Isoliquiritigenin preparation method combining enzyme inhibition and peeling treatment

Abstract

The invention discloses an isoliquiritigenin preparation method combining enzyme inhibition and peeling treatment, and relates to the technical field of natural product extraction. Enzyme deactivation treatment and peeling processes are innovatively introduced, so that enzymatic browning and pigment migration are inhibited from the source, the color depth of isoliquiritigenin is effectively reduced, the product is bright yellow, and the quality of isoliquiritigenin is improved. Meanwhile, due to the optimized decolorizing effect, the use of a decolorizing agent is reduced, the production efficiency is greatly improved, the cost is reduced, the environmental protection property and the industrial feasibility of the process are ensured, and compared with a traditional process, the process disclosed by the invention not only simplifies the flow, improves the product purity, but also remarkably enhances the market competitiveness; and a technical guarantee is provided for realizing green and efficient production and internationalized expansion of enterprises.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product extraction, and particularly to a method for preparing isoliquiritigenin by combining enzyme inhibition and peeling treatment. Background Art

[0002] Licorice (Glycyrrhizae Radix et Rhizoma) is the dried root and rhizome of the leguminous plant Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., or Glycyrrhiza glabra L., and is one of the important traditional Chinese medicines widely used in the field of traditional Chinese medicine. It is mild in nature and sweet in taste, and has various effects such as invigorating the spleen and replenishing qi, relieving spasm and pain, and coordinating various medicines, and has always been highly regarded and widely used in the medical field.

[0003] Isoliquiritigenin is a flavonoid compound with various biological activities and is widely present in licorice. In recent years, with the in-depth study of its pharmacological effects, it has been found that isoliquiritigenin has significant pharmacological effects such as anti-inflammatory, hepatoprotective, and anti-tumor effects, and thus has attracted wide attention in the pharmaceutical, cosmetic, and food industries. However, since isoliquiritigenin itself is yellow or orange-yellow, in some application scenarios, light-colored isoliquiritigenin better meets the product design requirements. Especially in the high-end market, light-colored isoliquiritigenin is gradually becoming a market demand due to its higher flexibility in formulation applications and better consumer acceptance. Therefore, developing a preparation process for high-purity and light-colored isoliquiritigenin has become a research direction with important application prospects.

[0004] Traditional methods for extracting isoliquiritigenin mainly rely on ethanol ultrasonic extraction. Although this method is simple to operate, it has certain limitations in extraction efficiency and purity. To improve the extraction efficiency of isoliquiritigenin, researchers have proposed various improvement methods. For example, Liu Xiaona introduced a new extraction process in the "Study on the High-Efficiency Extraction, Separation, and Purification Process of Isoliquiritigenin", which significantly improved the extraction rate of isoliquiritigenin by hydrolyzing isoliquiritin in licorice with hydrochloric acid or cellulase.

[0005] Chinese Patent, with the publication number CN101328115A, application date July 30, 2008, and patent name "Method for Extracting Isoliquiritigenin from Licorice", discloses a method for extracting isoliquiritigenin by acid hydrolysis and alkaline ring-opening extraction. It uses hydrochloric acid to hydrolyze glycyrrhizin and isoliquiritin into liquiritigenin and isoliquiritigenin, and converts liquiritigenin into isoliquiritigenin under alkaline conditions, further enriching the source of isoliquiritigenin.

[0006] However, the above methods all have some deficiencies in the process design. For example, these methods usually directly crush and extract licorice without considering the possible browning phenomenon that may occur during the storage and processing of licorice. In addition, there are significant color differences between the outer skin and the core of licorice, which can easily lead to a darker color of the final product during the extraction process and require additional decolorization treatment. Traditional decolorization methods rely on a large amount of decolorizing agents. Although they can improve the product appearance, they are often accompanied by problems such as increased costs, loss of active ingredients, and introduction of impurities, ultimately affecting the product purity and quality.

[0007] Therefore, how to prepare high-purity isoliquiritigenin while increasing the extraction rate and reducing or avoiding decolorization treatment has become an important research topic in this field. Summary of the Invention

[0008] To overcome the deficiencies in the background technology, the present invention provides a method for preparing isoliquiritigenin by combining enzyme inhibition and peeling treatment. By introducing enzyme inactivation treatment and peeling treatment, the present invention effectively reduces enzymatic browning and the dissolution and migration of pigments, thereby reducing the color depth of isoliquiritigenin, etc.

[0009] To achieve the above-mentioned invention purpose, the present invention adopts the following technical solutions: A method for preparing isoliquiritigenin by combining enzyme inhibition and peeling treatment, the preparation method specifically includes the following steps: The first step, enzyme inactivation treatment: First, dry the cleaned licorice, then cut it into small sections with a length of 10-20 cm, and then place the licorice sections in a superheated steam environment, or process them in a microwave device, or soak them in a solution containing an enzyme inhibitor for enzyme inactivation 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 in subsequent processing steps; The second step, licorice peeling: After completing the enzyme inactivation treatment, take out the licorice from the superheated steam or microwave device or enzyme inhibitor solution, and drain the water on its surface, and then remove the dark outer skin of the licorice by manual or mechanical or chemical methods to reduce the dissolution amount of pigments in the subsequent extraction process, thereby improving the purity and quality of the extract; The third step, drying and crushing: Put the peeled licorice into a microwave device or a hot air drying oven for drying treatment. After drying, cool the licorice core to room temperature, and then use a crusher to crush it and sieve it through a 10-100 mesh sieve to obtain licorice powder with uniform particle size for subsequent extraction operations; The fourth step, acid hydrolysis: Add the pulverized 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 reaction to fully hydrolyze the glycoside components into the corresponding aglycones. After the reaction is completed, filter while it is hot to obtain the acid water extract; Step 5, Conversion: Slowly add any one or a combination of two or more of ammonia water, potassium hydroxide, sodium hydroxide, or calcium hydroxide to the acid-hydrolyzed solution as a conversion reagent, and carry out the conversion under the condition of pH 12 - 13. After the conversion is completed, filter the conversion system, and add any one or a combination of two or more of sulfuric acid, hydrochloric acid, nitric acid, citric acid, or malic acid to the filtrate as a neutralizing reagent for neutralization. Then, obtain the base conversion product after concentration and drying; Step 6, Organic solvent extraction: Add 3 - 50 times the amount of 50% - 100% ethanol, methanol, or acetone solution to the base conversion product as an extraction solvent, and repeatedly extract the base conversion product 1 - 3 times to effectively extract isoliquiritigenin from the base conversion product. After concentration and drying, obtain the crude extract of isoliquiritigenin; Step 7, Hot water washing: Mix the crude extract of isoliquiritigenin with 1 - 5 times the amount of hot water at 70°C - 100°C, fully disperse it, and then filter. Repeat this process 1 - 5 times, and discard the filtrate to remove impurities and pigments; Step 8, Recrystallization: Use any two or more of water, methanol, ethanol, acetone, hydrochloric acid, or sulfuric acid as a recrystallization solvent for repeated recrystallization to further improve the purity of isoliquiritigenin, and finally obtain yellow isoliquiritigenin.

[0010] In the method for preparing isoliquiritigenin by combining enzyme inhibition and peeling treatment, when using superheated steam environment to inactivate enzymes in licorice in the first step, the temperature for enzyme inactivation treatment is 95°C - 160°C, and the time is 5 - 20 minutes.

[0011] In the method for preparing isoliquiritigenin by combining enzyme inhibition and peeling treatment, when using a microwave device to inactivate enzymes in licorice in the first step, the power for enzyme inactivation treatment is 0.4 - 32 kw, and the treatment time is 1 - 15 minutes.

[0012] In the method for preparing isoliquiritigenin by combining enzyme inhibition and peeling treatment, when treating the enzyme-inhibited licorice by chemical peeling method in the second step, utilize the strong alkalinity of sodium hydroxide and potassium hydroxide or the weak acidity of citric acid and malic acid to carry out peeling after moistening or soaking.

[0013] In the method for preparing isoliquiritigenin by combining enzyme inhibition and peeling treatment, the drying temperature of the hot air drying oven in the third step is 25°C - 100°C.

[0014] The preparation method of isoliquiritigenin combining enzyme inhibition and peeling treatment. During the acidolysis reaction in the fourth step, the acid hydrolysis temperature is 45 °C to 100 °C, and the hydrolysis time is 0.5 to 4 hours.

[0015] The preparation method of isoliquiritigenin combining enzyme inhibition and peeling treatment. During the conversion in the fifth step, the conversion temperature is 30 °C to 80 °C, and the conversion time is 20 to 90 min.

[0016] The preparation method of isoliquiritigenin combining enzyme inhibition and peeling treatment. During the extraction in the sixth step, the temperature is 4 °C to 82 °C.

[0017] The preparation method of isoliquiritigenin combining enzyme inhibition and peeling treatment. During the recrystallization in the eighth step, the temperature is controlled at 0 °C to 40 °C.

[0018] Adopting the above technical solution, the present invention has the following advantages: By innovatively introducing enzyme inactivation treatment and peeling process, the present invention inhibits enzymatic browning and pigment migration from the source, effectively reduces the color depth of isoliquiritigenin, makes the product present a bright yellow color, meets the needs of the high-end market. At the same time, the optimized decolorization effect reduces the use of decolorizing agents, greatly improves production efficiency and reduces costs, ensuring the environmental protection and industrial feasibility of the process. Compared with the traditional process, the present invention not only simplifies the process and improves the product purity, but also significantly enhances the market competitiveness, providing technical support for the enterprise to achieve green and efficient production and international expansion. Brief Description of the Drawings

[0019] Figure 1 It is the flow chart of the preparation of isoliquiritigenin in the embodiment of the present invention. Detailed Embodiments

[0020] The present invention can be more detailedly explained through the following embodiments, and the present invention is not limited to the following embodiments; Based on the principle of inhibiting enzymatic browning and blocking pigment migration, the present invention optimizes the color and purity of isoliquiritigenin from the root. The polyphenol oxidase in licorice raw materials is easy to react with polyphenolic substances to generate brown substances, which is the main reason for the deepening of the product color. The present invention quickly inactivates the enzyme activity through steam or microwave enzyme inactivation treatment to prevent the formation of browning substances. At the same time, by peeling, the surface tissue rich in pigments is removed, blocking the pigments from entering the extraction system and reducing the pigment content in the extraction solution.

[0021] A preparation method of isoliquiritigenin combining enzyme inhibition and peeling treatment according to the present invention, the preparation method specifically includes the following steps: The first step, enzyme inactivation treatment: First, dry the cleaned licorice, then cut it into small sections with a length of 10 - 20 cm. Next, place the licorice sections in a superheated steam environment, or process them in a microwave device, or soak them in a solution containing enzyme inhibitors for enzyme inactivation treatment 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 in subsequent processing steps; During implementation, when using a superheated steam environment for enzyme inactivation treatment of licorice, the temperature for enzyme inactivation treatment is 95°C - 160°C, and the time is 5 - 20 minutes. When using a microwave device for enzyme inactivation treatment of licorice, the power for enzyme inactivation treatment is 0.4 - 32 kw, and the treatment time is 1 - 15 minutes; Second step: Licorice skin peeling: After completing the enzyme inactivation treatment, take out the licorice from the superheated steam or microwave device or enzyme inhibitor solution, and drain the water on its surface. Then, remove the dark outer skin of the licorice by manual, mechanical or chemical methods to reduce the dissolution of pigments in the subsequent extraction process, thereby improving the purity and quality of the extract. During implementation, when treating the enzyme-inhibited licorice by chemical skin peeling, utilize the strong alkalinity of sodium hydroxide and potassium hydroxide or the weak acidity of citric acid and malic acid to perform skin peeling after moistening or soaking; Third step: Drying and grinding: Put the skinned licorice into a microwave device or a hot air drying oven for drying treatment. After drying, cool the licorice core to room temperature, then use a pulverizer to grind 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. During implementation, the drying temperature of the hot air drying oven is 25°C - 100°C; Fourth step: Acid hydrolysis: Add the ground 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 reaction to fully hydrolyze the glycoside components into the corresponding aglycones. After the reaction is completed, filter while it is hot to obtain an acid water extract. During implementation, during the acid hydrolysis reaction, the acid hydrolysis temperature is 45°C - 100°C, and the hydrolysis time is 0.5 - 4 hours; Fifth step: Conversion: Slowly add any one or a combination of two or more of ammonia water, potassium hydroxide, sodium hydroxide, or calcium hydroxide to the acid-hydrolyzed solution as a conversion reagent, and perform conversion under the condition of pH value 12 - 13. After the conversion is completed, filter the conversion system, and add any one or a combination of two or more of sulfuric acid, hydrochloric acid, nitric acid, citric acid, or malic acid to the filtrate as a neutralization reagent for neutralization, and then obtain an alkali conversion product after concentration and drying. During implementation, the conversion temperature during conversion is 30°C - 80°C, and the conversion time is 20 - 90 min.

[0022] Step 6: Organic solvent extraction: Adding 3 to 50 times the amount of 50% to 100% ethanol or methanol or acetone solution as an extraction solvent to the alkali conversion product, repeatedly extracting the alkali conversion product 1 to 3 times, thereby effectively extracting isoliquiritigenin in the alkali conversion product, and obtaining a crude isoliquiritigenin extract after concentration and drying; when implementing, the temperature during extraction is 4°C to 82°C; Step 7: Hot water washing: Mix the crude isoliquiritigenin extract with 1 to 5 times the amount of hot water at 70°C to 100°C, fully disperse it, and filter it. Repeat this process 1 to 5 times, and discard the filtrate to remove impurities and pigments. Step 8: Recrystallization: Use any two or more mixed solvents of water, methanol, ethanol, acetone, hydrochloric acid or sulfuric acid as recrystallization solvents, perform repeated recrystallization, further improve the purity of isoliquiritigenin, and finally obtain yellow isoliquiritigenin. During implementation, the temperature during recrystallization is controlled at 0°C to 40°C.

[0023] In specific practice, combined with the attached Figure 1 The present invention discloses a method for preparing isoliquiritigenin combined with enzyme inhibition and peeling treatment, and the preparation method specifically comprises the following steps: The first step, enzyme inactivation treatment: The licorice raw material is treated with enzyme inhibition by heating, pH adjustment, adding inhibitors or rapid drying to inhibit the activity of its endogenous enzymes and reduce the generation of pigments caused by enzymatic oxidation; During implementation, the cleaned licorice is first dried and then cut into small segments of 10 to 20 cm in length for subsequent processing. Next, the licorice segments are placed in a superheated steam environment of 95 to 160°C, or processed in a microwave device with a power of 0.4 to 32kW, or immersed in a solution containing enzyme inhibitors for enzyme inactivation. These treatment methods are all aimed at effectively inhibiting the activity of endogenous enzymes in licorice, thereby reducing the production of pigments caused by enzymatic oxidation reactions, and ensuring the quality and stability of licorice in subsequent processing links; During implementation, when the licorice raw material is subjected to enzyme inhibition treatment by heating, the licorice is subjected to enzyme inactivation treatment by steam, and the temperature of the enzyme inactivation treatment is 80° C. to 100° C., and the time is 1 to 30 minutes; When the licorice raw material is subjected to enzyme inhibition treatment by heating, the licorice is subjected to enzyme inactivation treatment by microwave method, the enzyme inactivation treatment power is 1 to 80 kw, and the treatment time is 1 to 45 minutes; In specific practice, steam, microwave, or other enzyme-inhibiting technologies can be used to perform enzyme inactivation treatment on licorice to obtain enzyme-inactivated licorice. Advantages of the enzyme inactivation treatment: The enzyme inhibition treatment is carried out on licorice raw materials to inhibit the activity of endogenous enzymes in them and reduce the generation of pigments caused by enzymatic oxidation. This step effectively prevents the accumulation of impurities and pigments generated by the action of enzymes during the extraction process, ensuring that the color of the extract remains stable and the purity is improved. This process provides reliable technical support for the subsequent production of light isoliquiritigenin, contributing to the quality performance and competitiveness of the final product in the market; During implementation, when using steam to perform enzyme inactivation treatment on licorice, the temperature of the enzyme inactivation treatment is 80°C to 100°C, and the temperature of the enzyme inactivation treatment is preferably 90°C to 100°C; when using the microwave method to perform enzyme inactivation treatment on licorice, the power of the enzyme inactivation treatment is 1 to 80 kw; further, during implementation, the treatment time is preferably 3 to 15 min; During implementation, the enzyme inactivation treatment methods include but are not limited to the steam method and the microwave method, and chemical methods or high-pressure methods can also be used for enzyme inactivation treatment; Second step, licorice skin removal: The enzyme-inhibited licorice is processed by manual skin removal, mechanical skin removal, or chemical skin removal methods to effectively remove the outer skin rich in pigments and reduce the dissolution of pigments in the outer skin into the extraction solution; During implementation, after the enzyme inactivation treatment is completed, the licorice is taken out from the superheated steam, microwave equipment, or enzyme inhibitor solution, and the moisture on its surface is drained. Then, the dark outer skin of the licorice is removed by manual, mechanical, or chemical methods. This outer skin contains a high concentration of pigments. Removing the outer skin helps to significantly reduce the dissolution amount of pigments during the subsequent extraction process, thereby improving the purity and quality of the extract; During implementation, when using the chemical skin removal method to process the enzyme-inhibited licorice, the strong alkalinity of sodium hydroxide and potassium hydroxide or the weak acidity of citric acid and malic acid are used for skin removal after moistening or infiltration; In specific practice, the enzyme-inactivated licorice is skinned by manual skin removal method, mechanical skin removal method, or chemical skin removal method to remove the darker outer skin part and obtain lighter-colored skinned licorice. Advantages of skin removal: Although the enzyme inhibition treatment can inhibit the generation of some pigments, licorice still contains a large amount of pigments, especially mainly concentrated in the outer skin part of licorice, while the pigment content in the core part is relatively low. Therefore, by skinning the enzyme-inhibited licorice, the outer skin rich in pigments can be effectively removed, reducing the dissolution of pigments in the outer skin into the extraction solution, thereby greatly reducing the chromaticity of the extraction solution. This not only improves the visual purity of the final extract but also significantly reduces the difficulty and cost required in the subsequent purification process; Third step, drying and pulverizing: The skinned licorice is dried, and after drying, it is pulverized by a pulverizer and screened to finally obtain dry licorice powder; During implementation, the peeled licorice is placed in a microwave device or a hot air drying oven and dried at a moderate temperature of 25-100 °C to prevent the effective components in the licorice from being damaged or degraded by excessive temperature. After drying, the licorice core is cooled to room temperature, then crushed using a crusher and screened through a sieve with a mesh size of 10-100 meshes to obtain licorice powder with uniform particle size for subsequent extraction operations; During implementation, the drying of the peeled licorice is carried out using an oven or microwave, and the drying temperature of the oven is 25 °C - 100 °C; In specific practice, the peeled licorice core is dried at 25 °C - 100 °C for 0-24 hours and then crushed. After crushing, it is sieved through a 10-100 mesh sieve to obtain dry licorice powder. Through drying, crushing, and sieving, relatively consistent dry licorice powder can be obtained, ensuring the maximum retention of effective components during the extraction process and improving process stability; Fourth step, acid hydrolysis: Add the acid hydrolysis solvent to the dry licorice powder for acid hydrolysis reaction; During implementation, add the crushed licorice powder to a 0.5-3 mol / L hydrochloric acid, sulfuric acid, or nitric acid solution with a volume 5-20 times that of the licorice powder, and carry out the acid hydrolysis reaction at 60-100 °C for 0.5-4 hours to ensure the full hydrolysis of glycoside components. After the reaction is completed, filter while it is hot to obtain the acid aqueous extract; During implementation, during the acidolysis reaction, the acid concentration is 0.5-3 mol / L, the dosage of the acid solution is 5-20 times the amount of the licorice powder, the acid hydrolysis temperature is 45 °C - 100 °C, and the hydrolysis time is 0.5-4 hours.

[0024] In specific practice, add 0.5-3 mol / L hydrochloric acid to the dry licorice powder for acidolysis reaction. The dosage of the acid solution is 5-20 times the amount of the licorice powder. The reaction temperature is controlled at 45 °C - 100 °C, and the time is 0.5-4 h. The acidolysis process helps to break the glycosidic bonds in the licorice tissue structure and components, releasing more isoliquiritigenin-related components (licoricidin, isoliquiritigenin, etc.); Fifth step, conversion: Slowly add the conversion reagent to the acidolysis solution, adjust the pH value to 12-13 for the conversion reaction. After the conversion is completed, filter the conversion system, add acid to the filtrate for neutralization, and obtain the base conversion product after concentration and drying; During implementation, any one or a combination of two or more of ammonia water, potassium hydroxide, sodium hydroxide, or calcium hydroxide is slowly added to the acid-hydrolyzed solution as a conversion reagent, and the pH value is adjusted to 12 - 13. The reaction is carried out at 30 °C - 80 °C for 20 - 90 min. After the conversion is completed, the conversion system is filtered, and the filtrate is neutralized with any one or a combination of two or more of sulfuric acid, hydrochloric acid, nitric acid, citric acid, or malic acid as a neutralizing reagent. Then, after concentration and drying, the base-converted product is obtained; During implementation, the conversion reagent is any one or a combination of two or more of ammonia water, potassium hydroxide, sodium hydroxide, or calcium hydroxide, the conversion temperature is 30 °C - 80 °C, and the conversion time is 20 - 90 min; the acid used to neutralize the alkaline solution is any one or a combination of two or more of sulfuric acid, hydrochloric acid, nitric acid, citric acid, or malic acid.

[0025] In specific practice, any one or a combination of two or more of ammonia water, potassium hydroxide, sodium hydroxide, and calcium hydroxide is slowly added to the acid-hydrolyzed solution, the pH value is adjusted to 12 - 13, and the conversion reaction is carried out. The reaction temperature is 30 °C - 80 °C, and the time is 20 - 90 min. The role of the alkaline environment: liquiritigenin and isoliquiritigenin can be dissolved in a strong alkaline solution, and to a certain extent, liquiritigenin is converted into isoliquiritigenin.

[0026] After the conversion is completed, the conversion system is filtered, and the filtrate is neutralized with sulfuric acid or hydrochloric acid, nitric acid, citric acid, or malic acid. After concentration and drying, the base-converted product is obtained. The role of adding acid for neutralization: terminate the conversion reaction and inhibit the ionization of isoliquiritigenin, facilitating the subsequent extraction of isoliquiritigenin.

[0027] Step 6: Organic solvent extraction: The base-converted product is repeatedly extracted with an extraction solvent to effectively dissolve isoliquiritigenin in the base-converted product. After concentration and drying, a crude extract of isoliquiritigenin is obtained; During implementation, 3 - 50 times the amount of a 50% - 100% ethanol, methanol, or acetone solution is added to the base-converted product as an extraction solvent, and the base-converted product is repeatedly extracted 1 - 3 times under the condition of 4 °C - 82 °C to effectively extract isoliquiritigenin in the base-converted product. After concentration and drying, a crude extract of isoliquiritigenin is obtained; During implementation, the extraction solvent is any one of a 50% - 100% ethanol, methanol, or acetone solution.

[0028] In specific practice, 3 - 50 times the amount of a 50% - 100% ethanol, methanol, or acetone solution is used to extract the base-converted product at 4 °C - 82 °C for 0.2 - 72 h. After extracting 1 - 3 times, the filtrates are combined, and after concentration and drying, a crude extract of isoliquiritigenin is obtained; Step 7, Hot water washing: Mix the crude liquiritigenin extract with hot water, filter it after full dispersion, repeat 1 - 5 times, and discard the filtrate to remove impurities and pigments. During implementation, mix the crude liquiritigenin extract with 1 - 5 times the amount of hot water at 70°C - 100°C, filter it after full dispersion, repeat 1 - 5 times, and discard the filtrate to remove impurities and pigments. In specific practice, mix 1 - 5 times the amount of hot water at 70°C - 100°C with the crude liquiritigenin extract, filter it after full dispersion, repeat 1 - 5 times, and discard the filtrate to remove impurities and pigments. Step 8, Recrystallization: Use a recrystallization solvent and perform repeated recrystallization under the condition of 0°C - 40°C to further improve the purity of liquiritigenin and finally obtain yellow liquiritigenin.

[0029] During implementation, use any one or a mixture of two or more of water, methanol, ethanol, acetone, hydrochloric acid, or sulfuric acid as the recrystallization solvent, and perform repeated recrystallization under the condition of 0°C - 40°C to further improve the purity of liquiritigenin and finally obtain yellow liquiritigenin.

[0030] During implementation, the recrystallization solvent is any one or a mixture of two or more of water, methanol, ethanol, acetone, hydrochloric acid, or sulfuric acid.

[0031] In specific practice, use water, methanol, ethanol, acetone, hydrochloric acid, sulfuric acid, or their mixed solvent, and perform repeated recrystallization under the condition of 0°C - 40°C to further improve the purity of liquiritigenin and finally obtain yellow liquiritigenin.

[0032] During specific implementation, the first step of enzyme inhibition treatment, the sixth step of extraction, and the eighth step of purification treatment can be optimized using different temperatures or solvent systems respectively to improve the purity of liquiritigenin or reduce the color depth.

[0033] Specific embodiments of the present invention are as follows: Example 1:

[0034] Take 1 kg of Glycyrrhiza uralensis Fisch., and perform enzyme inactivation treatment with microwave at 0.4 kw for 10 min. After enzyme inactivation, the licorice is manually peeled to remove the darker outer skin part, obtaining a lighter-colored licorice core. The licorice core is dried at 45 °C and then pulverized to 40 mesh. The licorice powder is mixed with 20 times the amount of 0.5 mol / L hydrochloric acid and acid-hydrolyzed at 90 °C for 2 h. After the acid hydrolysis is completed, sodium hydroxide particles are slowly added to adjust the pH to 12, and the reaction is carried out at 80 °C for 45 min. After the reaction is completed, it is filtered, and the filtrate is cooled to room temperature and neutralized with sulfuric acid. After concentration and drying, an alkali-converted product is obtained. 5 times the amount of 95% ethanol is added to the alkali-converted product and extracted at 78 °C for 1 h. After extraction 3 times, the filtrates are combined, concentrated and dried to obtain a crude extract of isoliquiritigenin. Finally, 5 times the amount of 70 °C hot water is mixed with the crude extract of isoliquiritigenin, fully dispersed and then filtered, and this is repeated 3 times, and the filtrate is discarded. Using a mixed solvent of water, ethanol and sulfuric acid, dissolution and crystallization are carried out at 4 °C to obtain 4.3527 g of yellow crystalline isoliquiritigenin with a purity of 95.51% (HPLC method). Example 2:

[0035] Take 1 kg of Glycyrrhiza uralensis Fisch., and perform enzyme inactivation treatment with microwave at 0.7 kw for 6 min. After enzyme inactivation, the licorice is manually peeled to remove the darker outer skin part, obtaining a lighter-colored licorice core. The licorice core is dried at 45 °C and then pulverized to 40 mesh. The licorice powder is mixed with 10 times the amount of 1 mol / L hydrochloric acid and acid-hydrolyzed at 90 °C for 2 h. After the acid hydrolysis is completed, sodium hydroxide particles are slowly added to adjust the PH to 12, and the reaction is carried out at 90 °C for 30 min. After the reaction is completed, it is filtered, and the filtrate is cooled to room temperature and neutralized with sulfuric acid. After concentration and drying, an alkali-converted product is obtained. 15 times the amount of 95% ethanol is added to the alkali-converted product and extracted at 78 °C for 1 h. After extraction 2 times, the filtrates are combined, concentrated and dried to obtain a crude extract of isoliquiritigenin. Finally, 5 times the amount of 70 °C hot water is mixed with the crude extract of isoliquiritigenin, fully dispersed and then filtered, and this is repeated 3 times, and the filtrate is discarded. Using a mixed solvent of water, methanol and sulfuric acid, dissolution and crystallization are carried out at 4 °C to obtain 3.9260 g of yellow crystalline isoliquiritigenin with a purity of 94.75% (HPLC method). Example 3:

[0036] Take 1 kg of Glycyrrhiza uralensis Fisch., and carry out enzyme inactivation treatment with microwave at 32 kw for 2 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 and then crushed to 40 mesh. The licorice powder is mixed with 4 times the amount of 3 mol / L hydrochloric acid and acid-hydrolyzed at 80 °C for 2.5 h. After the acid hydrolysis is completed, sodium hydroxide particles are slowly added to adjust the pH to 13, and the reaction is carried out at 45 °C for 2.5 h. After the reaction is completed, filtration is carried out. After the filtrate is cooled to room temperature, it is neutralized with sulfuric acid, concentrated and dried to obtain the base conversion product. 20 times the amount of 80% ethanol is added to the base conversion product and extracted at 80 °C for 1 h. After extraction twice, the filtrates are combined, concentrated and dried to obtain the crude extract of isoliquiritigenin. Finally, 3 times the amount of 70 °C hot water is mixed with the crude extract of isoliquiritigenin, fully dispersed and then filtered, and this is repeated 3 times, and the filtrate is discarded. Using a mixed solvent of water, methanol and sulfuric acid, dissolution and crystallization are carried out at 4 °C to obtain 4.0380 g of yellow crystalline isoliquiritigenin with a purity of 96.24% (HPLC method). Implementation: 4

[0037] Take 1 kg of Glycyrrhiza uralensis Fisch., and carry out enzyme inactivation treatment with steam at 98 °C for 20 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 and then crushed to 40 mesh. The licorice powder is mixed with 8 times the amount of 1.5 mol / L hydrochloric acid and acid-hydrolyzed at 90 °C for 2 h. After the acid hydrolysis is completed, sodium hydroxide particles are slowly added to adjust the pH to 13, and the reaction is carried out at 60 °C for 90 min. After the reaction is completed, filtration is carried out. After the filtrate is cooled to room temperature, it is neutralized with sulfuric acid, concentrated and dried to obtain the base conversion product. 10 times the amount of 80% ethanol is added to the base conversion product and extracted at 80 °C for 2 h. After extraction twice, the filtrates are combined, concentrated and dried to obtain the crude extract of isoliquiritigenin. Finally, 5 times the amount of 70 °C hot water is mixed with the crude extract of isoliquiritigenin, fully dispersed and then filtered, and this is repeated 3 times, and the filtrate is discarded. Using a mixed solvent of water, methanol and sulfuric acid, dissolution and crystallization are carried out at 4 °C to obtain 4.1586 g of yellow crystalline isoliquiritigenin with a purity of 95.76% (HPLC method). Implementation: 5

[0038] Take 1 kg of Glycyrrhiza inflata, and carry out enzyme inactivation treatment with steam at 105 °C for 15 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 and then crushed to 40 mesh. The licorice powder is mixed with 4 times the amount of 3 mol / L hydrochloric acid and acid-hydrolyzed at 80 °C for 2.5 h. After the acid hydrolysis is completed, sodium hydroxide particles are slowly added to adjust the pH to 13, and the reaction is carried out at 45 °C for 2.5 h. After the reaction is completed, it is filtered, and the filtrate is cooled to room temperature and then neutralized with sulfuric acid. After concentration and drying, the alkali-converted product is obtained. 20 times the amount of 70% ethanol is added to the alkali-converted product and extracted at 75 °C for 2 h. After extraction twice, the filtrates are combined, concentrated and dried to obtain the crude extract of isoliquiritigenin. Finally, 5 times the amount of 70 °C hot water is mixed with the crude extract of isoliquiritigenin, fully dispersed and then filtered, and the operation is repeated 5 times, and the filtrate is discarded. Using a mixed solvent of water, methanol and sulfuric acid, dissolution and crystallization are carried out at 4 °C to obtain 0.5883 g of yellow crystalline isoliquiritigenin with a purity of 96.13% (HPLC method). Example 6:

[0039] Take 1 kg of Glycyrrhiza glabra, 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 and then crushed to 40 mesh. The licorice powder is mixed with 5 times the amount of 2 mol / L hydrochloric acid and acid-hydrolyzed at 100 °C for 0.5 h. After the acid hydrolysis is completed, sodium hydroxide particles are slowly added to adjust the pH to 13, and the reaction is carried out at 80 °C for 45 min. After the reaction is completed, it is filtered, and the filtrate is cooled to room temperature and then neutralized with sulfuric acid. After concentration and drying, the alkali-converted product is obtained. 5 times the amount of 80% ethanol is added to the alkali-converted product and extracted at 80 °C for 2 h. After extraction three times, the filtrates are combined, concentrated and dried to obtain the crude extract of isoliquiritigenin. Finally, 5 times the amount of 70 °C hot water is mixed with the crude extract of isoliquiritigenin, fully dispersed and then filtered, and the operation is repeated 5 times, and the filtrate is discarded. Using a mixed solvent of water, methanol and sulfuric acid, dissolution and crystallization are carried out at 4 °C to obtain 0.9472 g of yellow crystalline isoliquiritigenin with a purity of 94.38% (HPLC method). Example 7:

[0040] Take 1 kg of Glycyrrhiza uralensis Fisch., and perform enzyme inactivation treatment with steam at 130 °C for 7 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 and then ground to 40 mesh. The licorice powder is mixed with 5 times the amount of 2 mol / L hydrochloric acid and acid-hydrolyzed at 70 °C for 3 h. After the acid hydrolysis is completed, sodium hydroxide particles are slowly added to adjust the pH to 13, and the reaction is carried out at 70 °C for 80 min. After the reaction is completed, it is filtered, and the filtrate is cooled to room temperature and neutralized with sulfuric acid, and then concentrated and dried to obtain the alkali-converted product. 10 times the amount of 70% ethanol is added to the alkali-converted product and extracted at 70 °C for 1.5 h. After extraction 2 times, the filtrates are combined, concentrated and dried to obtain the crude extract of isoliquiritigenin. Finally, 3 times the amount of 70 °C hot water is mixed with the crude extract of isoliquiritigenin, fully dispersed and then filtered, and this is repeated 5 times, and the filtrate is discarded. Using a mixed solvent of water, methanol and sulfuric acid, dissolution and crystallization are carried out at 25 °C to obtain 3.6469 g of yellow crystalline isoliquiritigenin with a purity of 96.82% (HPLC method). Example 8:

[0041] Take 1 kg of Glycyrrhiza uralensis Fisch., and perform 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 and then ground to 40 mesh. The licorice powder is mixed with 5 times the amount of 2 mol / L hydrochloric acid and acid-hydrolyzed at 60 °C for 4 h. After the acid hydrolysis is completed, sodium hydroxide particles are slowly added to adjust the pH to 13, and the reaction is carried out at 60 °C for 1.5 h. After the reaction is completed, it is filtered, and the filtrate is cooled to room temperature and neutralized with sulfuric acid, and then concentrated and dried to obtain the alkali-converted product. 10 times the amount of 80% ethanol is added to the alkali-converted product and extracted at 25 °C for 72 h. After extraction 3 times, the filtrates are combined, concentrated and dried to obtain the crude extract of isoliquiritigenin. Finally, 5 times the amount of 80 °C hot water is mixed with the crude extract of isoliquiritigenin, fully dispersed and then filtered, and this is repeated 4 times, and the filtrate is discarded. Using a mixed solvent of water, methanol and sulfuric acid, dissolution and crystallization are carried out at 4 °C to obtain 3.7086 g of yellow crystalline isoliquiritigenin with a purity of 95.63% (HPLC method). Example 9:

[0042] Take 1 kg of Glycyrrhiza uralensis Fisch., and carry out enzyme inactivation treatment with steam at 105 °C for 15 min. After enzyme inactivation, the color of the outer skin of the licorice was removed by manual peeling, and the lighter-colored licorice core was obtained. The licorice core was dried at 60 °C and then crushed to 40 mesh. The licorice powder was mixed with 5 times the amount of 2 mol / L hydrochloric acid and acid-hydrolyzed at 80 °C for 2.5 h. After the acid hydrolysis was completed, sodium hydroxide particles were slowly added to adjust the pH to 13, and the reaction was carried out at 80 °C for 80 min. After the reaction was completed, the mixture was filtered, and the filtrate was cooled to room temperature and neutralized with sulfuric acid. After concentration and drying, an alkali-converted product was obtained. 8 times the amount of 80% methanol was added to the alkali-converted product and extracted at 68 °C for 1 h. After extraction twice, the filtrates were combined, concentrated and dried to obtain a crude extract of isoliquiritigenin. Finally, 5 times the amount of hot water at 100 °C was mixed with the crude extract of isoliquiritigenin, and after sufficient dispersion, it was filtered and repeated 3 times, and the filtrate was discarded. A yellow crystalline isoliquiritigenin (4.0380 g, purity 92.28% (HPLC method)) was obtained by dissolving and crystallizing with a mixed solvent of water, methanol and sulfuric acid at 4 °C.

[0043] The parts not detailed in the present invention are prior art.

[0044] The embodiments selected herein for the purpose of disclosing the invention of the present invention are currently considered suitable. However, it should be understood that the present invention is intended to include all variations and improvements of all embodiments within the scope of this concept and invention.

Claims

1. A method for preparing isoliquiritigenin by combining enzyme inhibition and peeling treatment, characterized in that: The preparation method specifically comprises the following steps: The first step, enzyme inactivation treatment: First, the cleaned licorice is dried, and then cut into small segments of 10 to 20 cm in length. The licorice segments are then placed in a superheated steam environment, or processed in a microwave device, or immersed in a solution containing enzyme inhibitors for enzyme inactivation treatment, so as to effectively inhibit the activity of endogenous enzymes in licorice, thereby reducing the production of pigments caused by enzymatic oxidation reactions, and ensuring the quality and stability of licorice in subsequent processing links; Step 2: Licorice peeling: After the enzyme inactivation treatment is completed, the licorice is taken out from the superheated steam or microwave device or enzyme inhibitor solution, and the water on its surface is drained, and then the dark outer skin of the licorice is removed by manual, mechanical or chemical methods to reduce the amount of pigment dissolved in the subsequent extraction process, 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, and then crushed by a grinder, 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 to carry out acid hydrolysis reaction, so that the glycoside components are fully hydrolyzed into corresponding aglycones. After the reaction is completed, it is filtered while hot to obtain an acid water extract; Step 5: Conversion: Slowly add any one of ammonia water, potassium hydroxide, sodium hydroxide or calcium hydroxide, or a combination of two or more thereof as a conversion reagent to the solution after acid hydrolysis, and perform conversion at a pH value of 12 to 13. After the conversion is completed, filter the conversion system, and add any one of sulfuric acid, hydrochloric acid, nitric acid, citric acid or malic acid, or a combination of two or more thereof as a neutralizing reagent to the filtrate for neutralization, and then concentrate and dry to obtain an alkali conversion product; Step 6: Organic solvent extraction: Adding 3 to 50 times the amount of 50% to 100% ethanol or methanol or acetone solution as an extraction solvent to the alkali conversion product, repeatedly extracting the alkali conversion product 1 to 3 times, thereby effectively extracting isoliquiritigenin in the alkali conversion product, and concentrating and drying to obtain a crude isoliquiritigenin extract; Step 7: Hot water washing: Mix the crude isoliquiritigenin extract with 1 to 5 times the amount of hot water at 70°C to 100°C, fully disperse it, and filter it. Repeat this process 1 to 5 times, and discard the filtrate to remove impurities and pigments. Step 8: Recrystallization: Use any two or more mixed solvents of water, methanol, ethanol, acetone, hydrochloric acid or sulfuric acid as recrystallization solvents, perform repeated recrystallization, further improve the purity of isoliquiritigenin, and finally obtain yellow isoliquiritigenin.

2. The method for preparing isoliquiritigenin combined with enzyme inhibition and peeling treatment according to claim 1, characterized in that: In the first step, when the licorice is inactivated by superheated steam, the temperature of the inactivation treatment is 95° C. to 160° C., and the time is 5 to 20 minutes.

3. The method for preparing isoliquiritigenin combined with enzyme inhibition and peeling treatment according to claim 1, characterized in that: In the first step, when microwave equipment is used to inactivate enzymes in liquorice, the inactivation power is 0.4-32 kw, and the treatment time is 1-15 minutes.

4. The method for preparing isoliquiritigenin combined with enzyme inhibition and peeling treatment according to claim 1, characterized in that: In the second step, when the enzyme-inhibited licorice is treated by chemical peeling, the peeling is performed after moistening or soaking by using the strong alkalinity of sodium hydroxide and potassium hydroxide or the weak acidity of citric acid and malic acid.

5. The method for preparing isoliquiritigenin combined with enzyme inhibition and peeling treatment according to claim 1, characterized in that: The drying temperature of the hot air drying oven in the third step is 25°C to 100°C.

6. The method for preparing isoliquiritigenin combined with enzyme inhibition and peeling treatment according to claim 1, characterized in that: During the acid hydrolysis reaction in the fourth step, the acid hydrolysis temperature is 45°C to 100°C, and the hydrolysis time is 0.5 to 4 hours.

7. The method for preparing isoliquiritigenin combined with enzyme inhibition and peeling treatment according to claim 1, characterized in that: The conversion temperature during the conversion in the fifth step is 30° C. to 80° C., and the conversion time is 20 to 90 min.

8. The method for preparing isoliquiritigenin combined with enzyme inhibition and peeling treatment according to claim 1, characterized in that: The temperature during extraction in the sixth step is 4°C to 82°C.

9. The method for preparing isoliquiritigenin combined with enzyme inhibition and peeling treatment according to claim 1, characterized in that: The temperature during recrystallization in the eighth step is controlled at 0°C to 40°C.

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