic acid 5:4: 1.2: 1.2, the rest of the procedure is the same as in example 1.
Example 3
The difference from example 1 is that n-hexane/ethyl acetate/methanol/water 2: 0.8: 3:0.8, n-heptane/ethyl acetate/methanol/acetic acid 5:4: 1.2:0.8, the rest of the procedure is the same as in example 1.
Comparative example 1:
the difference from example 1 is that the n-hexane/ethyl acetate/methanol/water ratio was changed, i.e. n-hexane/ethyl acetate/methanol/water 2:3: 3: and 5, other operations are the same.
Comparative example 2:
the difference from example 1 is that the n-heptane/ethyl acetate/methanol/acetic acid ratio in example 1 was changed, i.e. n-hexane/ethyl acetate/methanol/water 3: 2: 4: and 2, other operations are the same.
Comparative example 3:
the difference from example 1 is that the n-heptane/ethyl acetate/methanol/acetic acid composition in example 1 was changed and acetic acid was replaced with water, i.e. n-heptane/ethyl acetate/methanol/water 5:4:1:1, other operations are the same.
Comparative example 4:
the difference from example 1 is that the n-hexane/ethyl acetate/methanol/water composition in example 1 was changed and water was replaced with acetic acid, i.e. n-heptane/ethyl acetate/methanol/acetic acid 2:1:3:1, other operations are the same.
Through research of comparative examples, it is found that changing the proportion of any one solvent system or changing the components of the solvent system in the invention can lead to the separation of effective components.
Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive faculty, based on the technical solutions of the present invention.
Claims (6)
1. A method for preparing ginkgolic acid components is characterized by comprising the following steps:
(1) separating semen Ginkgo from semen Ginkgo, drying the testa in shade, and extracting the dried testa with petroleum ether for three times to obtain total ginkgolic acid extract;
(2) separating and enriching total ginkgolic acid components by pH zone countercurrent chromatography to obtain corresponding components;
(3) separating and purifying the enriched components in a circulating countercurrent chromatography mode to obtain ginkgolic acid monomers with the purity higher than 95%;
wherein the boiling range of the petroleum ether extraction in the step (1) is 60-90 ℃;
the specific operation process of the pH zone countercurrent chromatography for separation and enrichment in the step (2) is as follows:
a) formulating a two-phase solvent system
Preparing a solvent system from n-hexane/ethyl acetate/methanol/water according to a volume ratio of 2:1:3:1, placing the solvent system in a separating funnel, shaking uniformly, standing and layering to obtain an upper phase and a lower phase, wherein the upper phase is a stationary phase, and the lower phase is a mobile phase;
b) dissolving the sample
Adding 10mmol hydrochloric acid as reserved acid into the upper phase, adding 10mmol triethylamine as eluting alkali into the lower phase, and dissolving the total ginkgolic acid extract extracted in the step (1) into 10mL of acidified upper phase and 10mL of non-alkalinized lower phase;
c) sample introduction and separation
Placing a sample injection valve of a semi-preparative high-speed countercurrent chromatograph in a sample injection state, filling a chromatographic separation column with a stationary phase at a certain flow velocity by using a pump, and stopping the pump; starting a speed controller to enable a chromatographic separation column of a high-speed flow chromatograph to rotate forwards, injecting a dissolved sample into a sample injection valve of the counter-current chromatograph by using an injector when the rotating speed reaches 800 revolutions per minute, and enabling the sample to enter the chromatographic separation column by rotating the sample injection valve to be in a column connection state; setting the flow rate of a mobile phase to be 2.0mL/min, starting pumping the mobile phase, and receiving components I, II, III, IV and V according to a detector ultraviolet spectrogram, wherein the component III is a peak 3 monomer in a total sample, the component V is a peak 5 monomer in the total sample, the component I is a mixture of peak 1 and peak 2 monomers in the total sample, and the component IV is a mixture of peak 4 and peak 5 monomers in the total sample;
the specific operation process of the separation and purification in the circulating countercurrent chromatography mode in the step (3) comprises the following steps:
a) switching the six-way valve to a conventional separation mode, carrying out hydrodynamic balance on a countercurrent chromatography two-phase solvent system, and after the balance is good, feeding a sample of the unseparated component I or IV prepared in the step (2);
wherein the two-phase solvent system is configured to: preparing a solvent system from n-heptane/ethyl acetate/methanol/acetic acid in a volume ratio of 5:4:1:1, placing the solvent system in a separating funnel, shaking uniformly, standing for layering, and separating into an upper phase and a lower phase;
b) switching the six-way valve to perform the circulating countercurrent chromatography of the sample, thereby realizing the internal circulating separation of the sample;
c) and after the separated samples are fully separated, switching the six-way valve, entering a conventional elution mode, and sequentially collecting the separated peak branch pipes according to the ultraviolet detection result.
2. The method as set forth in claim 1, wherein the total ginkgolic acid extract in the step (b) is 2.0g in the step (2).
3. The method according to claim 1, wherein in the step (3), the step a) of mixing sample injection is that the unseparated component I or IV sample prepared in the step (2) is dissolved in 5mL of the upper phase and 5mL of the lower phase.
4. The preparation method according to claim 1, wherein in the step (3), the sample injection process in the step a) is as follows: placing a sample injection valve of a semi-preparative high-speed countercurrent chromatograph in a sample injection state, filling a chromatographic separation column with a stationary phase at a certain flow velocity by using a pump, and stopping the pump; starting a speed controller to enable a chromatographic separation column of the high-speed flow chromatograph to rotate forwards, setting the flow rate of a mobile phase to be 2.0mL/min when the rotating speed reaches 800 revolutions per minute, starting pumping the mobile phase, injecting a dissolved sample into a sample injection valve of the counter-current chromatograph by using an injector after the hydrodynamic balance is achieved, and rotating the sample injection valve to be in a column connection state to enable the sample to enter the chromatographic separation column.
5. The preparation method according to claim 1, wherein the column volume of the high-speed countercurrent chromatography is 300mL, the sample loading amount is 500mg, the rotation speed is 800 rpm, the upper phase is a stationary phase, the lower phase is a mobile phase, the flow rate is 2.0mL/min, and the detection wavelength is 254 nm.
6. Use of the method of any one of claims 1 to 5 for the preparation of a ginkgolic acid monomer compound by isolation.
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