CN108176079B - A method for decolorizing Glycyrrhrizae radix extract - Google Patents
A method for decolorizing Glycyrrhrizae radix extract Download PDFInfo
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Abstract
The invention relates to the technical field of chemical extraction, and particularly relates to a method for decoloring a liquorice extract. The decoloring method comprises the following steps: preparing a chromatographic column filled with a molecularly imprinted polymer containing no licorice flavonoids; crushing, drying and extracting liquorice with alcohol to obtain a liquorice primary extract; passing the Glycyrrhrizae radix primary extract through decolorizing macroporous resin, collecting filtrate, concentrating to obtain extract, and dissolving the extract with ethanol water solution to obtain primarily decolorized Glycyrrhrizae radix extract; passing the primarily decolorized Glycyrrhrizae radix extract through chromatographic column filled with molecularly imprinted polymer without Glycyrrhrizae radix flavonoid, and collecting filtrate; concentrating the filtrate, centrifuging, collecting filtrate, and vacuum drying to obtain Glycyrrhrizae radix extract. The method can specifically remove the colored licorice flavonoid substances in the licorice extract, reserve other colorless or light-colored functional components, can be applied to the decolorization treatment of daily chemical products such as skin smearing and the like, and has the advantages of strong pertinence, high recovery rate of the extract and obvious decolorization effect.
Description
Technical Field
The invention relates to the technical field of chemical extraction, and particularly relates to a method for decoloring a liquorice extract.
Background
Licorice (academic name:Glycyrrhiza uralensis Fisch) And alias: radix Angelicae Dahuricae, radix Glycyrrhizae, and radix Glycyrrhizae. Perennial herbs have thick roots and rhizomes with a diameter of 1-3 cm, brown outer skin and light yellow inner surface, and have sweet taste. The functions are mainly used for clearing away heat and toxic material, eliminating phlegm and relieving cough, and treating abdominal cavity and the like. The licorice extract generally comprises: glycyrrhizin, glycyrrhizic acid, liquiritin, glycyrrhetin, formononetin, quercetin, etc., and is yellow to brown yellow powder. Has effects of invigorating spleen and qi, clearing away heat and toxic materials, eliminating phlegm and relieving cough, relieving spasm and pain, and harmonizing the drugs.
The flavonoid component of licorice is the main cause of the yellow color of licorice extract, and it has other effects. The flavonoid component in Glycyrrhrizae radix has spasmolytic effect. Licoflavone is the main flavonoid component, and has anti-inflammatory activity. The flavone has antioxidant and hepatoprotective effects. The preclinical test results show that the flavone can kill most of helicobacter pylori causing gastric ulcer and enteritis. Wherein the glabridin (whitening gold) belongs to flavonoid components, can penetrate into the skin, keep high activity, whiten skin, efficiently resist oxidation, and effectively inhibit the activity of various enzymes in the melanin generation process, especially inhibit the activity of tyrosine enzyme. Meanwhile, the whitening cream has the effects of preventing rough skin, resisting inflammation and resisting bacteria, and is a whitening component with good curative effect and comprehensive functions at present. However, the color of part of flavonoid components can affect the visual perception of the finished product of the cosmetic to a certain extent, so that the flavonoid components with darker colors in the licorice extract are removed, and the glabridin and other active components are reserved, so that the whitening functional components can be reserved, and the visual perception of the cosmetic can be effectively improved. However, the problem of how to remove the yellow component in the licorice extract has not been solved so far.
At present, many inventions extract liquorice, however, single extraction product is too single, liquorice resources are not utilized completely, and resources are wasted, for example, patent numbers 201610296645.3 and 201510102210.6, in both inventions, purified glabridin is extracted, the extraction methods mentioned in the inventions have more steps, repeated operation and long time, and the types of adopted organic solvents are too many, the final recovery rate is very low, and the methods can only extract single glabridin, cannot realize extraction of multiple extracts and decoloration of liquorice extracts, cause waste of other resources in liquorice, and cannot fully exert the whole efficacy of liquorice. Patent No. 201410289036.6 adopts reversed phase C18 chromatography to separate glabridin from Glycyrrhrizae radix, and the final separated product has glabridin content of > 99%, but the method has high cost and single separated product, and other effective components in Glycyrrhrizae radix can not be fully utilized. Patent No. 201310178727.4 adopts molecular imprinting technique to separate glabridin from radix Glycyrrhizae, but has the disadvantage of single substance, and other components except glabridin in radix Glycyrrhizae are not recovered sufficiently, which results in waste of resources.
Disclosure of Invention
In view of the above, the present invention provides a method for decoloring a licorice extract. The invention can realize the extraction and decoloration of multiple components, saves resources and simultaneously maintains a simple and convenient method.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for decoloring a liquorice extract, which comprises the following steps:
preparing a molecularly imprinted polymer chromatographic column: taking a colored licorice flavonoid component in licorice as a template, and carrying out polymerization reaction on the template, a functional monomer and a cross-linking agent in the presence of an initiator to obtain a molecularly imprinted polymer containing the licorice flavonoid; crushing the obtained molecularly imprinted polymer, and eluting a licorice flavonoid template in the molecularly imprinted polymer by using an eluent to obtain the molecularly imprinted polymer without the licorice flavonoid; loading the molecularly imprinted polymer without the glycyrrhiza flavonoid into a column to obtain a chromatographic column loaded with the molecularly imprinted polymer without the glycyrrhiza flavonoid;
extraction and primary decolorization: crushing, drying and extracting liquorice with alcohol to obtain a liquorice primary extract; passing the Glycyrrhrizae radix primary extract through decolorizing macroporous resin, collecting filtrate, concentrating to obtain extract, and dissolving the extract with ethanol water solution to obtain primarily decolorized Glycyrrhrizae radix extract;
column passing: passing the primarily decolorized Glycyrrhrizae radix extract through chromatographic column filled with molecularly imprinted polymer without Glycyrrhrizae radix flavonoid, and collecting filtrate; concentrating the filtrate, centrifuging, collecting filtrate, and vacuum drying to obtain Glycyrrhrizae radix extract.
In the invention, the flavonoid components with darker colors and higher content in the liquorice mainly comprise liquiritigenin, liquiritin, isoliquiritigenin, isoliquiritin and the like.
Preferably, the licoflavonoid is one or more of liquiritigenin, liquiritin, isoliquiritigenin and isoliquiritin.
In the specific embodiment provided by the invention, the licorice flavonoid is a mixture of liquiritigenin, liquiritin, isoliquiritigenin and isoliquiritin.
Preferably, the mass ratio of the liquiritigenin, the liquiritin and the isoliquiritin is (1-2): (1-2): (1-2): (1-2).
In the specific embodiment provided by the invention, the mass ratio of the liquiritigenin, the liquiritin and the isoliquiritigenin to the isoliquiritin is 1:1:1: 1.
The invention can realize the extraction and decoloration of multiple components, saves resources and simultaneously maintains a simple and convenient method. The invention does not use dangerous chemicals, is safe and reliable, does not need to use small glassware, and is suitable for large-scale commercial production.
Preferably, the polymerization conditions are: and (3) reacting for 11-13 hours at 75-85 ℃ under the protection of nitrogen.
In one embodiment of the present invention, the polymerization conditions are: the reaction was carried out at 75 ℃ for 13 hours.
Preferably, the functional monomer is styrene.
Preferably, the initiator is potassium persulfate.
Preferably, the crosslinking agent is one or more of divinylbenzene, polyethylene glycol, N-methylene bisacrylamide, zinc oxide, ethyl orthosilicate, ethylene glycol dimethacrylate, trimethoxypropyl trimethacrylate or hydroxypropyl methacrylate.
Preferably, in the step of preparing the molecularly imprinted polymer chromatographic column, crushing the molecularly imprinted polymer chromatographic column into 40-100 meshes; in the steps of extraction and primary decoloration, crushing is carried out until the powder is crushed to 10-30 meshes.
In one embodiment of the present invention, in the extraction and primary decolorization steps, the pulverization is carried out to 20 mesh.
Preferably, in the step of preparing the molecularly imprinted polymer chromatographic column, the eluent is a methanol solvent containing 40 vt% -60 vt% of acetic acid.
In one embodiment of the present invention, the eluent is methanol solvent containing 50vt% acetic acid in the step of preparing the molecularly imprinted polymer chromatographic column.
Preferably, the alcohol extraction is: extracting for 2-3 times by using 90-99.5 vt% ethanol water solution, combining the extracts, wherein the extraction temperature is 40-60 ℃, and the extraction time is 2-4 hours; concentrating to be vacuum distillation at 40-60 ℃.
In a specific embodiment provided by the present invention, the alcohol extraction is: extracting with 95 vt% ethanol water solution for 3 times, and mixing the extracts.
In a specific embodiment provided by the invention, the extraction temperature of the alcohol extraction is 40 ℃, and the extraction time is 2 hours.
In one embodiment of the present invention, the concentration is a reduced pressure distillation at 50 ℃.
Preferably, the flow rate of the sample passing through the column is 1-3 BV/h.
In one embodiment of the present invention, the sample flow rate through the column is 2 BV/h.
Preferably, the method further comprises the following steps after the filtrate is collected: eluting the column with eluent after the sample loading is finished, and collecting the eluent; concentrating the eluate, centrifuging, collecting filtrate, and vacuum drying to obtain licoflavonoid product.
Preferably, the eluent is 60-80 vt% ethanol water solution, and the flow rate of elution is 1-3 BV/h.
In one embodiment of the present invention, the eluent is 70 vt% ethanol water solution, and the elution flow rate is 2 BV/h.
Preferably, in the extraction and primary decolorization steps, the drying is carried out until the water content is less than 4%.
Preferably, in the steps of extraction and primary decoloration, the extract is dissolved by ethanol water solution, and the ethanol water solution is 50-70 vt% ethanol water solution.
Preferably, in the extraction and preliminary decolorization steps, the extract is dissolved with an aqueous ethanol solution of 60 vt%.
Preferably, the rotation speed of the centrifugation in the column passing step is 3500-4500 rpm, and the vacuum degree of vacuum drying is 0.09-0.10 MPa.
Preferably, the rotation speed of the centrifugation in the column passing step is 4000rpm, and the vacuum degree of vacuum drying is 0.093 MPa.
The invention provides a method for decoloring a liquorice extract. The decoloring method comprises the following steps:
preparing a chromatographic column filled with a molecularly imprinted polymer containing no licorice flavonoids; crushing, drying and extracting liquorice with alcohol to obtain a liquorice primary extract; passing the Glycyrrhrizae radix primary extract through decolorizing macroporous resin, collecting filtrate, concentrating to obtain extract, and dissolving the extract with ethanol water solution to obtain primarily decolorized Glycyrrhrizae radix extract; passing the primarily decolorized Glycyrrhrizae radix extract through chromatographic column filled with molecularly imprinted polymer without Glycyrrhrizae radix flavonoid, and collecting filtrate; concentrating the filtrate, centrifuging, collecting filtrate, and vacuum drying to obtain Glycyrrhrizae radix extract. The invention has the technical effects that:
1. the method takes the licorice flavonoid component as a template, can specifically remove the colored licorice flavonoid substance in the licorice extract, reserves other colorless or light-colored functional components, can be applied to the decolorization treatment of daily chemical products such as skin smearing and the like, and has strong pertinence, higher recovery rate of the extract and obvious decolorization effect.
2. The method has the advantages of high efficiency, environmental protection, high repeatability and easy product collection.
3. The invention can simultaneously realize the extraction and the decoloration of multiple components, save resources, reduce cost and simultaneously maintain a simple and convenient method.
4. The invention does not use dangerous chemicals, is safe and reliable, does not need to use small glassware, and is suitable for large-scale commercial production.
Drawings
FIG. 1 is a color chart.
Detailed Description
The invention discloses a method for decoloring liquorice extract, which can be realized by appropriately improving process parameters by referring to the content in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The reagents or instruments used in the method for decoloring the licorice extract provided by the invention can be purchased from the market.
The invention is further illustrated by the following examples:
example 1
(1) Preparing a molecularly imprinted polymer chromatographic column: adding styrene into 2mol/L sodium hydroxide solution, and stirring for 30min to remove impurities. 30mL of the styrene, 400mL of water and 1g of licorice flavonoid (comprising 0.25g of liquiritigenin, 0.25g of liquiritin, 0.25g of isoliquiritigenin, 0.25g of isoliquiritin and the same below) are heated and stirred at 75 ℃, then 0.2g of cross-linking agent divinylbenzene is added, finally 0.8g of initiator potassium persulfate is added, and the reaction is carried out for 13h under the protection of high-purity nitrogen, so as to obtain the molecular imprinting polymer containing the glabridin. And then crushing and sieving the polymer, taking polymer particles of 40-100 meshes, eluting a licorice flavonoid template in the molecular polymer by using a methanol solvent of 50% acetic acid, and finally obtaining the molecularly imprinted polymer without the licorice flavonoid. And finally, packing the molecular polymer without the licorice flavonoids into a column for later use.
(2) Extracting effective parts: pulverizing 400g Glycyrrhrizae radix, drying to water content less than 4%, extracting with 5L 95% ethanol for 3 times at 40 deg.C for 2 hr, passing the extract through macroporous resin, collecting decolorized filtrate, and spin drying to obtain extract.
(3) Column passing: dissolving the extract obtained in the step (2) with 100mL of 60% ethanol solution, then allowing the extract to pass through a chromatographic column filled with the molecularly imprinted polymer without the licoflavone in the step (1), wherein the sample loading flow rate is 2 BV/h, and collecting filtrate; after the reaction is finished, eluting the column by using 60 percent ethanol solution with the flow rate of 2 BV/h, and sequentially collecting the eluent containing the licoflavone.
(4) Collecting a product: spin-drying the filtrate and eluate to separate out components, centrifuging at high rotation speed, collecting components, and drying under vacuum degree of 0.093MPa to obtain decolorized Glycyrrhrizae radix extract and Glycyrrhrizae radix flavonoids (liquiritigenin, liquiritin, isoliquiritigenin, isoliquiritin).
Example 2
(1) Preparing a decolorized molecularly imprinted polymer chromatographic column: adding styrene into 2mol/L sodium hydroxide solution, and stirring for 30min to remove impurities. 30mL of the styrene, 400mL of water and 1g of licorice flavonoid (comprising 0.25g of liquiritigenin, 0.25g of liquiritin, 0.25g of isoliquiritigenin, 0.25g of isoliquiritin and the same below) are heated and stirred at 75 ℃, then 0.3g of cross-linking agent (0.1 g of polyethylene glycol 400 and 0.2g of ethyl orthosilicate) is added, and finally 0.8g of initiator potassium persulfate is added into the mixture to react for 13 hours under the protection of high-purity nitrogen, so that the molecular imprinting polymer containing the glabridin is obtained. And then crushing and sieving the polymer, taking polymer particles of 40-100 meshes, eluting a licorice flavonoid template in the molecular polymer by using a methanol solvent of 50% acetic acid, and finally obtaining the molecularly imprinted polymer without the licorice flavonoid. And finally, packing the molecular polymer without the licorice flavonoids into a column for later use.
(2) Extracting effective parts: pulverizing 400g Glycyrrhrizae radix, drying to water content less than 4%, extracting with 5L 95% ethanol for 3 times at 40 deg.C for 2 hr, passing the extract through macroporous resin, collecting decolorized filtrate, and spin drying to obtain extract.
(3) Column passing: dissolving the extract obtained in the step (2) with 100mL of 60% ethanol solution, then allowing the extract to pass through a chromatographic column filled with the molecularly imprinted polymer without the licoflavone in the step (1), wherein the sample loading flow rate is 2 BV/h, and collecting filtrate; after the reaction is finished, eluting the column by using 60 percent ethanol solution with the flow rate of 2 BV/h, and sequentially collecting the eluent containing the licoflavone.
(4) Collecting a product: spin-drying the filtrate and eluate to separate out components, centrifuging at high rotation speed, collecting components, and drying under vacuum degree of 0.093MPa to obtain decolorized Glycyrrhrizae radix extract and Glycyrrhrizae radix flavonoids (liquiritigenin, liquiritin, isoliquiritigenin, isoliquiritin).
Comparative example 1
(1) Preparing a decolorized molecularly imprinted polymer chromatographic column: adding styrene into 2mol/L sodium hydroxide solution, and stirring for 30min to remove impurities. 30mL of the styrene, 400mL of water and 1g of licorice flavonoid (comprising 0.25g of liquiritigenin, 0.25g of liquiritin, 0.25g of isoliquiritigenin, 0.25g of isoliquiritin and the same below) are heated and stirred at 75 ℃, then 0.3g of cross-linking agent (0.15 g of ethylene glycol dimethacrylate and 0.15g of N, N-methylene bisacrylamide) is added, finally 0.8g of initiator potassium persulfate is added, and the mixture reacts for 13 hours under the protection of high-purity nitrogen, so that the molecular imprinting polymer containing the glabridin is obtained. And then crushing and sieving the polymer, taking polymer particles of 40-100 meshes, eluting a licorice flavonoid template in the molecular polymer by using an ether solvent, and finally obtaining the molecularly imprinted polymer without the licorice flavonoid. And finally, packing the molecular polymer without the licorice flavonoids into a column for later use.
(2) Extracting effective parts: pulverizing 400g Glycyrrhrizae radix, drying to water content less than 4%, extracting with 5L 95% ethanol for 3 times at 40 deg.C for 2 hr, passing the extract through macroporous resin, collecting decolorized filtrate, and spin drying to obtain extract.
(3) Column passing: dissolving the extract obtained in the step (2) with 100mL of 60% ethanol solution, then allowing the extract to pass through a chromatographic column filled with the molecularly imprinted polymer without the licoflavone in the step (1), wherein the sample loading flow rate is 2 BV/h, and collecting filtrate; after the reaction is finished, eluting the column by using 60 percent ethanol solution with the flow rate of 2 BV/h, and sequentially collecting the eluent containing the licoflavone.
(4) Collecting a product: spin-drying the filtrate and eluate to separate out components, centrifuging at high rotation speed, collecting components, and drying under vacuum degree of 0.093MPa to obtain decolorized Glycyrrhrizae radix extract and Glycyrrhrizae radix flavonoid respectively.
Comparative example 2
(1) Preparing a decolorized molecularly imprinted polymer chromatographic column: adding styrene into 2mol/L sodium hydroxide solution, and stirring for 30min to remove impurities. 30mL of the styrene, 400mL of water and 1g of licorice flavonoid (comprising 0.25g of liquiritigenin, 0.25g of liquiritin, 0.25g of isoliquiritigenin, 0.25g of isoliquiritin and the same below) are heated and stirred at 75 ℃, then 0.3g of cross-linking agent (0.1 g of polyethylene glycol 400 and 0.2g of trimethoxypropyl trimethacrylate) is added, finally 0.8g of initiator potassium persulfate is added, and the mixture reacts for 13 hours under the protection of high-purity nitrogen, so that the molecular imprinting polymer containing the glabridin is obtained. And then crushing and sieving the polymer, taking polymer particles of 40-100 meshes, eluting a licorice flavonoid template in the molecular polymer by using a petroleum ether-chloroform (50: 50, V: V) solvent, and finally obtaining the molecularly imprinted polymer without the licorice flavonoid. And finally, packing the molecular polymer without the licorice flavonoids into a column for later use.
(2) Extracting effective parts: pulverizing 400g Glycyrrhrizae radix, drying to water content less than 4%, extracting with 5L 95% ethanol for 3 times at 40 deg.C for 2 hr, passing the extract through macroporous resin, collecting decolorized filtrate, and spin drying to obtain extract.
(3) Column passing: dissolving the extract obtained in the step (2) with 100mL of 60% ethanol solution, then allowing the extract to pass through a chromatographic column filled with the molecularly imprinted polymer without the licoflavone in the step (1), wherein the sample loading flow rate is 2 BV/h, and collecting filtrate; after the reaction is finished, eluting the column by using 60 percent ethanol solution with the flow rate of 2 BV/h, and sequentially collecting the eluent containing the licoflavone.
(4) Collecting a product: spin-drying the filtrate and eluate to separate out components, centrifuging at high rotation speed, collecting components, and drying under vacuum degree of 0.093MPa to obtain decolorized Glycyrrhrizae radix extract and Glycyrrhrizae radix flavonoid respectively.
Comparative example 3
(1) Taking 400g of Glycyrrhiza glabra, respectively carrying out reflux extraction on the 400g of Glycyrrhiza glabra with 40, 35 and 30kg of 95% ethanol for 2.0, 1.5 and 1.0h, mechanically using the third extracting solution, concentrating the first two extracting solutions, and concentrating until the specific gravity is 1.05-1.10.
(2) Sequentially extracting with petroleum ether, chloroform and ethyl acetate for 3-5 times, concentrating chloroform extract, mixing with 100g of diatomite, drying, extracting with Soxhlet extractor, sequentially extracting with petroleum ether → ethyl acetate → acetone → ethanol, recovering solvent, separating ethyl acetate part 200g with silica gel column chromatography, eluting with petroleum ether, eluting with petroleum ether-methanol gradient (6 gradients, 100:5, 100:10, 100:15, 100:20, 100:25, 100:30, V: V), separating 100:15 eluate by silica gel column chromatography, performing gradient elution with chloroform-methanol (100: 3-100: 10, V: V), separating the chloroform-methanol (100: 3-100: 6, V: V) eluate with silica gel thin layer, separation was performed with petroleum ether-ethyl acetate (100: 15, V: V) to obtain about 10mg of the compound.
(3) Dissolving the compound by chloroform-methanol (100: 7-100: 10, V: V), separating by a silica gel reduced pressure column, eluting by petroleum ether-acetone (100: 8, V: V), adding 3-5% of active carbon into eluent, refluxing and decoloring for 30min, and concentrating and crystallizing.
(4) Adding one of petroleum ether, n-hexane or diethyl ether into the crystal, stirring thoroughly, and dripping one or mixed solvent of small amount of methanol, ethanol, acetone, chloroform, dichloromethane or n-butanol until the crystal is rinsed to obtain white lustrous glabridin with 98.8% content.
Comparative example 4
The specific method of this comparative example is as follows:
(1) stock preparation
The components are proportioned according to the following dosage ratios: template molecules (licorice flavonoids including 0.25g liquiritigenin, 0.25g liquiritin, 0.25g isoliquiritigenin, 0.25g isoliquiritin, the same below) 0.97g, functional monomers (methacrylic acid) 1.63g, cross-linking agent (ethylene glycol dimethacrylate) 19.00g, initiator (azobisisobutyronitrile) 0.10g, and solvent (mixed solvent of acetonitrile and chloroform with the weight ratio of 1: 1) 42.00 g.
(2) Preparation of molecularly imprinted polymers with template molecules
Adding the template molecule licorice flavonoids, methacrylic acid, ethylene glycol dimethacrylate, azodiisobutyronitrile, acetonitrile and chloroform mixed solvent prepared in the step (1) into a reaction kettle, uniformly mixing, carrying out ultrasonic degassing for 15min, then filling nitrogen to remove oxygen, sealing, and polymerizing for 24 hours in a constant-temperature water bath at 60 ℃ to obtain a molecularly imprinted polymer with template molecules, and then grinding and sieving the molecularly imprinted polymer with 60-100 meshes to obtain the 60-100-mesh molecularly imprinted polymer particles with the template molecules.
(3) Purification of molecularly imprinted polymers with template molecules
And (3) eluting the molecularly imprinted polymer particles with the template molecules prepared in the step (2) by using a methanol solution with the acetic acid volume fraction of 10%, and drying at 60 ℃ in vacuum to obtain 18.4g of the molecularly imprinted polymer for purifying the licorice flavonoids.
(4) And (3) packing 15g of the molecularly imprinted polymer prepared in the step (2) for purifying the licorice flavonoids into a column. Extracting effective parts: pulverizing 400g Glycyrrhrizae radix, drying to water content less than 4%, extracting with 5L 95% ethanol for 3 times at 40 deg.C for 2 hr, passing the extract through macroporous resin, collecting decolorized filtrate, and spin drying to obtain extract. The column was loaded after redissolving 1L of chloroform.
After the solution on the column flows out, 300 ml of acetonitrile solution with the volume fraction of 5% of methanol is used for leaching, the chromatographic column is eluted by methanol solution with the volume fraction of 10% of acetic acid, and the filtrate and the eluent are collected. Spin-drying the filtrate and eluate to separate out components, centrifuging at high rotation speed, collecting components, and drying under vacuum degree of 0.093MPa to obtain decolorized Glycyrrhrizae radix extract and Glycyrrhrizae radix flavonoids (liquiritigenin, liquiritin, isoliquiritigenin, isoliquiritin).
Comparative example 5
This example describes the preparation of a molecularly imprinted monolithic column using metal ions as a bridging agent and glycyrrhiza flavonoid as a template. The method comprises the following specific steps:
preparing a licorice flavonoid imprinted monolithic column by an in-situ polymerization method:
a. dissolving 3.09 mass percent of template licorice flavonoids, 1.64 mass percent of bridging agent cobalt acetate, 2.76 mass percent of functional monomer 4-vinylpyridine, 0.29 mass percent of initiator azobisisobutyronitrile and 26.04 mass percent of cross-linking agent ethylene glycol dimethacrylate into a mixed pore-foaming agent solution containing 43.59 mass percent of 1-butyl-3-methylimidazolium tetrafluoroborate, 3.32 mass percent of N, N-dimethylformamide and 19.27 mass percent of dimethyl sulfoxide; ultrasonic dissolving for 30min, clarifying, introducing nitrogen gas, removing oxygen in the pre-polymerization mixed solution, injecting the pre-polymerization mixed solution into a stainless steel column (100 × 4.6 mm I.D.), sealing two ends, and reacting in 50-60 deg.C water bath for 16-18 h.
And (3) injecting the pre-polymerization mixed solution into a sealed container, and reacting in a water bath at 50-60 ℃ for 16-18 h to obtain the licorice flavonoid molecularly imprinted polymer taking metal ions as the bridging agent.
b. The reacted column was connected to a high pressure pump of HPLC, and washed with acetonitrile to remove the remaining porogen and soluble material from the whole column, the flow rate was gradually increased from 0.1 mL/min to 0.5 mL/min, and after washing to 100m L, the column was replaced with 150 mL of methanol/acetic acid (v/v, 7/3) mixed solution to remove the template molecules. Finally the system was equilibrated to baseline levels using the mobile phase.
The preparation method of the licorice flavonoid molecular imprinting monolithic column without metal ions comprises the following steps except that cobalt acetate is not added.
c. 15g of the prepared molecularly imprinted polymer for purifying the licorice flavonoids are packed into a column. Extracting effective parts: pulverizing 400g Glycyrrhrizae radix, drying to water content less than 4%, extracting with 5L 95% ethanol for 3 times at 40 deg.C for 2 hr, passing the extract through macroporous resin, collecting decolorized filtrate, and spin drying to obtain extract.
d. Redissolving the licorice extract with 1L chloroform and loading on the column. After the solution on the column flows out, 300 ml of acetonitrile solution with the volume fraction of 5% of methanol is used for leaching, the chromatographic column is eluted by methanol solution with the volume fraction of 10% of acetic acid, and the filtrate and the eluent are collected. Spin-drying the filtrate and eluate to separate out components, centrifuging at high rotation speed, collecting components, and drying under vacuum degree of 0.093MPa to obtain decolorized Glycyrrhrizae radix extract and Glycyrrhrizae radix flavonoids (liquiritigenin, liquiritin, isoliquiritigenin, isoliquiritin).
Test example 1 test of decoloring Effect
The decoloring effect is compared for the above examples and comparative examples. The standard colorimetric method adopted by the patent establishes a colorimetric table with the chromaticity of 5-450 as shown in figure 1.
The color data and recovery rates for the above examples and comparative examples are shown in the following table.
TABLE 1 color data and recovery results
| Item | Example 1 | Example 2 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | Comparative example 5 |
| Color value | 80 | 125 | 350 | 300 | 50 | 175 | 250 |
| Percent recovery% | 92.4 | 91.6 | 91.2 | 90.4 | ＜1 | 81.4 | 74.1 |
Note: the recovery rate refers to the overall recovery rate of the licorice extract.
According to the data in the table, the colorimetric values of comparative examples 1 and 2 are higher than those of the examples, which indicates that the darker components are not completely separated, and from the viewpoint of the whole process, the difference is that the eluent used in the preparation of the molecularly imprinted polymer is different from the examples, which indicates that three solvents of ether, petroleum ether and chloroform cannot effectively elute the darker components in the licorice extract; comparative example 3 adopts a traditional combined method of organic solvent dissolution elution, crystallization and activated carbon adsorption decoloration, although the final component glabridin content is very high and the decoloration effect is best, the whole operation steps are complex, the solvent types are various, and the recovery rate is too low (only glabridin is basically obtained finally, and other components are basically removed); comparative example 4 using a functional monomer different from that of example, the overall separation effect is closer to that of example, but the effect is less; comparative example 5 has a poor decoloring effect and a low recovery rate compared to examples, indicating that metal ions are generally effective as a bridging agent.
Test example 2 active ingredient detection
According to the compositional analysis of the examples and comparative examples, the composition of 7 licorice extracts is shown in the following table:
TABLE 2 analysis results of the composition of licorice extract
| Item(s) Eyes of a user | Example 1 | Example 2 | Comparative example 1 | Comparative example 2 | Comparison of Example 3 | Comparative example 4 | Comparative example 5 |
| Lifting device Get Article (A) Become into Is divided into | Glabridin, Other Flavonoids Composition (I) | Glabridin, Other Flavonoids Composition (I) | Glabridin, licorice Glycoside, isoliquiritin and their preparation The flavonoid component of Tata | Glabridin, licorice Glycoside, isoliquiritin and their preparation The flavonoid component of Tata | Guangan Ganmao Pralidoxime sodium | Glabridin, other classes Flavone component, small amount of sweet Grass glycoside, isoliquiritin | Glabridin, other classes Flavone component, partially sweet Grass glycoside, isoliquiritin |
| Is divided into Separation device Become into Is divided into | Liquiritigenin and isoliquiritigenin Liquiritigenin and licorice Phlomis grass glycoside and Isogan Grass glycoside | Liquiritigenin and isoliquiritigenin Liquiritigenin and licorice Phlomis grass glycoside and Isogan Grass glycoside | Liquiritigenin and isoliquiritigenin Vegetable extract | Liquiritigenin and isoliquiritigenin Vegetable extract | Others Yellow-like pigment Keto to Is divided into | Liquiritigenin and isoliquiritigenin Vegetable extract, liquiritin and isopropyl Grass glycoside | Liquiritigenin and isoliquiritigenin Vegetable extract, liquiritin and isopropyl Grass glycoside |
Because the main color-developing components are liquiritigenin, isoliquiritigenin, liquiritin and isoliquiritin, the solvents adopted in comparative examples 1 and 2 have better elution effect on liquiritigenin and isoliquiritin, but have poorer elution capability on liquiritin and isoliquiritin, only liquiritigenin and isoliquiritin are separated finally; comparative example 3 the flavonoid components except glabridin were separated, so the final glabridin content was high; comparative examples 4 and 5 were similar in the case where the darker components, glycyrrhizin and isoliquiritin, could not be completely separated, so that the separated extract remained.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A method for decoloring a licorice extract is characterized by comprising the following steps:
preparing a molecularly imprinted polymer chromatographic column: taking a colored licorice flavonoid component in licorice as a template, and carrying out polymerization reaction on the template, a functional monomer and a cross-linking agent in the presence of an initiator to obtain a molecularly imprinted polymer containing the licorice flavonoid; crushing the obtained molecularly imprinted polymer, and eluting a licorice flavonoid template in the molecularly imprinted polymer by using an eluent to obtain the molecularly imprinted polymer without the licorice flavonoid; loading the molecularly imprinted polymer without the licorice flavonoids into a column to obtain a chromatographic column loaded with the molecularly imprinted polymer without the licorice flavonoids;
extraction and primary decolorization: crushing, drying and extracting liquorice with alcohol to obtain a liquorice primary extract; passing the initial extract of Glycyrrhrizae radix through macroporous resin for decolorization, collecting filtrate, concentrating to obtain extract, and dissolving the extract with ethanol water solution to obtain primarily decolorized Glycyrrhrizae radix extract;
column passing: passing the primarily decolorized Glycyrrhrizae radix extract through chromatographic column filled with molecularly imprinted polymer without Glycyrrhrizae radix flavonoid, and collecting filtrate; concentrating the filtrate, centrifuging, collecting filtrate, and vacuum drying to obtain Glycyrrhrizae radix extract;
the licorice flavonoids are liquiritigenin, liquiritin, isoliquiritigenin and isoliquiritin;
in the step of preparing the molecularly imprinted polymer chromatographic column, the eluent is a methanol solvent containing 40 vt% -60 vt% of acetic acid;
the functional monomer is styrene; the initiator is potassium persulfate; the cross-linking agent is one or more of divinylbenzene, polyethylene glycol, N-methylene bisacrylamide, zinc oxide, ethyl orthosilicate, ethylene glycol dimethacrylate, trimethoxypropyl trimethacrylate or hydroxypropyl methacrylate.
2. The decolorization method according to claim 1, characterized in that the polymerization conditions are: and (3) reacting for 11-13 hours at 75-85 ℃ under the protection of nitrogen.
3. The decoloring method according to claim 1, wherein, in the step of preparing a molecularly imprinted polymer chromatography column, the pulverization is to 40 to 100 mesh; in the steps of extraction and primary decoloration, the crushing is to be 10-30 meshes.
4. The decolorization method according to claim 1, wherein said alcohol extraction is: extracting for 2-3 times by using 90-99.5 vt% ethanol water solution, and combining the extracts, wherein the extraction temperature is 40-60 ℃, and the extraction time is 2-4 hours; and the concentration in the extraction and primary decolorization steps is reduced pressure distillation at 40-60 ℃.
5. The decolorization method according to claim 1, wherein the sample flow rate of the column is 1-3 BV/h.
6. The decolorization method according to claim 1, further comprising, after collecting the permeate: eluting the column with eluent after the sample loading is finished, and collecting the eluent; concentrating and centrifuging the eluent, collecting filtrate, and vacuum drying to obtain the licorice flavonoid product.
7. The decolorization method according to claim 6, wherein an eluent used after the loading is finished is 60-80 vt% ethanol water solution, and the flow rate of the elution is 1-3 BV/h.
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Inventor after: Liu Xiaoying Inventor after: Liu Guangrong Inventor after: Che Biao Inventor after: Tang Jian Inventor after: Du Zhiyun Inventor after: Agee Akbar Esa Inventor after: He Fei Inventor after: Lin Li Inventor before: Liu Xiaoying Inventor before: Liu Guangrong Inventor before: Che Biao Inventor before: Tang Jian Inventor before: Du Zhiyun Inventor before: Agee Akbar Esa Inventor before: He Fei Inventor before: Lin Li |