Method for simultaneously separating three flavone monomeric compounds from glycyrrhiza glabra
By combining supercritical CO2 extraction and macroporous resin chromatography with a recrystallization solvent system, high-purity glycyrrhizin, glycyrrhizin, and isopentenylcoumarestrol were successfully separated from Glycyrrhiza glabra. This solved the problems of unclear separation and high cost in existing technologies, and achieved efficient and low-energy separation of flavonoid monomers.
Patent Information
- Application Number
- CN202511798233.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-02
- Publication Date
- 2026-03-06
AI Technical Summary
Existing technologies for separating glycyrrhizin flavonoids from Glycyrrhiza glabra suffer from problems such as high dependence on raw materials, unclear separation of target products, and insufficient integration and optimization of the entire process, resulting in unstable product quality, high costs, and low efficiency.
Three flavonoid monomers, glycyrrhizin, glycyrrhizin, and isopentenylcoumarestrol, were separated from Glycyrrhiza glabra by supercritical CO2 extraction combined with macroporous resin chromatography and recrystallization solvent system, and by ultrasonic dissolution, standing, column elution and recrystallization.
This method enables the efficient and low-cost simultaneous separation of three high-purity flavonoid monomers from licorice root, reducing energy and solvent consumption while improving product purity and yield. It is suitable for use in the cosmetics and pharmaceutical industries.
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Figure CN121609708A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of compound separation technology, and in particular to a method for simultaneously separating three flavonoid monomer compounds from licorice root. Background Technology
[0002] In recent years, studies have found that flavonoids have a variety of pharmacological effects, including anti-cancer, anti-tumor, antioxidant, anti-inflammatory, antibacterial, anti-cardiovascular disease, lipid-lowering, anti-osteoporosis, analgesia, prevention of Alzheimer's disease, and regulation of the immune system. Licorice glabra is one of the most important and commonly used licorice varieties in the world. As a "national elder" in traditional Chinese medicine prescriptions, it plays a role in harmonizing various medicines, clearing heat and detoxifying, relieving cough and resolving phlegm. Licorice flavonoids are an important class of active ingredients in licorice. Due to their complex composition, similar structure, and poor stability of some compounds, separation is difficult and costly. In general, the main disadvantages are as follows: (1) High dependence on raw materials: The final yield and product composition of all methods are heavily dependent on the variety, origin, harvesting season and part of licorice, resulting in large batch-to-batch differences in product quality and difficulty in standardization. (2) Unclear separation of target products: "Licorice flavonoids" is a mixture, and different monomers have different activities. Existing schemes mostly use the yield of "total flavonoids" as an indicator, and lack highly selective and low-cost preparation methods for specific highly active monomers. (3) Insufficient integration and optimization of the whole process: Most studies focus on the optimization of a single step, lacking the "whole process optimization" of extraction, separation and purification as a whole system, resulting in high energy consumption and material consumption, and low overall efficiency.
[0003] Supercritical CO2 extraction was used to replace traditional organic solvent extraction. Although some entrainers are added during the extraction of licorice flavonoids due to the polarity of the target components, the amount of solvent used is negligible compared to traditional organic solvent extraction methods. Therefore, this study presents a method for simultaneously separating three high-value-added flavonoid monomers—glabridin, glabridin, and isopentenylcoumarestrol—from Glycyrrhiza glabra using supercritical CO2 extraction, which has certain technological innovation and economic value. Summary of the Invention
[0004] The purpose of this invention is to provide a method for simultaneously separating three flavonoid monomer compounds from licorice root extract, addressing the shortcomings of existing technologies.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for simultaneously isolating three flavonoid monomers from licorice root, comprising the following steps: 1) Mix licorice powder with ethanol, extract with supercritical CO2 and then evaporate to dryness to obtain an extract; 2) The extract and ethanol are mixed and then subjected to ultrasonic dissolution and allowed to stand to obtain the supernatant fraction; 3) The supernatant fraction was added from the top into the macroporous resin chromatography column, and then the macroporous resin chromatography column was eluted with ethanol. The eluted fraction was desolventized to obtain the main fraction extract. 4) Mix the main fraction extract and recrystallization solvent, and let the mixture stand at 23~27℃ to obtain solid 1 and filtrate 1; let filtrate 1 stand at 12~17℃ to obtain solid 2 and filtrate 2; let filtrate 2 stand at 2~6℃ to obtain solid 3. Step 4) The recrystallization solvent is petroleum ether and ethyl acetate, with a volume ratio of 3~5:1; solid 1 is glycyrrhizin, solid 2 is glycyrrhizin, and solid 3 is isopentenylcoumarin.
[0006] Preferably, the moisture content of the licorice powder in step 1) is 0.5-1%; the mass-to-volume ratio of licorice powder to ethanol is 600-800g:3-4L, and the volume fraction of ethanol is 20-40%.
[0007] Preferably, in step 1), the supercritical CO2 extraction is performed at a temperature of 45-55°C, a pressure of 28-35 MPa, a time of 2-4 h, and a CO2 flow rate of 100-200 kg / h.
[0008] As a preferred embodiment, the preparation process of the licorice root powder is as follows: the dried licorice root and stem are sequentially pulverized, passed through an 80-100 mesh sieve, and dried to obtain licorice root powder; the drying temperature is 45-55℃.
[0009] Preferably, in step 2), the mass ratio of the extract to ethanol is 1:2.5~3.5, and the volume fraction of ethanol is 30~40%; the ultrasonic dissolution time is 10~30 min, and the standing time is 25~35 min.
[0010] Preferably, the packing material in the macroporous resin chromatography column in step 3) is macroporous resin D101, the ratio of column inner diameter to column height is 1:5~6; the loading volume of the supernatant fraction is 1 column volume V, and the loading flow rate is 0.008~0.012V / min.
[0011] Preferably, the volume fraction of ethanol in step 3) is 60-70%, and the elution volume in the ethanol elution is 1-1.5 column volumes V, and the liquid flow rate is 0.008-0.012 V / min.
[0012] Preferably, in step 4), the mass ratio of the main distillate extract to the recrystallization solvent is 1:2.5~3.5, the mixture is allowed to stand for 25~35 minutes, filtrate 1 is allowed to stand for 5~7 hours, and filtrate 2 is allowed to stand for 5~7 hours.
[0013] The beneficial effects of this invention are: 1) This invention employs supercritical CO2 extraction to replace traditional solvent extraction. Its core technological advantages lie in its green, safe, efficient, and high-quality nature. Using CO2 avoids the use of large amounts of flammable and explosive organic solvents, resulting in extremely high safety. The product has no solvent residue and high purity, and exhibits extremely high selectivity. By changing pressure and temperature, the density and solubility of CO2 can be adjusted for targeted extraction of specific components. This invention boasts high extraction efficiency; supercritical CO2 has low viscosity, high diffusivity, fast mass transfer rate, strong permeability, and relatively short extraction time. The operating cost of this invention is low. While the initial equipment investment is slightly higher, the overall operating cost and energy consumption are relatively low. There is no need for solvent recovery, resulting in a long-term cost advantage.
[0014] 2) This invention employs supercritical CO2 carrying ethanol for extraction, uses macroporous resin D101 as the packing material for separation, and uses a mixed solvent of petroleum ether and ethyl acetate for continuous recrystallization of the three target products. This recrystallization process allows for the continuous crystallization and purification of multiple substances using a single solvent system, yielding three high-purity target products in a single step. Compared to traditional processes, this avoids the cumbersome steps of multiple desolvation, redissolution, and reprecipitation, significantly reducing energy consumption and the difficulty of solvent recovery.
[0015] 3) This invention successfully obtained three glycyrrhizin flavonoids: glycyrrhizin, glycyrrhizin, and isopentenylcoumarestrol. The glycyrrhizin content reached over 97%, with a yield of over 85%; the glycyrrhizin content reached over 98%, with a yield of over 86%; and the isopentenylcoumarestrol content reached over 99%, with a yield of over 88%. Glycyrrhizin has a strong whitening effect and can be added to cosmetics. The tyrosinase activity inhibition rate of glycyrrhizin is C... 50 =3.5μM, isopentenylcoumarestrol can be used to prevent osteoporosis in the elderly and inhibit the growth of breast tumors and breast cancer cells. Attached Figure Description
[0016] Figure 1 The 1H-NMR spectrum of the solid 1-glycyrrhizin separated in Example 1; Figure 2 The image shows the 1H-NMR spectrum of the solid 2-glycyrrhizin isolated in Example 1. Figure 3 The 13C-NMR spectrum of solid 2-glycyrrhizin isolated in Example 1; Figure 4 The 1H-NMR spectrum of solid 3-isopentenylcoumarestrol isolated in Example 1; Figure 5 The image shows the 13C-NMR spectrum of the solid 3-isopentenylcoumarestrol isolated in Example 1. Detailed Implementation
[0017] This invention provides a method for simultaneously isolating three flavonoid monomers from licorice root, comprising the following steps: 1) Mix licorice powder with ethanol, extract with supercritical CO2 and then evaporate to dryness to obtain an extract; 2) The extract and ethanol are mixed and then subjected to ultrasonic dissolution and allowed to stand to obtain the supernatant fraction; 3) The supernatant fraction was added from the top into the macroporous resin chromatography column, and then the macroporous resin chromatography column was eluted with ethanol. The eluted fraction was desolventized to obtain the main fraction extract. 4) Mix the main fraction extract and recrystallization solvent, and let the mixture stand at 23~27℃ to obtain solid 1 and filtrate 1; let filtrate 1 stand at 12~17℃ to obtain solid 2 and filtrate 2; let filtrate 2 stand at 2~6℃ to obtain solid 3. Step 4) The recrystallization solvent is petroleum ether and ethyl acetate, with a volume ratio of 3~5:1; solid 1 is glycyrrhizin, solid 2 is glycyrrhizin, and solid 3 is isopentenylcoumarin.
[0018] In this invention, the moisture content of the licorice powder in step 1) is preferably 0.5-1%, more preferably 0.6-0.9%, and even more preferably 0.7-0.8%; the mass-to-volume ratio of licorice powder to ethanol is preferably 600-800g:3-4L, more preferably 650-750g:3.2-3.8L, and even more preferably 680-700g:3.5-3.6L; the volume fraction of ethanol is preferably 20-40%, more preferably 25-35%, and even more preferably 30%.
[0019] In this invention, during the supercritical CO2 extraction in step 1), the temperature is preferably 45~55℃, more preferably 48~52℃, and even more preferably 50℃; the pressure is preferably 28~35MPa, more preferably 30~33MPa, and even more preferably 31~32MPa; the time is preferably 2~4h, more preferably 2.5~3.5h, and even more preferably 3h; and the CO2 flow rate is preferably 100~200kg / h, more preferably 120~180kg / h, and even more preferably 150~160kg / h.
[0020] In this invention, the preferred preparation process of the licorice root powder is as follows: the dried licorice root and stem are sequentially pulverized, passed through an 80-100 mesh sieve, and dried to obtain licorice root powder; the drying temperature is preferably 45-55℃, more preferably 47-52℃, and even more preferably 50℃.
[0021] In this invention, the mass ratio of the extract to ethanol in step 2) is preferably 1:2.5~3.5, more preferably 1:2.7~3.2, and even more preferably 1:3; the volume fraction of ethanol is preferably 30~40%, more preferably 32~38%, and even more preferably 35~36%; the ultrasonic dissolution time is preferably 10~30 min, more preferably 15~25 min, and even more preferably 20 min; the standing time is preferably 25~35 min, more preferably 27~32 min, and even more preferably 30 min.
[0022] In this invention, the packing material in the macroporous resin chromatography column in step 3) is preferably macroporous resin D101, and the ratio of column inner diameter to column height is preferably 1:5~6, more preferably 1:5.2~5.8, and even more preferably 1:5.5~5.6; the loading volume of the supernatant fraction is preferably 1 column volume V, and the loading flow rate is preferably 0.008~0.012V / min, more preferably 0.009~0.011V / min, and even more preferably 0.01V / min.
[0023] In this invention, the volume fraction of ethanol in step 3) is preferably 60-70%, more preferably 62-68%, and even more preferably 65-66%; in the ethanol elution, the elution volume is preferably 1-1.5 column volumes V, more preferably 1.2-1.3 column volumes V, and the liquid flow rate is preferably 0.008-0.012 V / min, more preferably 0.009-0.011 V / min, and even more preferably 0.01 V / min.
[0024] In step 4) of the present invention, the volume ratio of petroleum ether to ethyl acetate is preferably 3~5:1, more preferably 3.5~4.5:1, and even more preferably 4:1.
[0025] In this invention, the mass ratio of the main distillate extract to the recrystallization solvent in step 4) is preferably 1:2.5~3.5, more preferably 1:2.7~3.2, and even more preferably 1:3~3.1.
[0026] In this invention, the preferred temperature for settling the mixture is 24-26°C, more preferably 25°C, and the preferred time is 25-35 min, more preferably 27-32 min, and even more preferably 30 min. The preferred temperature for settling filtrate 1 is 14-16°C, more preferably 15°C, and the preferred time is 5-7 h, more preferably 5.5-6.5 h, and even more preferably 6 h. The preferred temperature for settling filtrate 2 is 3-5°C, more preferably 4°C, and the preferred time is 5-7 h, more preferably 5.5-6.5 h, and even more preferably 6 h.
[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0028] Example 1
[0029] After rinsing the rhizome of Glycyrrhiza glabra with tap water and air-drying it, the powder was pulverized and passed through a 100-mesh sieve. The sieved powder was then dried at 50°C to obtain Glycyrrhiza glabra powder with a moisture content of 0.7%. 600g of the Glycyrrhiza glabra powder was added to a 10L extraction vessel, along with 3L of 20% (v / v) ethanol. Supercritical CO2 extraction was performed at 55°C, 31MPa, and a CO2 flow rate of 200kg / h for 3 hours. After extraction, the extract was evaporated to dryness, yielding 60g of extract.
[0030] The extract and 40% ethanol (by volume) were mixed at a feed-to-liquid ratio of 1:3, sonicated at 40 kHz for 30 min, and allowed to stand for 30 min. The supernatant fraction was collected for later use. Macroporous resin D101 was used as the packing material, and the column was packed with an inner diameter to height ratio of 1:6. The supernatant fraction was slowly and evenly added to the macroporous resin column from the top, with a loading volume of 1 column volume V and a flow rate of 0.01 V / min. The macroporous resin column was slowly washed with 70% ethanol (by volume) at a flow rate of 0.01 V / min and a washing volume of 1.5 column volumes V. The fraction washed down by ethanol was desolventized to obtain the main fraction extract. Petroleum ether and ethyl acetate were prepared as a recrystallization solvent at a volume ratio of 4:1. The main fraction extract and the recrystallization solvent were mixed at a material-to-liquid ratio of 1:3. After being stirred and dispersed evenly at room temperature, the mixture was allowed to stand at 25°C for 30 minutes. The precipitated crystals were filtered and then allowed to evaporate naturally to obtain solid 1 and filtrate 1. Filtrate 1 was placed at 15°C for 6 hours, and the precipitated crystals were filtered and then allowed to evaporate naturally to obtain solid 2 and filtrate 2. Filtrate 2 was placed at 4°C for 6 hours, and the precipitated crystals were filtered and then allowed to evaporate to obtain solid 3.
[0031] In this embodiment, the extract contains 15.89% glycyrrhizin, 11.63% glycyrrhizin, and 0.96% isopentenylcoumarestrol; the main fraction extract contains 45% glycyrrhizin, 38% glycyrrhizin, and 3.96% isopentenylcoumarestrol.
[0032] HPLC quantitative analysis was performed on solids 1, 2, and 3 of this embodiment. The mass content of glycyrrhizin in solid 1 was 97%, the mass content of glycyrrhizin in solid 2 was 98%, and the mass content of isopentenylcoumarestrol in solid 3 was 99%. The yield of solid 1 was 70%, the yield of solid 2 was 75%, and the yield of solid 3 was 90%. The 1H-NMR spectrum of solid 1-glycyrrhizin separated in Example 1 is shown below. Figure 1As shown; the 1H-NMR and 13C-NMR spectra of the solid 2-glycyrrhizin isolated in Example 1 are as follows. Figure 2 , Figure 3 As shown; the 1H-NMR and 13C-NMR spectra of the solid 3-isopentenylcoumarestrol isolated in Example 1 are as follows. Figure 4 , Figure 5 As shown.
[0033] Example 2
[0034] After rinsing the rhizome of Glycyrrhiza glabra with tap water and air-drying it, the powder was pulverized and passed through an 80-mesh sieve. The sieved powder was then dried at 55°C to obtain Glycyrrhiza glabra powder with a moisture content of 0.9%. 700g of the Glycyrrhiza glabra powder was added to a 10L extraction vessel, along with 3.5L of 40% (v / v) ethanol. Supercritical CO2 extraction was performed at 50°C, 33MPa, and a CO2 flow rate of 150kg / h for 2.5h. After extraction, the extract was evaporated to dryness, yielding 65g of extract.
[0035] The extract and 30% ethanol (by volume) were mixed at a feed-to-liquid ratio of 1:2.5, and dissolved by sonication at 40 kHz for 20 min, followed by standing for 35 min. The supernatant fraction was collected for later use. Macroporous resin D101 was used as the packing material, and the column was packed with an inner diameter to height ratio of 1:5. The supernatant fraction was slowly and evenly added to the macroporous resin column from the top, with a loading volume of 1 column volume V and a flow rate of 0.011 V / min. The macroporous resin column was slowly washed with 65% ethanol (by volume) at an elution rate of 0.011 V / min and a washing volume of 1.3 column volumes V. The fraction washed down by ethanol was desolventized to obtain the main fraction extract. Petroleum ether and ethyl acetate were prepared as a recrystallization solvent at a volume ratio of 3.5:1. The main fraction extract and the recrystallization solvent were mixed at a material-to-liquid ratio of 1:2.7. After being stirred and dispersed evenly at room temperature, the mixture was allowed to stand at 26°C for 33 min. The precipitated crystals were filtered and then allowed to evaporate naturally to obtain solid 1 and filtrate 1. Filtrate 1 was placed at 14°C for 6.5 h, and the precipitated crystals were filtered and then allowed to evaporate naturally to obtain solid 2 and filtrate 2. Filtrate 2 was placed at 5°C for 6.5 h, and the precipitated crystals were filtered and then allowed to evaporate to obtain solid 3.
[0036] In this embodiment, the extract contains 18.02% glycyrrhizin, 14.36% glycyrrhizin, and 1.02% isopentenylcoumarestrol; the main fraction extract contains 50% glycyrrhizin, 46% glycyrrhizin, and 4.62% isopentenylcoumarestrol.
[0037] HPLC quantitative analysis was performed on solids 1, 2, and 3 in this embodiment. The mass content of glycyrrhizin in solid 1 was 98%, the mass content of glycyrrhizin in solid 2 was 98.5%, and the mass content of isopentenylcoumarestrol in solid 3 was 99%.
[0038] The method of this invention features high extraction efficiency and good selectivity, controlling the content of high-content crude extract to less than ten components (the number of components is determined by the number of liquid phase peaks), reducing the pressure on subsequent separation work. This invention uses macroporous resin for column chromatography separation, which, compared to silica gel separation, uses only alcohol-water mixtures as solvents, resulting in less environmental pressure. The solid packing material can be repeatedly activated and reused, eliminating solid waste pressure. It also boasts high separation efficiency and good selectivity, controlling the high content of desired fractions to approximately five substances (the number of substances is determined by the number of liquid phase peaks). The final purification method employs continuous recrystallization using a single solvent system. The main product extract is completely dissolved and dispersed using a prepared recrystallization mixed solvent, followed by low-temperature static recrystallization, allowing for complete solvent recovery. This invention can simultaneously obtain three high-value-added components—glabridin, glabridin, and isopentenylcoumarestrol—in a single recrystallization solvent system, achieving high preparation efficiency and significantly lower costs than other methods.
[0039] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for simultaneous isolation of three flavonoid monomer compounds from Glycyrrhiza glabra, characterized in that, The method comprises the following steps: 1) mixing glycyrrhiza glabra powder and ethanol, and performing supercritical CO2 extraction and rotary drying to obtain an extract; 2) mixing the extract and ethanol, and sequentially performing ultrasonic dissolution and standing to obtain a supernatant fraction; 3) adding the supernatant fraction from the upper part into a macroporous resin chromatographic column, and then eluting the macroporous resin chromatographic column with ethanol, and performing desolventization on the eluted fraction to obtain a main fraction extract; 4) mixing the main fraction extract and a recrystallization solvent, and standing the mixture at 23-27 DEG C to obtain solid 1 and filtrate 1; standing the filtrate 1 at 12-17 DEG C to obtain solid 2 and filtrate 2; and standing the filtrate 2 at 2-6 DEG C to obtain solid 3. In step 4), the recrystallization solvent is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 3-5:1; the solid 1 is glyyrrhizin, the solid 2 is glabridin, and the solid 3 is isoprenyl coumestans.
2. The method of claim 1, wherein, In step 1), the water content of the glycyrrhiza glabra powder is 0.5-1%, and the mass-volume ratio of the glycyrrhiza glabra powder to ethanol is 600-800 g:3-4 L, and the volume fraction of ethanol is 20-40%.
3. The method according to claim 1 or 2, characterized in that, In the supercritical CO2 extraction in step 1), the temperature is 45-55 DEG C, the pressure is 28-35 MPa, the time is 2-4 h, and the CO2 flow rate is 100-200 kg / h.
4. The method of claim 3, wherein, The preparation process of the glycyrrhiza glabra powder comprises the following steps: sequentially performing crushing, 80-100 mesh sieving and drying on dried glycyrrhiza glabra roots and stems to obtain the glycyrrhiza glabra powder; and the drying temperature is 45-55 DEG C.
5. The method of claim 3, wherein, In step 2), the mass ratio of the extract to ethanol is 1:2.5-3.5, the volume fraction of ethanol is 30-40%, the ultrasonic dissolution time is 10-30 min, and the standing time is 25-35 min.
6. The method according to claim 1 or 5, characterized in that, In step 3), the filler in the macroporous resin chromatographic column is macroporous resin D101, the ratio of the column inner diameter to the column height is 1:5-6, the sample volume of the supernatant fraction is 1 column volume V, and the sample flow rate is 0.008-0.012 V / min.
7. The method of claim 6, wherein, In step 3), the volume fraction of ethanol is 60-70%, and in the ethanol elution, the elution volume is 1-1.5 column volumes V, and the liquid flow rate is 0.008-0.012 V / min.
8. The method of claim 7, wherein, In step 4), the mass ratio of the main fraction extract to the recrystallization solvent is 1:2.5-3.5, the standing time of the mixture is 25-35 min, the standing time of the filtrate 1 is 5-7 h, and the standing time of the filtrate 2 is 5-7 h.