Method for removing ginkgolic acid in water-soluble ginkgo leaf extract

Abstract

The invention discloses a method for removing ginkgolic acid in a water-soluble ginkgo leaf extract, which comprises the following steps: mixing the ginkgo leaf extract with a compound enzyme solution, fully stirring for enzymolysis, adding a modified fullerene-graphene oxide compound for adsorption, then carrying out electrodialysis separation, then carrying out chromatographic column adsorption, refrigerating, crystallizing and removing impurities, thus obtaining the ginkgolic acid in the water-soluble ginkgo leaf extract. And performing membrane concentration and drying to obtain the ginkgo biloba extract without ginkgolic acid. According to the method, a revolutionary breakthrough is realized in the field of purification of the water-soluble ginkgo leaf extract by constructing a synergistic system of biological enzymolysis, nano material adsorption, electrochemical separation and a molecular recognition technology. Compared with a traditional process, the scheme creatively adopts a composite enzymolysis pretreatment technology, and creates favorable conditions for subsequent purification; the innovatively designed modified fullerene-graphene oxide nano composite material can be used for efficiently capturing free ginkgolic acid under a mild condition; gradient removal of ginkgolic acid is achieved through a unique electrodialysis-molecular imprinting coupling technology, the removal efficiency is guaranteed, and flavonoid active ingredients are protected.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural extract purification, in particular, relates to a method for removing ginkgo acid in water-soluble ginkgo leaf extract. BACKGROUND

[0002] Ginkgo tea is a health product refined from ginkgo leaves. There are more than one hundred kinds of ginkgo tea on the market. The main medicinal components of ginkgo tea, flavonoids and lactones, have the effects of improving cardiovascular and cerebrovascular circulation, antagonizing platelet activation factor and resisting thrombosis, and can be used for preventing and treating senile dementia and cerebral thrombosis. However, ginkgo acid is the main toxic substance in ginkgo leaf extract and ginkgo tea, and its safety has attracted much attention.

[0003] Ginkgo acid is a derivative of salicylic acid. The biological toxicity of ginkgo acid mainly includes the following aspects: 1. Sensitization, excessive intake of ginkgo acid can cause allergic reactions, nerve damage and gene mutation. The sensitization mechanism may be enzyme activity inhibition, cytotoxicity, allergy, etc. 2. Ginkgo acid has the effect of inhibiting gene recombination activity and can inhibit the catalytic activity of CYP enzyme. 3. Embryonic toxicity, nephrotoxicity, carcinogenicity, etc. In the above cases, the use range of ginkgo leaf extract is limited, and poisoning often occurs after a long time.

[0004] In order to solve the above problems, the existing technology for reducing the content of ginkgo acid uses ordinary ginkgo leaf extract as raw material, dissolves it in an organic solvent aqueous solution, and then removes ginkgo acid by gel-type hydrogen bond adsorption resin and rinses. The ultra-low acid ginkgo leaf extract can be obtained by concentrating and drying the effluent and rinsing liquid. This method requires harsh separation technology conditions and high cost, and the use of organic solvent extraction requires a large amount of solvent and is easy to cause environmental pollution. The selectivity of resin adsorption is poor, the process technology is difficult to operate, and the regeneration of resin is difficult. In addition, when ginkgo acid is separated in the prior art, the main medicinal component flavonoids in ginkgo leaf extract is often oxidized, resulting in the loss of effective components and affecting the efficacy of ginkgo leaf extract. SUMMARY

[0005] The present application relates to a method for removing ginkgo acid in water-soluble ginkgo leaf extract, which comprises the following steps: S1, enzyme pretreatment: mixing ginkgo leaf extract with a complex enzyme solution composed of equal amounts of cellulase and pectinase, and enzyme hydrolysis at 35-45℃ for 30-60 minutes to obtain an enzyme hydrolysis solution after inactivation; S2, nanocarrier adsorption: adding modified fullerene-graphene oxide composite to the enzymatic hydrolysate, 80-100 kHz ultrasonic assisted dispersion for 10 minutes, NaHCO3 to adjust pH to 5.0-6.0, stirring adsorption for 0.8-1.2 hours; S3, electrodialysis separation: injecting the mixed solution into an electrodialysis device, using an anion exchange membrane, electrodialysis separation of ginkgo acid, collecting the solution in the cathode chamber; S4, chromatography column adsorption: passing the solution in the cathode chamber through a chromatography column filled with ginkgo acid molecularly imprinted polymer, and after adsorbing the ginkgo acid, eluting the target component with an ethanol aqueous solution; S5, refrigeration crystallization impurity removal: after refrigerating the eluate at 2-8°C for 12-48 hours, removing the crystallized impurities by centrifugation; S6, membrane concentration and drying: concentrating the filtrate using an 8000-12000 Da ultrafiltration membrane, and vacuum drying to obtain a low-acid ginkgo leaf extract.

[0006] Further, in step S1, the enzyme activity concentration of the cellulase is 50-100 U / g, and the enzyme activity concentration of the pectinase is 20-50 U / g. The composite enzymatic hydrolysis replaces organic solvent extraction, and the molecularly imprinted chromatography replaces high-pollution resin adsorption, realizing water-phase operation throughout the whole process, eliminating the use of toxic solvents, reducing environmental load by more than 90%, and avoiding technical bottlenecks such as resin regeneration difficulties.

[0007] Further, the particle size of the modified fullerene-graphene oxide composite in step S2 is 50-100 nm, and the addition amount is 0.5-2 wt%. Based on the synergistic effect of the targeted adsorption of the modified fullerene-graphene oxide composite and the specific molecularly imprinted technology of ginkgo acid, high-precision capture of toxic components is realized, and the removal selectivity of ginkgo acid is improved to an industrial limit, with a residual amount far below the international safety threshold.

[0008] Further, the modified fullerene-graphene oxide composite in step S2 is prepared by the following method: (1) Fullerene C60 is reacted with 3-aminopropyltriethoxysilane at a molar ratio of 1:3 to obtain aminated fullerene; (2) mixing graphene oxide and aminated fullerene at a mass ratio of 1:2, crosslinking for 5 minutes under the assistance of 800W microwave to obtain.

[0009] Further, in step S3, the conditions for electrodialysis separation of ginkgo acid are 15-25V voltage, 10-15°C temperature, and separation for 30-60 minutes.

[0010] Further, in step S4, the pore size of the chromatography column is 2-5 nm, the adsorption flow rate is 2-5 BV / h, and the volume ratio of ethanol to water in the ethanol aqueous solution is 1:9.

[0011] Further, the molecularly imprinted polymer of step S4 is prepared by using ginkgolic acid as a template molecule, methacrylic acid as a functional monomer, and ethylene glycol dimethacrylic acid ester as a crosslinking agent, with a molar ratio of 1:4:20, and light polymerization at 60 DEG C for 6 hours.

[0012] Further, the ultrafiltration membrane of step S6 is a polyether sulfone / titanium dioxide composite membrane, and the operating pressure is 0.5-1.0 MPa.

[0013] Further, the whole process is carried out under the protection of an inert atmosphere, and the oxygen content is controlled to be ≤50 ppm. Through the whole process of inert atmosphere protection and low-temperature electrodialysis separation technology, the oxidative degradation path of ginkgo flavones is completely blocked, the active ingredient activity retention rate is ensured to be close to the natural state, and the loss of effective components caused by oxidation in the traditional process is overcome.

[0014] The present application realizes a revolutionary breakthrough in the field of water-soluble ginkgo leaf extract purification by constructing a synergistic system of biological enzymolysis, nanomaterial adsorption, electrochemical separation and molecular recognition technology. Compared with the traditional process, the present application creatively uses a composite enzymolysis pretreatment technology to effectively break the wrapping barrier of plant cell walls to ginkgolic acid, creating favorable conditions for subsequent purification; the innovatively designed modified fullerene-graphene oxide nanocomposite material exhibits excellent targeted adsorption performance and can efficiently capture free ginkgolic acid under mild conditions; the unique electrodialysis-molecular imprinting combined technology realizes the gradient removal of ginkgolic acid, which not only ensures the removal efficiency but also maximally protects the flavone active ingredients; the whole process inert atmosphere protection system and low-temperature operation strategy fundamentally inhibit the oxidative degradation of effective components, so that the product quality reaches the pharmaceutical grade standard. The technical system completely gets rid of the dependence on organic solvents, the production process is green and environmentally friendly, the equipment investment and operation cost are significantly reduced, and the obtained ultra-low acid ginkgo leaf extract can be widely used in the fields of medicines, health products and functional foods, and has a broad market prospect, which opens up a new way for the deep development and efficient utilization of ginkgo resources. DETAILED DESCRIPTION

[0015] The technical solutions of the present application will be further described below in combination with examples and comparative examples, but the protection scope of the present application is not limited thereto.

[0016] The ginkgolic acid content in the commercially available ordinary ginkgo leaf extract without ginkgolic acid removal is generally 100-3000 ppm. The ginkgo leaf extract used in the examples of the present application is ordinary ginkgo leaf extract without ginkgolic acid removal (purchased from Xuzhou Junyuan Ginkgo Products Co., Ltd.), and the ginkgolic acid content thereof is detected to be 300 ppm.

[0017] The whole process of the present application is carried out under the protection of an inert atmosphere, and the oxygen content is controlled to be ≤50 ppm.

[0018] Preparation of modified fullerene-graphene oxide composite: (1) 500 g of fullerene C60 was reacted with an equal amount of 3-aminopropyl triethoxysilane to obtain aminated fullerene; (2) 500 g of graphene oxide was mixed with the aminated fullerene obtained in step (1) to prepare by crosslinking under the assistance of 800 W microwave for 5 min.

[0019] Preparation of ginkgolic acid molecularly imprinted polymer: 50 g of ginkgolic acid was used as a template molecule, 50 g of methacrylic acid was used as a functional monomer, and 500 g of ethylene glycol dimethacrylate was used as a crosslinking agent. The polymer was prepared by photopolymerization at 60°C for 6 hours.

[0020] Example Example 1: (1) Raw material weighing: 25 g of commercially available ginkgo leaf extract was weighed; (2) Enzymatic pretreatment: The ginkgo leaf extract was mixed with 250 ml of a composite enzyme solution, the composite enzyme was composed of 50 g of 75 U / g cellulase and 35 U / g pectinase, and the mixture was enzymatically treated at 40°C for 45 min. After inactivation, an enzymatic solution was obtained; (3) Nanocarrier adsorption: 1.2 wt% of modified fullerene-graphene oxide composite with a particle size of 75 nm was added to the enzymatic solution, and ultrasonic dispersion was performed at 90 kHz for 10 min. NaHCO3 was used to adjust the pH to 5.5, and stirring adsorption was performed for 1 h; (4) Electrodialysis separation: The mixture was injected into an electrodialysis device, an anion exchange membrane was used, and ginkgolic acid was separated at a voltage of 20 V and a temperature of 13°C for 45 min. The cathode chamber solution was collected; (5) Chromatography column adsorption: The cathode chamber solution was passed through a chromatography column with a pore size of 4 nm filled with ginkgolic acid molecularly imprinted polymer, and the adsorption flow rate was 3 BV / h. After the adsorption of ginkgolic acid was completed, the target component was eluted with an ethanol-water solution, and the volume ratio of ethanol to water was 1:9; (6) Cold storage crystallization and impurity removal: After the eluate was stored at 5°C for 30 h, the crystalline impurities were removed by centrifugation; (7) Membrane concentration and drying: The filtrate was concentrated using a 10000 Da ultrafiltration membrane, the ultrafiltration membrane was a polyether sulfone / titanium dioxide composite membrane, the ultrafiltration pressure was 0.8 MPa, and vacuum drying was performed to obtain 22.74 g of low-acid ginkgo leaf extract.

[0021] Example 2: (1) Raw material weighing: 30 g of commercially available ginkgo leaf extract was weighed; (2) Enzymatic pretreatment: The ginkgo leaf extract was mixed with 300 ml of a composite enzyme solution, the composite enzyme was composed of 60 g of 50 U / g cellulase and 50 U / g pectinase, and the mixture was enzymatically treated at 35°C for 60 min. After inactivation, an enzymatic solution was obtained; (3) Nanometer carrier adsorption: 0.5wt% modified fullerene-graphene oxide composite with a particle size of 100 nm was added to the enzymatic solution, and 800 kHz ultrasonic assisted dispersion was performed for 10 minutes. NaHCO3 was used to adjust the pH to 6.0, and stirring adsorption was performed for 0.8 hours; (4) Electrodialysis separation: The mixed solution was injected into the electrodialysis device, and an anion exchange membrane was used. The separation of ginkgo acid was performed at a voltage of 25V and a temperature of 10℃ for 60 minutes, and the cathode chamber solution was collected; (5) Chromatography column adsorption: The cathode chamber solution was passed through a chromatography column with a pore size of 2nm filled with ginkgo acid molecularly imprinted polymer, and the adsorption flow rate was 5BV / h. After the adsorption of ginkgo acid was completed, the target component was eluted with an ethanol aqueous solution, and the volume ratio of ethanol to water was 1:9; (6) Refrigeration crystallization impurity removal: After the eluent was refrigerated at 2℃ for 12 hours, the crystallization impurities were removed by centrifugation; (7) Membrane concentration and drying: The filtrate was concentrated using an 8000Da ultrafiltration membrane, the ultrafiltration membrane was a polyether sulfone / titanium dioxide composite membrane, the ultrafiltration pressure was 1.0MPa, and vacuum drying was performed to obtain 26.03g of low-acid ginkgo leaf extract.

[0022] Example 3: (1) Raw material weighing: 40g of commercially available ginkgo leaf extract was weighed; (2) Enzymatic pretreatment: The ginkgo leaf extract was mixed with 400ml of a composite enzyme solution composed of 80g of 100U / g cellulase and 20U / g pectinase. Enzymatic hydrolysis was performed at 45℃ for 30 minutes, and the inactivated solution was obtained; (3) Nanometer carrier adsorption: 2wt% modified fullerene-graphene oxide composite with a particle size of 50nm was added to the enzymatic solution, and 100kHz ultrasonic assisted dispersion was performed for 10 minutes. NaHCO3 was used to adjust the pH to 5.0, and stirring adsorption was performed for 1.2 hours; (4) Electrodialysis separation: The mixed solution was injected into the electrodialysis device, and an anion exchange membrane was used. The separation of ginkgo acid was performed at a voltage of 15V and a temperature of 15℃ for 30 minutes, and the cathode chamber solution was collected; (5) Chromatography column adsorption: The cathode chamber solution was passed through a chromatography column with a pore size of 5nm filled with ginkgo acid molecularly imprinted polymer, and the adsorption flow rate was 2BV / h. After the adsorption of ginkgo acid was completed, the target component was eluted with an ethanol aqueous solution, and the volume ratio of ethanol to water was 1:9; (6) Refrigeration crystallization impurity removal: After the eluent was refrigerated at 8℃ for 48 hours, the crystallization impurities were removed by centrifugation; (7) Membrane concentration and drying: The filtrate was concentrated using an 12000Da ultrafiltration membrane, the ultrafiltration membrane was a polyether sulfone / titanium dioxide composite membrane, the ultrafiltration pressure was 0.5MPa, and vacuum drying was performed to obtain 33.45g of low-acid ginkgo leaf extract.

[0023] Comparative Example 1 Comparative Example 1 The difference between this comparative example and Example 1 is that in step (2), the complex enzyme solution is replaced by ordinary distilled water.

[0024] Comparative Example 2 The difference between this comparative example and Example 1 is that step (3) is cancelled, and the enzymatic solution is directly subjected to electrodialysis separation.

[0025] Comparative Example 3 The difference between this comparative example and Example 1 is that step (5) is cancelled, and the solution in the cathode chamber is directly subjected to refrigeration crystallization impurity removal.

[0026] Comparative Example 4 The difference between this comparative example and Example 1 is that step (6) is cancelled, and the solution in the cathode chamber is directly subjected to membrane concentration vacuum drying.

[0027] Performance Test The total flavone content and ginkgo acid content of the ginkgo leaf extracts prepared in Examples 1-3 and Comparative Examples 1-4 were detected, and the detection methods were as follows: 1. Detection of ginkgo acid according to Chinese Pharmacopoeia: octadecylsilane bonded silica gel was used as the filler (column length 150 mm, column inner diameter 4.6 mm, particle size 5 μm); acetonitrile containing 0.1% trifluoroacetic acid was used as the mobile phase A, and water containing 0.1% trifluoroacetic acid was used as the mobile phase B, gradient elution; the detection wavelength was 310 nm; 2. Detection of total flavone glycoside according to Chinese Pharmacopoeia: octadecylsilane bonded silica gel was used as the filler; methanol-0.4% phosphoric acid solution (50:50) was used as the mobile phase, the flow rate was 1.0 mL / min, the column temperature was 30°C, and the detection wavelength was 360 nm. The detection results are as follows: Table 1. Partial component content of ginkgo leaf extracts prepared in Examples 1-3 and Comparative Examples 1-4 Test Example Total flavone content / % Gingko acid content / ppm Example 1 31.84 0.13 Example 2 30.42 0.11 Example 3 28.86 0.12 Comparative Example 1 23.43 0.29 Comparative Example 2 29.65 0.27 Comparative Example 3 30.94 0.28 Comparative Example 4 31.36 0.26 From the above table, it can be concluded that the total flavonoid content of Examples 1-3 is 30.31% on average, and the ginkgo acid content is 0.12 ppm on average. In addition, from the table data, it can also be found that in Comparative Example 1, the complex enzyme solution is replaced with ordinary distilled water, and it is found that the total flavonoid content in the ginkgo leaf extract is significantly reduced, and the ginkgo acid content is significantly increased; in Comparative Example 2, the nano-carrier adsorption is cancelled, and the enzyme solution is directly subjected to electrodialysis separation, and it is found that the ginkgo acid content in the ginkgo leaf extract is significantly increased; in Comparative Example 3, the chromatography column adsorption is cancelled, and the solution in the cathode chamber is directly subjected to refrigeration crystallization impurity removal, and it is found that the ginkgo acid content in the ginkgo leaf extract is significantly increased; in Comparative Example 4, the refrigeration crystallization impurity removal in step (6) is cancelled, and the membrane concentration vacuum drying is directly performed, and it is found that the ginkgo acid content in the ginkgo leaf extract is significantly increased. In summary, it can be concluded that the adjusted or cancelled steps have a significant technical effect on achieving the purpose of the present application.

[0028] It should be understood that the above examples are only for illustrating the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and implement it, and the specific implementation of the present application cannot be limited to these descriptions. For ordinary skilled persons in the art to which the present application belongs, without departing from the concept of the present application, some simple deductions or substitutions can be made, and any equivalent changes or modifications made according to the spirit and essence of the present application should be covered within the protection scope of the present application.

Claims

1. A method for removing ginkgolic acid from water-soluble ginkgo leaf extract, characterized in that, Includes the following steps: S1. Enzymatic hydrolysis pretreatment: Ginkgo biloba extract is mixed with a compound enzyme solution, wherein the compound enzyme is composed of equal masses of cellulase and pectinase. The mixture is hydrolyzed at 35-45°C for 30-60 minutes, and the hydrolysate is obtained after inactivation. S2. Adsorption on nanocarriers: Add modified fullerene-graphene oxide composite to the enzymatic hydrolysate, disperse with ultrasonic assistance at 80-100 kHz for 10 minutes, adjust the pH to 5.0-6.0, and stir for adsorption for 0.8-1.2 hours; S3. Electrodialysis separation: The mixture is injected into the electrodialysis device, and ginkgolic acid is separated by electrodialysis using an anion exchange membrane. The solution in the cathode chamber is collected. S4. Column adsorption: The cathode chamber solution is passed through a chromatography column filled with ginkgolic acid molecularly imprinted polymer to adsorb ginkgolic acid, and then the target component is eluted with an ethanol aqueous solution. S5. Cold Crystallization and Impurity Removal: After refrigerating the eluent at 2-8°C for 12-48 hours, centrifuge to remove crystallized impurities; S6. Membrane Concentration and Drying: The filtrate is concentrated using an 8000-12000 Da ultrafiltration membrane and then vacuum dried to obtain a low-acid ginkgo leaf extract.

2. The method for removing ginkgolic acid from water-soluble ginkgo leaf extract according to claim 1, characterized in that: In step S1, the enzyme activity concentration of the cellulase is 50-100 U / g, and the enzyme activity concentration of the pectinase is 20-50 U / g.

3. The method for removing ginkgolic acid from water-soluble ginkgo leaf extract according to claim 1, characterized in that: The modified fullerene-graphene oxide composite in step S2 has a particle size of 50-100 nm and an addition amount of 0.5-2 wt%.

4. The method for removing ginkgolic acid from water-soluble ginkgo leaf extract according to claim 1, characterized in that: The modified fullerene-graphene oxide composite of step S2 was prepared by the following method: (1) Fullerene C60 was reacted with 3-aminopropyltriethoxysilane at a molar ratio of 1:3 to obtain aminated fullerene; (2) Graphene oxide and aminated fullerene were mixed at a mass ratio of 1:2 and crosslinked by microwave assisted by 800W for 5 minutes.

5. The method for removing ginkgolic acid from water-soluble ginkgo leaf extract according to claim 1, characterized in that: In step S3, the conditions for electrodialysis separation of ginkgolic acid are: separation for 30 to 60 minutes at a voltage of 15 to 25V and a temperature of 10 to 15℃.

6. The method for removing ginkgolic acid from water-soluble ginkgo leaf extract according to claim 1, characterized in that: In step S4, the pore size of the chromatography column is 2-5 nm, the adsorption flow rate is 2-5 BV / h, and the volume ratio of ethanol to water in the ethanol-water solution is 1:

9.

7. The method for removing ginkgolic acid from water-soluble ginkgo leaf extract according to claim 1, characterized in that: The molecularly imprinted polymer in step S4 was prepared by photopolymerization at 60°C for 6 hours, using ginkgolic acid as the template molecule, methacrylic acid as the functional monomer, and ethylene glycol dimethacrylate as the crosslinking agent, with a molar ratio of 1:4:

20.

8. The method for removing ginkgolic acid from water-soluble ginkgo leaf extract according to claim 1, characterized in that: The ultrafiltration membrane in step S6 is a polyethersulfone / titanium dioxide composite membrane with an operating pressure of 0.5–1.0 MPa.

9. The method for removing ginkgolic acid from water-soluble ginkgo leaf extract according to claim 1, characterized in that: The entire process is carried out under an inert atmosphere, with the oxygen content controlled at ≤50ppm.