Preparation method of licoflavone

Abstract

The invention relates to the technical field of medicine preparation, in particular to a preparation method of licoflavone. The preparation method comprises the following steps: providing a licorice raw material, crushing, sieving, and carrying out solid-state fermentation to obtain licorice powder after solid-state fermentation; the preparation method comprises the following steps: adding a first mixed organic solution into a reaction kettle, soaking, adding a second mixed organic solution into the first mixed aqueous solution again, soaking, adding a second mixed organic solution, extracting, collecting an organic phase, carrying out enzymolysis enzyme deactivation and impurity removal on a solid product, and adding the second mixed organic solution again for extracting; and combining the organic phases, dialyzing, purifying and concentrating to obtain the licoflavone.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and in particular to a method for preparing glycyrrhiza flavonoids. Background Art

[0002] Glycyrrhiza is a perennial herb belonging to the subfamily Papilionoideae of the family Leguminosae. Licorice is a traditional Chinese medicine with antitussive, heat-clearing, anti-inflammatory and anti-tumor effects. Studies have shown that glycyrrhizic flavonoids play an important role in the above effects. The traditional extraction methods of glycyrrhizic flavonoids mainly include solvent extraction and steam distillation. Both methods have the defects of low extraction efficiency, incomplete extraction of effective ingredients, and high solvent consumption, which wastes licorice resources. Therefore, there is an urgent need for a method for preparing and extracting glycyrrhizic flavonoids that can maximize the utilization of licorice resources. Summary of the invention

[0003] The present invention provides a method for preparing licorice flavonoids to solve the deficiencies in the related art.

[0004] According to a first aspect of an embodiment of the present disclosure, a method for preparing glycyrrhiza flavonoids is provided, the preparation method comprising the following steps: Step 1: providing a licorice raw material, drying the licorice raw material, crushing and sieving the licorice raw material to obtain licorice powder; Step 2: subjecting the licorice powder to a solid-state fermentation process to obtain solid-state fermented licorice powder; Step 3: adding the solid-state fermented licorice powder into the first mixed organic solution for soaking, wherein the soaking process is performed under ultrasound to obtain pretreated licorice powder; Step 4: adding the pretreated licorice powder to a first mixed aqueous solution for soaking, and then adding a second mixed organic solution to the first mixed aqueous solution for extraction, to obtain a first organic phase, a first aqueous phase and a first solid product after extraction; collecting the first organic phase, and putting the first solid product into the reaction of steps 5 and 6; Step 5: subjecting the first solid product to an enzymatic hydrolysis process, wherein the enzymatic hydrolysis process is performed under ultrasound; the enzymatic hydrolysis process uses a composite enzyme to obtain an enzymatic hydrolyzate; Step 6: After the enzymatic hydrolysate has been subjected to an enzyme inactivation and impurity removal process, a second mixed organic solution is added thereto, and after extraction, a second organic phase, a second aqueous phase and a second solid product are obtained; and the second organic phase is collected; Step 7: The first organic phase and the second organic phase are combined and dialyzed, purified and concentrated to obtain the glycyrrhizin flavonoids.

[0005] In one aspect of the embodiments of the present disclosure, the licorice raw material is licorice waste residue after decoction.

[0006] In one aspect of the embodiments of the present disclosure, specifically, step 1 comprises: providing decocted licorice waste residue, drying the licorice waste residue at 60° C. for 12 hours, and then crushing the licorice waste residue and passing it through a 40-mesh sieve to obtain licorice powder.

[0007] In one aspect of the embodiments of the present disclosure, specifically, step 2 includes the following steps: Step 2-1: providing a bacterial strain, and activating 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 niger, Mucor or Aspergillus fumigatus; Step 2-2: inoculating the licorice powder obtained in step 1 with the activated and diluted strain, controlling the fermentation temperature to be 25° C.-30° C., the water content in the fermentation process to be 70%-80%, and the fermentation time to be 2-4 days, to obtain licorice powder after solid-state fermentation.

[0008] In one aspect of the embodiments of the present disclosure, preferably, in step 2-1, the bacterial species is selected from one of Rhizopus oryzae, Aspergillus oryzae, Rhizopus niger or Aspergillus fumigatus; further preferably, the bacterial species is selected from Rhizopus oryzae.

[0009] In one aspect of the embodiments of the present disclosure, further specifically, step 2-1 includes: providing a strain, wherein the strain is selected from one of Rhizopus oryzae, Aspergillus oryzae, Rhizopus nigromaculus, Mucor or Aspergillus fumigatus; preparing a PDA culture medium, adding an appropriate amount of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt, i.e., ABTS, thereto, so that the final concentration of ABTS is 0.01%-0.03%; applying the strain to the PDA culture medium, culturing at 25°C-30°C for 4-6 days, eluting after the cultivation to obtain a suspension containing spores of the strain, filtering the suspension and adding an appropriate amount of water to dilute it to obtain the activated and diluted strain.

[0010] In one aspect of the embodiments of the present disclosure, in step 3, the first mixed organic solution is selected from any one of the following two combinations: a) chloroform, and any one of methanol, ethanol, propanol, isopropanol, ether or acetone; b) n-hexane, and any one of ethanol, acetone, and ethyl acetate.

[0011] In one aspect of an embodiment of the present disclosure, specifically, in step 3, the first mixed organic solution can 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.

[0012] In one aspect of the embodiments of the present disclosure, it is further preferred that in step 3, the first mixed organic solution can 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.

[0013] In one aspect of the embodiments of the present disclosure, in step 4, the first mixed aqueous solution is an aqueous solution comprising a surfactant and acetylacetone; the surfactant is selected from fatty alcohol polyoxyethylene ether.

[0014] In one aspect of the embodiments of the present 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.

[0015] In one aspect of the embodiments of the present disclosure, glacial acetic acid may be added to the first mixed aqueous solution, and the concentration of the glacial acetic acid may be selected from 0.3-0.6 g / L.

[0016] In one aspect of the embodiments of the present disclosure, in step 4, the second mixed organic solution is ethyl acetate containing a quaternary ammonium salt compound. In one aspect of the embodiments of the present 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.

[0017] In one aspect of the disclosed embodiment, in the second mixed organic solution, the content of the quaternary ammonium salt compound is 10-25 g / L.

[0018] In one aspect of the disclosed embodiment, step 4 includes the following steps: Step 4-1: adding the pretreated licorice powder to a first mixed aqueous solution, and soaking the mixture at room temperature for 1-3 hours, wherein the first mixed aqueous solution is an aqueous solution containing a surfactant and acetylacetone; Step 4-2: adding 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; the first organic phase is collected, and the first solid product is put into the reaction of steps 5 and 6.

[0019] In one aspect of the embodiments of the present disclosure, the volume ratio of the first mixed aqueous solution to the second mixed organic solution is selected from (0.5-1.5):1.

[0020] In one aspect of the disclosed embodiments, in step 5, the enzymatic hydrolysis process is performed using a composite enzyme comprising papain and neutral protease.

[0021] In one aspect of the embodiments of the present disclosure, step 5 and step 6 include the following steps: Step 5: After drying the first solid product, add 0.01%-0.05% of a composite enzyme solution thereto, adjust the pH value of the mixed solution to 5-6, and then ultrasonicate at 60° C.-70° C. and 200 kHz for 1-1.5 hours to obtain an enzymatic solution; wherein the composite enzyme comprises papain and neutral protease; Step 6: After the enzyme is inactivated by boiling water, the enzymatic hydrolyzate is cooled to room temperature, and then impurities are removed by macroporous resin adsorption and chromatography separation process, and a second mixed organic solution is added thereto for extraction, and after extraction, a second organic phase, a second aqueous phase and a second solid product are obtained; the second organic phase is collected; wherein the second mixed organic solution is ethyl acetate containing a quaternary ammonium salt compound.

[0022] In one aspect of the disclosed embodiment, the amount of the complex enzyme solution added is selected from: 5-15 ml of the complex enzyme solution / 1 g of the first solid product.

[0023] According to a second aspect of the embodiments of the present disclosure, there is provided a glycyrrhizin flavonoid, characterized in that the glycyrrhizin flavonoid is obtained by the aforementioned preparation method.

[0024] The technical solution provided by the embodiments of the present disclosure may include the following beneficial effects: It can be seen from the above embodiments that the present disclosure makes the most of licorice waste residue and extracts as much licorice flavonoids as possible compared to other extraction methods.

[0025] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present disclosure. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. The relevant embodiments described herein are illustrative and are used to provide a basic understanding of the present invention. The embodiments of the present invention should not be interpreted as limiting the present invention.

[0027] For simplicity, only some numerical ranges are specifically disclosed herein. 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, and any upper limit can be combined with any other upper limit to form an unspecified range. In addition, each separately disclosed point or single value can itself be combined as a lower limit or upper limit with any other point or single value or with other lower limits or upper limits to form an unspecified range.

[0028] In this article, the terms "comprises", "comprising" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of more restrictions, an element defined by the sentence "comprising a ..." does not exclude the presence of other identical elements in the process, method, article or device that includes the element.

[0029] In the description herein, unless otherwise specified, “above” and “below” include the number.

[0030] Unless otherwise specified, the terms used in the present invention have the commonly known meanings generally understood by those skilled in the art. Unless otherwise specified, the numerical values ​​of the various parameters mentioned in the present invention can be measured by various measurement methods commonly used in the art (for example, they can be tested according to the methods given in the embodiments of the present invention).

[0031] The term "about" is used to describe and illustrate small changes. When used in conjunction with an event or situation, the term may refer to an example in which the event or situation occurs precisely and an example in which the event or situation occurs very approximately. For example, when used in conjunction with a numerical value, 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%. In addition, amounts, ratios, and other numerical values ​​are sometimes presented in this article in a range format. It should be understood that such a range format is for convenience and simplicity, and should be flexibly understood to include not only numerical values ​​explicitly designated as range limits, but also all individual numerical values ​​or subranges encompassed within the range, as if each numerical value and subrange were explicitly designated.

[0032] A list of items connected by the terms "at least one of," "at least one of," "at least one of," or other similar terms may mean any combination of the listed items. For example, if items A and B are listed, the phrase "at least one of A and B" means only A; only B; or A and B. In another example, if items A, B, and C are listed, 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.

[0033] In the present disclosure, in step 2, the solid-state fermentation process utilizes the lignocellulose in the medicinal material as a support and nutrient source for the growth of microorganisms; lignin in the medicinal material is a rigid barrier that is not conducive to the extraction of effective active substances in the medicinal material; the solid-state fermentation process can effectively degrade lignin and promote the hydrolysis of cellulose components. In addition, through the action of microorganisms, the medicinal material is fermented under appropriate temperature, humidity and moisture conditions to enhance its original properties or produce new effects. Therefore, selecting a suitable strain for solid-state fermentation can not only improve the utilization rate of glycyrrhizin in licorice waste residue, thereby increasing the content of glycyrrhizin, but also give the prepared glycyrrhizin better antioxidant properties.

[0034] In the present disclosure, in step 3, the role of soaking licorice powder with an organic solution (accompanied by ultrasound) is to destroy the cell wall and cell membrane (mainly the cell wall) of licorice, so as to further maximize the extraction of licorice flavonoids. Among common organic solutions, chloroform, acetone, n-hexane, toluene and carbon tetrachloride can be used for soaking to destroy the cell wall of the plant; among them, toluene and carbon tetrachloride have a strong destructive effect on the cell wall, which can significantly reduce the potential of the cell wall and increase the permeability; chloroform and acetone can dissolve the lipid components in the cell wall, which can better swell or dissolve the cell structure; n-hexane has a relatively weak destructive effect on the cell membrane, but can increase the permeability of the cell wall, making it easier to release substances in the cell.

[0035] In the present disclosure, in step 4, the role of the surfactant is to further destroy the cell membrane. The surfactant has a hydrophilic group and a hydrophobic group, which is similar to the phospholipid bilayer structure of the cell membrane; therefore, the surfactant can be integrated into the cell membrane and destroy the integrity of the cell membrane by aggregation or reaction. The surfactant can be adsorbed on the surface of the cell membrane by electrostatic action, interact with the phospholipids on the membrane, causing the phospholipids and proteins to separate, thereby destroying the cell membrane; or, the hydrophobic group of the surfactant can interact with the lipids of the cell membrane, change the structure of the cell membrane, and cause the cell membrane to dissolve; or, the surfactant interacts with the phospholipid bilayer of the cell membrane to form a micellar structure, thereby dissolving the cell membrane; in short, the role of the surfactant is to further destroy the cell membrane of licorice so as to further maximize the extraction of licorice flavonoids. In the present disclosure, a fatty alcohol polyoxyethylene ether surfactant is used because the molecular structure of its polymer is easy to be removed by dialysis during post-treatment, which is its superiority over other surfactants.

[0036] The present application is further described below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application.

[0037] Embodiment 1: Embodiment 1 comprises the following steps: 1. Provide 50 g of decocted licorice residue, dry it at 60° C. for 12 h, then crush it and pass it through a 40-mesh sieve to obtain licorice powder; 2. Provide Rhizopus oryzae; prepare PDA culture medium, add an appropriate amount of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt, i.e., ABTS, thereto, so that the final concentration of ABTS is 0.02%; apply Rhizopus oryzae strains on PDA culture medium, culture at 28°C for 5 days, and after the culture is completed, wash to obtain a suspension containing spores of the strain, filter the suspension and add an appropriate amount of water to dilute, to obtain an activated and diluted strain. Inoculate licorice powder with the activated and diluted strain, control the fermentation temperature to be selected from 28°C, the water content of the fermentation process to be selected from 70%, and the fermentation time to be selected from 5 days, to obtain licorice powder after solid-state fermentation.

[0038] 3. Add the licorice powder after solid-state fermentation into the first mixed organic solution and soak it for 5 hours. The soaking process is carried out under ultrasound at 200kHz to obtain pretreated licorice powder; the first mixed organic solution is a mixed solution of n-hexane and acetone, wherein n-hexane and acetone are mixed in a volume ratio of 45mL:100mL.

[0039] 4. Add the pretreated licorice powder to the first mixed aqueous solution and soak it at room temperature for 2 hours, wherein the first mixed aqueous solution is 200 mL of an aqueous solution containing surfactant AEO-7, acetylacetone and glacial acetic acid (the concentration of AEO-7 is 20 g / L, the concentration of acetylacetone is 8 g / L, and the concentration of glacial acetic acid is 0.5 g / L). Then add the second mixed organic solution to the first mixed aqueous solution for extraction; wherein the second mixed organic solution is ethyl acetate (200 mL) containing tetrabutylammonium bromide, and the concentration of tetrabutylammonium bromide is selected from 15 g / L. Obtain a first organic phase, a first aqueous phase and a first solid product; collect the first organic phase, and put the first solid product into the subsequent reaction.

[0040] 5. After the first solid product is dried (weighed to be 34 g), 0.03% of the complex enzyme solution is added thereto, and then the pH value of the mixed solution is adjusted to 5.5, and then ultrasonicated at 60°C and 200 kHz for 1.5 hours to obtain an enzymatic solution; wherein the complex enzyme comprises papain and neutral protease (the amount added is selected from 1:1); the amount of the complex enzyme solution added is 12 ml of the complex enzyme solution / 1 g of the first solid product.

[0041] 6. After the enzymatic hydrolysate is inactivated by boiling water, it is cooled to room temperature, and then impurities are removed by macroporous resin adsorption (D101). A second mixed organic solution is added thereto for extraction (the same as in 4). After extraction, a second organic phase, a second aqueous phase and a second solid product are obtained; the second organic phase is collected.

[0042] 7. After combining the first organic phase and the second organic phase, the glycyrrhizin of Example 1 is obtained through dialysis, purification and concentration.

[0043] Embodiment 2: Embodiment 2 comprises the following steps: 1. Provide 50 g of decocted licorice residue, dry it at 60° C. for 12 h, then crush it and pass it through a 40-mesh sieve to obtain licorice powder; 2. Provide Aspergillus fumigatus; prepare PDA culture medium, add an appropriate amount of 2,2-azo-di(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt, i.e., ABTS, thereto, so that the final concentration of ABTS is 0.02%; apply Aspergillus fumigatus strains (with the same mass as Rhizopus oryzae used in Example 1) on PDA culture medium, culture at 28°C for 5 days, and after culturing, wash to obtain a suspension containing spores of the strain, filter the suspension and add an appropriate amount of water to dilute, to obtain an activated and diluted strain. Inoculate licorice powder with the activated and diluted strain, control the fermentation temperature to be selected from 28°C, the water content of the fermentation process to be selected from 70%, and the fermentation time to be selected from 5 days, to obtain licorice powder after solid-state fermentation.

[0044] 3. Add the licorice powder after solid-state fermentation into the first mixed organic solution and soak it for 5 hours. The soaking process is carried out under ultrasound at 200kHz to obtain pretreated licorice powder; the first mixed organic solution is a mixed solution of n-hexane and acetone, wherein n-hexane and acetone are mixed in a volume ratio of 45mL:100mL.

[0045] 4. Add the pretreated licorice powder to the first mixed aqueous solution and soak it at room temperature for 2 hours, wherein the first mixed aqueous solution is 200 mL of an aqueous solution containing surfactant AEO-7, acetylacetone and glacial acetic acid (the concentration of AEO-7 is 20 g / L, the concentration of acetylacetone is 8 g / L, and the concentration of glacial acetic acid is 0.5 g / L). Then add the second mixed organic solution to the first mixed aqueous solution for extraction; wherein the second mixed organic solution is ethyl acetate (200 mL) containing tetrabutylammonium bromide, and the concentration of tetrabutylammonium bromide is selected from 15 g / L. Obtain a first organic phase, a first aqueous phase and a first solid product; collect the first organic phase, and put the first solid product into the subsequent reaction.

[0046] 5. After the first solid product is dried (weighed to be 34 g), 0.03% of the complex enzyme solution is added thereto, and then the pH value of the mixed solution is adjusted to 5.5, and then ultrasonicated at 60°C and 200 kHz for 1.5 hours to obtain an enzymatic solution; wherein the complex enzyme comprises papain and neutral protease (the amount added is selected from 1:1); the amount of the complex enzyme solution added is 12 ml of the complex enzyme solution / 1 g of the first solid product.

[0047] 6. After the enzymatic hydrolysate is inactivated by boiling water, it is cooled to room temperature, and then impurities are removed by macroporous resin adsorption (D101). A second mixed organic solution is added thereto for extraction (the same as in 4). After extraction, a second organic phase, a second aqueous phase and a second solid product are obtained; the second organic phase is collected.

[0048] 7. After combining the first organic phase and the second organic phase, the glycyrrhizin of Example 2 is obtained through dialysis, purification and concentration.

[0049] Example 3: The steps of Example 3 are substantially the same as those of Example 1, except that an equal mass of Rhizopus nigromaculus is used to replace the Rhizopus oryzae used in Example 1.

[0050] Example 4: The steps of Example 4 are substantially the same as those of Example 1, except that an equal mass of Aspergillus oryzae species is used to replace the Rhizopus oryzae species used in Example 1.

[0051] Example 5: The steps of Example 5 are substantially the same as those of Example 1, except that an equal mass of Mucor species is used to replace the Rhizopus oryzae species used in Example 1.

[0052] Comparative Example 1: Comparative Example 1 comprises the following steps: 1. Provide 50 g of decocted licorice residue, dry it at 60° C. for 12 h, then crush it and pass it through a 40-mesh sieve to obtain licorice powder; 2. Add the crushed licorice powder into the first mixed organic solution and soak it for 5 hours. The soaking process is carried out under ultrasound at 200kHz to obtain pretreated licorice powder; the first mixed organic solution is a mixed solution of n-hexane and acetone, wherein n-hexane and acetone are mixed in a volume ratio of 45mL:100mL.

[0053] 3. Add the pretreated licorice powder to the first mixed aqueous solution and soak it at room temperature for 2 hours, wherein the first mixed aqueous solution is 200 mL of an aqueous solution containing surfactant AEO-7, acetylacetone and glacial acetic acid (the concentration of AEO-7 is 20 g / L, the concentration of acetylacetone is 8 g / L, and the concentration of glacial acetic acid is 0.5 g / L). Then add the second mixed organic solution to the first mixed aqueous solution for extraction; wherein the second mixed organic solution is ethyl acetate (200 mL) containing tetrabutylammonium bromide, and the concentration of tetrabutylammonium bromide is selected from 15 g / L. Obtain a first organic phase, a first aqueous phase and a first solid product; collect the first organic phase, and put the first solid product into the subsequent reaction.

[0054] 4. After the first solid product is dried (weighed to be 34 g), 0.03% of the complex enzyme solution is added thereto, and then the pH value of the mixed solution is adjusted to 5.5, and then ultrasonicated at 60°C and 200 kHz for 1.5 hours to obtain an enzymatic solution; wherein the complex enzyme comprises papain and neutral protease (the amount added is selected from 1:1); the amount of the complex enzyme solution added is 12 ml of the complex enzyme solution / 1 g of the first solid product.

[0055] 5. After the enzymatic hydrolysate is inactivated by boiling water, it is cooled to room temperature, and then impurities are removed by macroporous resin adsorption (D101). A second mixed organic solution is added thereto for extraction (the same as in 3). After extraction, a second organic phase, a second aqueous phase and a second solid product are obtained; the second organic phase is collected.

[0056] 6. After combining the first organic phase and the second organic phase, the glycyrrhizin of Comparative Example 1 was obtained through dialysis, purification and concentration.

[0057] The main difference between Comparative Example 1 and Examples 1-5 is that Comparative Example 1 does not include a solid-state fermentation process.

[0058] 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 the product was in a paste state. The product was collected and then placed in a freeze dryer for 36 hours. The product was re-dissolved with 70% ethanol (the dilution factor was 10 times), and the absorbance at 510 nm was measured to calculate the flavonoid content. The values ​​of the flavonoid content of the products of Examples 1-5 and Comparative Example 1 are shown in Table 1: Table 1:

[0059] It can be seen that the flavonoids yields of Examples 1-5 after solid-state fermentation are higher than those of Comparative Example 1, indicating that the solid-state fermentation process can effectively degrade lignin and promote the hydrolysis of cellulose components. However, different types of strains have different abilities to hydrolyze cellulose components in licorice. The ability of Example 5 using Mucor species to hydrolyze cellulose components in licorice is significantly weaker than that of Examples 1-4.

[0060] Embodiment 6: Embodiment 6 comprises the following steps: 1. Provide 50 g of decocted licorice residue, dry it at 60° C. for 12 h, then crush it and pass it through a 40-mesh sieve to obtain licorice powder; 2. Provide Rhizopus oryzae; prepare PDA culture medium, add an appropriate amount of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt, i.e., ABTS, thereto, so that the final concentration of ABTS is 0.02%; apply Rhizopus oryzae strains on PDA culture medium, culture at 28°C for 5 days, and after the culture is completed, wash to obtain a suspension containing spores of the strain, filter the suspension and add an appropriate amount of water to dilute, to obtain an activated and diluted strain. Inoculate licorice powder with the activated and diluted strain, control the fermentation temperature to be selected from 28°C, the water content of the fermentation process to be selected from 70%, and the fermentation time to be selected from 5 days, to obtain licorice powder after solid-state fermentation.

[0061] 3. Add the licorice powder after solid-state fermentation into the first mixed organic solution and soak it for 5 hours. The soaking process is carried out under ultrasound at 200kHz to obtain pretreated licorice powder; the first mixed organic solution is a mixed solution of n-hexane and acetone, wherein n-hexane and acetone are mixed in a volume ratio of 100mL:45mL.

[0062] 4. Add the pretreated licorice powder to the first mixed aqueous solution and soak it at room temperature for 2 hours, wherein the first mixed aqueous solution is 200 mL of an aqueous solution containing surfactant AEO-7, acetylacetone and glacial acetic acid (the concentration of AEO-7 is 20 g / L, the concentration of acetylacetone is 8 g / L, and the concentration of glacial acetic acid is 0.5 g / L). Then add the second mixed organic solution to the first mixed aqueous solution for extraction; wherein the second mixed organic solution is ethyl acetate (200 mL) containing tetrabutylammonium bromide, and the concentration of tetrabutylammonium bromide is selected from 15 g / L. Obtain a first organic phase, a first aqueous phase and a first solid product; collect the first organic phase, and put the first solid product into the subsequent reaction.

[0063] 5. After the first solid product is dried (weighed to be 34 g), 0.03% of the complex enzyme solution is added thereto, and then the pH value of the mixed solution is adjusted to 5.5, and then ultrasonicated at 60°C and 200 kHz for 1.5 hours to obtain an enzymatic solution; wherein the complex enzyme comprises papain and neutral protease (the amount added is selected from 1:1); the amount of the complex enzyme solution added is 12 ml of the complex enzyme solution / 1 g of the first solid product.

[0064] 6. After the enzymatic hydrolysate is inactivated by boiling water, it is cooled to room temperature, and then impurities are removed by macroporous resin adsorption (D101). A second mixed organic solution is added thereto for extraction (the same as in 4). After extraction, a second organic phase, a second aqueous phase and a second solid product are obtained; the second organic phase is collected.

[0065] 7. After combining the first organic phase and the second organic phase, the glycyrrhizin of Example 6 was obtained through dialysis, purification and concentration.

[0066] Example 7: The steps of Example 7 are substantially the same as those of Example 1, except that a first mixed organic solution of n-hexane and ethanol mixed in a volume ratio of 45 mL:100 mL is used to replace the first mixed organic solution used in Example 1.

[0067] Example 8: The steps of Example 8 are substantially the same as those of Example 1, except that a first mixed organic solution of n-hexane and ethanol mixed in a volume ratio of 100 mL:45 mL is used to replace the first mixed organic solution used in Example 1.

[0068] Example 9: The steps of Example 9 are substantially the same as those of Example 1, except that a first mixed organic solution of chloroform and methanol in a volume ratio of 45 mL:100 mL is used to replace the first mixed organic solution used in Example 1.

[0069] Example 10: The steps of Example 10 are substantially the same as those of Example 1, except that a first mixed organic solution of chloroform and ethanol in a volume ratio of 45 mL:100 mL is used to replace the first mixed organic solution used in Example 1.

[0070] Example 11: The steps of Example 11 are substantially the same as those of Example 1, except that a first mixed organic solution of toluene and ethanol mixed in a volume ratio of 45 mL:100 mL is used to replace the first mixed organic solution used in Example 1.

[0071] Comparative Example 2: Comparative Example 2 comprises the following steps: 1. Provide 50 g of decocted licorice residue, dry it at 60° C. for 12 h, then crush it and pass it through a 40-mesh sieve to obtain licorice powder; 2. Provide Rhizopus oryzae; prepare PDA culture medium, add an appropriate amount of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt, i.e., ABTS, thereto, so that the final concentration of ABTS is 0.02%; apply Rhizopus oryzae strains on PDA culture medium, culture at 28°C for 5 days, and after the culture is completed, wash to obtain a suspension containing spores of the strain, filter the suspension and add an appropriate amount of water to dilute, to obtain an activated and diluted strain. Inoculate licorice powder with the activated and diluted strain, control the fermentation temperature to be selected from 28°C, the water content of the fermentation process to be selected from 70%, and the fermentation time to be selected from 5 days, to obtain licorice powder after solid-state fermentation.

[0072] 3. Add the solid-state fermented licorice powder to the first mixed aqueous solution and soak it at room temperature for 2 hours, wherein the first mixed aqueous solution is 200 mL of an aqueous solution containing surfactant AEO-7, acetylacetone and glacial acetic acid (the concentration of AEO-7 is 20 g / L, the concentration of acetylacetone is 8 g / L, and the concentration of glacial acetic acid is 0.5 g / L). Then add the second mixed organic solution to the first mixed aqueous solution for extraction; wherein the second mixed organic solution is ethyl acetate (200 mL) containing tetrabutylammonium bromide, and the concentration of tetrabutylammonium bromide is selected from 15 g / L. Obtain a first organic phase, a first aqueous phase and a first solid product; collect the first organic phase, and put the first solid product into the subsequent reaction.

[0073] 4. After the first solid product is dried (weighed to be 34 g), 0.03% of the complex enzyme solution is added thereto, and then the pH value of the mixed solution is adjusted to 5.5, and then ultrasonicated at 60°C and 200 kHz for 1.5 hours to obtain an enzymatic solution; wherein the complex enzyme comprises papain and neutral protease (the amount added is selected from 1:1); the amount of the complex enzyme solution added is 12 ml of the complex enzyme solution / 1 g of the first solid product.

[0074] 5. After the enzymatic hydrolysate is inactivated by boiling water, it is cooled to room temperature, and then impurities are removed by macroporous resin adsorption (D101). A second mixed organic solution is added thereto for extraction (the same as in 3). After extraction, a second organic phase, a second aqueous phase and a second solid product are obtained; the second organic phase is collected.

[0075] 6. After combining the first organic phase and the second organic phase, the mixture was dialyzed, purified and concentrated to obtain the glycyrrhizin of Comparative Example 2.

[0076] The main difference between Comparative Example 2 and the embodiment is that, in Comparative Example 2, no first organic solution is used for soaking to further destroy the cell wall.

[0077] Embodiment 12: Embodiment 12 comprises the following steps: 1. Provide 50 g of decocted licorice residue, dry it at 60° C. for 12 h, then crush it and pass it through a 40-mesh sieve to obtain licorice powder; 2. Provide Rhizopus oryzae; prepare PDA culture medium, add an appropriate amount of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt, i.e., ABTS, thereto, so that the final concentration of ABTS is 0.02%; apply Rhizopus oryzae strains on PDA culture medium, culture at 28°C for 5 days, and after the culture is completed, wash to obtain a suspension containing spores of the strain, filter the suspension and add an appropriate amount of water to dilute, to obtain an activated and diluted strain. Inoculate licorice powder with the activated and diluted strain, control the fermentation temperature to be selected from 28°C, the water content of the fermentation process to be selected from 70%, and the fermentation time to be selected from 5 days, to obtain licorice powder after solid-state fermentation.

[0078] 3. Add the licorice powder after solid-state fermentation into the first mixed organic solution and soak it for 5 hours. The soaking process is carried out under ultrasound at 200kHz to obtain pretreated licorice powder; the first mixed organic solution is a mixed solution of n-hexane and acetone, wherein n-hexane and acetone are mixed in a volume ratio of 45mL:100mL.

[0079] 4. Add the pretreated licorice powder to the first mixed aqueous solution and soak it at room temperature for 2 hours, wherein the first mixed aqueous solution is 200 mL of an aqueous solution containing acetylacetone and glacial acetic acid (the concentration of acetylacetone is 8 g / L, and the concentration of glacial acetic acid is 0.5 g / L). Then add the second mixed organic solution to the first mixed aqueous solution for extraction; wherein the second mixed organic solution is ethyl acetate (200 mL) containing tetrabutylammonium bromide, and the concentration of tetrabutylammonium bromide is selected from 15 g / L. Obtain a first organic phase, a first aqueous phase and a first solid product; collect the first organic phase, and put the first solid product into the subsequent reaction.

[0080] 5. After the first solid product is dried (weighed to be 34 g), 0.03% of the complex enzyme solution is added thereto, and then the pH value of the mixed solution is adjusted to 5.5, and then ultrasonicated at 60°C and 200 kHz for 1.5 hours to obtain an enzymatic solution; wherein the complex enzyme comprises papain and neutral protease (the amount added is selected from 1:1); the amount of the complex enzyme solution added is 12 ml of the complex enzyme solution / 1 g of the first solid product.

[0081] 6. After the enzymatic hydrolysate is inactivated by boiling water, it is cooled to room temperature, and then impurities are removed by macroporous resin adsorption (D101). A second mixed organic solution is added thereto for extraction (the same as in 4). After extraction, a second organic phase, a second aqueous phase and a second solid product are obtained; the second organic phase is collected.

[0082] 7. After combining the first organic phase and the second organic phase, the glycyrrhizin of Example 12 is obtained through dialysis, purification and concentration.

[0083] The main difference between Example 12 and other examples is that Example 12 does not use a surfactant in the first mixed aqueous solution.

[0084] Example 13: The steps of Example 13 are substantially the same as those of Example 1, except that the first mixed aqueous solution does not contain acetylacetone.

[0085] Example 14: The steps of Example 14 are basically the same as those of Example 1, except that in the composite enzyme, papain is replaced by pectinase, and neutral protease is replaced by cellulase.

[0086] Comparative Example 3: Comparative Example 3 comprises the following steps: 1. Provide 50 g of decocted licorice residue, dry it at 60° C. for 12 h, then crush it and pass it through a 40-mesh sieve to obtain licorice powder; 2. Provide Rhizopus oryzae; prepare PDA culture medium, add an appropriate amount of 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt, i.e., ABTS, thereto, so that the final concentration of ABTS is 0.02%; apply Rhizopus oryzae strains on PDA culture medium, culture at 28°C for 5 days, and after the culture is completed, wash to obtain a suspension containing spores of the strain, filter the suspension and add an appropriate amount of water to dilute, to obtain an activated and diluted strain. Inoculate licorice powder with the activated and diluted strain, control the fermentation temperature to be selected from 28°C, the water content of the fermentation process to be selected from 70%, and the fermentation time to be selected from 5 days, to obtain licorice powder after solid-state fermentation.

[0087] 3. Add the solid-state fermented licorice powder to water, then add 0.03% of the complex enzyme solution thereto, then adjust the pH value of the mixed solution to 5.5, and then ultrasonicate at 60°C and 200kHz for 1.5h to obtain an enzymatic solution; wherein the complex enzyme comprises cellulase and pectinase (the amount added is selected from 1:1); the amount of the complex enzyme solution added is 20ml of the complex enzyme solution / 1g of the solid product. Then, after enzyme inactivation, impurity removal, dialysis, purification and concentration, the licorice flavonoids of Comparative Example 3 are obtained.

[0088] Comparative Example 4: Comparative Example 4 comprises the following steps: 1. Provide 50 g of decocted licorice residue, dry it at 60° C. for 12 h, then crush it and pass it through a 40-mesh sieve to obtain licorice powder; 2. Add 800 mL of 70% ethanol to the licorice powder, heat and reflux to extract for 1.5 h, filter, add 700 mL of 70% ethanol to the residue and extract again, combine the filtrate; remove the solvent by reduced pressure distillation to obtain the glycyrrhiza flavonoids of Comparative Example 4.

[0089] 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 the product was in a paste state. The product was collected and then placed in a freeze dryer for 36 hours. The product was re-dissolved with 70% ethanol (the dilution factor was 10 times), and the absorbance at 510 nm was measured to calculate the flavonoid content. The values ​​of the flavonoid content of the products of Examples 6-14 and Comparative Examples 2-4 are shown in Table 2: Table 2:

[0090] By comparing Examples 1, 6-11 and Comparative Example 2, it can be seen that the use of organic solution to soak licorice powder can better destroy the cell wall of licorice and further extract licorice flavonoids. The destructive effect of n-hexane on the cell membrane is relatively weak, but it can increase the permeability of the cell wall, making it easier to release substances in the cell, and its combination with acetone has the best effect; chloroform and toluene have too strong destructive ability on the cell wall of licorice, which will cause the loss of flavonoids, and considering the toxicity of the two, the combination of n-hexane and acetone is the best.

[0091] By comparing Examples 12 and 13 with Example 1, it can be seen that the fatty alcohol polyoxyethylene ether surfactant can further destroy the cell membrane of licorice, thereby further maximizing the extraction of licorice flavonoids, and the introduction of acetylacetone can also further destroy the cell membrane of licorice.

[0092] By comparing Example 14 with Example 1, it can be seen 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 time, during the previous multiple soaking processes, the structures of licorice such as lignin, cellulose, and cell walls have been basically destroyed. At this time, the solid formed by glycyrrhizin and the alkaloids in licorice will hinder the extraction of the last remaining glycyrrhizin flavonoids. Therefore, the use of a composite enzyme of neutral protease and papain is better than the use of a composite enzyme of cellulase and pectinase.

[0093] Those skilled in the art will readily appreciate other embodiments of the present invention after considering the specification and practicing the disclosure disclosed herein. The present invention is intended to cover any variations, uses or adaptations of the present invention that follow the general principles of the present invention and include common knowledge or customary technical means in the art that are not disclosed by the present invention.

Claims

1. A method for preparing glycyrrhiza flavonoids, characterized in that: The preparation method comprises the following steps: Step 1: providing a licorice raw material, drying the licorice raw material, crushing and sieving the licorice raw material to obtain licorice powder; Step 2: subjecting the licorice powder to a solid-state fermentation process to obtain solid-state fermented licorice powder; Step 3: adding the solid-state fermented licorice powder into the first mixed organic solution for soaking, wherein the soaking process is performed under ultrasound to obtain pretreated licorice powder; Step 4: adding the pretreated licorice powder to the first mixed aqueous solution for soaking, then adding the second mixed organic solution to the first mixed aqueous solution, and obtaining a first organic phase, a first aqueous phase and a first solid product after extraction; collecting the first organic phase, and putting the first solid product into the reaction of step 5 and step 6; Step 5: subjecting the first solid product to an enzymatic hydrolysis process, wherein the enzymatic hydrolysis process is performed under ultrasound; the enzymatic hydrolysis process uses a composite enzyme to obtain an enzymatic hydrolyzate; Step 6: After the enzymatic hydrolysate has been subjected to an enzyme inactivation and impurity removal process, a second mixed organic solution is added thereto, and after extraction, a second organic phase, a second aqueous phase and a second solid product are obtained; and the second organic phase is collected; Step 7: The first organic phase and the second organic phase are combined and dialyzed, purified and concentrated to obtain the glycyrrhizin flavonoids.

2. The preparation method according to claim 1, characterized in that: The liquorice raw material is liquorice waste residue after decoction.

3. The preparation method according to claim 1, characterized in that: Step 2 includes the following steps: Step 2-1: providing a bacterial strain, and activating the bacterial strain to obtain an activated and diluted bacterial strain; wherein the bacterial strain is selected from Rhizopus oryzae, Aspergillus oryzae, Rhizopus niger, Mucor or Aspergillus fumigatus; Step 2-2: inoculating the licorice powder obtained in step 1 with the activated and diluted strain, controlling the fermentation temperature to be 25° C.-30° C., the water content in the fermentation process to be 70%-80%, and the fermentation time to be 2-4 days, to obtain licorice powder after solid-state fermentation.

4. The preparation method according to claim 1, characterized in that: In step 3, the first mixed organic solution is selected from any one of the following two combinations: a) chloroform, and any one of methanol, ethanol, propanol, isopropanol, ether or acetone; b) n-hexane, and any one of ethanol, acetone, and ethyl acetate.

5. The preparation method according to claim 1, characterized in that: 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.

6. 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.

7. The preparation method according to claim 5 or 6, characterized in that: Step 4 includes the following steps: Step 4-1: adding the pretreated licorice powder to a first mixed aqueous solution, and soaking the mixture at room temperature for 1-3 hours, wherein the first mixed aqueous solution is an aqueous solution containing a surfactant and acetylacetone; Step 4-2: adding 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; the first organic phase is collected, and the first solid product is put into the reaction of steps 5 and 6.

8. The preparation method according to claim 1, characterized in that: In step 5, the enzymatic hydrolysis process is performed using a composite enzyme comprising papain and neutral protease.

9. The preparation method according to claim 8, characterized in that: Step 5 and step 6 include the following steps: Step 5: After drying the first solid product, add 0.01%-0.05% of a composite enzyme solution thereto, adjust the pH value of the mixed solution to 5-6, and then ultrasonicate at 60°C-70°C and 200kHz for 1-1.5h to obtain an enzymatic solution; wherein the composite enzyme comprises papain and central protease; Step 6: After the enzyme is inactivated by boiling water, the enzymatic hydrolyzate is cooled to room temperature, and then impurities are removed by macroporous resin adsorption and chromatography separation process, and a second mixed organic solution is added thereto for extraction, and after extraction, a second organic phase, a second aqueous phase and a second solid product are obtained; the second organic phase is collected; wherein the second mixed organic solution is ethyl acetate containing a quaternary ammonium salt compound.

10. The preparation method according to claim 9, characterized in that: The amount of the complex enzyme solution added is selected from: 5-15 ml of the complex enzyme solution / 1 g of the first solid product.

Images