Liquiritigenin preparation method based on enzyme inhibition treatment and peeling process

Abstract

The invention discloses a liquiritigenin preparation method based on enzyme inhibition treatment and a peeling process, relates to the technical field of natural product extraction, effectively solves the problems of pigment impurities and low purity in the existing liquiritigenin extraction process by combining enzyme inhibition and peeling technologies, and improves the yield of liquiritigenin by inhibiting the activity of endogenous enzyme of liquorice and reducing pigment generation. The method comprises the following steps: firstly, extracting liquiritigenin from glycyrrhizin, removing a pigment-rich outer skin part through peeling, remarkably improving the purity of a liquiritigenin extracting solution, then converting liquiritin into liquiritigenin through acid hydrolysis, extracting with solvents such as ethanol and ethyl acetate, and further purifying through repeated recrystallization, thereby finally obtaining high-purity liquiritigenin. The method has the characteristics of simple process and low cost, is suitable for large-scale industrial production, and can effectively improve the production efficiency, simplify the subsequent purification step and obviously reduce the production cost.

Description

Technical Field

[0001] The invention relates to the technical field of natural product extraction, and in particular to a method for preparing glycyrrhizin based on enzyme inhibition treatment and peeling process. Background Art

[0002] Liquiritigenin, also known as 4',7-dihydroxyflavone, is a dihydroflavonoid compound in licorice. It has multiple pharmacological activities such as anti-inflammatory, antioxidant, antibacterial and anti-tumor, and is widely used in medicine, food and health products. However, the existing licorice extraction technology still has many problems, especially in reducing pigment impurities and improving extraction purity.

[0003] At present, the extraction and purification methods of glycyrrhizin mainly include ethanol hot extraction, acid hydrolysis, solvent extraction and column chromatography. For example, Niu Ben used 95% ethanol hot extraction in the "Research on Separation of Licoricein and Isoliquiritigenin" and purified it by silica gel column chromatography. Although a high extraction rate was obtained, there were more pigment residues in the extract, which increased the difficulty and cost of subsequent purification. Han Bo and others used hydrochloric acid hydrolysis to improve the extraction efficiency of glycyrrhizin in licorice powder, but the use of strong acid is not only corrosive to the equipment, but also does not solve the problem of removing pigment impurities.

[0004] In addition, a number of related patents are also committed to improving the extraction efficiency of glycyrrhizin. For example: Chinese patent, publication number CN102391232A, application date September 26, 2011, patent name Method for extracting glycyrrhizin from licorice, authorization announcement number CN102391232B, and Chinese patent, publication number CN106916859A, application date February 13, 2017, patent name A method for rapidly extracting glycyrrhizin from licorice waste residue, authorization announcement number CN106916859B, the two patents respectively introduce the methods of using acid hydrolysis and cellulase enzymolysis, although these methods improve the extraction efficiency to a certain extent, the strong acid or enzymolysis process fails to effectively solve the problem of pigment impurities.

[0005] Chinese patent, publication number CN107459503A, application date May 10, 2017, patent name is a method for separation and purification of glycyrrhizin, authorization announcement number CN107459503B, which utilizes the solubility difference of solvents for extraction and purification, but involves a large amount of harmful solvents, affecting the application safety of samples.

[0006] Chinese Patent, with the publication number CN111202764A, the application date being January 10, 2020, the patent title being "A Process for Increasing the Content of Liquiritin", the authorization announcement number being CN111202764B, and Chinese Patent, with the publication number CN117660199A, the application date being December 7, 2023, the patent title being "A Species of Psathyrella candolleana, a Bacterial Agent, a Method and Application for Converting Liquiritin into Liquiritin", the authorization announcement number being CN117660199B. The two patents respectively improve the extraction rate through specific enzymatic hydrolysis methods and strain fermentation, but the problems of pigment interference and insufficient extraction purity remain the key unsolved problems.

[0007] In summary, the existing extraction processes of liquiritin still have significant limitations in aspects such as pigment interference, low purity, and high costs, especially lacking effective methods for solving pigment generation and reducing impurities. Therefore, there is an urgent need for an innovative technology that can effectively prevent pigment generation and simplify the extraction and purification process to provide a more efficient and economical solution for the large-scale application of liquiritin. Summary of the Invention

[0008] To overcome the deficiencies in the background technology, the present invention provides a method for preparing liquiritin based on enzyme inhibition treatment and peeling process. By innovatively combining enzyme inhibition treatment and peeling process, the browning reaction and pigment generation are effectively reduced in the pretreatment stage of licorice raw materials, and the outer skin part rich in pigments is removed. This method reduces the content of pigment impurities at the source and significantly improves the purity of the liquiritin extract. By simplifying the subsequent purification steps, not only the extraction efficiency is effectively improved, but also the production cost is reduced. The purity of the final product, liquiritin, can reach 96%.

[0009] To achieve the above-mentioned invention purpose, the present invention adopts the following technical scheme: A method for preparing liquiritin based on enzyme inhibition treatment and peeling process, the preparation method specifically includes the following steps: The first step, enzyme inhibition treatment: After drying the cleaned licorice, cut it into small sections with a length of 10 - 20 cm, then place the licorice sections in a superheated steam environment, or process them in a microwave environment, or soak the licorice sections in a solution containing an enzyme inhibitor for enzyme inhibition treatment to effectively inhibit the activity of endogenous enzymes in licorice, thereby reducing the pigment generation caused by enzymatic oxidation reaction and ensuring the quality and stability of licorice in the subsequent processing process; The second step, peeling: After taking out the enzyme-inhibited licorice from superheated steam, microwave, or enzyme inhibitor solution, drain the excess moisture on its surface, and then remove the dark outer skin of the licorice by manual peeling, mechanical peeling, or chemical peeling. This part of the outer skin contains a large amount of pigments. By removing the outer skin, the dissolution of dark pigments in the subsequent extraction process is reduced, thereby improving the purity and quality of the extract. Step 3: Drying and Crushing: Place the peeled licorice in a microwave device or a hot air drying oven for drying. After drying, cool the licorice core to room temperature, and then use a crusher to crush it and screen it through a sieve with a mesh size of 10 - 100 meshes to obtain licorice powder with uniform particle size for subsequent extraction operations. Step 4: Acid Hydrolysis: Add the crushed licorice powder to 5 - 20 times the amount of 0.5 - 3 mol / L hydrochloric acid, sulfuric acid, or nitric acid solution for acid hydrolysis to ensure sufficient hydrolysis and avoid the conversion and degradation of liquiritin. After the reaction, filter while it is hot to obtain acid hydrolysis residue and acid hydrolysis solution. Step 5: Extraction: Extract the acid hydrolysis residue with at least one solvent of ethanol, methanol, ethyl acetate, or water, and extract the acid hydrolysis solution with ethyl acetate. After recovering the solvents of the extraction solution and the extraction liquid, combine the solid components to obtain the liquiritin extract. Step 6: Recrystallization: Perform repeated recrystallization on the extract with any one or a combination of two or more of water, ethanol, methanol, or acetone, and detect the distribution and content of liquiritin by HPLC to finally obtain liquiritin.

[0010] In the method for preparing liquiritin based on enzyme inhibition treatment and peeling process, when using a superheated steam environment to perform enzyme inhibition treatment on licorice in the first step, the treatment temperature is 95°C - 160°C, and the treatment time is 5 - 20 minutes.

[0011] In the method for preparing liquiritin based on enzyme inhibition treatment and peeling process, when using a microwave environment to perform enzyme inhibition treatment on licorice in the first step, the power of the microwave is 0.4 - 32 kw, and the treatment time is 1 - 15 min.

[0012] In the method for preparing liquiritin based on enzyme inhibition treatment and peeling process, when using a chemical peeling method to peel the enzyme-inhibited licorice in the second step, utilize the strong alkalinity of sodium hydroxide and potassium hydroxide, or the weak acidity of citric acid and malic acid, and perform moistening or infiltration treatment.

[0013] In the method for preparing liquiritin based on enzyme inhibition treatment and peeling process, the drying temperature of licorice in the third step is 25°C - 100°C.

[0014] The preparation method of liquiritin based on enzyme inhibition treatment and peeling process. When performing acid hydrolysis in the fourth step, the acid hydrolysis temperature is 60°C to 100°C, and the hydrolysis time is 0.5 to 4 hours.

[0015] The preparation method of liquiritin based on enzyme inhibition treatment and peeling process. In the fifth step, the material-liquid ratio during extraction is 1:3 to 1:50, the extraction temperature is 4°C to 82°C, the extraction time is 0.2 to 72 h, and the solvent ratio (aqueous phase: organic phase) during extraction is 1:0.5 to 1:3.

[0016] The preparation method of liquiritin based on enzyme inhibition treatment and peeling process. In the sixth step, the temperature for recrystallization is controlled at 0°C to 40°C.

[0017] Adopting the above technical solution, the present invention has the following advantages: By combining enzyme inhibition and peeling technologies, the present invention effectively solves the problems of pigment impurities and low purity existing in the existing liquiritin extraction process. By inhibiting the activity of endogenous enzymes in licorice, the generation of pigments is reduced, and by peeling, the outer skin part rich in pigments is removed, significantly improving the purity of the liquiritin extraction solution. Subsequently, liquiritin is obtained by converting liquiritin glycoside through acid hydrolysis and extracting with solvents such as ethanol and ethyl acetate, and further purified by repeated recrystallization. Finally, high-purity liquiritin is obtained. The present invention has the characteristics of simple process and low cost, and is suitable for large-scale industrial production, which can effectively improve production efficiency, simplify subsequent purification steps, and significantly reduce production costs. Brief Description of the Drawings

[0018] Figure 1 It is the flowchart for the preparation of liquiritin in the embodiment of the present invention. Detailed Description of the Invention

[0019] The present invention can be more detailedly explained through the following embodiments, and the present invention is not limited to the following embodiments; By inhibiting the mechanism of enzymatic browning and blocking pigment migration, the present invention improves the color and purity of liquiritin from the source. The reaction between polyphenol oxidase and polyphenolic substances in licorice raw materials to form brown compounds is one of the main reasons for the darkening of product color. The present invention rapidly inactivates the enzyme activity through enzyme inactivation treatment, avoiding the formation of browning substances. At the same time, by using peeling treatment to remove the surface tissue containing pigments, pigment entry into the extraction process is effectively blocked, thereby significantly reducing the pigment content in the extraction solution. By reducing pigment interference, the purification process is simplified, not only improving the purity of liquiritin, but also enhancing the stability and feasibility of large-scale production. After optimizing the key steps, a white liquiritin product is finally prepared, meeting the requirements of the high-end market and enhancing the market competitiveness of the product.

[0020] A method for preparing liquiritin based on enzyme inhibition treatment and peeling process, the preparation method specifically includes the following steps: The first step, enzyme inhibition treatment: After drying the cleaned licorice and cutting it into small sections with a length of 10-20 cm, then placing the licorice sections in a superheated steam environment, or treating them in a microwave environment, or soaking the licorice sections in a solution containing an enzyme inhibitor for enzyme inhibition treatment to effectively inhibit the activity of endogenous enzymes in licorice, thereby reducing the generation of pigments caused by enzymatic oxidation reactions and ensuring the quality and stability of licorice during subsequent processing; When implemented, when using a superheated steam environment to perform enzyme inhibition treatment on licorice, the treatment temperature is 95°C-160°C, and the treatment time is 5-20 minutes. When using a microwave environment to perform enzyme inhibition treatment on licorice, the power of the microwave is 0.4-32 kw, and the treatment time is 1-15 min; The second step, peeling: After taking out the licorice treated by enzyme inhibition from the superheated steam or microwave or enzyme inhibitor solution, draining the excess water on its surface, and then using manual peeling or mechanical peeling or chemical peeling methods to remove the dark outer skin of the licorice. This part of the outer skin contains a large amount of pigments. By removing the outer skin, the dissolution of dark pigments in the subsequent extraction process is reduced, thereby improving the purity and quality of the extract; When implemented, when using the chemical peeling method to peel the licorice treated by enzyme inhibition, the strong alkalinity of sodium hydroxide and potassium hydroxide, or the weak acidity of citric acid and malic acid are utilized, and after moistening or infiltration treatment; The third step, drying and pulverizing: Place the peeled licorice in a microwave device or a hot air drying oven for drying. After drying is completed, cool the licorice core to room temperature, and then use a pulverizer to pulverize it and screen it through a 10-100 mesh sieve to obtain licorice powder with uniform particle size for subsequent extraction operations. When implemented, the drying temperature of the licorice is 25°C-100°C; The fourth step, acid hydrolysis: Add the pulverized licorice powder to 5-20 times the amount of 0.5-3 mol / L hydrochloric acid or sulfuric acid or nitric acid solution for acid hydrolysis to ensure sufficient hydrolysis and avoid the conversion and degradation of liquiritin. After the reaction ends, filter while it is hot to obtain acidolysis residue and acidolysis solution. When implemented, during acid hydrolysis, the acid hydrolysis temperature is 60°C-100°C, and the hydrolysis time is 0.5-4 hours; The fifth step, extraction: Extract the acidolysis residue with at least one solvent of ethanol, methanol, ethyl acetate or water, and extract the acidolysis solution with ethyl acetate. After recovering the solvents of the extract and the extractant, combine the solid components to obtain the liquiritigenin extract. During implementation, the material-liquid ratio during extraction is 1:3 to 1:50, the extraction temperature is 4°C to 82°C, the extraction time is 0.2 to 72 h, and the solvent ratio (aqueous phase: organic phase) during extraction is 1:0.5 to 1:3; Step 6, recrystallization: Use any one or a combination of two or more of water, ethanol, methanol, or acetone as a mixed solvent to repeatedly recrystallize the extract, and detect the distribution and content of liquiritigenin by HPLC to finally obtain liquiritigenin. During implementation, the temperature of recrystallization is controlled at 0°C to 40°C.

[0021] In the actual practice of the present invention, in combination with the attached Figure 1 , a method for preparing liquiritigenin based on enzyme inhibition treatment and peeling process according to the present invention, the preparation method specifically includes the following steps: Step 1, enzyme inhibition treatment: Use heating, adjusting PH, adding inhibitors, or rapid drying methods to inhibit the activity of endogenous enzymes in licorice and reduce pigment formation; During implementation, after drying the cleaned licorice, cut it into small sections with a length of 10 to 20 cm for subsequent processing. Subsequently, place the licorice sections in a superheated steam environment at 95 to 160 °C, or in a microwave environment of 0.4 to 32 kW for treatment, or soak the licorice sections in a solution containing enzyme inhibitors for enzyme inhibition treatment. These treatment methods are designed to effectively inhibit the activity of endogenous enzymes in licorice, thereby reducing pigment formation caused by enzymatic oxidation reactions and ensuring the quality and stability of licorice during subsequent processing; In specific practice, when using heating to inhibit the activity of endogenous enzymes in licorice, use steam to perform enzyme inhibition treatment on licorice, the treatment temperature is 95°C to 160°C, and the treatment time is 5 to 20 minutes; When using heating to inhibit the activity of endogenous enzymes in licorice, use microwave technology to perform enzyme inhibition treatment on licorice, the power of the microwave is 0.4 to 32 kw, and the treatment time is 1 to 15 min; Use steam, microwave or other enzyme inhibition technologies to perform enzyme inhibition treatment on licorice to inhibit the activity of endogenous enzymes, thereby reducing pigment formation caused by enzymatic oxidation reactions. The role of enzyme inhibition treatment: This step effectively prevents the accumulation of impurities and pigments caused by enzyme activity, ensures the color and purity of the subsequent extract, and provides a basic guarantee for the preparation of high-purity liquiritigenin; Step 2, peeling: The deglycyrrhizinated licorice after enzyme inhibition treatment is peeled by manual peeling method, mechanical peeling method or chemical peeling method to remove the outer skin part rich in pigments; During implementation, after taking out the licorice after enzyme inhibition treatment from superheated steam, microwave or enzyme inhibitor solution, drain the excess water on its surface. Subsequently, use manual peeling, mechanical peeling or chemical peeling methods to remove the dark outer skin of the licorice. This part of the outer skin contains a large amount of pigments. By removing the outer skin, the dissolution of dark pigments during the subsequent extraction process can be significantly reduced, thereby improving the purity and quality of the extract; During specific implementation, when using the chemical peeling method to peel the licorice after enzyme inhibition treatment, utilize the strong alkalinity of sodium hydroxide and potassium hydroxide, or the weak acidity of citric acid and malic acid, and carry out moistening or soaking treatment.

[0022] In specific practice, the deglycyrrhizinated licorice after enzyme inhibition treatment is peeled by manual peeling method, mechanical peeling method or chemical peeling method. The function of peeling: There are still a large number of pigments in the licorice after enzyme inhibition treatment, and the pigments are mainly concentrated in the outer skin part of the licorice, and less in the core part of the licorice. Therefore, peeling can remove the deep pigments in the outer skin of the licorice, reduce the dissolution of pigments in the outer skin, thereby significantly reducing the chromaticity in the extraction solution and improving the visual purity of the final extract, and at the same time helping to reduce the difficulty and cost of the subsequent purification process; Step 3: Drying and pulverizing: After drying the peeled licorice, use a pulverizer to pulverize it and sieve to obtain dry licorice powder; During implementation, place the peeled licorice in a microwave device or a hot air drying oven and dry it under mild conditions of 25 - 100 °C to avoid damage or degradation of the active ingredients in the licorice caused by too high temperature or heat. After drying is completed, cool the licorice core to room temperature, and then use a pulverizer to pulverize it and sieve it through a sieve with a mesh size of 10 - 100 meshes to obtain licorice powder with uniform particle size for subsequent extraction operations; Furthermore, the drying temperature of the licorice is 25 °C - 100 °C, and the mesh number of the sieve during sieving is 10 - 100 meshes.

[0023] In specific practice, the peeled licorice core is dried at 25 °C - 100 °C for 0 - 24 hours and then pulverized, and after pulverization, it is sieved with a 10 - 100 mesh sieve to obtain dry licorice powder. The advantages of this process: Drying treatment is carried out under milder conditions, which can avoid the destruction of the active ingredients of the licorice caused by too high temperature; pulverizing and sieving can ensure the uniformity of particle size, improve the extraction rate and ensure the stability of the process.

[0024] Step 4: Acid hydrolysis: The pulverized licorice powder is subjected to acid hydrolysis with hydrochloric acid, sulfuric acid or nitric acid solution, and after the reaction ends, it is filtered to obtain acidolysis residue and acidolysis solution; During implementation, the pulverized licorice powder is added to 5 - 20 times the amount of 0.5 - 3 mol / L hydrochloric acid, sulfuric acid or nitric acid solution, and hydrolyzed at 60 - 100 °C for 0.5 - 4 hours to ensure sufficient hydrolysis and avoid the conversion and degradation of liquiritin. After the reaction, filter while it is hot to obtain the acid hydrolysis residue and acid hydrolysis solution; During implementation, the acid concentration during acid hydrolysis is 0.5 - 3 mol / L, the amount of acid solution used is 5 - 20 times the amount of licorice powder, the acid hydrolysis temperature is 60 °C - 100 °C, and the hydrolysis time is 0.5 - 4 hours.

[0025] In specific practice, 5 - 20 times the amount of 0.5 - 3 mol / L hydrochloric acid, sulfuric acid or nitric acid is added to the dried licorice powder, and an acid hydrolysis reaction is carried out at a temperature of 60 °C - 100 °C for a duration of 0.5 - 4 hours. After the acid hydrolysis is completed, filter to obtain the acid hydrolysis residue and acid hydrolysis solution. The role of acid hydrolysis: convert liquiritin in licorice into its corresponding aglycone (liquiritigenin), thereby improving the extraction efficiency; Step Five, Extraction: The acid hydrolysis residue and acid hydrolysis solution are respectively extracted with an extraction solvent and an extraction solvent, the solvents are recovered and the components are combined to obtain a liquiritigenin extract; During implementation, at least one solvent of ethanol, methanol, ethyl acetate or water in an amount of 3 - 50 times is used to extract the acid hydrolysis residue at 4 - 82 °C for 1 - 3 times, each extraction for 0.2 - 72 h, and the extraction solutions are combined. The acid hydrolysis solution is extracted with 0.5 - 3 times the amount of ethyl acetate for 1 - 3 times, and the extraction solutions are combined. After the solvents of the extraction solution and the extraction solution are recovered, the solid components are combined to obtain the liquiritigenin extract; In specific implementation, the extraction solvent during the extraction process is at least one of ethanol, methanol, ethyl acetate or water, and the extraction solvent is ethyl acetate; the solid - liquid ratio during extraction is 1:3 - 1:50, the extraction temperature is 4 °C - 82 °C, the extraction time is 0.2 - 72 h, and the solvent ratio (aqueous phase: organic phase) during extraction is 1:0.5 - 1:3.

[0026] In specific practice, the acid hydrolysis residue is repeatedly extracted 1 - 3 times with 50% - 95% ethanol, methanol or ethyl acetate to effectively dissolve liquiritigenin in licorice; the acid hydrolysis solution is extracted with ethyl acetate 1 - 3 times. As a highly efficient organic solvent, ethyl acetate can selectively extract the liquiritigenin - like components in the acid hydrolysis solution. After extraction, the solvent is recovered by vacuum distillation or other recovery techniques to obtain the combined liquiritigenin extract, laying a foundation for the subsequent purification steps; During implementation, the solid - liquid ratio during the extraction process is 1:3 - 1:50, the extraction temperature is 4 °C - 82 °C, and the extraction time is 0.2 - 72 h; Step Six, Recrystallization: The extract was recrystallized repeatedly with a recrystallization solvent to finally obtain high-purity liquiritin.

[0027] During implementation, the extract was recrystallized repeatedly with a mixed solvent of any one or two or more of water, ethanol, methanol, or acetone at 0 °C to 40 °C, and the distribution and content of liquiritin were detected by HPLC to finally obtain liquiritin.

[0028] During specific implementation, the recrystallization solvent is a mixed solvent of any one or two or more of water, ethanol, methanol, or acetone; the temperature of the recrystallization is controlled at 0 °C to 40 °C.

[0029] In specific practice, repeated recrystallization treatment was carried out with water, ethanol, methanol, acetone, or their mixed solvent to further remove impurities and obtain high-purity white liquiritin; During implementation, the temperature of the recrystallization is controlled at 0 °C to 40 °C.

[0030] During specific implementation, the first step of enzyme inhibition treatment, the fifth step of extraction, and the sixth step of purification treatment can be optimized with different temperatures or solvent systems respectively to improve the purity of liquiritin or reduce the color depth.

[0031] Specific embodiments of the present invention are as follows: Example 1:

[0032] Take 1 kg of Glycyrrhiza uralensis Fisch., and perform microwave enzyme inactivation treatment at 32 kw for 1 min. After enzyme inactivation, the color of the outer skin of the licorice was removed by manual peeling to obtain a lighter-colored licorice core. The licorice core was dried at 45 °C, pulverized, and sieved through a 40-mesh sieve. 10 times the amount of 1 mol / L hydrochloric acid was added to the dried licorice powder, and acid hydrolysis was carried out at 90 °C for 2 h. After acid hydrolysis was completed, filtration was carried out to obtain acid hydrolysis residue and acid hydrolysis solution. The acid hydrolysis residue was extracted with 5 times the amount of 95% ethanol at 78 °C for 2 h, and the extraction was repeated 3 times. The acid hydrolysis solution was extracted with ethyl acetate 3 times. The solvents were recovered respectively, and the alcohol extract of the acid hydrolysis residue and the ethyl acetate extract of the acid hydrolysis solution were combined to obtain a liquiritin extract. Recrystallization was carried out with methanol-water to obtain 2.9274 g of white liquiritin, and the purity was 96.59% (HPLC method). Example 2:

[0033] Take 1 kg of Glycyrrhiza uralensis Fisch., and treat it with 0.4 kw microwave for 5 min to inactivate enzymes. After enzyme inactivation, the licorice is manually peeled to remove the darker outer skin part, and the lighter-colored licorice core is obtained. The licorice core is dried at 45 °C, pulverized and sieved through a 60-mesh sieve. Add 20 times the amount of 0.5 mol / L hydrochloric acid to the dried licorice powder, and hydrolyze at 90 °C for 2 h. After hydrolysis is completed, filter to obtain the acid hydrolysis residue and the acid hydrolysis solution. The acid hydrolysis residue is extracted with 8 times the amount of 95% ethanol at 70 °C for 3 h, and the extraction is repeated 3 times. The acid hydrolysis solution is extracted with ethyl acetate 3 times. The solvents are recovered respectively, and the alcohol extract of the acid hydrolysis residue and the ethyl acetate extract of the acid hydrolysis solution are combined to obtain the liquiritin extract. Recrystallize with methanol-water to obtain 2.9341 g of white liquiritin, with a purity of 96.17% (HPLC method). Example 3:

[0034] Take 1 kg of Glycyrrhiza uralensis Fisch., and treat it with steam at 98 °C for 20 min to inactivate enzymes. After enzyme inactivation, the licorice is manually peeled to remove the darker outer skin part, and the lighter-colored licorice core is obtained. The licorice core is dried at 60 °C, pulverized and sieved through a 60-mesh sieve. Add 4 times the amount of 3 mol / L hydrochloric acid to the dried licorice powder, and hydrolyze at 80 °C for 2.5 h. After hydrolysis is completed, filter to obtain the acid hydrolysis residue and the acid hydrolysis solution. The acid hydrolysis residue is extracted with 10 times the amount of 95% ethanol at 78 °C for 2 h, and the extraction is repeated 3 times. The acid hydrolysis solution is extracted with ethyl acetate 3 times. The solvents are recovered respectively, and the alcohol extract of the acid hydrolysis residue and the ethyl acetate extract of the acid hydrolysis solution are combined to obtain the liquiritin extract. Recrystallize with methanol-water to obtain 2.9419 g of white liquiritin, with a purity of 96.22% (HPLC method). Example 4:

[0035] Take 1 kg of Glycyrrhiza uralensis Fisch., and treat it with steam at 105 °C for 15 min to inactivate enzymes. After enzyme inactivation, the licorice is manually peeled to remove the darker outer skin part, and the lighter-colored licorice core is obtained. The licorice core is dried at 60 °C, pulverized and sieved through a 60-mesh sieve. Add 8 times the amount of 1.5 mol / L hydrochloric acid to the dried licorice powder, and hydrolyze at 90 °C for 2 h. After hydrolysis is completed, filter to obtain the acid hydrolysis residue and the acid hydrolysis solution. The acid hydrolysis residue is extracted with 10 times the amount of methanol at 65 °C for 3 h, and the extraction is repeated 3 times. The acid hydrolysis solution is extracted with ethyl acetate 3 times. The solvents are recovered respectively, and the alcohol extract of the acid hydrolysis residue and the ethyl acetate extract of the acid hydrolysis solution are combined to obtain the liquiritin extract. Recrystallize with methanol-water to obtain 2.9458 g of white liquiritin, with a purity of 96.53% (HPLC method). Example 5:

[0036] Take 1 kg of Glycyrrhiza uralensis Fisch., and carry out enzyme inactivation treatment with steam at 110 °C for 9 min. After enzyme inactivation, the licorice is manually peeled to remove the darker outer skin part, and the lighter-colored licorice core is obtained. The licorice core is dried at 60 °C, pulverized and sieved through a 60-mesh sieve. Add 5 times the amount of 2 mol / L hydrochloric acid to the dried licorice powder, and carry out acid hydrolysis at 100 °C for 0.5 h. After acid hydrolysis is completed, filter to obtain acid hydrolysis residue and acid hydrolysis solution. The acid hydrolysis residue is extracted with 3 times the amount of 95% ethanol at 25 °C for 72 h, and extracted repeatedly 2 times. The acid hydrolysis solution is extracted with ethyl acetate 3 times. The solvents are recovered respectively, and the ethanol extract of the acid hydrolysis residue and the ethyl acetate extract of the acid hydrolysis solution are combined to obtain liquiritigenin extract. Recrystallize with methanol-water to obtain 0.3521 g of white liquiritigenin, with a purity of 96.03% (HPLC method). Example 6:

[0037] Take 1 kg of Glycyrrhiza glabra L., and carry out enzyme inactivation treatment with steam at 160 °C for 6 min. After enzyme inactivation, the licorice is manually peeled to remove the darker outer skin part, and the lighter-colored licorice core is obtained. The licorice core is dried at 60 °C, pulverized and sieved through a 60-mesh sieve. Add 5 times the amount of 2 mol / L hydrochloric acid to the dried licorice powder, and carry out acid hydrolysis at 60 °C for 4 h. After acid hydrolysis is completed, filter to obtain acid hydrolysis residue and acid hydrolysis solution. The acid hydrolysis residue is extracted with 5 times the amount of 95% ethanol at 78 °C for 2 h, and extracted repeatedly 3 times. The acid hydrolysis solution is extracted with ethyl acetate 3 times. The solvents are recovered respectively, and the ethanol extract of the acid hydrolysis residue and the ethyl acetate extract of the acid hydrolysis solution are combined to obtain liquiritigenin extract. Recrystallize with methanol-water to obtain 1.1227 g of white liquiritigenin, with a purity of 97.85% (HPLC method).

[0038] The parts not detailed in the present invention are prior art.

[0039] The examples selected herein for disclosing the invention object of the present invention are considered suitable at present. However, it should be understood that the present invention is intended to include all changes and improvements of all examples falling within the scope of this concept and invention.

Claims

1. A method for preparing glycyrrhizin based on enzyme inhibition treatment and peeling process, characterized in that: The preparation method specifically comprises the following steps: The first step, enzyme inhibition treatment: The cleaned licorice is dried and cut into small segments of 10 to 20 cm in length, and then the licorice segments are placed in a superheated steam environment, or processed in a microwave environment, or immersed in a solution containing enzyme inhibitors for enzyme inhibition treatment, so as to effectively inhibit the activity of endogenous enzymes in licorice, thereby reducing the generation of pigments caused by enzymatic oxidation reactions, and ensuring the quality and stability of licorice in subsequent processing; Step 2: Peeling: After the licorice treated with enzyme inhibition is taken out from the superheated steam or microwave or enzyme inhibitor solution, the excess water on the surface is drained, and then the dark outer skin of the licorice is removed by manual peeling, mechanical peeling or chemical peeling. This part of the outer skin contains a large amount of pigment. By removing the outer skin, the dissolution of the dark pigment in the subsequent extraction process is reduced, thereby improving the purity and quality of the extract; Step 3: Drying and crushing: The peeled licorice is placed in a microwave device or a hot air drying oven for drying. After drying, the licorice core is cooled to room temperature, then pulverized using a pulverizer, and sieved through a 10-100 mesh screen to obtain licorice powder with uniform particle size for subsequent extraction operations; Step 4: Acid hydrolysis: The crushed licorice powder is added to 5 to 20 times the amount of 0.5 to 3 mol / L hydrochloric acid, sulfuric acid or nitric acid solution for acid hydrolysis to ensure sufficient hydrolysis and avoid the conversion and degradation of glycyrrhizin. After the reaction is completed, the powder is filtered while hot to obtain an acid hydrolysis residue and an acid hydrolysis solution; Step 5: Extraction: Extracting the acid hydrolysis residue with at least one solvent selected from ethanol, methanol, ethyl acetate or water, extracting the acid hydrolysis solution with ethyl acetate, recovering the extract and the solvent of the extract, and combining the solid components to obtain a glycyrrhizin extract; Step 6: Recrystallization: The extract is repeatedly recrystallized using any one of water, ethanol, methanol or acetone or a mixed solvent of any combination of two or more, and the distribution and content of glycyrrhizin are detected by HPLC to finally obtain glycyrrhizin.

2. The method for preparing glycyrrhizin based on enzyme inhibition treatment and peeling process according to claim 1, characterized in that: In the first step, when the liquorice is treated with superheated steam to inhibit enzymes, the treatment temperature is 95° C. to 160° C. and the treatment time is 5 to 20 minutes.

3. The method for preparing glycyrrhizin based on enzyme inhibition treatment and peeling process according to claim 1, characterized in that: In the first step, when the licorice is treated with a microwave environment to inhibit enzymes, the microwave power is 0.4-32 kw and the treatment time is 1-15 min.

4. The method for preparing glycyrrhizin based on enzyme inhibition treatment and peeling process according to claim 1, characterized in that: In the second step, when the licorice after enzyme inhibition treatment is peeled by chemical peeling, the strong alkalinity of sodium hydroxide and potassium hydroxide, or the weak acidity of citric acid and malic acid is used for moistening or soaking treatment.

5. The method for preparing glycyrrhizin based on enzyme inhibition treatment and peeling process according to claim 1, characterized in that: The drying temperature of the liquorice in the third step is 25°C to 100°C.

6. The method for preparing glycyrrhizin based on enzyme inhibition treatment and peeling process according to claim 1, characterized in that: During the acid hydrolysis in the fourth step, the acid hydrolysis temperature is 60° C. to 100° C., and the hydrolysis time is 0.5 to 4 hours.

7. The method for preparing glycyrrhizin based on enzyme inhibition treatment and peeling process according to claim 1, characterized in that: The solid-liquid ratio during extraction in the fifth step is 1:3-1:50, the extraction temperature is 4°C-82°C, the extraction time is 0.2-72 h, and the solvent ratio (aqueous phase: organic phase) during extraction is 1:0.5-1:

3.

8. The method for preparing glycyrrhizin based on enzyme inhibition treatment and peeling process according to claim 1, characterized in that: The recrystallization temperature in the sixth step is controlled at 0°C to 40°C.

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