Extraction method and water-soluble application of high-purity glabridin

Abstract

The invention belongs to the field of separation and purification, and discloses an extraction method and application of high-purity glabridin, and the extraction method specifically comprises the following steps: a glycyrrhiza glabra raw material and a pH response type DES reagent are mixed, stirred and extracted for 1-2 hours, centrifugation is carried out, a supernatant is collected, and the pH response type DES reagent is composed of hydrogen bond receptor choline chloride and hydrogen bond donor malonic acid; and adding an alkali solution into the supernate to adjust the pH value to 7.0-8.0 for phase separation, separating into an upper-layer solution phase and a lower-layer precipitation phase, collecting the lower-layer precipitation phase, centrifuging, washing and drying to obtain the glabridin. According to the invention, the integration of extraction and primary separation is realized, the extraction and primary enrichment are combined into one in the process of'alkali addition, namely precipitation ', and meanwhile, the DES component can be almost quantitatively recovered, so that the production cost and the environmental burden are greatly reduced, the purity of the obtained product is higher, and the load of the subsequent refining process is reduced.

Description

A method for extracting high-purity glycyrrhizin and its water-soluble applications Technical Field

[0001] This invention belongs to the field of separation and purification, specifically relating to a method for extracting high-purity glycyrrhizin and its water-soluble applications. Background Technology

[0002] Glycyrrhizin is a hydrophobic flavonoid compound extracted from the root of Glycyrrhiza glabra, with the chemical formula C. 20 H 20 O4, with a molecular weight of 324.3704, inhibits tyrosinase activity to reduce melanin production and is known as "whitening gold" due to its skin-whitening effects, thus possessing a large potential market. However, due to the poor water solubility of glycyrrhizin, industrial extraction mainly employs organic solvent extraction methods (such as methanol, ethanol, and ethyl acetate). These methods have the following drawbacks: 1) Low extraction efficiency: limited ability to damage cell walls, requiring large amounts of solvent and long extraction times; 2) Poor selectivity: simultaneous dissolution of large amounts of pigments, sugars, proteins, and other impurities, placing a heavy burden on subsequent separation and purification, resulting in high costs; 3) Environmental pollution: the use of volatile organic solvents is unfriendly to operators and the environment; 4) Safety issues: the residue of toxic solvents such as methanol limits its application in high-end products. Therefore, developing a new, efficient, green, and highly selective extraction method is urgently needed.

[0003] Deep eutectic solvents (DES) are green solvents discovered in recent years by Abbott et al., which can replace traditional organic solvents and ionic liquids. They refer to two- or three-component eutectic mixtures composed of hydrogen bond acceptors (such as quaternary ammonium salts) and hydrogen bond donors (such as amides, carboxylic acids, and polyols) in a certain stoichiometric ratio, with a freezing point significantly lower than the melting point of each pure component. As a highly efficient and green solvent, DES are increasingly being used in the extraction of small-molecule resource components from traditional Chinese medicine. However, the extraction of glycyrrhizin or the separation of flavonoids using DES are still under investigation.

[0004] CN114685523A discloses a method for extracting and purifying glycyrrhizin from *Glycyrrhiza glabra* residue using DES solvent, comprising the following steps: (1) mixing propionic acid and choline chloride to prepare a eutectic solvent, extracting under ultrasonic action, filtering and collecting the extract to obtain a crude glycyrrhizin extract; (2) extracting the obtained crude extract with ethyl acetate as the extractant, the extractant phase being evaporated by gradient rotary evaporation and dissolved in ethanol; (3) separating and purifying with a macroporous resin to obtain a glycyrrhizin extract. However, the subsequent steps of crude extraction using DES solvent in this invention are relatively complex, and the recovery rate needs to be improved.

[0005] In summary, most existing studies on the extraction of flavonoids from traditional Chinese medicine or plants using eutectic solvents focus on the extraction rate of flavonoids by the eutectic solvent, while neglecting the optimization of the separation and purification of flavonoids in the extract and the recovery and utilization of the eutectic solvent. Summary of the Invention

[0006] The purpose of this invention is to overcome at least one deficiency of the prior art. By utilizing the specific binding of DES solution with glycyrrhizin, the target component can be efficiently enriched during the extraction process. When alkali is added to increase the pH of the DES system, the hydrogen bond network between DES molecules is destroyed, leading to spontaneous phase separation of the system. This spontaneous phase separation allows glycyrrhizin to precipitate from the DES phase, achieving rapid separation of the product from the solvent. Thus, this invention provides a method for extracting high-purity glycyrrhizin and its application.

[0007] The technical solution adopted by the present invention is as follows: Firstly, the present invention provides a method for extracting high-purity glycyrrhizin, including the following steps: Step (1) DES enhanced extraction: Glycyrrhiza glabra raw material is mixed and stirred with pH-responsive DES reagent for 1-2 hours, and the supernatant is collected by centrifugation, wherein the pH-responsive DES reagent is composed of hydrogen bond acceptor choline chloride and hydrogen bond donor malonic acid; Step (2) Induced phase separation: The supernatant obtained in step 1 is added to an alkaline solution to adjust the pH to 7.0-8.0 and the phases are separated into an upper solution phase and a lower precipitate phase. The lower precipitate phase is collected and centrifuged, washed and dried to obtain the glycyrrhizin.

[0008] In some embodiments, the alkaline solution in step (2) is selected from at least one of ammonia water, sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate solution.

[0009] Optionally, the concentration of the alkaline solution used can be adjusted according to actual production conditions. While ensuring the efficiency of pH adjustment, it is also necessary to avoid local pH abrupt changes due to excessively high alkaline concentration. In practical applications, a single alkaline solution or a mixture of multiple alkaline solutions can be flexibly selected according to production needs to achieve stable pH adjustment and further improve phase separation.

[0010] In some embodiments, the phase separation processing time in step (2) is 10-20 minutes.

[0011] If the phase separation time is too short, the oil phase, water phase, and any solid phases in the system will not be fully separated and layered, which can easily lead to phase mixing and cross-contamination of components, thus affecting the purity and yield of subsequent products. If the phase separation time is too long, it will not only prolong the overall process cycle and increase production energy consumption, but may also cause slight re-emulsification of the already layered phases due to prolonged standing, or cause irreversible adsorption and loss of the target component at the phase interface.

[0012] Preferably, in step (2), an alkaline solution is added to adjust the pH to 7.0-7.5. Glycyrrhizin maintains good molecular structural stability in this neutral to weakly alkaline environment, thus preserving its biological activity and physicochemical properties to the greatest extent.

[0013] In some embodiments, the method for recovering the upper solution phase in the induced phase separation of step (2) is as follows: the upper solution phase is concentrated under reduced pressure, and water is removed to recover choline chloride and malonic acid reagents. The recovered mixture can be directly used for the preparation of a new round of pH-responsive DES reagents without additional purification treatment, realizing the recycling of DES components and significantly reducing production costs.

[0014] In some embodiments, the pH-responsive DES reagent in step (1) is prepared by mixing choline chloride, a hydrogen bond acceptor, with malonic acid, a hydrogen bond donor, and stirring at 70-85 °C until a homogeneous and transparent liquid is formed, thereby obtaining the pH-responsive DES reagent.

[0015] In some embodiments, the molar ratio of hydrogen bond acceptor choline chloride to hydrogen bond donor malonic acid in the pH-responsive DES reagent is 1:1 to 1:1.5.

[0016] In some embodiments, the ratio of the licorice root extract to the pH-responsive DES reagent is 1:15 to 1:25 (g / mL).

[0017] In some embodiments, the temperature for mixing and stirring the licorice root extract with the pH-responsive DES reagent is 50-65 °C.

[0018] Secondly, the present invention provides a glycyrrhizin prepared by the extraction method described in the first aspect.

[0019] Thirdly, the present invention provides a method for preparing water-soluble glycyrrhizin, comprising the following steps: adding the glycyrrhizin described in the second aspect to a cyclodextrin aqueous solution and stirring at 50-60°C until the solution is clear and transparent, drying and pulverizing to obtain water-soluble glycyrrhizin, wherein the cyclodextrin composition ratio is 85-95% and the glycyrrhizin composition ratio is 5%-15%.

[0020] The key feature of this invention is that it fully considers the fact that glycyrrhizin is completely insoluble in water and extremely difficult to be absorbed by the skin. By utilizing the fact that cyclodextrin is highly soluble in water, glycyrrhizin is encapsulated to form a glycyrrhizin cyclodextrin encapsulation, which increases the water solubility of glycyrrhizin, thereby making it easier for glycyrrhizin to be absorbed by the skin and exert a good whitening effect. A water-soluble glycyrrhizin preparation scheme has been established, and the use of toxic organic solvents is avoided in the process. Compared with the existing technology, it is simpler, greener and safer.

[0021] The cyclodextrins used in this invention are all pharmaceutical-grade cyclodextrins, which are safer and more reliable. The cyclodextrins used are highly water-soluble, which allows for the encapsulation of a large amount of glycyrrhizin. Therefore, the content of water-soluble glycyrrhizin is high, with a mass concentration of 10%. Using 99% pure glycyrrhizin as the active ingredient ensures the purity and content of glycyrrhizin, allowing it to better exert its whitening effect.

[0022] This invention uses purified water as a solvent, thus the cost is relatively low, making it suitable for large-scale production. It can greatly improve the comprehensive utilization of glycyrrhizin, save resources, and generate good social and economic benefits.

[0023] In some embodiments, the cyclodextrin used is any one of hydroxypropyl β-cyclodextrin, methyl β-cyclodextrin, β-cyclodextrin, α-cyclodextrin, and hydroxypropyl α-cyclodextrin, and the mass ratio of the cyclodextrin to purified water is 1:1 to 4 to prepare an aqueous cyclodextrin solution.

[0024] In some embodiments, the preparation method of cyclodextrin aqueous solution is as follows: dissolve cyclodextrin in purified water, heat to 50-60℃, and stir at a speed of 1000-2500 r / min until the cyclodextrin is completely dissolved in the purified water to obtain a clear and transparent cyclodextrin aqueous solution.

[0025] In some embodiments, the amount of glycyrrhizin added is calculated based on the amount of cyclodextrin used, and glycyrrhizin is added to the cyclodextrin aqueous solution. The temperature is heated to 50-60°C, and the stirring speed is 1000-2500 r / min until the glycyrrhizin is completely dissolved in the cyclodextrin aqueous solution. The resulting solution is clear and transparent, and the encapsulation of glycyrrhizin is completed.

[0026] In some embodiments, the obtained glycyrrhizin cyclodextrin encapsulation solution is vacuum dried at a temperature of 60-70°C, and then pulverized after complete drying to obtain 10% (wt%) water-soluble glycyrrhizin.

[0027] The beneficial effects of this invention are: 1) This invention effectively realizes the recycling of solvents. Through the ingenious pH-responsive DES design and simple alkali addition operation, the product and solvent are efficiently and quickly separated, and the DES components can be recovered almost quantitatively, which greatly reduces production costs and environmental burden.

[0028] 2) This invention integrates extraction and initial separation. The process of "adding alkali to precipitate" combines extraction and initial enrichment into one, simplifying the process flow and shortening the production cycle.

[0029] 3) The product of the present invention has higher purity. The functionalized DES itself has better selectivity. In addition, water washing can effectively remove water-soluble impurities, resulting in higher purity of crude product and reducing the load on subsequent refining processes. Attached Figure Description

[0030] Figure 1 is a liquid chromatogram of the standard of glycyrrhiza glabra.

[0031] Figure 2 is a liquid chromatogram of the pure glycyrrhizin obtained in Example 1.

[0032] Figure 3 is a liquid chromatogram of the final pure glycyrrhizin obtained in Comparative Example 1.

[0033] Figure 4 is the liquid chromatogram of the pure glycyrrhizin obtained in Comparative Example 2.

[0034] Figure 5 is the liquid chromatogram of the pure glycyrrhizin obtained in Comparative Example 3.

[0035] Figure 6 shows images of the precipitate obtained by induced phase separation, with the left side being Comparative Example 4 and the right side being Example 1. Detailed Implementation

[0036] To provide a clearer description of the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Unless otherwise indicated, all solvents and reagents used in the following embodiments and comparative examples are commercially available and used as is.

[0037] The extraction method of glycyrrhizin in this invention is summarized as follows: 1. The hydrogen bond acceptor choline chloride and the hydrogen bond donor malonic acid are mixed and stirred until a homogeneous and transparent liquid is formed to obtain a pH-responsive DES reagent.

[0038] 2. Take the raw material of Glycyrrhiza glabra and mix it with the pH-responsive DES reagent mentioned above, stir and extract, and centrifuge to collect the supernatant.

[0039] 3. Slowly add alkaline solution to the supernatant while stirring until the pH of the system rises to 7.0-8.0, at which point a large amount of precipitate will immediately appear in the system.

[0040] 4. Centrifuge to collect the crude glycyrrhizin precipitate, and wash the precipitate with a small amount of deionized water to remove residual DES reagent.

[0041] 5. Combine the upper aqueous phase generated in step 3 with the washing water in step 4, and concentrate under reduced pressure to remove excess water. Recycle the regenerated choline chloride and malonic acid, which can be directly used for the preparation of the next batch of DES.

[0042] 6. Dissolve the crude glycyrrhizin obtained in step 4 in ethanol, slowly add ice water and keep it for a period of time, then centrifuge and dry to obtain pure glycyrrhizin.

[0043] In the embodiments and comparative examples of this invention, the extraction rate is calculated as follows: Extraction rate (mg / g) = (finished product mass of glycyrrhizin (mg) * purity) / raw material glycyrrhiza powder (g).

[0044] Example 1 This example provides a method for extracting glycyrrhizin, the specific operation is as follows: 1. Mix choline chloride, a hydrogen bond acceptor, and malonic acid, a hydrogen bond donor, at a molar ratio of 1:1.5, and stir at 75°C until a homogeneous and transparent liquid is formed to obtain a pH-responsive DES reagent.

[0045] 2. Add 500g of Glycyrrhiza glabra powder to 10 L of the above pH-responsive DES reagent and mix. Stir and extract at 55°C for 1 hour.

[0046] 3. Centrifuge the DES extract obtained in step 2 and collect the supernatant. Slowly add ammonia solution to the supernatant while stirring until the pH of the system rises to 7.0, at which point a large amount of precipitate will immediately appear in the system.

[0047] 4. Centrifuge to collect the crude glycyrrhizin precipitate, and wash the precipitate with a small amount of deionized water to remove residual DES reagent.

[0048] 5. Combine the upper aqueous phase generated in step 3 with the washing water in step 4, and concentrate under reduced pressure to remove excess water. Recycle the regenerated choline chloride and malonic acid, which can be directly used for the preparation of the next batch of DES.

[0049] 6. Dissolve the crude glycyrrhizin obtained in step 4 in ethanol, slowly add 10 mL of ice water, keep at 0°C for 3 hours, centrifuge and dry to obtain 0.9 g of pure glycyrrhizin.

[0050] The results of HPLC analysis are shown in Figure 2. Compared with the standard shown in Figure 1, the purity of glycyrrhizin is >99%, and the extraction rate of this example is 1.78 mg / g of licorice powder.

[0051] Example 2 This example provides a method for preparing water-soluble glycyrrhizin: Take 895 g of hydroxypropyl β-cyclodextrin and 895 mL of purified water. Heat the water to 50-60°C and stir at 1000-2500 r / min. After the cyclodextrin is completely dissolved in the water, add 105 g of 99% pure glycyrrhizin. Maintain the water temperature at 50-60°C and stir at 1000-2500 r / min for 45 min. The encapsulation is complete when the solution becomes clear and transparent. Vacuum dry the above water-soluble glycyrrhizin solution at 60-70°C until completely dried. Pulverize to obtain 1 kg of 10% (wt%) water-soluble glycyrrhizin.

[0052] Example 3 This example provides a method for preparing water-soluble glycyrrhizin: Take 947 g of hydroxypropyl β-cyclodextrin and 947 mL of purified water. Heat the water to 50-60°C and stir at 1000-2500 r / min. After the cyclodextrin is completely dissolved in the water, add 53 g of 99% pure glycyrrhizin. Maintain the water temperature at 50-60°C and stir at 1000-2500 r / min for 45 min. The encapsulation is complete when the solution becomes clear and transparent. Vacuum dry the above water-soluble glycyrrhizin solution at 60-70°C until completely dried. Pulverize to obtain 1 kg of 5% (wt%) water-soluble glycyrrhizin.

[0053] Example 4 The difference between this example and Example 1 is that in step 1, the hydrogen bond acceptor choline chloride and the hydrogen bond donor malonic acid are mixed in a 1:1 molar ratio, while the rest is the same as in Example 1.

[0054] Finally, the crude glycyrrhizin was dissolved in ethanol, precipitated in water, centrifuged and dried to obtain 0.85g of pure glycyrrhizin. HPLC analysis showed that the purity of glycyrrhizin was >99%, and the extraction rate was 1.68 mg / g of Glycyrrhiza glabra powder.

[0055] Comparative Example 1 differs from Example 1 in that, in step 3, the ammonia solution is replaced with pure water, and the pH of the system is 6.0. All other aspects are the same as in Example 1.

[0056] Finally, the crude glycyrrhizin was dissolved in ethanol, precipitated in water, centrifuged and dried to obtain 0.75 g of glycyrrhizin. The results of HPLC analysis are shown in Figure 3. The purity of glycyrrhizin was 70%, and the extraction rate was 1.05 mg / g of Glycyrrhiza glabra powder.

[0057] The difference between Comparative Example 2 and Example 1 is that in step 3, after adding ammonia solution until the pH of the system rises to 7.0, dilute sulfuric acid solution is added until the pH of the system drops to 4.0. The rest is the same as in Example 1.

[0058] Finally, the crude glycyrrhizin was dissolved in ethanol, precipitated in water, centrifuged and dried to obtain 0.82 g of glycyrrhizin. The results of HPLC analysis are shown in Figure 4. The purity of glycyrrhizin was 82%, and the extraction rate was 1.34 mg / g of Glycyrrhiza glabra powder.

[0059] Comparative Example 3: The comparative example followed the technical solution disclosed in CN114685523A and performed the following operations: 1) Propionic acid and choline chloride were mixed at a molar ratio of 1.5:1 and stirred at 75°C until a homogeneous and transparent liquid was formed to obtain a eutectic solvent; 2) 500g of licorice powder was added to 10L of the above eutectic solvent and mixed, and stirred and extracted at 55°C for 1 hour. 1) Filter and collect the extract to obtain crude glycyrrhizin extract; 2) Extract the crude glycyrrhizin extract twice with 3 times the volume of ethyl acetate, combine the ethyl acetate, evaporate the ethyl acetate under reduced pressure, and dissolve in ethanol; 3) Separate and purify using D101 macroporous resin, elute with 50-60% ethanol solution, collect the eluent, concentrate, and recover the ethanol to obtain crude glycyrrhizin; 4) Dissolve the crude glycyrrhizin obtained in step 4 in ethanol, slowly add 10 mL of ice water, keep at 0℃ for 3 hours, centrifuge and dry to obtain 0.4 g pure glycyrrhizin.

[0060] HPLC analysis showed the results, as shown in Figure 5, that the purity of glycyrrhizin was >95%, and the extraction rate was 0.76 mg / g of Glycyrrhiza glabra powder.

[0061] The difference between Comparative Example 4 and Example 1 is that in step 3, an ammonia solution was added until the pH of the system rose to 8.2. The rest of the steps were the same as in Example 1.

[0062] As shown in Figure 6, compared to Example 1, the lower precipitate in Comparative Example 4 turned pink after the addition of ammonia, suggesting that a higher pH value may affect the stability of glycyrrhizin.

[0063] The above is a further detailed description of the present invention and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A method for extracting high-purity glycyrrhizin, characterized in that, Includes the following steps: Step (1) DES enhanced extraction: Mix and stir the raw material of Glycyrrhiza glabra with pH-responsive DES reagent for 1-2 hours, centrifuge and collect the supernatant. The pH-responsive DES reagent is composed of hydrogen bond acceptor choline chloride and hydrogen bond donor malonic acid. Step (2) Induced phase separation: Add alkaline solution to the supernatant obtained in step 1 to adjust the pH to 7.0-8.0 and separate the phases into an upper solution phase and a lower precipitate phase. Collect the lower precipitate phase and centrifuge, wash and dry to obtain the glycyrrhizin.

2. The extraction method according to claim 1, characterized in that, In step (2), an alkaline solution is added to adjust the pH to 7.0-7.5 for phase separation.

3. The extraction method according to claim 1, characterized in that, The alkaline solution in step (2) is selected from at least one of ammonia water, sodium carbonate, potassium carbonate, sodium bicarbonate, and potassium bicarbonate solution.

4. The extraction method according to claim 1, characterized in that, The method for recovering the upper solution phase in the induced phase separation of step (2) is as follows: take the upper solution phase and concentrate it under reduced pressure, and recover choline chloride and malonic acid reagent by removing water.

5. The extraction method according to claim 1, characterized in that, The method for preparing the pH-responsive DES reagent in step (1) is as follows: choline chloride, the hydrogen bond acceptor, is mixed with malonic acid, the hydrogen bond donor, and stirred at 70-85 °C until a uniform and transparent liquid is formed, thus obtaining the pH-responsive DES reagent.

6. The extraction method according to claim 1 or 4, characterized in that, In the pH-responsive DES reagent, the molar ratio of hydrogen bond acceptor choline chloride to hydrogen bond donor malonic acid is 1:1 to 1:1.

5.

7. The extraction method according to claim 1 or 4, characterized in that, The ratio of the raw material of Glycyrrhiza glabra to the pH-responsive DES reagent is 1:15 to 1:25 (g / mL).

8. The extraction method according to claim 1, characterized in that, In step (1), the temperature for mixing and extracting the raw material of Glycyrrhiza glabra with pH-responsive DES reagent is 50~65 ℃.

9. A glycyrrhizin, characterized in that, It is prepared by the extraction method described in claim 1.

10. A method for preparing water-soluble glycyrrhizin, characterized in that, The process includes the following steps: adding the glycyrrhizin described in claim 9 to an aqueous cyclodextrin solution and stirring at 50-60°C until the solution is clear and transparent, then drying and pulverizing to obtain water-soluble glycyrrhizin, wherein the cyclodextrin composition ratio is 85-95% and the glycyrrhizin composition ratio is 5%-15%.

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