Licochalcone A preparation method based on enzyme inhibition and peeling technology

Abstract

The invention discloses a licochalcone A preparation method based on an enzyme inhibition and peeling technology, and relates to the technical field of natural product extraction and separation. By combining the enzyme inhibition and peeling technology, oxidation generation of polyphenol and pigment precursors in liquorice is reduced from the source, enzymatic browning is remarkably reduced, the initial purity of an extracting solution is improved, and the yield of the licochalcone A is increased. According to the technical improvement, the complex decolorizing step in the traditional process is effectively simplified, the use amount of a decolorizing agent and the discharge of chemical substances in wastewater are effectively reduced, the environmental protection requirement is met, and the production cost is reduced. Compared with the prior art, through innovative process optimization, comprehensive consideration of environmental protection, cost and efficiency is reflected in the whole process from the source to purification, the production period is shortened, the product purity is improved, and the capacity of adapting to industrial large-scale production is remarkably improved. The method has higher application value and advantages and the like in market competition due to the advantages.

Description

Technical Field

[0001] The present invention relates to the technical field of extraction and separation of natural products, and particularly relates to a preparation method of Glycyrrhiza chalcone A based on enzyme inhibition and peeling technology. Background Art

[0002] Glycyrrhiza chalcone A is a natural flavonoid compound derived from Glycyrrhiza, which has significant anti-inflammatory, whitening and anti-tumor pharmacological effects and is widely used in the fields of medicine, health products and cosmetics. With the rapid growth of the market demand for high-value-added natural products, the research on methods for efficiently and economically extracting and separating Glycyrrhiza chalcone A has attracted much attention. However, there are still multiple technical bottlenecks in the existing processes, including the generation of pigments affecting product purity, the complication of process steps, the high production cost, and the environmental problems brought about by solvent recovery.

[0003] In the prior art, for the Chinese patent with the publication number CN101117311A, the application date of June 20, 2007, the authorization publication number CN100586920C, and the title "Preparation method of high-purity Glycyrrhiza chalcone A", it discloses a method of purification by organic solvent extraction, concentration and combination of macroporous resin and polyamide column chromatography. Although this method can extract and purify Glycyrrhiza chalcone A, the high dependence on organic solvents increases the environmental burden and the difficulty of solvent recovery. In addition, the problem of pigment generation has not been effectively solved, resulting in a darker color of the extract and increasing the complexity of subsequent separation.

[0004] For the Chinese patent with the publication number CN105859538A, the application date of June 8, 2016, the authorization publication number CN105859538B, and the title "A purification method of Glycyrrhiza chalcone A", it uses silica gel column chromatography and recrystallization technology for separation and purification. Although this method has made progress in improving the purity of Glycyrrhiza chalcone A, due to the failure to effectively inhibit pigment generation, its separation process is complex and costly. In addition, the use of a large amount of organic solvents further increases the production cost and environmental risks.

[0005] For the Chinese patent with the publication number CN113880706A, the application date of September 27, 2021, the authorization publication number CN113880706B, and the title "A preparation and detection method of high-purity Glycyrrhiza chalcone A", it proposes a scheme using macroporous resin AB-8, high-speed countercurrent chromatography technology and methanol recrystallization method. Although this method has made progress in improving the purity, its equipment requirements are high and the throughput is small, which limits its application in large-scale industrial production.

[0006] The existing extraction and purification processes still face problems such as pigment generation, complex processes, high production costs and heavy environmental burdens, and are in urgent need of innovation and improvement. Therefore, developing simplified extraction and purification processes to reduce pigment generation, reduce costs and reduce environmental burdens has become a key issue that needs to be urgently addressed in the technical field. Summary of the invention

[0007] In order to overcome the shortcomings of the background technology, the present invention provides a method for preparing licorice chalcone A based on enzyme inhibition and peeling technology. The present invention reduces the generation and migration of pigments by inhibiting oxidase activity and removing the licorice peel, thereby simplifying the subsequent separation and purification steps and improving the production efficiency. The purity of the final product reaches more than 96%.

[0008] In order to achieve the above-mentioned purpose of the invention, the present invention adopts the following technical solution: A method for preparing licorice chalcone A based on enzyme inhibition and peeling technology, the preparation method specifically comprising the following steps: The first step, enzyme inhibition treatment: The cleaned licorice is dried, and then the dried licorice is cut into small segments of 10 to 20 cm in length, and the licorice segments are placed in a superheated steam or microwave environment, or the licorice segments are immersed in a solution containing an enzyme inhibitor for enzyme inhibition treatment, so as to inhibit the activity of endogenous enzymes, thereby reducing the generation of pigments caused by enzymatic oxidation reactions; Step 2: Peeling: After the licorice treated with enzyme inhibition is taken out from the superheated steam or microwave or enzyme inhibitor, the surface water is drained, and the dark outer skin containing a large amount of pigment is removed by manual peeling, mechanical peeling or chemical peeling to reduce the dissolution of the dark pigment during the extraction process; Step 3: Drying and crushing: After the licorice is peeled, it is placed in a microwave device or a hot air drying oven for drying. After the dried peeled licorice core is cooled to room temperature, it is crushed with a pulverizer and sieved with a 10-100 mesh screen to obtain licorice powder with uniform particle size for extraction; Step 4: Extraction: Take an appropriate amount of crushed and sieved licorice powder, add 3 to 50 times the amount of 50% to 100% ethanol or methanol solution to extract it, and combine the extracts after repeated extraction for 1 to 3 times. Repeated extraction can more effectively dissolve the licorice chalcone A component in the licorice; Step 5: Preliminary purification: The extract was concentrated until the alcohol smell disappeared, and then loaded onto an AB-8 or D-101 or HP-20 or XAD-4 or ADS-8 chromatographic column for purification in portions. After adsorption overnight, the chromatographic column was first eluted with pure water to elute macromolecular substances and large-polarity components, and then gradient elution was performed with a 0% to 100% ethanol or methanol solution, and the content of liquiritigenin A in the eluate was monitored by HPLC. The alcohol elution fraction rich in liquiritigenin A was collected. The eluate rich in liquiritigenin A was concentrated and then loaded onto a polyamide column with a mesh size of 30 to 60. After adsorption overnight, gradient elution was performed with 0% to 100% ethanol or methanol, and the content of liquiritigenin A in the eluate was monitored by HPLC. The alcohol elution fraction rich in liquiritigenin A was collected, combined, concentrated, and dried to obtain the crude product of liquiritigenin A; Step 6, recrystallization: Using the solubility characteristics of liquiritigenin A, repeated recrystallization was performed using a mixed solvent composed of any two or more combinations of water, methanol, ethanol, acetone, hydrochloric acid, or sulfuric acid. The content of liquiritigenin A was detected by HPLC to further improve the purity of liquiritigenin A, and bright yellow needle-shaped crystals of liquiritigenin A were obtained.

[0009] In the method for preparing liquiritigenin A based on enzyme inhibition and peeling technology, when using superheated steam to perform enzyme inhibition treatment on licorice in the first step, the treatment temperature is 95°C to 160°C, and the treatment time is 5 to 20 minutes.

[0010] In the method for preparing liquiritigenin A based on enzyme inhibition and peeling technology, when using a microwave environment to perform enzyme inhibition treatment on licorice in the first step, the power of the microwave is 0.4 to 32 kw, and the treatment time is 1 to 15 min.

[0011] In the method for preparing liquiritigenin A based on enzyme inhibition and peeling technology, when using a chemical peeling method to peel licorice in the second step, the strong alkalinity of sodium hydroxide and potassium hydroxide or the acidity of citric acid and malic acid is utilized for peeling after moistening or infiltration.

[0012] In the method for preparing liquiritigenin A based on enzyme inhibition and peeling technology, the drying temperature of the hot air drying oven in the third step is set at 25°C to 100°C.

[0013] In the method for preparing liquiritigenin A based on enzyme inhibition and peeling technology, the temperature during extraction in the fourth step is 5 to 80 °C.

[0014] In the method for preparing liquiritigenin A based on enzyme inhibition and peeling technology, the temperature during recrystallization in the sixth step is controlled at 0 °C to 40 °C.

[0015] Adopting the above-mentioned technical solution, the present invention has the following advantages: By combining enzyme inhibition and peeling technology, the present invention reduces the oxidative generation of polyphenols and pigment precursors in licorice from the source, significantly reduces the occurrence of enzymatic browning, and improves the initial purity of the extract. This technological improvement effectively simplifies the complex decolorization step in the traditional process, effectively reduces the usage amount of decolorizing agents and the discharge of chemical substances in wastewater, meets the environmental protection requirements and reduces the production cost, etc.

[0016] Specifically, the peeling treatment can effectively remove the pigments and impurities on the surface of licorice, further improve the extraction purity of licorice chalcone A, and make the purity of the final product stably reach more than 96%. At the same time, combined with the optimized enzyme inactivation process, the present invention improves the production efficiency on the basis of shortening the process time, effectively reduces the production cost, and shows significant economic benefits and environmental protection advantages.

[0017] Compared with the prior art, through innovative process optimization, the present invention reflects the comprehensive consideration of environmental protection, cost and efficiency from the source to the purification process. It not only shortens the production cycle, improves the product purity, but also significantly improves the ability to adapt to large-scale industrial production. These advantages make the present invention have higher application value and advantages in the market competition, etc. Brief Description of the Drawings

[0018] Figure 1 It is the preparation flow chart of licorice chalcone A in the embodiment of the present invention. Detailed Embodiments

[0019] The present invention can be more specifically explained through the following embodiments, and the present invention is not limited to the following embodiments; The principle of the present invention mainly combines enzyme inhibition and peeling technology to optimize the extraction process of licorice chalcone A. Through enzyme inactivation treatment, the polyphenol oxidase in licorice is quickly inactivated, effectively inhibiting the oxidative generation of polyphenols and pigment precursors, and significantly reducing the color depth of the extract. At the same time, the peeling treatment removes the outer layer of licorice, reduces the entry of pigments and impurities, and further reduces the risk of pigment migration.

[0020] A preparation method of licorice chalcone A based on enzyme inhibition and peeling technology, the preparation method specifically includes the following steps: The first step, enzyme inhibition treatment: The cleaned licorice is dried, and then the dried licorice is cut into small sections with a length of 10 - 20 cm. The licorice sections are placed in an environment of superheated steam or microwave, or the licorice sections are soaked in a solution containing an enzyme inhibitor for enzyme inhibition treatment to inhibit the activity of its endogenous enzymes, thereby reducing the pigment formation caused by enzymatic oxidation reaction; during implementation, when using superheated steam 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; Step 2: Skin peeling: After taking out the enzyme-inhibited licorice from superheated steam or microwave or enzyme inhibitor, drain the surface moisture, and use the method of manual skin peeling, mechanical skin peeling or chemical skin peeling to remove the dark outer skin containing a large amount of pigment, so as to reduce the dissolution of deep pigment during the extraction process; during implementation, when using the chemical skin peeling method to perform skin peeling on licorice, utilize the strong alkalinity of sodium hydroxide and potassium hydroxide or the acidity of citric acid and malic acid to perform skin peeling after moistening or soaking; Step 3: Drying and pulverizing: After the licorice is skinned, place it in a microwave device or a hot air drying oven for drying. After the dried skinned licorice core is cooled to room temperature, it is pulverized with a pulverizer and screened with a sieve of 10 - 100 meshes to obtain licorice powder with uniform particle size for extraction use; during implementation, the drying temperature of the hot air drying oven is set at 25°C - 100°C; Step 4: Extraction: Take an appropriate amount of pulverized and screened licorice powder, add 3 - 50 times the amount of 50% - 100% ethanol or methanol solution to it for extraction, and combine the extraction solutions after extracting 1 - 3 times repeatedly. Repeated extraction can more effectively dissolve the licorachalcone A component in licorice; during implementation, the temperature during extraction is 5 - 80 °C; Step 5: Preliminary purification: The extraction solution is concentrated until it has no alcohol smell, and then is loaded onto an AB-8 or D-101 or HP-20 or XAD-4 or ADS-8 chromatographic column for purification in batches. After adsorption overnight, first wash the chromatographic column with pure water to elute macromolecular substances and large polar components, and then perform gradient elution with 0% - 100% ethanol or methanol solution, and monitor the content of licorachalcone A in the eluate with HPLC. Collect the alcohol elution components rich in licorachalcone A, concentrate the eluate rich in licorachalcone A and then load it onto a polyamide column of 30 - 60 meshes, adsorb overnight, and then perform gradient elution with 0% - 100% ethanol or methanol, and monitor the content of licorachalcone A in the eluate with HPLC. Collect the alcohol elution components rich in licorachalcone A, combine, concentrate and dry to obtain the crude product of licorachalcone A; Step 6: Recrystallization: Utilize the solubility characteristics of liquiritigenin A, and perform repeated recrystallization using a mixed solvent composed of any two or more of water, methanol, ethanol, acetone, hydrochloric acid, or sulfuric acid in any combination. Detect the content of liquiritigenin A by HPLC method to further improve the purity of liquiritigenin A, and obtain needle-shaped crystals of liquiritigenin A in bright yellow. During implementation, the temperature during recrystallization is controlled at 0 °C to 40 °C.

[0021] In the specific practice of the present invention, in combination with the attached Figure 1 As shown, the present invention is carried out according to the following steps: The first step: enzyme inhibition treatment: Perform enzyme inhibition treatment on licorice by heating, adjusting the pH value, adding inhibitors, or rapid drying. During implementation, dry the cleaned licorice, and then cut it into small sections with a length of 10 to 20 cm for subsequent treatment. Place the licorice sections in a superheated steam environment at 95 to 160 °C, or in a microwave environment with a power of 0.4 to 32 kW for treatment, or soak the licorice sections in a solution containing enzyme inhibitors for enzyme inhibition treatment to inhibit the activity of its endogenous enzymes, thereby reducing the pigment formation caused by enzymatic oxidation reactions; Advantages of enzyme inhibition treatment: It not only prevents the accumulation of impurities and pigments caused by enzyme activity, but also ensures excellent performance in color and purity of subsequent extracts. In addition, these treatment methods provide reliable technical guarantees for the preparation of high-purity liquiritigenin A by reducing the pigment content in the extraction solution.

[0022] During implementation, the licorice is preferably Glycyrrhiza inflata Bat. When using the heating method for enzyme inhibition treatment of licorice, the heating method is steam heating. When using steam heating to inhibit the activity of oxidase in licorice, the treatment temperature is 95 °C to 160 °C, and the treatment time is 5 to 20 minutes; When using the heating method for enzyme inhibition treatment of licorice, the heating method is microwave heating. When using microwave heating for enzyme inhibition treatment of licorice, the power of the microwave is 0.4 to 32 kW, and the treatment time is 1 to 15 min; The second step: peeling: After taking out the licorice after enzyme inhibition treatment from superheated steam, microwave, or enzyme inhibitor, drain the surface moisture, and perform peeling on the licorice after enzyme inhibition treatment by manual peeling, mechanical peeling, or chemical peeling, which can remove the deep pigments in the outer skin and reduce their dissolution during the extraction process; During implementation, use manual peeling, mechanical peeling, or chemical peeling to remove the dark outer skin containing a large amount of pigments to reduce the dissolution of deep pigments during the extraction process; Advantages of peeling treatment: A large amount of pigments still exist in the enzyme-inhibited licorice, and these pigments are mainly concentrated in the outer skin part, while the pigments in the core part of licorice are relatively less. Therefore, peeling treatment can effectively remove the deep pigments in the outer skin and reduce their dissolution during the extraction process. This measure significantly reduces the chromaticity of the extract and improves the visual purity of the final extract. In addition, by removing the pigment-rich outer layer, not only the efficiency of the subsequent purification process is optimized, but also the related difficulty and cost are reduced, creating good conditions for obtaining high-purity licorice components; Step 3: Drying and pulverizing: The peeled licorice core is dried under mild conditions to avoid damage to the active ingredients by high temperature. After drying, the licorice core is pulverized and screened to obtain licorice powder with uniform particle size; During implementation, when the peeled licorice core is dried under mild conditions, it is selected to be dried in an oven, and the drying temperature of the oven is set at 25°C to 100°C; When screening the pulverized licorice core, a sieve mesh with 10 to 100 meshes is selected for screening; That is, the peeled licorice core is dried at 25°C to 100°C for 0 to 24 hours and then pulverized. After pulverization, it is processed with a 10 to 100-mesh sieve to obtain dry licorice powder; this process ensures that the finally obtained dry licorice powder has a consistent particle size, which is convenient for subsequent extraction and application; During specific implementation, after the licorice is peeled, it is placed in a microwave device or a hot air drying oven and dried under mild conditions of 25 to 100°C to avoid damage or degradation of the licorachalcone A component in the licorice by excessive temperature or heat. After the dried peeled licorice core is cooled to room temperature, it is pulverized with a pulverizer and screened with a 10 to 100-mesh sieve to obtain licorice powder with uniform particle size for extraction use; Step 4: Extraction: A 50% to 100% ethanol or methanol solution is used for 1 to 3 times of repeated extraction to effectively dissolve the licorachalcone A in the licorice; During implementation, an appropriate amount of the pulverized and screened licorice powder is taken, and 3 to 50 times the amount of a 50% to 100% ethanol or methanol solution is added thereto. After stirring evenly, extraction is carried out at 5 to 80°C. After repeated extraction for 1 to 3 times, the extraction solutions are combined. Repeated extraction can more effectively dissolve the licorachalcone A component in the licorice; In specific practice, 50% - 95% ethanol or methanol in an amount of 3 - 50 times the weight of the defatted licorice powder is used for extraction at 5 - 80 °C. Each extraction lasts for 1 - 72 h, and after repeating the extraction 1 - 3 times, the extraction solutions are combined to obtain an extraction solution containing licochalcone A. This extraction process utilizes the polarity and solubility of the solvent, enabling the full extraction of the target components in licorice, thereby improving the extraction efficiency and purity. Through multiple extractions, it can ensure that as many active ingredients as possible are dissolved and provide a higher - concentration extraction solution for subsequent separation and purification steps; Step 5, Preliminary purification: The extraction solution is concentrated until it has no alcohol smell and then loaded onto an AB - 8 or D - 101 or HP - 20 or XAD - 4 or ADS - 8 chromatographic column for purification. Gradient elution is carried out with 0% - 100% ethanol or methanol. The content of licochalcone A in the eluate is monitored by HPLC. The alcohol - washed fractions rich in licochalcone A are collected, concentrated, and then loaded onto a polyamide column. Gradient elution is carried out with 0% - 100% ethanol or methanol, and the alcohol - washed fractions rich in licochalcone A are collected, concentrated, and dried to obtain the crude product of licochalcone A; During implementation, the extraction solution is concentrated until it has no alcohol smell and then loaded onto an AB - 8 or D - 101 or HP - 20 or XAD - 4 or ADS - 8 chromatographic column for purification in batches. After adsorption overnight, first, the chromatographic column is eluted with pure water to elute macromolecular substances and large - polarity components. Then, gradient elution is carried out with a 0% - 100% ethanol or methanol solution, and the content of licochalcone A in the eluate is monitored by HPLC. The alcohol - washed fractions rich in licochalcone A are collected. The eluate rich in licochalcone A is concentrated and then loaded onto a polyamide column with a mesh size of 30 - 60. After adsorption overnight, gradient elution is carried out with 0% - 100% ethanol or methanol, and the content of licochalcone A in the eluate is monitored by HPLC. The alcohol - washed fractions rich in licochalcone A are collected, combined, concentrated, and dried to obtain the crude product of licochalcone A; In specific implementation, the extraction solution is concentrated until it has no alcohol smell and then purified through an AB - 8 or D101 or HP - 20 or XAD - 4 or ADS - 8 chromatographic column. Gradient elution is carried out with 2.5 - 5 column volumes of 0% - 100% methanol or ethanol. The content of licochalcone A in the components is monitored by HPLC. The elution fractions rich in licochalcone A are collected. The solution is concentrated and then loaded onto a polyamide column. Elution is carried out with 0 - 100% methanol or ethanol. The alcohol - washed solution is collected and the content of licochalcone A is monitored, and then concentrated and dried to obtain the crude product of licochalcone A; Step 6, Recrystallization: Water or methanol or ethanol or acetone or hydrochloric acid or sulfuric acid, or any one or two or a combination of two or more of them are used as a mixed solvent for repeated recrystallization to further remove impurities and improve the purity of licochalcone A, obtaining needle - shaped crystals of licochalcone A in bright yellow; In specific implementation, taking advantage of the solubility characteristics of liquiritigenin A, a mixed solvent formed by any two or more combinations of water, methanol, ethanol, acetone, hydrochloric acid, or sulfuric acid is used for repeated recrystallization. The content of liquiritigenin A is detected by HPLC method to further improve the purity of liquiritigenin A, and bright yellow needle-shaped crystalline liquiritigenin A is obtained.

[0023] Furthermore, the first enzyme inhibition step, the fourth extraction step, the fifth purification step, and the sixth recrystallization step can be optimized using different temperatures or solvent systems respectively to improve the purity of liquiritigenin A or reduce the color depth.

[0024] Specific embodiments of the present invention are as follows: Example 1:

[0025] Put the Glycyrrhiza inflata Bat. into a microwave device, set the power to 400 W, and control the treatment time at 10 min. After enzyme inhibition treatment, remove the outer skin of the licorice by manual peeling method. Crush the peeled licorice and pass it through a 10-mesh sieve. Then, reflux extract the peeled licorice powder with 5 times the amount of 80% ethanol at 80 °C for 2 h, repeat 3 times and combine the extracts. After the extract is concentrated to no alcohol smell, pass it through macroporous resin AB-8, discard the water and 30% ethanol elution fractions, collect the 75% ethanol elution fraction, concentrate it and then pass it through a polyamide column, discard the water wash fraction, collect the 50% ethanol eluate, concentrate and dry it, and then use methanol-water for repeated recrystallization to obtain 2.1527 g of bright yellow liquiritigenin A with a purity of 96.67% (HPLC method). Example 2:

[0026] Put the Glycyrrhiza inflata Bat. into a microwave device, set the power to 700 W, and control the treatment time at 6 min. After enzyme inhibition treatment, remove the outer skin of the licorice by manual peeling method. Crush the peeled licorice and pass it through a 40-mesh sieve. Then, reflux extract the peeled licorice powder with 10 times the amount of 80% ethanol at 75 °C for 2 h, repeat three times and combine the extracts. After the extract is concentrated to no alcohol smell, pass it through macroporous resin AB-8, discard the water and 30% ethanol elution fractions, collect the 75% ethanol elution fraction, concentrate it and then pass it through a polyamide column, discard the water wash fraction, collect the 50% ethanol eluate, concentrate and dry it, and then use methanol-water for repeated recrystallization to obtain 2.0787 g of bright yellow liquiritigenin A with a purity of 96.24% (HPLC method). Example 3:

[0027] Put Glycyrrhiza inflata into a microwave device, set the power to 32 kW, and control the treatment time at 2 min. After the enzyme inhibition treatment, remove the outer skin of the licorice by the manual peeling method. Crush the peeled licorice and pass it through an 80-mesh sieve. Then, reflux extract the peeled licorice powder with 15 times the amount of 80% ethanol at 70 °C for 2 h, repeat three times and combine the extracts. Concentrate the extract until it has no alcohol smell, then pass it through macroporous resin AB-8, discard the water and 30% ethanol elution fractions, collect the 75% ethanol elution fraction, concentrate it, pass it through a polyamide column, discard the water washing fraction, collect the 50% ethanol eluate, concentrate and dry it, and then perform repeated recrystallization with methanol-water to obtain 2.2948 g of bright yellow licorinchalcone A with a purity of 96.82% (HPLC method). Example 4:

[0028] Heat Glycyrrhiza inflata in steam at 98 °C for 20 min to inhibit the activity of oxidase. After the enzyme inhibition treatment, remove the outer skin of the licorice by the manual peeling method. Crush the peeled licorice and pass it through a 100-mesh sieve. Then, reflux extract the peeled licorice powder with 15 times the amount of 80% ethanol at 80 °C for 2 h, repeat three times and combine the extracts. Concentrate the extract until it has no alcohol smell, then pass it through macroporous resin AB-8, discard the water and 30% ethanol elution fractions, collect the 75% ethanol elution fraction, concentrate it, pass it through a polyamide column, discard the water washing fraction, collect the 50% ethanol eluate, concentrate and dry it, and then perform repeated recrystallization with methanol-water to obtain 2.2517 g of bright yellow licorinchalcone A with a purity of 98.37% (HPLC method). Example 5:

[0029] Heat Glycyrrhiza inflata in steam at 105 °C for 15 min to inhibit the activity of oxidase. After the enzyme inhibition treatment, remove the outer skin of the licorice by the manual peeling method. Crush the peeled licorice and pass it through a 10-mesh sieve. Then, reflux extract the peeled licorice powder with 15 times the amount of 80% ethanol at 80 °C for 2 h, repeat three times and combine the extracts. Concentrate the extract until it has no alcohol smell, then pass it through macroporous resin AB-8, discard the water and 30% ethanol elution fractions, collect the 75% ethanol elution fraction, concentrate it, pass it through a polyamide column, discard the water washing fraction, collect the 50% ethanol eluate, concentrate and dry it, and then perform repeated recrystallization with methanol-water to obtain 2.0522 g of bright yellow licorinchalcone A with a purity of 96.21% (HPLC method). Example 6:

[0030] Heat Glycyrrhiza inflata in steam at 110 °C for 9 min to inhibit the activity of oxidase. After enzyme inhibition treatment, remove the outer skin of the licorice by manual peeling method. Crush the peeled licorice and sieve it through a 40-mesh sieve. Then, reflux extract the peeled licorice powder with 15 times the amount of 95% ethanol at 80 °C for 2 h, repeat three times and combine the extracts. Concentrate the extract until the alcohol smell disappears, then pass it through macroporous resin AB-8, discard the water and 30% ethanol elution fractions, collect the 75% ethanol elution fraction, concentrate it, pass it through a polyamide column, discard the water-washed fraction, collect the 50% ethanol eluate, concentrate and dry it, and then perform repeated recrystallization with methanol-water to obtain 1.9101 g of bright yellow licorinchalcone A with a purity of 96.60% (HPLC method). Example 7:

[0031] Heat Glycyrrhiza inflata in steam at 160 °C for 6 min to inhibit the activity of oxidase. After enzyme inhibition treatment, remove the outer skin of the licorice by manual peeling method. Crush the peeled licorice and sieve it through a 100-mesh sieve. Then, reflux extract the peeled licorice powder with 15 times the amount of 60% ethanol at 25 °C for 72 h, repeat three times and combine the extracts. Concentrate the extract until the alcohol smell disappears, then pass it through macroporous resin AB-8, discard the water and 30% ethanol elution fractions, collect the 75% ethanol elution fraction, concentrate it, pass it through a polyamide column, discard the water-washed fraction, collect the 50% ethanol eluate, concentrate and dry it, and then perform repeated recrystallization with methanol-water to obtain 1.3221 g of bright yellow licorinchalcone A with a purity of 96.54% (HPLC method).

[0032] The parts not detailed in the present invention are prior art.

[0033] The examples selected herein for disclosing the object of the present invention are considered suitable at present. However, it should be understood that the present invention is intended to include all variations and improvements of all examples falling within the scope of this concept and invention.

Claims

1. A method for preparing licorice chalcone A based on enzyme inhibition and peeling technology, characterized in that: The preparation method specifically comprises the following steps: The first step, enzyme inhibition treatment: The cleaned licorice is dried, and then the dried licorice is cut into small segments of 10 to 20 cm in length, and the licorice segments are placed in a superheated steam or microwave environment, or the licorice segments are immersed in a solution containing an enzyme inhibitor for enzyme inhibition treatment, so as to inhibit the activity of endogenous enzymes, thereby reducing the generation of pigments caused by enzymatic oxidation reactions; Step 2: Peeling: After the licorice treated with enzyme inhibition is taken out from the superheated steam or microwave or enzyme inhibitor, the surface water is drained, and the dark outer skin containing a large amount of pigment is removed by manual peeling, mechanical peeling or chemical peeling to reduce the dissolution of the dark pigment during the extraction process; Step 3: Drying and crushing: After the licorice is peeled, it is placed in a microwave device or a hot air drying oven for drying. After the dried peeled licorice core is cooled to room temperature, it is crushed with a pulverizer and sieved with a 10-100 mesh screen to obtain licorice powder with uniform particle size for extraction; Step 4: Extraction: Take an appropriate amount of crushed and sieved licorice powder, add 3 to 50 times the amount of 50% to 100% ethanol or methanol solution to extract it, and combine the extracts after repeated extraction for 1 to 3 times. Repeated extraction can more effectively dissolve the licorice chalcone A component in the licorice; Step 5: Preliminary purification: The extract is concentrated until there is no alcohol smell, and then loaded onto an AB-8 or D-101 or HP-20 or XAD-4 or ADS-8 chromatographic column for purification. After adsorption overnight, the chromatographic column is first eluted with pure water to elute macromolecular substances and large polar components, and then gradient elution is performed with 0% to 100% ethanol or methanol solution, and the content of licorice chalcone A in the eluate is monitored by HPLC, and the alcohol-washed fractions rich in licorice chalcone A are collected. The eluate rich in licorice chalcone A is concentrated and loaded onto a 30-60 mesh polyamide column, adsorbed overnight, and then gradient eluted with 0% to 100% ethanol or methanol, and the content of licorice chalcone A in the eluate is monitored by HPLC, and the alcohol-washed fractions rich in licorice chalcone A are collected, combined, concentrated, and dried to obtain a crude licorice chalcone A product. Step 6: Recrystallization: The solubility characteristics of Licorice Chalcone A are utilized, and a mixed solvent of any two or more of water, methanol, ethanol, acetone, hydrochloric acid or sulfuric acid in any combination is used for repeated recrystallization, and the content of Licorice Chalcone A is detected by HPLC to further improve the purity of Licorice Chalcone A and obtain bright yellow needle-shaped crystals of Licorice Chalcone A.

2. The method for preparing licorice chalcone A based on enzyme inhibition and peeling technology according to claim 1, characterized in that: In the first step, when superheated steam is used to treat the licorice 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 licorice chalcone A based on enzyme inhibition and peeling technology according to claim 1, wherein in the first step, when the licorice is treated with a microwave environment to inhibit the enzyme, the microwave power is 0.4 to 32 kw and the treatment time is 1 to 15 minutes.

4. The method for preparing licorice chalcone A based on enzyme inhibition and peeling technology according to claim 1, wherein in the second step, when the licorice is peeled by a chemical peeling method, the peeling is performed after moistening or soaking by utilizing the strong alkalinity of sodium hydroxide or potassium hydroxide or the acidity of citric acid or malic acid.

5. The method for preparing licorice chalcone A based on enzyme inhibition and peeling technology according to claim 1, wherein the drying temperature of the hot air drying oven in the third step is set to 25°C to 100°C. 6 . The method for preparing licorice chalcone A based on enzyme inhibition and peeling technology according to claim 1 , wherein the temperature during the extraction in the fourth step is 5 to 80° C.

7. The method for preparing licorice chalcone A based on enzyme inhibition and peeling technology according to claim 1, wherein the temperature during recrystallization in the sixth step is controlled at 0°C to 40°C.

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