The application relates to a technology for preparing high-purity glabridin by reverse purification with a carbon dioxide supercritical extraction method and application thereof.

Abstract

The present application relates to the technical field of preparing glabridin, and particularly relates to a technology and application of preparing high-purity glabridin by using carbon dioxide supercritical extraction method reverse purification. The present application firstly adopts as many extraction methods of extracting glabridin as possible, but at the same time, many impurities are extracted, which are difficult to separate from glabridin. The present application further proposes to use carbon dioxide supercritical reverse extraction method to extract the difficult-to-separate impurities in the glabridin crude extract, so that high-purity glabridin can be obtained through recrystallization finally.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of preparing glabridin, and particularly relates to a technical method and application of preparing high-purity glabridin by using carbon dioxide supercritical extraction reverse purification. BACKGROUND

[0002] Glycyrrhiza glabra is a dicotyledonous plant of the Leguminosae family, and contains glycyrrhizin, triterpene saponins, glycyrrhizin, isoglycyrrhizin, polysaccharides and other substances. Among them, there are many small molecule functional components such as flavonoids, and glabridin is one of the main flavonoids in Glycyrrhiza glabra and only exists in Glycyrrhiza glabra.

[0003] Glabridin is known as 'whitening gold' due to its strong whitening effect. It can eliminate free radicals and melanin in the skin, inhibit tyrosinase, and is often used for skin whitening and melanin inhibition. Glabridin shows strong anti-free radical oxidation in the cytochrome oxidation system, can significantly inhibit free radicals generated in the metabolic process of the body, so as to prevent the oxidation-sensitive biological macromolecules (low-density lipoprotein LDL, DNA) and cell walls from being damaged by free radical oxidation. Thus, it can prevent and treat some pathological changes related to free radical oxidation, such as atherosclerosis and cell aging. In the medical application field, it has pharmacological effects such as anti-oxidation, blood pressure reduction, blood lipid reduction and anti-inflammatory.

[0004] At present, the process for extracting glabridin has problems such as the use of organic solvents and the need for complex chromatographic purification steps, and there is still a lot of room for optimization in efficiency and cost control. SUMMARY

[0005] To solve the above technical problems, the present application provides a method for preparing glabridin by carbon dioxide supercritical reverse purification. The content of glabridin in Glycyrrhiza glabra is not high, and there are many impurities, so the final yield of glabridin extracted from Glycyrrhiza glabra is not high.

[0006] The first extraction method selected by the present application is to extract as much glabridin as possible. Although this method can maximize the extraction of glabridin, it also extracts many impurities. These impurities are difficult to separate from glabridin by general methods. The present application creatively uses carbon dioxide supercritical reverse extraction method to remove the impurities in the glabridin crude extract. After removing these impurities, high-purity and high-yield glabridin can be obtained by recrystallization. The whole process does not use chromatographic purification separation method and a large amount of environmentally unfriendly solvents. The overall process is simple, the purification yield is high, and the cost is low.

[0007] The specific method comprises the following steps:

[0008] (1) Preparation of glabridin crude extract;

[0009] (2) Carbon dioxide supercritical reverse purification preparation: using carbon dioxide supercritical extraction method to extract impurities in the glabriden crude extract obtained in step (1).

[0010] In some embodiments, the carbon dioxide supercritical extraction method described in step 2 comprises: placing the glabriden crude extract into a carbon dioxide supercritical extraction kettle, introducing carbon dioxide, stirring extraction at 10-25 MPa, 40-55℃ for 30-60 minutes, then cooling and reducing pressure to normal temperature and pressure, and collecting the residue after extraction.

[0011] In some embodiments, the carbon dioxide supercritical extraction method described in step (2) adds an entrainer or does not add an entrainer; the entrainer is one or a combination of ethanol, capric acid triglyceride.

[0012] In some embodiments, the collected residue after extraction is further purified by recrystallization.

[0013] In some embodiments, the recrystallization solvent is selected from one or more of the following: ethanol, n-butanol, isobutyl alcohol, n-pentanol, isoamyl alcohol, and a mixture of one or more of ethyl acetate, petroleum ether, butyl acetate, isobutyl acetate, and methyl tert-butyl ether.

[0014] In some embodiments, the preparation of the glabriden crude extract described in step (1) comprises:

[0015] S1: crushing the roots of glabrous kudzu vine to obtain glabrous kudzu vine powder;

[0016] S2: alcohol extraction of the glabrous kudzu vine powder 1 to multiple times, and combining to obtain glabrous kudzu vine alcohol extract;

[0017] S3: filtering the glabrous kudzu vine alcohol extract, and concentrating the filtrate to obtain glabrous kudzu vine extraction concentrate;

[0018] S4: adding the glabrous kudzu vine extraction concentrate to an extraction liquid, and collecting the supernatant by standing or collecting the centrifugal liquid by centrifugation, which is the glabriden crude extract.

[0019] In some embodiments, the crushed glabrous kudzu vine roots are obtained as 20-100 mesh glabrous kudzu vine powder.

[0020] In some embodiments, multiple times can be 2, 3, etc. After each extraction, filtration is performed, and the filter residue is extracted again. Finally, the filtrates from multiple extractions are combined to form the alcohol extract.

[0021] In some embodiments, the alcohol is one or more of ethanol, butanediol, and propylene glycol. Specifically, it can be 95% ethanol, so the product is more commonly used in cosmetics, and therefore 95% pure food-grade ethanol is commonly used in industry.

[0022] In some embodiments, the solvent used in the alcohol extraction is 5-15 times the volume of the powdered liquorice.

[0023] In some embodiments, the extraction can be heated to around 50-80°C for reflux extraction.

[0024] In some embodiments, the extraction time is 0.5-2 hours.

[0025] In some embodiments, the preparation of the glycyrrhizin crude extract in step (1) comprises:

[0026] S1: Collect liquorice, take the roots, dry thoroughly, and grind to 20-100 mesh;

[0027] S2: Put the ground powder into a reaction kettle, add 5-15 times the volume of 95% purity food-grade ethanol, and heat to around 50-80°C for 0.5-2 hours of reflux extraction;

[0028] S3: Filter through a 20-mesh sieve and collect the residue;

[0029] S4: Add 5-10 times the volume of 95% purity food-grade ethanol to the residue, and heat to around 50-80°C for 0.5-2 hours of reflux extraction;

[0030] S5: Filter through a 20-mesh sieve and collect the residue;

[0031] S6: Combine the two filtrates and filter through a 100-mesh filter;

[0032] S7: Direct the combined filtrate into a concentration tank and concentrate under reduced pressure at a temperature of 50-70°C;

[0033] S8: Direct the concentrated liquid into an extraction tank and add 1-3 times the volume of extraction liquid for extraction;

[0034] S9: Cool, stand, settle, collect the supernatant, and obtain the glycyrrhizin crude extract.

[0035] In some embodiments, the steps between grinding (S1) and alcohol extraction (S2) also include a step of ultrasonic cell disruption and homogenization; 95% purity food-grade ethanol solution is used.

[0036] In some embodiments, the preparation of the glycyrrhizin crude extract in step (1) comprises:

[0037] S1: Collect liquorice, take the roots, dry thoroughly, and grind to 20-100 mesh;

[0038] S2: The crushed powder is soaked with 5-15 times the volume of water, and after filtration, the filter residue is added with 1-3 times the volume of 95% purity food-grade ethanol, and then the cell is broken by ultrasonic wave with frequency higher than 15-20 KHz under high-intensity sound energy input, and then homogenate is performed;

[0039] S3: The homogenized material is fed into a reaction kettle, 5-15 times the volume of 95% purity food-grade ethanol is added, heated to reflux at about 50-80℃ for 0.5-2 hours; filtration is performed, and the filter residue is collected;

[0040] S4: 5-10 times the volume of 95% purity food-grade ethanol is added to the filter residue, heated to reflux at about 50-80℃ for 0.5-2 hours;

[0041] S5: 20-mesh screen filtration is performed, and the filter residue is collected;

[0042] S6: The two filtrates are combined, and 100-mesh filter filtration is performed;

[0043] S7: The combined filtrate is introduced into a concentration tank, and concentrated under reduced pressure at a temperature of 50-70℃;

[0044] S8: The concentrated liquid is introduced into an extraction tank, and 1-3 times the volume of extraction liquid is added for extraction;

[0045] S9: Cooling, standing, sedimentation, collection of supernatant, and glabridin crude extract is obtained.

[0046] In some embodiments, the extraction liquid is ethyl acetate.

[0047] Beneficial effects

[0048] 1. The preparation of glabridin crude product according to the present application can maximize the retention of glabridin and avoid loss;

[0049] 2. The carbon dioxide supercritical extraction method breaks the conventional method, and reversely extracts impurities from the glabridin crude product to maximize the retention of glabridin, removes some impurities that are difficult to remove, maximizes the retention of the product, and greatly improves the purity.

[0050] 3. Further, according to the product requirements, the extract after supercritical extraction can be directly recrystallized, avoiding the chromatography step.

[0051] Term explanation

[0052] Certain embodiments of the application are now described with reference to the following examples. Various modifications and changes can be made thereto by those skilled in the art without departing from the scope of the application as defined in the appended claims. The disclosure of all patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

[0053] It should be further recognized that certain features of the application, described in detail herein, are described in the context of multiple separate embodiments for clarity, but can also be provided in combination in a single embodiment. Conversely, various features of the application, described in the context of a single embodiment for brevity, can also be provided separately or in any appropriate subcombination.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All patents and publications cited herein are incorporated by reference in their entirety.

[0055] In the description of the specification, the description using the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the particular feature, structure, material, or characteristic following the term is included in at least one embodiment or example of the application. The illustrative appearances of the above-mentioned terms in various places in the specification are not necessarily referred to the same embodiment or example. Also, the particular features, structures, materials, or characteristics can be combined in any suitable manner in one or more embodiments or examples. Furthermore, the terminology "comprising" is used as equivalent to "including", "containing", "characterized by", "comprised of", "characterized by", "having", "including", "containing", "characterized by", "comprised of", or "comprising", and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0056] In the following disclosure, all numbers disclosed herein are approximations that can vary depending on the desired properties sought to be obtained by those of ordinary skill in the art. The numerical values in some embodiments can vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20% and the like. Whenever a numerical range is disclosed, any number or any combination of numbers within the range is expressly stated to be disclosed. Numbers within the range can be expressly recited or can be implicitly supported by the language that the range is recited in. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 is an HPLC profile of the crude extract of Example 1.

[0058] Figure 2 is the HPLC profile of the crude extract of Example 4 after back extraction.

[0059] Figure 3 is the HPLC profile of glabridin after recrystallization of Example 5.

[0060] Figure 4 is the HPLC profile of glabridin after recrystallization of Comparative Example 2. DETAILED DESCRIPTION

[0061] In order to make the objectives, technical solutions, and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. The specific embodiments described herein are only used to explain the present application and should not be used to constitute any limitation on the present application. In addition, in the following description, the description of well-known structures and techniques is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and techniques are also described in many publications.

[0062] The reagents used in the present application can be purchased from the market or can be prepared by the methods described in the present application. Preparation of glabridin crude extract:

[0063] Example 1:

[0064] 9 kg of glabridin root was cut into powder and fed into a reaction kettle. 10 times the volume (90 L) of 95% pure food-grade ethanol was added. At about 60°C, it was heated to reflux for 1.5 hours and filtered. 9 times the volume of 95% content food-grade ethanol (81 L) was added to the filter residue again, heated to reflux at about 60°C for 1.5 hours, and filtered through a 20 mesh screen. The two filtrates (about 171 L) were combined and filtered through a 100 mesh filter. The filtrate was introduced into a concentration tank and concentrated under reduced pressure at a temperature of 65°C until the volume was reduced to 8.5 L. The concentrated solution was transferred to an extraction tank, 9 L of ethyl acetate was added, and after stirring, it was cooled overnight. The supernatant was drawn off to obtain the glabridin crude extract. The HPLC profile of the crude extract is shown in Figure 1. Figure 1 .

[0065] Example 2:

[0066] The 9 kg of glabridin root was cut into powder and soaked in 10 times volume (90 L) of water for 2 hours, filtered and the filtrate was removed. The residue was added with 2 times volume (18 L) of 95% purity food grade ethanol, and then the ultrasonic wave with frequency higher than 15-20 KHz was used to break the cells under high intensity sound energy input, followed by homogenization. The homogenized sample was added into a reactor, and then 8 times volume (72 L) of 95% purity food grade ethanol was added, heated to about 60 °C and refluxed for 1.5 hours, and then filtered. Again, 9 times volume of 95% purity food grade ethanol (81 L) was added to the residue, heated to about 60 °C and refluxed for 1.5 hours, and then filtered. The filtrates obtained from the two times of ethanol extraction (about 171 L) were combined and filtered through a 100 mesh filter. The filtrate was introduced into a concentration tank and concentrated under reduced pressure at 65 °C until the volume was reduced to 8.5 L. The concentrated solution was transferred into an extraction tank, 9 L of ethyl acetate was added, stirred and then cooled overnight. The supernatant was drawn to obtain the glabridin crude extract.

[0067] Removal of impurities by supercritical carbon dioxide extraction:

[0068] Example 3

[0069] The glabridin crude extract obtained in Example 1 was added into a supercritical carbon dioxide extraction instrument. The extraction temperature was 50 °C, the extraction pressure was 20 MPa, the extraction time was 30 min, and the entrainer was 100 g of glyceryl tricaprylate (GTCC) with an entrainer feeding speed of 10 HZ.

[0070] Example 4

[0071] The glabridin crude extract obtained in Example 1 was added into a supercritical carbon dioxide extraction instrument. The extraction temperature was 40 °C, the extraction pressure was 15 MPa, the extraction time was 45 min, and the entrainer was 200 mL of anhydrous ethanol with an entrainer feeding speed of 10 HZ. The HPLC analysis of the crude extract was as follows: Figure 2 .

[0072] Further purification by recrystallization

[0073] Example 5

[0074] The glabridin sample after extraction in Example 4 was added with 100 mL of 95% ethanol, redissolved and concentrated to 10 mL. It was placed at 4 °C for 24 h to crystallize. The white powder crystals were precipitated at the bottom of the container. The crystallized sample was introduced into a filter paper lined funnel, washed and filtered. It was redissolved in ethanol and recrystallized for 2 more times. The crystals were collected and placed in a vacuum drying apparatus for 12 hours. The purity was as follows: Figure 3 and the yield was 95.8%

[0075] Comparative Example 1

[0076] Nine kilograms of *Glycyrrhiza glabra* roots were chopped into powder and added to a reaction vessel. Ten times the volume (90 L) of 95% pure food-grade ethanol was added. The mixture was heated under reflux at approximately 60°C for 1.5 hours and then filtered. Nine times the volume (81 L) of 95% pure food-grade ethanol was added to the residue again, and the mixture was heated under reflux at approximately 60°C for 1.5 hours and then filtered through a 20-mesh sieve. The two filtrates (approximately 171 L) were combined and filtered through a 100-mesh filter. The filtrate was transferred to a concentration tank and concentrated under reduced pressure at 65°C. The concentrate was redissolved with 100 mL of 95% ethanol and concentrated to 10 mL. The mixture was then allowed to stand at 4°C for 24 hours to crystallize. No crystals precipitated; the resulting substance was a dark brown, thick liquid.

[0077] Comparative Example 2

[0078] Nine kilograms of *Glycyrrhiza glabra* root roots were chopped into powder and added to a reaction vessel. Ten times the volume (90 L) of 95% pure food-grade ethanol was added. The mixture was heated under reflux at approximately 60°C for 1.5 hours and then filtered. Nine times the volume (81 L) of 95% pure food-grade ethanol was added to the residue again, and the mixture was heated under reflux at approximately 60°C for 1.5 hours and then filtered through a 20-mesh sieve. The two filtrates (approximately 171 L) were combined and filtered through a 100-mesh filter. The filtrate was transferred to a concentration tank and concentrated under reduced pressure at 65°C until the volume became 8.5 L. The concentrate was transferred to an extraction tank, 9 L of ethyl acetate was added, and the mixture was stirred and cooled overnight. The supernatant was collected to obtain the crude extract of glycyrrhiza glabra. The crude extract was concentrated, and 100 mL of 95% ethanol was added to redissolve the extract, which was then concentrated to 10 mL. The mixture was placed at 4°C and allowed to stand for 24 hours to crystallize. A small amount of yellow powdery crystals settled at the bottom of the container. The purity was as follows. Figure 4 The yield was 87.6%. Further recrystallization can be performed to improve purity, but this results in significant product loss.

[0079] glycyrrhizin determination

[0080] 1. Preparation of standard solutions and curves:

[0081] Weigh 50 mg of glycyrrhizin standard sample, add 30 mL of methanol and stir. Place the mixture in an ultrasonic cleaner for ultrasonic extraction at room temperature. After complete dissolution, transfer the solution to a 50 mL volumetric flask, dilute with water to the mark, and shake well. This solution serves as the standard solution control sample.

[0082] Take 50, 100, 300, 500, and 800 μL of standard solution and add them to 10 mL volumetric flasks, respectively, and dilute to volume to prepare concentrations of 5 mg / L, 10 mg / L, 30 mg / L, 50 mg / L, and 80 mg / L to establish a standard curve.

[0083] 2. Sample preparation:

[0084] Take the appropriate amount (accurate to 0.0001 g) of sample in a 25 mL stoppered colorimetric tube, add methanol to dissolve and dilute to 25 mL, vortex to disperse the sample completely, ultrasonic extraction for 15-30 min, shake well after dissolving, and prepare the sample to be tested. Take the supernatant and filter it with a 0.45 μm membrane filter, and transfer the filtrate to a 2 mL sample bottle for testing.

[0085] 3. Determination:

[0086] Under the reference chromatographic conditions, the standard solution and the sample are determined respectively, and the area of the peak at about 5.9 min is recorded. The standard curve is prepared according to the measured standard solution, and the sample concentration C (mg / L) is quantified by external standard method. The following formula is used for calculation.

[0087]

[0088] In the formula:

[0089] X1 - the content of glabridin in the sample, %;

[0090] m - the mass of the sample taken, g;

[0091] C - the sample concentration obtained by standard curve quantification, mg / L;

[0092] f - the dilution multiple of the sample to be tested;

[0093] V - the constant volume of the sample to be tested, mL.

[0094] Chromatographic conditions

[0095] a) Chromatographic column: ZORBAX StableBond C18, 4.6 x 250 mm, 5 μm;

[0096] b) Flow rate: 1.0 mL / min;

[0097] c) Mobile phase: acetonitrile: water = 66.5: 33.5, filtered with a 0.22 μm membrane filter;

[0098] d) Injection volume: 10 μL;

[0099] e) Column temperature: 25°C;

[0100] f) Wavelength: 280 nm;

[0101] The methods of the present application have been described by way of preferred embodiments, and modifications or suitable variations and adaptations of the methods and applications described herein can be made by those skilled in the art in the light of the content, spirit and scope of the present application, to implement and apply the present technology. Those skilled in the art can make appropriate modifications to the process parameters based on the content herein to implement. It is particularly important to note that all such obvious substitutions and modifications are within the scope of the present application.

Claims

1. A method for preparing glabridin by supercritical reverse phase purification of carbon dioxide, characterized by, The method comprises: (1) Preparation of a glabridin crude extract; (2) Preparation by carbon dioxide supercritical reverse purification: impurities in the glabridin crude extract obtained in step (1) are removed by a carbon dioxide supercritical extraction method; the carbon dioxide supercritical extraction method adds an entrainer, and the entrainer is one or a combination of ethanol, capric acid triglyceride; The carbon dioxide supercritical extraction method comprises: placing the glabridin crude extract into a carbon dioxide supercritical extraction kettle, introducing carbon dioxide, stirring and extracting under the conditions of 10-25 MPa and 40-55 ℃ for 30-60 minutes, then reducing the temperature and pressure to normal temperature and pressure, and collecting the residue after extraction.

2. The method of claim 1, wherein, The residue after extraction is further purified by recrystallization.

3. The method of claim 2, wherein, The recrystallization solvent is selected from one or a combination of the following: n-butanol, isobutyl alcohol, n-pentanol, isopentyl alcohol, n-hexanol, heptanol, n-octanol, and one or a combination of the following: ethyl acetate, butyl acetate, isobutyl acetate, and methyl tert-butyl ether.

4. The method according to any one of claims 1 to 3, characterized in that, Step (1) the preparation of the glabridin crude extract comprises: S1: crushing the roots of glabra to obtain glabra root powder; S2: alcohol extraction of the glabra root powder 1 to multiple times to obtain glabra alcohol extract; S3: filtering the glabra alcohol extract, concentrating the filtrate to obtain glabra extraction concentrate; S4: adding the glabra extraction concentrate to an extraction liquid, standing to collect the supernatant, or centrifuging to collect the centrifugate, i.e. the glabridin crude extract.

5. The method of claim 4, wherein, The crushed glabra roots are 20-100 mesh glabra powder; and / or Multiple times are 2 or 3 times; after each extraction, the filtrate is filtered, and the filter residue is extracted again; and finally, the filtrates obtained by multiple extractions are combined to form the alcohol extract; and / or The alcohol is one or a combination of the following: ethanol, butanediol, and propylene glycol; and / or The alcohol extraction uses a solvent that is 5-15 times the volume of the glabra powder; and / or The extraction can be heated to about 50-80 °C for reflux extraction; and / or The extraction time is 0.5-2 hours.

6. The method of claim 5, wherein, The alcohol is 95% ethanol.

7. The method of claim 5, wherein, Step (1) the preparation of the glabridin crude extract comprises: S1: collecting glabra, taking the roots, drying thoroughly, and crushing to 20-100 mesh; S2: feeding the crushed powder into a reaction kettle, adding 5-15 times the volume of 95% purity food-grade ethanol, and heating to about 50-80 °C for reflux extraction for 0.5-2 hours; S3: filtering through a 20-mesh sieve to collect the filter residue; S4: adding 5-10 times the volume of 95% purity food-grade ethanol to the filter residue, heating to about 50-80 °C for reflux extraction for 0.5-2 hours; S5: filtering through a 20-mesh sieve to collect the filter residue; S6: combining the two filtrates and filtering through a 100-mesh filter; S7: introducing the combined filtrate into a concentration tank and concentrating under reduced pressure at a temperature of 50-70 °C; S8: introducing the concentrated liquid into an extraction tank, adding 1-3 times the volume of an extraction liquid for extraction; S9: cooling, standing, settling, collecting the supernatant, and obtaining the glabridin crude extract.

8. The method according to any one of claims 5-7, characterized in that, The method further comprises the steps of ultrasonic breaking and homogenizing between the steps of pulverizing and alcohol extraction; using 95% purity food-grade ethanol solution; and / or the extraction liquid is ethyl acetate.

Images