A method for preparing glabridin

Abstract

The present disclosure relates to the technical field of pharmaceutical preparation, and particularly relates to a preparation method of glabriden; the preparation method comprises the following steps: providing licorice raw materials, crushing and sieving, and then performing a solid state fermentation process to obtain licorice powder after solid state fermentation; then adding into a first mixed organic solution for soaking, and then adding into a first mixed aqueous solution for soaking, and then adding a second mixed organic solution for extraction, collecting the organic phase, and then adding the solid product into the second mixed organic solution for extraction after enzyme inactivation and impurity removal; combining the organic phases and performing dialysis, purification and concentration to obtain a licorice flavone extract; and then performing extraction of the licorice flavone extract by a third mixed organic solution containing imidazolyl sulfonate to obtain a product containing a higher proportion of glabriden.

Description

Technical Field

[0001] This disclosure relates to the field of pharmaceutical preparation technology, and in particular to a method for preparing glycyrrhizin. Background Technology

[0002] Glabridine is an isoflavone compound, a natural product extracted from a plant called licorice root (or European licorice). The structural formula of glabridine is as follows: .

[0003] Glycyrrhizin is present in licorice root glabridin at a concentration of 0.1%-3%, accounting for approximately 11% of the total flavonoids in licorice root glabridin. Due to its scarcity of natural resources and wide range of applications, it is in high demand and short supply, resulting in a high price. Pure glycyrrhizin is a white powder or white crystals, insoluble in water, but readily soluble in some organic solvents (acetone, toluene, ethanol). Its natural source, licorice root glabridin, is a plant belonging to the genus Glycyrrhiza in the legume family (Fabaceae).

[0004] Currently, the main methods for separating and purifying glycyrrhizin include: repeated extraction with multiple organic solvents, gradient elution by alumina column chromatography, gradient elution by silica gel column chromatography, or repeated extraction with organic solvents, resin adsorption and mixed solvent elution, and recrystallization. However, these methods generally suffer from poor specificity and selectivity of glycyrrhizin, numerous impurities, and difficulty in separating it with a single extraction or a single resin. Multiple extractions or the combined use of multiple resins are often required, resulting in complicated procedures, high separation costs, and environmental pollution caused by the large amount of organic waste liquid generated during elution.

[0005] Since the flavonoids in licorice are mainly isoflavone alkyl compounds, and the flavonoids in licorice root are mainly flavone alkyl compounds, among which glycyrrhizin accounts for a large proportion; therefore, the polarity difference of glycyrrhizin relative to other fat-soluble flavonoids can be utilized to select different extractants for extraction, thereby achieving effective separation of glycyrrhizin. Summary of the Invention

[0006] This disclosure provides a method for preparing glycyrrhizin to address the shortcomings of related technologies.

[0007] According to a first aspect of the present disclosure, a method for preparing glycyrrhizin is provided, the method comprising the following steps:

[0008] Step 1: Provide licorice raw material, dry the licorice raw material, pulverize and sieve it to obtain licorice powder;

[0009] Step 2: The licorice powder is subjected to a solid-state fermentation process to obtain solid-state fermented licorice powder;

[0010] Step 3: The licorice powder after solid-state fermentation is added to the first mixed organic solution for soaking. The soaking process is carried out under ultrasonic action to obtain pretreated licorice powder.

[0011] Step 4: The pretreated licorice powder is added to the first mixed aqueous solution for soaking. Then, a second mixed organic solution is added to the first mixed aqueous solution for extraction. After extraction, a first organic phase, a first aqueous phase, and a first solid product are obtained. The first organic phase is collected, and the first solid product is added to the reactions in steps 5 and 6.

[0012] Step 5: The first solid product is subjected to an enzymatic hydrolysis process, which is carried out under ultrasonication; the enzymatic hydrolysis process uses a compound enzyme to obtain an enzymatic hydrolysate;

[0013] Step 6: After the enzyme hydrolysate has undergone enzyme inactivation and impurity removal processes, a second mixed organic solution is added to it. After extraction, a second organic phase, a second aqueous phase, and a second solid product are obtained; the second organic phase is collected.

[0014] Step 7: Combine the first organic phase and the second organic phase, and then perform dialysis, purification and concentration to obtain the licorice flavonoid extract;

[0015] Step 8: The licorice flavonoid extract is extracted with a third mixed organic solution to obtain the glycyrrhizin.

[0016] In one aspect of this disclosure, the licorice raw material is licorice residue after decoction.

[0017] In one aspect of the embodiments of this disclosure, specifically, step 1 includes: providing licorice residue after decoction, drying the licorice residue at 60°C for 12 hours, then pulverizing it and passing it through a 40-mesh sieve to obtain licorice powder.

[0018] In one aspect of this disclosure, specifically, step 2 includes the following steps:

[0019] Step 2-1: Provide a bacterial strain, and activate the bacterial strain to obtain an activated and diluted bacterial strain; wherein the bacterial strain is selected from one of Rhizopus oryzae, Aspergillus oryzae, Rhizopus nigricans, Mucor, or Aspergillus fumigatus;

[0020] Step 2-2: Inoculate the licorice powder obtained in Step 1 with the activated and diluted bacterial strain, control the fermentation temperature to be selected from 25℃-30℃, the water content during the fermentation process to be selected from 70%-80%, and the fermentation time to be selected from 2-4 days, to obtain licorice powder after solid-state fermentation.

[0021] In one aspect of the present disclosure, preferably, in step 2-1, the strain is selected from Rhizopus oryzae.

[0022] In one aspect of the embodiments of this disclosure, more specifically, step 2-1 includes: providing a bacterial strain selected from one of Rhizopus oryzae, Aspergillus oryzae, Rhizopus nigricans, Mucor, or Aspergillus fumigatus; preparing a PDA medium and adding an appropriate amount of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt, i.e., ABTS, such that the final concentration of ABTS is 0.01%-0.03%; spreading the bacterial strain on the PDA medium and culturing it at 25℃-30℃ for 4-6 days; after culturing, eluting to obtain a suspension containing spores of the bacterial strain; filtering the suspension and diluting it with an appropriate amount of water to obtain the activated and diluted bacterial strain.

[0023] In one aspect of this disclosure, in step 3, the first mixed organic solution is selected from any combination of the following two:

[0024] a) Chloroform, and any one of methanol, ethanol, propanol, isopropanol, diethyl ether or acetone;

[0025] b) hexane, and any one of ethanol, acetone, or ethyl acetate.

[0026] In one aspect of the embodiments of this disclosure, specifically, in step 3, the first mixed organic solution may be selected from a mixed solution of chloroform and methanol, a mixed solution of chloroform and ethanol, a mixed solution of n-hexane and ethanol, or a mixed solution of n-hexane and acetone.

[0027] In one aspect of the embodiments of this disclosure, more preferably, in step 3, the first mixed organic solution may be selected from a mixed solution of n-hexane and acetone, wherein n-hexane and acetone are mixed in a volume ratio of (0.3-0.7):1.

[0028] In one aspect of this disclosure, in step 4, the first mixed aqueous solution is an aqueous solution containing a surfactant and acetylacetone; the surfactant is selected from fatty alcohol polyoxyethylene ethers.

[0029] In one aspect of this disclosure, in step 4, the fatty alcohol polyoxyethylene ether is selected from AEO-7, AEO-9, AEO-10, or AEO15; but is not limited thereto.

[0030] In one aspect of this disclosure, in the first mixed aqueous solution, the concentration of the surfactant is selected from 15-25 g / L, and the concentration of acetylacetone is selected from 5-10 g / L.

[0031] In one aspect of this disclosure, glacial acetic acid may also be added to the first mixed aqueous solution, wherein the concentration of the glacial acetic acid is selected from 0.3-0.6 g / L.

[0032] In one aspect of this disclosure, in step 4, the second mixed organic solution is ethyl acetate containing a quaternary ammonium salt compound. In another aspect of this disclosure, preferably, the quaternary ammonium salt compound is selected from tetrabutylammonium chloride, tetrabutylammonium bromide, tetrabutylammonium iodide, tetrabutylammonium hydrogen sulfate, or tetrabutylammonium hydroxide; specifically, the quaternary ammonium salt compound is selected from tetrabutylammonium bromide.

[0033] In one aspect of this disclosure, the content of the quaternary ammonium salt compound in the second mixed organic solution is 10-25 g / L.

[0034] In one aspect of this disclosure, step 4 includes the following steps:

[0035] Step 4-1: Add the pretreated licorice powder to the first mixed aqueous solution and soak it at room temperature for 1-3 hours, wherein the first mixed aqueous solution is an aqueous solution containing surfactant and acetylacetone;

[0036] Step 4-2: Add a second mixed organic solution to the first mixed aqueous solution for extraction; wherein the second mixed organic solution is ethyl acetate containing a quaternary ammonium salt compound;

[0037] Step 4-3: After extraction in step 4-2, a first organic phase, a first aqueous phase, and a first solid product are obtained; the first organic phase is collected, and the first solid product is added to the reactions in steps 5 and 6.

[0038] In one aspect of the embodiments of this disclosure, the volume ratio of the first mixed aqueous solution and the second mixed organic solution is selected from (0.5-1.5):1.

[0039] In one aspect of this disclosure, in step 5, the enzymatic hydrolysis process is performed using a complex enzyme comprising papain and a central protease.

[0040] In one aspect of this disclosure, steps 5 and 6 include the following steps:

[0041] Step 5: After drying the first solid product, add 0.01%-0.05% of a complex enzyme solution to it, then adjust the pH of the mixture to 5-6, and then sonicate at 60℃-70℃ and 200kHz for 1-1.5h to obtain an enzymatic hydrolysate; wherein the complex enzyme contains papain and central protease;

[0042] Step 6: After the enzyme hydrolysate is inactivated by boiling water, it is cooled to room temperature and then purified by macroporous resin adsorption and chromatography separation. A second mixed organic solution is added for extraction. After extraction, a second organic phase, a second aqueous phase and a second solid product are obtained. The second organic phase is collected. The second mixed organic solution is ethyl acetate containing quaternary ammonium salt compounds.

[0043] In one aspect of this disclosure, the amount of the added complex enzyme solution is selected from: 5-10 ml of complex enzyme solution / 1 g of the first solid product.

[0044] In one aspect of this disclosure, in step 8, the third mixed organic solution is a dipropanol solution containing imidazolium sulfonate; the imidazolium sulfonate is selected from any one of 1-butyl-3-methylimidazolium methanesulfonate, 1-butyl-3-methylimidazolium p-toluenesulfonate, 1-pentyl-3-methylimidazolium trifluoromethanesulfonate, or 1-hexyl-3-methylimidazolium methanesulfonate.

[0045] In one aspect of this disclosure, the content of the imidazolyl sulfonate in the third mixed organic solution is selected from 15-45 mg / mL.

[0046] In one aspect of this disclosure, in step 8, the third mixed organic solution is a dipropanol solution containing 1-butyl-3-methylimidazolium methanesulfonate.

[0047] In one aspect of this disclosure, in step 8, the third mixed organic solution is a dipropanol solution containing 1-butyl-3-methylimidazolium methanesulfonate; the content of 1-butyl-3-methylimidazolium methanesulfonate is selected from 20-30 mg / mL.

[0048] According to a second aspect of the present disclosure, a glycyrrhizin extract is provided, which is obtained by the aforementioned preparation method.

[0049] The technical solutions provided by the embodiments of this disclosure may include the following beneficial effects:

[0050] As can be seen from the above embodiments, this disclosure makes the most of licorice waste residue, first extracting as much licorice flavonoid extract as possible, and then using the difference in solubility and polarity to obtain a glycyrrhizin extract with a high content. The preparation process provided by this disclosure saves raw materials, reaction steps and reaction materials.

[0051] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit this disclosure. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. The embodiments described herein are illustrative in nature and are intended to provide a basic understanding of this invention. The embodiments of this invention should not be construed as limiting the invention.

[0053] For the sake of brevity, this article only discloses a few specific numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, just as any upper limit can be combined with any other upper limit to form an unspecified range. Furthermore, each individually disclosed point or single value can itself serve as a lower or upper limit and be combined with any other point or single value or with other lower or upper limits to form an unspecified range.

[0054] In this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0055] In this description, unless otherwise stated, "above" and "below" include the stated number.

[0056] Unless otherwise stated, the terminology used in this invention has the common meanings understood by those skilled in the art. Unless otherwise stated, the values ​​of the parameters mentioned in this invention can be measured using various measurement methods commonly used in the art (e.g., they can be tested according to the methods given in the embodiments of this invention).

[0057] The term "about" is used to describe and indicate small variations. When used in conjunction with an event or situation, the term may refer to examples in which the event or situation occurred precisely or in examples in which the event or situation occurred very approximately. For example, when used in conjunction with numerical values, the term may refer to a range of variation less than or equal to ±10% of the numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%. Additionally, quantities, ratios, and other numerical values ​​are sometimes presented in range format herein. It should be understood that such range format is for convenience and brevity and should be interpreted flexibly to include not only numerical values ​​explicitly specified as range limits but also all individual numerical values ​​or subranges covered within the range, as if each numerical value and subrange were explicitly specified.

[0058] The list of items connected by the terms "at least one of," "at least one of," "at least one of," or other similar terms can mean any combination of the listed items. For example, if items A and B are listed, then the phrase "at least one of A and B" means only A; only B; or A and B. In another instance, if items A, B, and C are listed, then the phrase "at least one of A, B, and C" means only A; or only B; only C; A and B (excluding C); A and C (excluding B); B and C (excluding A); or all of A, B, and C. Item A may contain a single component or multiple components. Item B may contain a single component or multiple components. Item C may contain a single component or multiple components.

[0059] The present application is further illustrated below with reference to embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application.

[0060] The present application is further illustrated below with reference to embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application.

[0061] Example 1: Example 1 includes the following steps:

[0062] 1. Provide 50g of licorice residue after decoction, dry it at 60℃ for 12h, then pulverize it and pass it through a 40-mesh sieve to obtain licorice powder;

[0063] 2. Provide *Rhizopus oryzae*; prepare PDA medium by adding an appropriate amount of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) to achieve a final ABTS concentration of 0.02%; spread the *Rhizopus oryzae* inoculum onto the PDA medium and incubate at 28°C for 5 days. After incubation, wash to obtain a suspension containing spores. Filter the suspension and dilute it with an appropriate amount of water to obtain activated and diluted inoculum. Inoculate licorice powder with the activated and diluted inoculum, controlling the fermentation temperature at 28°C, the moisture content during fermentation at 70%, and the fermentation time at 5 days to obtain licorice powder after solid-state fermentation.

[0064] 3. The licorice powder after solid-state fermentation is added to the first mixed organic solution and soaked for 5 hours. The soaking process is carried out under ultrasound at 200 kHz to obtain pretreated licorice powder. The first mixed organic solution is a mixture of n-hexane and acetone, wherein n-hexane and acetone are mixed at a volume ratio of 45 mL: 100 mL.

[0065] 4. Add the pretreated licorice powder to the first mixed aqueous solution and soak at room temperature for 2 hours. The first mixed aqueous solution is 200 mL of an aqueous solution containing surfactant AEO-7, acetylacetone, and glacial acetic acid (AEO-7 concentration: 20 g / L, acetylacetone concentration: 8 g / L, glacial acetic acid concentration: 0.5 g / L). Then, add a second mixed organic solution to the first mixed aqueous solution for extraction. The second mixed organic solution is 200 mL of ethyl acetate containing tetrabutylammonium bromide, with the concentration of tetrabutylammonium bromide selected from 15 g / L. A first organic phase, a first aqueous phase, and a first solid product are obtained. The first organic phase is collected, and the first solid product is added to subsequent reactions.

[0066] 5. After drying the first solid product (weighed to 34g), add 0.03% of the compound enzyme solution to it, then adjust the pH of the mixture to 5.5, and then sonicate at 60℃ and 200kHz for 1.5h to obtain the enzymatic hydrolysate; wherein the compound enzyme contains papain and neutral protease (the amount added is selected from 1:1); the amount of compound enzyme solution added is 12ml of compound enzyme solution / 1g of the first solid product.

[0067] 6. After the enzyme hydrolysate is inactivated by boiling water, it is cooled to room temperature and then purified by adsorption with macroporous resin (D101). The second mixed organic solution is added for extraction (same as in step 4). After extraction, a second organic phase, a second aqueous phase and a second solid product are obtained. The second organic phase is collected.

[0068] 7. After merging the first and second organic phases, the glycyrrhizin was obtained by dialysis, purification and concentration.

[0069] Example 2: Example 2 includes the following steps:

[0070] 1. Provide 50g of licorice residue after decoction, dry it at 60℃ for 12h, then pulverize it and pass it through a 40-mesh sieve to obtain licorice powder;

[0071] 2. Provide *Aspergillus fumigatus*; prepare PDA medium, adding an appropriate amount of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt, i.e., ABTS, to achieve a final ABTS concentration of 0.02%; spread the *Aspergillus fumigatus* strain (with the same mass as *Rhizopus oryzae* used in Example 1) onto the PDA medium and incubate at 28°C for 5 days. After incubation, wash to obtain a suspension containing spores of the strain. Filter the suspension and dilute it with an appropriate amount of water to obtain an activated and diluted strain. Inoculate licorice powder with the activated and diluted strain, controlling the fermentation temperature to 28°C, the water content during fermentation to 70%, and the fermentation time to 5 days, to obtain licorice powder after solid-state fermentation.

[0072] 3. The licorice powder after solid-state fermentation is added to the first mixed organic solution and soaked for 5 hours. The soaking process is carried out under ultrasound at 200 kHz to obtain pretreated licorice powder. The first mixed organic solution is a mixture of n-hexane and acetone, wherein n-hexane and acetone are mixed at a volume ratio of 45 mL: 100 mL.

[0073] 4. Add the pretreated licorice powder to the first mixed aqueous solution and soak at room temperature for 2 hours. The first mixed aqueous solution is 200 mL of an aqueous solution containing surfactant AEO-7, acetylacetone, and glacial acetic acid (AEO-7 concentration: 20 g / L, acetylacetone concentration: 8 g / L, glacial acetic acid concentration: 0.5 g / L). Then, add a second mixed organic solution to the first mixed aqueous solution for extraction. The second mixed organic solution is 200 mL of ethyl acetate containing tetrabutylammonium bromide, with the concentration of tetrabutylammonium bromide selected from 15 g / L. A first organic phase, a first aqueous phase, and a first solid product are obtained. The first organic phase is collected, and the first solid product is added to subsequent reactions.

[0074] 5. After drying the first solid product (weighed to 34g), add 0.03% of the compound enzyme solution to it, then adjust the pH of the mixture to 5.5, and then sonicate at 60℃ and 200kHz for 1.5h to obtain the enzymatic hydrolysate; wherein the compound enzyme contains papain and neutral protease (the amount added is selected from 1:1); the amount of compound enzyme solution added is 12ml of compound enzyme solution / 1g of the first solid product.

[0075] 6. After the enzyme hydrolysate is inactivated by boiling water, it is cooled to room temperature and then purified by adsorption with macroporous resin (D101). The second mixed organic solution is added for extraction (same as in step 4). After extraction, a second organic phase, a second aqueous phase and a second solid product are obtained. The second organic phase is collected.

[0076] 7. After merging the first and second organic phases, the glycyrrhizin was obtained by dialysis, purification and concentration.

[0077] Example 3: The steps of Example 3 are basically the same as those of Example 1, except that an equal mass of Rhizopus nigricans is used instead of the Rhizopus oryzae strain used in Example 1.

[0078] Example 4: The steps of Example 4 are basically the same as those of Example 1, except that an equal mass of Aspergillus oryzae strain is used instead of the Rhizopus oryzae strain used in Example 1.

[0079] Example 5: The steps of Example 5 are basically the same as those of Example 1, except that an equal mass of Mucor strain is used instead of the Rhizopus strain used in Example 1.

[0080] Comparative Example 1: Comparative Example 1 includes the following steps:

[0081] 1. Provide 50g of licorice residue after decoction, dry it at 60℃ for 12h, then pulverize it and pass it through a 40-mesh sieve to obtain licorice powder;

[0082] 2. The pulverized licorice powder is added to the first mixed organic solution and soaked for 5 hours. The soaking process is carried out under ultrasound at 200 kHz to obtain pretreated licorice powder. The first mixed organic solution is a mixture of n-hexane and acetone, wherein n-hexane and acetone are mixed at a volume ratio of 45 mL: 100 mL.

[0083] 3. Add the pretreated licorice powder to the first mixed aqueous solution and soak at room temperature for 2 hours. The first mixed aqueous solution is 200 mL of an aqueous solution containing surfactant AEO-7, acetylacetone, and glacial acetic acid (AEO-7 concentration: 20 g / L, acetylacetone concentration: 8 g / L, glacial acetic acid concentration: 0.5 g / L). Then, add a second mixed organic solution to the first mixed aqueous solution for extraction; the second mixed organic solution is 200 mL of ethyl acetate containing tetrabutylammonium bromide, with the tetrabutylammonium bromide concentration selected from 15 g / L. A first organic phase, a first aqueous phase, and a first solid product are obtained. The first organic phase is collected, and the first solid product is added to subsequent reactions.

[0084] 4. After drying the first solid product (weighed to 34g), add 0.03% of the compound enzyme solution to it, then adjust the pH of the mixture to 5.5, and then sonicate at 60℃ and 200kHz for 1.5h to obtain the enzymatic hydrolysate; wherein the compound enzyme contains papain and neutral protease (the amount added is selected from 1:1); the amount of compound enzyme solution added is 12ml of compound enzyme solution / 1g of the first solid product.

[0085] 5. After the enzyme hydrolysate is inactivated by boiling water, it is cooled to room temperature and then purified by adsorption with macroporous resin (D101). The second mixed organic solution is added for extraction (same as in step 3). After extraction, a second organic phase, a second aqueous phase and a second solid product are obtained. The second organic phase is collected.

[0086] 6. After merging the first and second organic phases, the glycyrrhizin was obtained by dialysis, purification and concentration to obtain the glycyrrhizin of Comparative Example 1.

[0087] The main difference between Comparative Example 1 and Examples 1-5 is that Comparative Example 1 does not include the solid-state fermentation process.

[0088] Determination of flavonoid content in Examples 1-5 and Comparative Example 1: The products of Examples 1-5 and Comparative Example 1 were added to a rotary evaporator and evaporated until a paste-like consistency was achieved. The product was collected and then dried in a freeze dryer for 36 hours. It was then reconstituted with 70% ethanol (diluted 10 times), and the absorbance at 510 nm was measured to calculate the flavonoid content. The flavonoid content values ​​of the products of Examples 1-5 and Comparative Example 1 are shown in Table 1.

[0089] Table 1:

[0090] As can be seen, the flavonoid yields of Examples 1-5 after solid-state fermentation were all higher than those of Comparative Example 1, indicating that solid-state fermentation can effectively degrade lignin and promote the hydrolysis of cellulose components. However, the ability of different strains to hydrolyze cellulose components in licorice varied. The ability of Example 5, which used Mucor strain, to hydrolyze cellulose components in licorice was significantly weaker than that of Examples 1-4.

[0091] Example 6: Example 6 includes the following steps:

[0092] 1. Provide 50g of licorice residue after decoction, dry it at 60℃ for 12h, then pulverize it and pass it through a 40-mesh sieve to obtain licorice powder;

[0093] 2. Provide *Rhizopus oryzae*; prepare PDA medium by adding an appropriate amount of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) to achieve a final ABTS concentration of 0.02%; spread the *Rhizopus oryzae* inoculum onto the PDA medium and incubate at 28°C for 5 days. After incubation, wash to obtain a suspension containing spores. Filter the suspension and dilute it with an appropriate amount of water to obtain activated and diluted inoculum. Inoculate licorice powder with the activated and diluted inoculum, controlling the fermentation temperature at 28°C, the moisture content during fermentation at 70%, and the fermentation time at 5 days to obtain licorice powder after solid-state fermentation.

[0094] 3. The licorice powder after solid-state fermentation is added to the first mixed organic solution and soaked for 5 hours. The soaking process is carried out under ultrasound at 200 kHz to obtain pretreated licorice powder. The first mixed organic solution is a mixture of n-hexane and acetone, wherein n-hexane and acetone are mixed at a volume ratio of 100 mL: 45 mL.

[0095] 4. Add the pretreated licorice powder to the first mixed aqueous solution and soak at room temperature for 2 hours. The first mixed aqueous solution is 200 mL of an aqueous solution containing surfactant AEO-7, acetylacetone, and glacial acetic acid (AEO-7 concentration: 20 g / L, acetylacetone concentration: 8 g / L, glacial acetic acid concentration: 0.5 g / L). Then, add a second mixed organic solution to the first mixed aqueous solution for extraction. The second mixed organic solution is 200 mL of ethyl acetate containing tetrabutylammonium bromide, with the concentration of tetrabutylammonium bromide selected from 15 g / L. A first organic phase, a first aqueous phase, and a first solid product are obtained. The first organic phase is collected, and the first solid product is added to subsequent reactions.

[0096] 5. After drying the first solid product (weighed to 34g), add 0.03% of the compound enzyme solution to it, then adjust the pH of the mixture to 5.5, and then sonicate at 60℃ and 200kHz for 1.5h to obtain the enzymatic hydrolysate; wherein the compound enzyme contains papain and neutral protease (the amount added is selected from 1:1); the amount of compound enzyme solution added is 12ml of compound enzyme solution / 1g of the first solid product.

[0097] 6. After the enzyme hydrolysate is inactivated by boiling water, it is cooled to room temperature and then purified by adsorption with macroporous resin (D101). The second mixed organic solution is added for extraction (same as in step 4). After extraction, a second organic phase, a second aqueous phase and a second solid product are obtained. The second organic phase is collected.

[0098] 7. After merging the first and second organic phases, the glycyrrhizin was obtained by dialysis, purification and concentration.

[0099] Example 7: The steps of Example 7 are basically the same as those of Example 1, except that the first mixed organic solution used in Example 1 is replaced with a first mixed organic solution of hexane and ethanol in a volume ratio of 45 mL:100 mL.

[0100] Example 8: The steps of Example 8 are basically the same as those of Example 1, except that the first mixed organic solution used in Example 1 is replaced with a first mixed organic solution of hexane and ethanol in a volume ratio of 100 mL: 45 mL.

[0101] Example 9: The steps of Example 9 are basically the same as those of Example 1, except that the first mixed organic solution used in Example 1 is replaced with a first mixed organic solution of chloroform and methanol in a volume ratio of 45 mL:100 mL.

[0102] Example 10: The steps of Example 10 are basically the same as those of Example 1, except that the first mixed organic solution used in Example 1 is replaced with a first mixed organic solution of chloroform and ethanol at a volume ratio of 45 mL:100 mL.

[0103] Example 11: The steps of Example 11 are basically the same as those of Example 1, except that the first mixed organic solution used in Example 1 is replaced with a first mixed organic solution of toluene and ethanol in a volume ratio of 45 mL:100 mL.

[0104] Comparative Example 2: Comparative Example 2 includes the following steps:

[0105] 1. Provide 50g of licorice residue after decoction, dry it at 60℃ for 12h, then pulverize it and pass it through a 40-mesh sieve to obtain licorice powder;

[0106] 2. Provide *Rhizopus oryzae*; prepare PDA medium by adding an appropriate amount of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) to achieve a final ABTS concentration of 0.02%; spread the *Rhizopus oryzae* inoculum onto the PDA medium and incubate at 28°C for 5 days. After incubation, wash to obtain a suspension containing spores. Filter the suspension and dilute it with an appropriate amount of water to obtain activated and diluted inoculum. Inoculate licorice powder with the activated and diluted inoculum, controlling the fermentation temperature at 28°C, the moisture content during fermentation at 70%, and the fermentation time at 5 days to obtain licorice powder after solid-state fermentation.

[0107] 3. The licorice powder after solid-state fermentation was added to the first mixed aqueous solution and soaked at room temperature for 2 hours. The first mixed aqueous solution was 200 mL of an aqueous solution containing surfactant AEO-7, acetylacetone, and glacial acetic acid (AEO-7 concentration: 20 g / L, acetylacetone concentration: 8 g / L, glacial acetic acid concentration: 0.5 g / L). Then, a second mixed organic solution was added to the first mixed aqueous solution for extraction. The second mixed organic solution was 200 mL of ethyl acetate containing tetrabutylammonium bromide, with the concentration of tetrabutylammonium bromide selected from 15 g / L. A first organic phase, a first aqueous phase, and a first solid product were obtained. The first organic phase was collected, and the first solid product was added to subsequent reactions.

[0108] 4. After drying the first solid product (weighed to 34g), add 0.03% of the compound enzyme solution to it, then adjust the pH of the mixture to 5.5, and then sonicate at 60℃ and 200kHz for 1.5h to obtain the enzymatic hydrolysate; wherein the compound enzyme contains papain and neutral protease (the amount added is selected from 1:1); the amount of compound enzyme solution added is 12ml of compound enzyme solution / 1g of the first solid product.

[0109] 5. After the enzyme hydrolysate is inactivated by boiling water, it is cooled to room temperature and then purified by adsorption with macroporous resin (D101). The second mixed organic solution is added for extraction (same as in step 3). After extraction, a second organic phase, a second aqueous phase and a second solid product are obtained. The second organic phase is collected.

[0110] 6. After merging the first and second organic phases, the glycyrrhizin was obtained by dialysis, purification and concentration to obtain the glycyrrhizin of Comparative Example 2.

[0111] The main difference between Comparative Example 2 and the Example is that Comparative Example 2 does not use the first organic solution for soaking to further disrupt the cell wall.

[0112] Example 12: Example 12 includes the following steps:

[0113] 1. Provide 50g of licorice residue after decoction, dry it at 60℃ for 12h, then pulverize it and pass it through a 40-mesh sieve to obtain licorice powder;

[0114] 2. Provide *Rhizopus oryzae*; prepare PDA medium by adding an appropriate amount of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) to achieve a final ABTS concentration of 0.02%; spread the *Rhizopus oryzae* inoculum onto the PDA medium and incubate at 28°C for 5 days. After incubation, wash to obtain a suspension containing spores. Filter the suspension and dilute it with an appropriate amount of water to obtain activated and diluted inoculum. Inoculate licorice powder with the activated and diluted inoculum, controlling the fermentation temperature at 28°C, the moisture content during fermentation at 70%, and the fermentation time at 5 days to obtain licorice powder after solid-state fermentation.

[0115] 3. The licorice powder after solid-state fermentation is added to the first mixed organic solution and soaked for 5 hours. The soaking process is carried out under ultrasound at 200 kHz to obtain pretreated licorice powder. The first mixed organic solution is a mixture of n-hexane and acetone, wherein n-hexane and acetone are mixed at a volume ratio of 45 mL: 100 mL.

[0116] 4. Add the pretreated licorice powder to the first mixed aqueous solution and soak at room temperature for 2 hours. The first mixed aqueous solution is 200 mL of an aqueous solution containing acetylacetone and glacial acetic acid (acetylacetone concentration is 8 g / L, and glacial acetic acid concentration is 0.5 g / L). Then, add the second mixed organic solution to the first mixed aqueous solution for extraction; the second mixed organic solution is 200 mL of ethyl acetate containing tetrabutylammonium bromide, and the concentration of tetrabutylammonium bromide is selected from 15 g / L. A first organic phase, a first aqueous phase, and a first solid product are obtained. Collect the first organic phase and add the first solid product to subsequent reactions.

[0117] 5. After drying the first solid product (weighed to 34g), add 0.03% of the compound enzyme solution to it, then adjust the pH of the mixture to 5.5, and then sonicate at 60℃ and 200kHz for 1.5h to obtain the enzymatic hydrolysate; wherein the compound enzyme contains papain and neutral protease (the amount added is selected from 1:1); the amount of compound enzyme solution added is 12ml of compound enzyme solution / 1g of the first solid product.

[0118] 6. After the enzyme hydrolysate is inactivated by boiling water, it is cooled to room temperature and then purified by adsorption with macroporous resin (D101). The second mixed organic solution is added for extraction (same as in step 4). After extraction, a second organic phase, a second aqueous phase and a second solid product are obtained. The second organic phase is collected.

[0119] 7. After merging the first and second organic phases, the glycyrrhizin was obtained by dialysis, purification and concentration.

[0120] The main difference between Example 12 and other examples is that no surfactant is used in the first mixed aqueous solution in Example 12.

[0121] Example 13: The steps of Example 13 are basically the same as those of Example 1, except that the first mixed aqueous solution does not contain acetylacetone.

[0122] Example 14: The steps of Example 14 are basically the same as those of Example 1, except that in the compound enzyme, papain is replaced with pectinase and neutral protease is replaced with cellulase.

[0123] Comparative Example 3: Comparative Example 3 includes the following steps:

[0124] 1. Provide 50g of licorice residue after decoction, dry it at 60℃ for 12h, then pulverize it and pass it through a 40-mesh sieve to obtain licorice powder;

[0125] 2. Provide *Rhizopus oryzae*; prepare PDA medium by adding an appropriate amount of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) to achieve a final ABTS concentration of 0.02%; spread the *Rhizopus oryzae* inoculum onto the PDA medium and incubate at 28°C for 5 days. After incubation, wash to obtain a suspension containing spores. Filter the suspension and dilute it with an appropriate amount of water to obtain activated and diluted inoculum. Inoculate licorice powder with the activated and diluted inoculum, controlling the fermentation temperature at 28°C, the moisture content during fermentation at 70%, and the fermentation time at 5 days to obtain licorice powder after solid-state fermentation.

[0126] 3. The licorice powder after solid-state fermentation was added to water, followed by the addition of 0.03% compound enzyme solution. The pH of the mixture was then adjusted to 5.5, and the solution was sonicated at 60℃ and 200kHz for 1.5 hours to obtain an enzymatic hydrolysate. The compound enzyme consisted of cellulase and pectinase (in a 1:1 ratio). The amount of compound enzyme solution added was 20 ml of compound enzyme solution per 1 g of solid product. After enzyme inactivation, impurity removal via dialysis, purification, and concentration, licorice flavonoids (Comparative Example 3) were obtained.

[0127] Comparative Example 4: Comparative Example 4 includes the following steps:

[0128] 1. Provide 50g of licorice residue after decoction, dry it at 60℃ for 12h, then pulverize it and pass it through a 40-mesh sieve to obtain licorice powder;

[0129] 2. Add 800 mL of 70% ethanol to the licorice powder, heat and reflux for 1.5 h, filter, add 700 mL of 70% ethanol to the filter residue and extract again, combine the filtrates; remove the solvent by vacuum distillation to obtain the licorice flavonoids of Comparative Example 4.

[0130] Determination of flavonoid content in Examples 6-14 and Comparative Examples 2-4: The products of Examples 6-14 and Comparative Examples 2-4 were added to a rotary evaporator and evaporated until a paste-like consistency was achieved. The product was collected and then dried in a freeze dryer for 36 hours. It was then reconstituted with 70% ethanol (diluted 10 times), and the absorbance at 510 nm was measured to calculate the flavonoid content. The flavonoid content values ​​of the products of Examples 6-14 and Comparative Examples 2-4 are shown in Table 2.

[0131] Table 2:

[0132] Comparative Examples 1, 6-11, and 2 show that soaking licorice powder in an organic solvent effectively disrupts the cell walls of licorice, thus facilitating the extraction of licorice flavonoids. Hexane has a relatively weak effect on cell membrane disruption but increases cell wall permeability, making it easier to release intracellular substances; its combination with acetone yields the best results. Chloroform and toluene are too potent at disrupting licorice cell walls, leading to flavonoid loss. Considering their toxicity, the combination of hexane and acetone is therefore the best choice.

[0133] A comparison of Examples 12 and 13 with Example 1 shows that fatty alcohol polyoxyethylene ether surfactants can further disrupt the cell membrane of licorice, thereby maximizing the extraction of licorice flavonoids, and the introduction of acetylacetone can also further disrupt the cell membrane of licorice.

[0134] A comparison between Example 14 and Example 1 shows that during the previous multiple soaking processes, glycyrrhizin can form a supramolecular complex structure with the alkaloids in licorice, which will hinder the further extraction of glycyrrhizin flavonoids. At this point, the lignin, cellulose, cell wall and other structures of licorice have been basically destroyed during the previous multiple soaking processes. The solid formed by glycyrrhizin and the alkaloids in licorice will hinder the extraction of the last remaining glycyrrhizin flavonoids. Therefore, the combined enzyme of neutral protease and papain is more effective than the combined enzyme of cellulase and pectinase.

[0135] Example 15: The steps of Example 15 are to extract the glycyrrhizin obtained in Example 1 with a third mixed organic solvent to prepare a higher concentration of glycyrrhizin.

[0136] The glycyrrhizin from Example 1 was dissolved in 2 mol / L water to obtain a salt solution, and 1-butyl-3-methylimidazolium methanesulfonate was dissolved in dipropanol to prepare a dipropanol solution with a concentration of 20 mg / mL. The solution was mixed with the dipropanol solution at a ratio of 1:1 and extracted. The supernatant was collected and centrifuged at 4000 r / min for 5 min. The supernatant was collected to obtain an alcoholic solution containing glycyrrhizin.

[0137] Example 16: The steps of Example 16 are the same as those of Example 15, except that 1-butyl-3-methylimidazolium methanesulfonate is replaced with 1-butyl-3-methylimidazolium p-toluenesulfonate.

[0138] Example 17: The steps of Example 17 are the same as those of Example 15, except that 1-butyl-3-methylimidazolium methanesulfonate is replaced with 1-pentyl-3-methylimidazolium trifluoromethanesulfonate.

[0139] Example 18: The steps of Example 18 are the same as those of Example 15, except that 1-butyl-3-methylimidazolium methanesulfonate is replaced with 1-hexyl-3-methylimidazolium methanesulfonate.

[0140] Detection of glycyrrhizin content: The products of Examples 15-18 were freeze-dried and then diluted with methanol. The glycyrrhizin content of Examples 15-18 was detected by high performance liquid chromatography (HPLC) under the following conditions: Agilent ODS-C18 column (250 mm × 4.6 mm, 5 μm); column temperature: 30 °C; injection volume: 10 μL; mobile phase: acetonitrile: ultrapure water containing 2% acetic acid = 45:55 (V / V); flow rate: 1 mL / min; detection wavelength: 280 nm.

[0141] The extraction rate of glycyrrhizin was calculated using the following formula: Extraction rate = C × V / m; where C is the concentration of glycyrrhizin measured in the diluent; V is the volume of the diluent; and m is the mass of the flavonoid extract from Example 1 after freeze-drying. The extraction rate values ​​for Examples 15-18 are shown in Table 3 below.

[0142] Table 3:

[0143] As can be seen from Table 1, using 1-butyl-3-methylimidazolium methanesulfonate as a eutectic solvent can effectively extract glycyrrhizin from glycyrrhiza flavonoids.

[0144] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This invention is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein.

Claims

1. A method for preparing glycyrrhizin, characterized in that, The preparation method includes the following steps: Step 1: Provide licorice raw material, dry the licorice raw material, pulverize and sieve it to obtain licorice powder; Step 2: Subject the licorice powder to a solid-state fermentation process to obtain solid-state fermented licorice powder; Step 3: Add the solid-state fermented licorice powder to a first mixed organic solution for soaking, the soaking process is carried out under ultrasonic action to obtain pretreated licorice powder; Step 4: Add the pretreated licorice powder to a first mixed aqueous solution for soaking, then add a second mixed organic solution to the first mixed aqueous solution, and after extraction, obtain a first organic phase, a first aqueous phase and a first solid product; collect the first organic phase and add the first solid product to the reactions in steps 5 and 6; Step 5: Subject the first solid product to an enzymatic hydrolysis process, the enzymatic hydrolysis process is carried out under ultrasonic action. The process is carried out under acoustic action; the enzymatic hydrolysis process uses a compound enzyme to obtain an enzymatic hydrolysate; Step 6: After the enzymatic hydrolysate undergoes enzyme inactivation and impurity removal, a second mixed organic solution is added to it, and after extraction, a second organic phase, a second aqueous phase, and a second solid product are obtained; the second organic phase is collected; Step 7: The first organic phase and the second organic phase are combined and subjected to dialysis, purification, and concentration to obtain a glycyrrhizin extract; Step 8: The glycyrrhizin extract is extracted with a third mixed organic solution to obtain the glycyrrhizin; In Step 3, the first mixed organic solution is selected from a mixed solution of n-hexane and acetone, wherein n-hexane and acetone are mixed in a volume ratio of (0.3-0.7):1; In Step 4, the first mixed aqueous solution is an aqueous solution containing a surfactant and acetylacetone; the surfactant is selected from fatty alcohol polyoxyethylene ether.

2. The preparation method according to claim 1, characterized in that, Step 2 includes the following steps: Step 2-1: Provide a microbial strain, activate the microbial strain to obtain an activated and diluted microbial strain; wherein, the microbial strain is selected from Rhizopus oryzae, Aspergillus oryzae, Rhizopus nigricans, Mucor, or Aspergillus fumigatus; Step 2-2: Inoculate the licorice powder obtained in Step 1 with the activated and diluted microbial strain, control the fermentation temperature to be selected from 25℃-30℃, the water content during the fermentation process to be selected from 70%-80%, and the fermentation time to be selected from 2-4 days, to obtain licorice powder after solid-state fermentation.

3. The preparation method according to claim 1, characterized in that, In step 4, the second mixed organic solution is ethyl acetate containing a quaternary ammonium salt compound.

4. The preparation method according to claim 1 or 3, characterized in that, Step 4 includes the following steps: Step 4-1: Add the pretreated licorice powder to the first mixed aqueous solution and soak at room temperature for 1-3 hours; Step 4-2: Add a second mixed organic solution to the first mixed aqueous solution for extraction; wherein, the second mixed organic solution is ethyl acetate containing a quaternary ammonium salt compound; Step 4-3: After extraction in Step 4-2, a first organic phase, a first aqueous phase, and a first solid product are obtained; collect the first organic phase and add the first solid product to the reactions in Steps 5 and 6.

5. The preparation method according to claim 1, characterized in that, In step 5, the enzymatic hydrolysis process is carried out using a complex enzyme, which is composed of papain and a central protease.

6. The preparation method according to claim 1 or 5, characterized in that, Steps 5 and 6 The process includes the following steps: Step 5: After drying the first solid product, add 0.01%-0.05% of a complex enzyme solution to it, then adjust the pH of the mixture to 5-6, and then sonicate at 60℃-70℃ and 200kHz for 1-1.5h to obtain an enzymatic hydrolysate; wherein the complex enzyme contains papain and a central protease; Step 6: After boiling water to inactivate the enzyme in the enzymatic hydrolysate, cool it to room temperature, and then remove impurities through macroporous resin adsorption and chromatography separation. Add a second mixed organic solution to it for extraction, and after extraction, obtain a second organic phase, a second aqueous phase, and a second solid product; collect the second organic phase; wherein the second mixed organic solution is ethyl acetate containing a quaternary ammonium salt compound.

7. The preparation method according to claim 5, characterized in that, The amount of the compound enzyme solution added is selected from: 5-10 ml of compound enzyme solution / 1 g of the first solid product.

8. The preparation method according to claim 1, characterized in that, In step 8, the third mixed organic solution is a dipropanol solution containing imidazolium sulfonate; the imidazolium sulfonate is selected from any one of 1-butyl-3-methylimidazolium methanesulfonate, 1-butyl-3-methylimidazolium p-toluenesulfonate, 1-pentyl-3-methylimidazolium trifluoromethanesulfonate or 1-hexyl-3-methylimidazolium methanesulfonate.

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