Method for simultaneously extracting and purifying glabridin, glycyrrhizic acid and isoliquiritigenin

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a method for simultaneously extracting and purifying glabridin, glycyrrhizic acid and isoliquiritigenin. The method comprises the following steps: (1) adding ethanol into glycyrrhiza glabra, and extracting to obtain an extracting solution; (2) extracting the extracting solution with ethyl acetate to obtain an organic phase and a water phase; and (3) purifying the organic phase by using a rapid preparative liquid chromatograph, carrying out gradient elution by using 30%-70% of ethyl acetate-cyclohexane as a mobile phase, and collecting the eluent, thereby obtaining the glabridin. (4) carrying out acidification and centrifugal precipitation on the water phase to obtain a glycyrrhizic acid crude product and a supernatant, adding absolute ethyl alcohol into the glycyrrhizic acid crude product, and carrying out ultrasonic extraction and centrifugation to obtain a glycyrrhizic acid ethanol solution; and (5) extracting and purifying the supernate to obtain the isoliquiritigenin.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a method for simultaneously extracting and purifying glycyrrhizin, glycyrrhizic acid and isoglycyrrhizin. Background Technology

[0002] Licorice root (Glycyrrhiza glabra) Glycyrrhiza glabra L. Licorice root (also known as European licorice or glycyrrhiza glabra) is one of the three types of licorice listed in the 2015 edition of the Chinese Pharmacopoeia. Its roots and rhizomes are used medicinally. Licorice is one of the most frequently used medicinal herbs in Chinese herbal prescriptions, used to treat typhoid fever, palpitations, sore throat, and lung heat with sore throat. Licorice root is not only rich in glycyrrhizic acid and glycyrrhizin, but also contains a unique flavonoid—glabridin. Glycyrrhizin has strong antioxidant, anti-atherosclerotic, anti-cancer, anti-malarial, and melanin-inhibiting functions. In particular, glabridin has very strong anti-inflammatory and melanin-inhibiting effects, earning it the reputation of "whitening gold" in cosmetics. It is used by many internationally renowned cosmetic manufacturers in whitening skincare products, possessing extremely high economic value.

[0003] Licorice is one of the most frequently used and widely consumed herbs in traditional Chinese medicine, rich in glycyrrhizic acid and glycyrrhizin flavonoids. However, when used to prepare herbal extracts, only glycyrrhizic acid is extracted, while the glycyrrhizin flavonoids are discarded in the residue. Traditional methods for extracting active plant components mainly include water extraction, ultrasound-assisted extraction, and organic solvent extraction.

[0004] Chinese invention patent application CN200910213748.9 discloses a method for producing high-purity glycyrrhizin. The method includes: drying and pulverizing *Glycyrrhiza glabra*, decocting it in alkaline water, filtering, and concentrating to obtain an alkaline decoction; loading the decoction onto a resin chromatography column and eluting sequentially with water and ethanol; collecting the ethanol eluent with a volume percentage concentration of 45-55% and concentrating it to obtain glycyrrhizic acid; collecting the ethanol eluent with a volume percentage concentration of 85-95% and concentrating it; loading the concentrated solution onto a polyamide chromatography column and eluting sequentially with ethanol of different volume percentage concentrations; collecting the ethanol eluent with a volume percentage concentration of 70-75% and concentrating it to dryness to obtain glycyrrhizin; refluxing the glycyrrhizin in acetone, cooling to crystallize, filtering, and drying to obtain the final product. This invention uses alkaline water instead of organic solvents for extraction, a macroporous resin column instead of a silica gel chromatography column for separation, and an ethanol solution instead of a mixed eluent such as chloroform, resulting in lower costs, better product safety, and higher product purity and yield.

[0005] Another Chinese invention patent application, CN201310327397.0, discloses a method for extracting high-purity glycyrrhizin from Glycyrrhiza glabra, comprising the following steps: (1) adding a mixed solution to dried Glycyrrhiza glabra powder and ultrasonically extracting for 10-30 min at 10-50℃, 30-80Hz, and 80-200w; the mixed solution is a mixture of ethyl acetate, acetone, and anhydrous ethanol; (2) filtering, evaporating the filtrate to dry the mixed solution, freeze-drying the solid, dissolving it in an ethanol aqueous solution, and centrifuging to remove the precipitate to obtain a clear and transparent solution; (3) adding the solution obtained in step (2) to a macroporous resin column and eluting with an ethanol aqueous solution; collecting the ethanol aqueous solution eluent; (4) concentrating the ethanol aqueous solution eluent and freeze-drying to obtain high-purity glycyrrhizin powder. This invention has a simple operation, short process flow, and low cost: using a mixed solution of ethyl acetate, anhydrous ethanol, and acetone as the extractant, and using a mixed resin for extraction and purification, high-purity glycyrrhizin is obtained.

[0006] Although existing technologies have been explored and achieved certain results in the extraction and separation of glycyrrhizin, the methods are generally cumbersome, labor-intensive, and have low extraction efficiency, and the purity of the separated compounds still needs to be further improved. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the column chromatography extraction method developed in this invention is a simple and efficient new extraction technology with low energy consumption, low solvent usage, and high extraction rate. The aim is to simultaneously extract glycyrrhizic acid and glycyrrhizin from Glycyrrhiza glabra. After separating glycyrrhizic acid and glycyrrhizin, the remaining part can be separated into isoglycyrrhizin through simple conversion, thereby achieving comprehensive development and utilization of Glycyrrhiza glabra.

[0008] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution: A method for simultaneously extracting and purifying glycyrrhizin, glycyrrhizic acid, and isoglycyrrhizin, characterized by comprising the following steps: (1) Extract the licorice root with ethanol to obtain an extract; (2) Extract the extract with ethyl acetate to obtain an organic phase and an aqueous phase; (3) The organic phase was purified by rapid preparative liquid chromatography with ethyl acetate-cyclohexane as the mobile phase and gradient elution of 30%-70%. The eluent was collected to obtain glycyrrhizin. (4) Acidify the aqueous phase, centrifuge and precipitate to obtain crude glycyrrhizic acid and supernatant. Add anhydrous ethanol to the crude glycyrrhizic acid, extract by ultrasonication and centrifuge to obtain glycyrrhizic acid ethanol solution. (5) The supernatant was extracted and purified to obtain isoliquiritin.

[0009] Preferably, the volume concentration of ethanol in step (1) is 55-65% ethanol solution, and the mass ratio of licorice root to ethanol is 1:1.5-3.5.

[0010] Preferably, in step (1), the ethanol is added and soaked for 3.5-4.5 hours. The extraction is carried out by column chromatography, which specifically includes soaking the licorice root in ethanol, placing it in a chromatography column, and eluting with 55-65% ethanol. The volume of the eluent is 1-2 times the amount of ethanol used.

[0011] Preferably, in step (2), the extract is concentrated to remove ethanol, and then extracted 1-2 times with an equal volume of ethyl acetate.

[0012] Preferably, the column packing material of the rapid preparation liquid chromatograph in step (3) is silica gel with a diameter-to-height ratio of 1:7. The mobile phase A is ethyl acetate and the mobile phase B is cyclohexane. Gradient elution is performed with 30%, 40%, 50%, 60%, and 70% of mobile phase A. The amount of mobile phase used is 15 BV, and the eluent is collected.

[0013] Preferably, in step (3), the eluent is dried, dissolved in methanol, and then water is added to make an 80% methanol solution, which is then crystallized and dried to obtain the final product.

[0014] Preferably, in step (3), the eluent is distilled under reduced pressure until dry, the crystallization temperature is 0-6℃, the crystallization time is 18-28 h, and the crystallization is dried under vacuum.

[0015] Preferably, in step (4), the aqueous phases are combined, the pH is adjusted to 2-3 with concentrated hydrochloric acid, and after precipitation at -15--20℃ for 20-40 minutes, the mixture is centrifuged and the precipitation is repeated 1-3 times to obtain crude glycyrrhizic acid.

[0016] Preferably, in step (4), the crude glycyrrhizic acid is dissolved in anhydrous ethanol, ultrasonically extracted at 40-50℃ for 10-30 min, and then centrifuged to obtain a glycyrrhizic acid ethanol solution.

[0017] Preferably, step (4) further includes converting glycyrrhizic acid into tripotassium glycyrrhizate by adding an alkaline ethanol solution to the glycyrrhizic acid ethanol solution until the solution pH reaches 8-8.5, centrifuging to obtain a precipitate, thereby obtaining tripotassium glycyrrhizate.

[0018] Preferably, step (4) further includes converting glycyrrhizic acid into glycyrrhizic acid monopotassium salt, wherein the alkaline ethanol solution is a 95% ethanol solution of 20% KOH, the water bath heating temperature is 55-65℃, and the heating time is 0.5-2h.

[0019] Preferably, in step (4), the tripotassium glycyrrhizate is added dropwise to glacial acetic acid until the solution boils slightly and is stirred. Then, it is dissolved in a glacial acetic acid-ethanol mixture, activated carbon is added for adsorption, the activated carbon is removed by centrifugation, and the centrifuged liquid crystallizes to obtain the monopotassium glycyrrhizate.

[0020] Preferably, in step (4), glacial acetic acid is added dropwise to the tripotassium glycyrrhizate at 60-75°C until the solution boils slightly and is stirred. The solution is then placed at 0-6°C for 10-15 h, centrifuged to collect the precipitate, and then washed with anhydrous ethanol. The precipitate is then dissolved in a mixed solution of glacial acetic acid and ethanol.

[0021] Preferably, in step (4), the precipitate obtained is dissolved by heating with 50% glacial acetic acid-95% ethanol solution at 75-85℃, activated carbon is added, and adsorption is carried out at 75-85℃ for 20-40 min. After centrifugation, the activated carbon is removed, and the centrifuged liquid crystallizes at 0-6℃ to obtain glycyrrhizic acid monopotassium salt.

[0022] Preferably, the purification in step (5) includes first adjusting the pH of the supernatant to 5-7, then heating it for 0.5-1.5 hours to hydrolyze it, adjusting the pH of the hydrolysate to neutral, adding ethanol, and adding alkali to adjust the pH to 14 to obtain the alkali-converted solution, then extracting the alkali-converted solution, purifying it using a rapid preparative liquid chromatograph, collecting the eluent, and drying it to obtain the final product.

[0023] Preferably, in step (5), the solution after alkali conversion is concentrated to remove ethanol, and ethyl acetate is added for extraction to obtain ethyl acetate phase. The purification is carried out by elution with ethyl acetate as the mobile phase, and the eluent containing isoglycyrrhizin is combined and dried to obtain the final product.

[0024] Compared with existing technologies, the present invention exhibits the following significant advantages: (1) This patent significantly improves the extraction efficiency of the target product by systematically optimizing the extraction process conditions, effectively shortens the purification cycle, and further improves the purity of the final product.

[0025] (2) Multi-component simultaneous extraction technology: This method innovatively realizes the simultaneous and efficient extraction of three target components, significantly improving the extraction efficiency and realizing the comprehensive utilization of raw materials.

[0026] (3) Compliant solvent system: The system uses safe solvents approved by the National Cosmetic Raw Material Catalog (2021 Edition), which fully comply with industry regulatory requirements and avoids compliance risks caused by prohibited solvents.

[0027] (4) Rapid preparative liquid chromatography purification process: By optimizing the parameters of preparative liquid chromatography, a rapid purification process that can complete the collection of target components within 20 minutes is established, which can effectively shorten the purification cycle compared with conventional methods. Attached Figure Description

[0028] Figure 1The HPLC chromatogram of glycyrrhizin is shown.

[0029] Figure 2 This is the HPLC chromatogram of isoliquiritigenin standard.

[0030] Figure 3 This is the HPLC chromatogram of the filtrate of Glycyrrhiza glabra before acid hydrolysis.

[0031] Figure 4 This is the HPLC chromatogram of the filtrate of Glycyrrhiza glabra after acid hydrolysis.

[0032] Figure 5 This is the HPLC chromatogram of purified isoliquiritigenin. Detailed Implementation

[0033] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0034] The numerical ranges used in this article should be understood as including all numbers within that range. For example, the range 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0035] As used herein, the term "comprising" or "including" means "including, but not limited to." This term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "substantially consisting of." In one embodiment, the term "comprising" as used throughout the application, particularly in the claims, may be replaced by the term "consisting of."

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0037] Example 1 A method for simultaneously extracting and purifying glycyrrhizin, glycyrrhizic acid, and isoglycyrrhizin comprises the following steps: (1) Column chromatography extraction method for simultaneous extraction of glycyrrhizin and glycyrrhizic acid: 150g of licorice powder was weighed and added to 60% ethanol solution at a material-to-liquid ratio of 1:2. The mixture was left to stand in a beaker at room temperature for 4 h. The mixture of licorice and 60% ethanol was loaded into a chromatography column with a diameter-to-height ratio of 1:5. The column was eluted with 60% ethanol and 1.6 MV of extract was collected. The content and extraction rate of glycyrrhizin and glycyrrhizic acid were calculated by HPLC.

[0038] (2) Separation of glycyrrhizin: The extracts obtained by column chromatography were combined, concentrated under reduced pressure to the aqueous phase, and extracted once with an equal volume of ethyl acetate. The organic and aqueous extracts were collected separately. The distribution of glycyrrhizin in the organic phase after extraction was detected by HPLC.

[0039] (3) Purification of glycyrrhizin: The extracted organic phases were combined and concentrated by rotary evaporation, and dried to obtain crude glycyrrhizin. The crude glycyrrhizin was purified using a rapid preparative liquid chromatograph (the column packing of the rapid preparative liquid chromatograph was silica gel with a diameter-to-height ratio of 1:7). Ethyl acetate (mobile phase A) and cyclohexane (mobile phase B) were used as the elution system. 5 BV of isocratic elution was performed with 30% mobile phase A and discarded. Then, gradient elution was performed with 30%, 40%, 50%, 60%, and 70% mobile phase A for 10 BV each, with 2 BV for each concentration gradient. The eluent was collected and the purity of glycyrrhizin was detected by HPLC.

[0040] (4) Purification of glycyrrhizic acid: The aqueous extract obtained by extracting glycyrrhizin was adjusted to pH 2-3 with concentrated hydrochloric acid, and the precipitate was placed at -20℃ for 30 min. After centrifugation, the precipitation was repeated twice to obtain crude glycyrrhizic acid and filtrate. The crude product can be dried by rotary evaporation to remove the influence of solvent. The filtrate is retained.

[0041] Conversion of tripotassium glycyrrhizate: The crude glycyrrhizic acid obtained by acid precipitation was dissolved in a minimum amount of anhydrous ethanol, extracted with ultrasonic assistance at 45°C for 20 minutes, and then centrifuged to obtain the supernatant.

[0042] Prepare a 20% KOH solution in 95% ethanol. Slowly add the 20% KOH solution in 95% ethanol to the supernatant obtained above until the pH of the supernatant reaches 8-8.5. Centrifuge to obtain the precipitate, namely tripotassium glycyrrhizate. Use nitrogen blowing to remove as much residual ethanol as possible.

[0043] Conversion of monopotassium glycyrrhizate: The obtained moist tripotassium glycyrrhizate was transferred to a beaker, and glacial acetic acid was added dropwise at 70°C to bring the solution to a gentle boil while stirring, resulting in a black solution. The solution was placed at 4°C for 12 h, centrifuged to collect the precipitate, and washed twice with anhydrous ethanol. The precipitate was dissolved in a minimum amount of 50% glacial acetic acid-95% ethanol solution at 80°C by heating. Activated carbon was added (approximately 1 g of activated carbon per 50 mL of solution), and adsorption was carried out at 80°C for 30 min. The activated carbon was removed by suction filtration, and the solution was filtered while hot. If the solution was taken out in small batches, it could be placed in an 80°C water bath and crystallized at 4°C. After removing the supernatant, the crystals were washed with pure water and then washed again with 95% ethanol and dried under vacuum.

[0044] (5) Purification of isoglycyrrhizin: After adjusting the pH of the filtrate retained in (4) above to 6, prepare a 2 mol / L hydrochloric acid aqueous solution and heat it at 90℃ for 1 h. After standing at room temperature, adjust the pH of the hydrolysate to neutral and add 95% ethanol to prepare a 60% ethanol solution. Add 12.5 mol / L sodium hydroxide solution to adjust the pH to 14 to obtain a 60% ethanol solution after alkali conversion. HPLC was used to detect the conversion between glycyrrhizin and isoglycyrrhizin. The 60% ethanol solution after alkali conversion was concentrated to the aqueous phase under reduced pressure and extracted with ethyl acetate. The resulting ethyl acetate phase was purified using a rapid preparative liquid chromatograph (the column packing was silica gel with a diameter-to-height ratio of 1:7). Isocratic elution was performed using ethyl acetate as the mobile phase, and 20 BV of eluent was collected. After vacuum distillation and vacuum drying, the purity was determined by HPLC.

[0045] Example 2 The only difference between this embodiment and Embodiment 1 is that the material-to-liquid ratio in step 1 is 1:3.2.

[0046] Comparative Example 1 The only difference between this comparative example and Example 1 is that the material-to-liquid ratio in step 1 is 1:4.8.

[0047] Comparative Example 2 The only difference between this comparative example and Example 1 is that the extraction solvent in step (2) is dichloromethane.

[0048] Comparative Example 3 The only difference between this comparative example and Example 1 is that in step (5), the 60% ethanol solution after alkali conversion is concentrated to the aqueous phase under reduced pressure, extracted with ethyl acetate, adsorbed with silica gel column, eluted with pure chloroform for 2 BV, and then eluented with chloroform:methanol = 1:100 (V / V), checked by TLC, and the same fractions are combined. The same fractions are then checked by TLC, concentrated, and crystallized to obtain isoglycyrrhizin.

[0049] Experimental Example 1: Determination of the content of glycyrrhizin and glycyrrhizic acid in extract and pure product The contents of glycyrrhizin and glycyrrhizic acid in Example 2 and Comparative Example 1 of the present invention were determined by the following HPLC method: Glycyrrhizin and glycyrrhizic acid were quantitatively analyzed by HPLC. Both compounds were analyzed under the same conditions using an Agilent HPLC system and an Agilent C10 column. 18 The chromatographic column (4.6 × 250 mm, 5 μm) was used, with an elution flow rate of 1 mL / min, a column temperature of 30 °C, and a detection wavelength of 230 nm. Gradient elution was performed using mobile phases A (acetonitrile) and B (0.05% aqueous phosphoric acid) according to Table 1. Table 1

[0050] Qualitative analysis of the two compounds was determined based on the retention times of their respective standards and an additive experiment of the standards and samples. Quantitative analysis of the compounds was calculated based on their peak areas and quantitative standard curves.

[0051] The contents of glycyrrhizin and glycyrrhizic acid in the extract of step (1) were detected by the above HPLC method, and the detection results are shown in Table 2 below. In the early stage, the contents of glycyrrhizin and glycyrrhizic acid in the medicinal material were obtained by ultrasound as 1.59 mg / g and 42.5 mg / g, respectively. The extraction rate of glycyrrhizin % = glycyrrhizin content in column chromatography extract / (mass of raw powder of licorice root * 1.59) * 100%; the extraction rate of glycyrrhizic acid % = glycyrrhizic acid content in column chromatography extract / (mass of raw powder of licorice root * 42.5) * 100%.

[0052] Table 2. Contents of glycyrrhizin and glycyrrhizic acid in *Glycyrrhiza glabra* extracted by column chromatography.

[0053] Results Analysis: Based on column chromatography extraction technology, this invention developed a process for the efficient simultaneous extraction of glycyrrhizin and glycyrrhizic acid from *Glycyrrhiza glabra* by systematically optimizing chromatographic experimental parameters, significantly improving the extraction efficiency of both compounds. The results are shown in Table 2. Under the conditions of Comparative Example 1, the extraction efficiency was high in the small-scale experiment, but it decreased significantly after pilot-scale expansion (Comparative Example 1 shows the pilot-scale expansion results). After repeated adjustments to the process, the inventors unexpectedly discovered that optimizing only the material-liquid ratio resulted in extraction rates of both target compounds exceeding 97%, an increase of 12% compared to the unoptimized method (Example 1). This result demonstrates that the optimized column chromatography method can achieve efficient simultaneous extraction of glycyrrhizin and glycyrrhizic acid, with a higher extraction rate than the unoptimized method.

[0054] In addition, the content of glycyrrhizin purified in step (3) of Example 1 was detected by the above HPLC method, and the results are as follows: Figure 1As shown, the purity of glycyrrhizin was calculated to be 98.90%.

[0055] Experimental Example 2: Determination of Glycyrrhizic Acid Monopotassium Salt Content The content of monopotassium glycyrrhizate was determined according to the content of monopotassium glycyrrhizate in GB 1886.240-2016, the National Standard of the People's Republic of China.

[0056] Accurately weigh 80 mg of the sample to be tested and dilute to 100 mL in a volumetric flask with 80% ethanol solution. Accurately transfer 2 mL to a 50 mL volumetric flask, dilute to the mark with 80% ethanol solution, and mix well to obtain the sample solution.

[0057] Take 1 mL of the sample solution and place it in a quartz cuvette. Measure the absorbance at 248 nm using a UV spectrophotometer. The purity of potassium glycyrrhizate is calculated according to the following formula: Potassium glycyrrhizate content =

[0058] In the formula: A represents the absorbance value of the sample solution.

[0059] The content of glycyrrhizic acid monopotassium salt in step (4) of Example 1 was determined by the above method, and the purity of the prepared glycyrrhizic acid monopotassium salt was 95.03%.

[0060] Experiment Example 3: Analysis of the Transformation and Purification Results of Isoliquiritin The content of isoglycyrrhizin in this invention was determined using the following HPLC method: Testing conditions: Yili Special ODS-AP C 18 The chromatographic column (4.6 × 250 mm, 5 μm), column temperature was 31℃, detection wavelength was 230 nm, flow rate was 1 mL / min, mobile phase A was acetonitrile, and mobile phase B was 0.026% phosphoric acid aqueous solution. Gradient elution method was used. Table 3 Gradient Elution Table

[0061] Compare the HPLC results of the solutions before and after acid hydrolysis of the filtrate of Glycyrrhiza glabra in step (5) of Example 1, as follows: Figures 2-4 As shown, isoliquiritin was not observed in the extract before acid hydrolysis, but its content increased after acid hydrolysis.

[0062] The acid-hydrolyzed isoliquiritigenin was then purified by rapid preparative liquid chromatography (HPLC), collected, concentrated under reduced pressure, and vacuum dried. HPLC analysis showed the isoliquiritigenin purity to be 85.60%. The HPLC chromatogram of the purified isoliquiritigenin is shown below. Figure 5 As shown.

[0063] Experimental Example 4: Determination of Product Yield and Purity Example 1: The pure products obtained from Comparative Examples 2-3 were subjected to content determination. The yields and purity results of glycyrrhizin, glycyrrhizic acid and isoglycyrrhizin are summarized in Table 4 below.

[0064] Table 4 Summary of Extraction Effects and Purity of Different Processes

[0065] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for simultaneously extracting and purifying glycyrrhizin, glycyrrhizic acid, and isoglycyrrhizin, characterized in that, Includes the following steps: (1) Extract the licorice root with ethanol to obtain an extract; (2) Extract the extract with ethyl acetate to obtain an organic phase and an aqueous phase; (3) The organic phase was purified by rapid preparative liquid chromatography with ethyl acetate-cyclohexane as the mobile phase and gradient elution of 30%-70%. The eluent was collected to obtain glycyrrhizin. (4) The aqueous phase is acidified and centrifuged to precipitate crude glycyrrhizic acid and filtrate. The crude glycyrrhizic acid is extracted with anhydrous ethanol by ultrasonic extraction and centrifugation to obtain glycyrrhizic acid ethanol solution. (5) The filtrate was extracted and purified to obtain isoliquiritin.

2. The method according to claim 1, characterized in that, The volume concentration of ethanol in step (1) is 55-65% ethanol solution, and the mass ratio of licorice root to ethanol is 1:1.5-3.

5.

3. The method according to claim 1, characterized in that, In step (1), the ethanol is added and soaked for 3.5-4.5 hours. The extraction is carried out by column chromatography. Specifically, after soaking the licorice root in ethanol, it is placed in a chromatography column and eluted with 55-65% ethanol. The volume of the eluent is 1-2 times the amount of ethanol used.

4. The method according to claim 1, characterized in that, In step (2), the extract is concentrated to remove ethanol, and then extracted 1-2 times with an equal volume of ethyl acetate.

5. The method according to claim 1, characterized in that, In step (3), the column packing material of the rapid liquid chromatograph is silica gel with a diameter-to-height ratio of 1:

7. Mobile phase A is ethyl acetate and mobile phase B is cyclohexane. Gradient elution is performed with mobile phase A at 30%, 40%, 50%, 60%, and 70% and the amount of mobile phase used is 15 BV. The eluent is collected.

6. The method according to claim 1, characterized in that, In step (4), the aqueous phases are combined, the pH is adjusted to 2-3 with concentrated hydrochloric acid, and the precipitation is carried out at -15--20℃ for 20-40 minutes. After centrifugation, the precipitation is repeated 1-3 times to obtain crude glycyrrhizic acid.

7. The method according to claim 6, characterized in that, In step (4), the crude glycyrrhizic acid is dissolved in anhydrous ethanol, and after ultrasonic extraction at 40-50℃ for 10-30 min, it is centrifuged to obtain glycyrrhizic acid ethanol solution.

8. The method according to claim 7, characterized in that, Step (4) also includes converting glycyrrhizic acid into tripotassium glycyrrhizate by adding an alkaline ethanol solution to the glycyrrhizic acid ethanol solution until the solution pH reaches 8-8.5, centrifuging to obtain a precipitate, thus obtaining tripotassium glycyrrhizate.

9. The method according to claim 8, characterized in that, Step (4) also includes converting glycyrrhizic acid into glycyrrhizic acid monopotassium salt, wherein the alkaline ethanol solution is a 95% ethanol solution of 20% KOH, the water bath heating temperature is 55-65℃, and the heating time is 0.5-2h; And / or, in step (4), the tripotassium glycyrrhizate is added dropwise to glacial acetic acid until the solution boils slightly and is stirred, then dissolved in a glacial acetic acid-ethanol mixture, activated carbon is added for adsorption, activated carbon is removed by centrifugation, and crystals are obtained to obtain the monopotassium glycyrrhizate. And / or, in step (4), add glacial acetic acid dropwise to the tripotassium glycyrrhizate at 60-75℃ until the solution boils slightly and stirs, place at 0-6℃ for 10-15 h, centrifuge to collect the precipitate, then wash with anhydrous ethanol, and dissolve the precipitate in a glacial acetic acid-ethanol mixture. And / or, in step (4), the precipitate obtained is dissolved by heating with a 1:1 mixture of glacial acetic acid and 95% ethanol at 75-85°C, activated carbon is added, and after adsorption at 75-85°C for 20-40 min, the activated carbon is removed by centrifugation, and the centrifuged liquid crystallizes at 0-6°C to obtain glycyrrhizic acid monopotassium salt.

10. The method according to claim 1, characterized in that, In step (5), the supernatant is first adjusted to pH 5-7 by adding acid, then heated for 0.5-1.5 h for hydrolysis. The pH of the hydrolysate is adjusted to neutral, ethanol is added until the solution contains 55-65% ethanol, and alkali is added to adjust the pH to 13-14 to obtain the alkali-converted solution. The alkali-converted solution is then extracted and purified using a rapid preparative liquid chromatograph. The chromatographic column is packed with silica gel with a diameter-to-height ratio of 1:

7. Ethyl acetate is used as the mobile phase for isocratic elution. 20 BV of eluent is collected and dried to obtain the final product. And / or, in step (5), the solution after alkali conversion is concentrated to remove ethanol, extracted with ethyl acetate to obtain ethyl acetate phase, purified by rapid preparative liquid chromatography, eluted with ethyl acetate as mobile phase, detected by TLC, and the eluent containing isoglycyrrhizin is combined and dried to obtain the final product.

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