Glabridin inclusion compound as well as preparation method and application thereof

Abstract

The invention discloses a glabridin inclusion compound as well as a preparation method and application thereof, and belongs to the technical field of medicines and cosmetics. The preparation method comprises the following steps: S1, carrying out water extraction treatment on liquorice to obtain liquorice residues and filtrate; s2, obtaining a precipitate A in the filtrate; extracting the licorice root residues to obtain an extract B containing glabridin; s3, respectively mixing the precipitate A and the extract B with a solvent to obtain a precipitate A solution and an extract B solution; and mixing the precipitate A solution and the extract B solution, and drying to obtain the glabridin inclusion compound. Compared with the prior art, the glabridin is wrapped by the components of the liquorice, the formed clathrate compound has excellent dissolving property and water dispersibility, irritant auxiliary materials such as solubilizers do not need to be added, the potential allergy risk and the production cost of the product can be effectively reduced, and the application field of the glabridin clathrate compound is widened.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine and cosmetics, and particularly relates to a glabridin inclusion compound and a preparation method and application thereof. BACKGROUND

[0002] Glycyrrhiza, a perennial herbaceous plant of the genus Glycyrrhiza in the family Fabaceae, is a variety of Glycyrrhiza glabra. It tastes sweet and is flat in nature, has the effects of tonifying the spleen and replenishing qi, relieving cough and phlegm, clearing heat and detoxifying, and regulating the properties of drugs, and is used to treat spleen and stomach weakness, deficiency of middle qi, cough and shortness of breath, palpitation and shortness of breath, sore throat, and carbuncle and sore, etc. The roots and rhizomes of Glycyrrhiza glabra can be used for medicine, can relieve eye inflammation, treat various inflammations of the digestive system such as oral ulcer, gastritis, peptic ulcer and excessive gastric acid, and chest pain, arthritis and some skin diseases, and can also be used as a laxative.

[0003] The roots of Glycyrrhiza glabra mainly contain a large amount of flavonoids, isoflavones, triterpenoid saponins, polysaccharides, phytosterols, coumarins and asparagines, etc. Studies have shown that glycyrrhizic acid has the same anti-inflammatory and anti-arthritic effects as corticosteroids or other corticosteroid hormones; it can also stimulate the production of hormones in the adrenal glands and slow down the degradation of hormones in the liver and kidneys. Glabridin, which is well known to people, is a kind of isoflavone in Glycyrrhiza glabra, and is known as "whitening gold" and has very good tyrosinase inhibitory activity. However, it has poor water solubility and low bioavailability, which directly affects its wide application.

[0004] There are many other methods for glabridin inclusion or solubilization at present, such as: using Glycyrrhiza glabra extract, apple extract, rice bran extract, oil, emulsifier, co-emulsifier and water as materials to prepare glabridin plant source microcapsule package; using exosome to prepare glabridin; using cyclodextrin derivative to prepare glabridin multiple package solid preparation; using gelatin and gum arabic as wall materials to prepare glabridin microcapsules, however, 0.5% of a solubilizer is needed as an auxiliary. The above-mentioned methods can increase the solubility or bioavailability of glabridin in water, however, these methods use some emulsifiers, solubilizers or alcohol solvents and other auxiliary materials with irritant or allergic properties to different degrees, which have potential risks of causing allergy and limited use field; or need to use expensive equipment, which has high production cost. SUMMARY

[0005] In order to overcome at least one problem existing in the prior art, one of the purposes of the present application is to provide a preparation method of glabridin inclusion compound, which can prepare glabridin inclusion compound with good water solubility and high bioavailability, and does not need to use irritant or allergic auxiliary materials, and has low potential risk of causing allergy.

[0006] The second object of the present application is to provide a glabridin inclusion compound prepared by the above preparation method.

[0007] The third object of the present application is to provide a glabridin preparation comprising the above glabridin inclusion compound.

[0008] The fourth object of the present application is to provide an application of the above glabridin inclusion compound or glabridin preparation.

[0009] To achieve the above objects, the technical solution adopted by the present application is as follows: The first aspect of the present application provides a preparation method of a glabridin inclusion compound, comprising the following steps: S1, water extraction treatment is performed on liquorice to obtain liquorice residue and filtrate; S2, a precipitate A is obtained in the filtrate; the liquorice residue is extracted to obtain an extract B containing glabridin; S3, the precipitate A and the extract B are mixed with solvents respectively to obtain a precipitate A solution and an extract B solution; the precipitate A solution and the extract B solution are mixed, and after drying, the glabridin inclusion compound is obtained.

[0010] The preparation method of the glabridin inclusion compound according to the first aspect of the present application has at least the following beneficial effects: Liquorice contains a large amount of flavonoids, isoflavones, triterpenoid saponins, polysaccharides, phytosterols, coumarins and asparagines, etc. These components contain a variety of different structures, among which there are mainly two types of hydrophilic structure and hydrophobic structure. By process control, different proportions of flavonoids, saponins, polysaccharides, polypeptides, etc. are obtained, forming a natural-like low eutectic system derived from liquorice, i.e. the precipitate A. The precipitate A contains water-soluble components such as glycyrrhizic acid and liquorice polysaccharides, which form an inclusion compound with the extract B containing glabridin, increasing the water solubility and bioavailability of glabridin. In addition, the components such as flavonoids, isoflavones, saponins and polysaccharides contained in liquorice have certain anti-inflammatory and anti-allergic effects, which can reduce the risk of allergy during product application. The present application uses the components of liquorice itself to wrap glabridin and form an inclusion compound, which has excellent solubility and water dispersibility. In the use process, the use of various solubilizers, surfactants and emulsifiers, etc. can be effectively and greatly reduced, the potential risk of allergy can be effectively reduced, the production cost can be reduced, and the use field of the product can be expanded.

[0011] In some specific embodiments of the present application, in step S2, the precipitate A is obtained in the filtrate by adjusting the filtrate to be acidic. In some more specific embodiments of the present application, the pH value of the filtrate is adjusted to 1-6.

[0012] In some specific embodiments of the present application, the licorice residue is extracted with an organic solvent in step S2; in some more specific embodiments of the present application, the organic solvent is selected from ester solvents.

[0013] In some specific embodiments of the present application, the licorice residue is extracted to obtain a pre-extract, and the pre-extract is further separated and purified, specifically including: mixing the pre-extract with an ethanol solution to obtain an ethanol extract; subjecting the ethanol extract to adsorption treatment by a print resin, and eluting the adsorbed print resin to obtain an eluate, and drying the eluate to obtain an extract B containing glabridin.

[0014] In some specific embodiments of the present application, the content of ethanol in the ethanol solution is 30-99.9wt% in the separation and purification process.

[0015] In some specific embodiments of the present application, the eluent used in the elution includes at least one of ethyl acetate, methanol, ethanol or chloroform in the separation and purification process.

[0016] In some specific embodiments of the present application, the mass ratio of the precipitate A to the extract B is 1: (0.01-30) in step S3.

[0017] In some specific embodiments of the present application, the solvent includes an alcohol solvent and water in step S3.

[0018] The second aspect of the present application provides a glabridin inclusion compound prepared by the preparation method of the first aspect of the present application.

[0019] The glabridin inclusion compound according to the second aspect of the present application has at least the following beneficial effects: In the glabridin inclusion compound prepared by the method of the first aspect of the present application, the surface of glabridin is coated with a specific inclusion layer, so that the glabridin inclusion compound has good water solubility and bioavailability, and the inclusion compound also contains anti-inflammatory, anti-allergic components such as flavonoids, isoflavones, saponins and polysaccharides, which is conducive to reducing the risk of allergy during use of the inclusion compound.

[0020] In some specific embodiments of the present application, the solubility of the glabridin inclusion compound is 100-20000μg / mL.

[0021] In some specific embodiments of the present application, the average Zeta potential of the glabridin inclusion compound is 1-5mV.

[0022] In some specific embodiments of the present application, the average particle size of the glabridin inclusion compound is 100-400nm.

[0023] The third aspect of the present application provides a formulation of glabridin, comprising the glabridin inclusion complex of the second aspect of the present application.

[0024] The formulation of glabridin according to the third aspect of the present application has at least the following beneficial effects: The formulation of glabridin of the present application can fully utilize the biological activity of glabridin, has high bioavailability, high tyrosinase inhibition rate and melanin production inhibition rate, has good whitening effect, and does not need to add solubilizers, surfactants and emulsifiers and other auxiliary materials with irritant or allergic properties, thus having the advantages of low potential allergic risk, low production cost and wide application field.

[0025] In some embodiments of the present application, the formulation of glabridin is a liquid formulation of glabridin.

[0026] In some embodiments of the present application, the solvent in the liquid formulation of glabridin comprises water.

[0027] The fourth aspect of the present application provides a product comprising the glabridin inclusion complex of the second aspect of the present application or the formulation of glabridin of the third aspect of the present application, wherein the product comprises a cosmetic, a pharmaceutical, a food or a health product.

[0028] The product according to the fourth aspect of the present application has at least the following beneficial effects: The glabridin inclusion complex and the formulation of glabridin of the present application have high bioavailability, high tyrosinase inhibition rate and melanin production inhibition rate, good whitening effect, low irritancy and low allergic risk, and can be widely applied in the fields of cosmetics, pharmaceuticals, foods or health products, etc. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is a physical diagram of the dissolution of the glabridin inclusion complex powder of Example 3 and its aqueous solution.

[0030] Figure 2 is a characterization diagram of the water dispersibility of the glabridin inclusion complex of Example 3.

[0031] Figure 3 is an XRD spectrum of the extract B and the glabridin inclusion complex in Example 3.

[0032] Figure 4 is a SEM image of the extract B and the glabridin inclusion complex in Example 3.

[0033] Figure 5 is a mass spectrometry imaging diagram of the extract B and the glabridin inclusion complex of Example 3 and the glabridin mixture of Comparative Example 1.

[0034] Figure 6 Tyrosinase inhibition rate of the glabridin inclusion complex of Example 3.

[0035] Figure 7 Melanin production inhibition rate of the glabridin inclusion complex of Example 3.

[0036] Figure 8 Comparison chart of tyrosinase inhibition rates of the glabridin inclusion complex of Example 3 and the glabridin mixture of Comparative Example 1. DETAILED DESCRIPTION

[0037] The content of the present application is further illustrated in detail by specific examples. It should also be understood that the following examples are only used to further illustrate the present application and should not be construed as limiting the scope of the present application. Some non-essential improvements and adjustments made by those skilled in the art based on the principles set forth in the present application are within the scope of the present application. The following examples specifically illustrate the process parameters, etc. which are only one example in the appropriate range, i.e. those skilled in the art can make appropriate selection within the range through the description herein, and are not limited to the specific data of the following examples. The raw materials, reagents or devices used in the following examples and comparative examples can be obtained from conventional commercial channels or can be obtained by known methods unless otherwise specified.

[0038] The first aspect of the embodiment of the present application provides a preparation method of a glabridin inclusion complex, comprising the following steps: S1, water extraction treatment is performed on licorice to obtain licorice residue and filtrate; S2, precipitate A is obtained in the filtrate; the licorice residue is extracted to obtain extract B containing glabridin; S3, precipitate A and extract B are mixed with solvents respectively to obtain precipitate A solution and extract B solution; the precipitate A solution and the extract B solution are mixed, and after drying, a glabridin inclusion complex is obtained.

[0039] Glycyrrhiza contains a large amount of flavonoids, isoflavones, triterpenoid saponins, polysaccharides, phytosterols, coumarin and asparagine and the like, which contain a plurality of different structures, wherein the main part is divided into two types of hydrophilic structure and hydrophobic structure, and different proportions of flavonoids, saponins, polysaccharides, polypeptides and the like are obtained by process control, forming a natural-like eutectic system derived from Glycyrrhiza, i.e. precipitate A, which contains water-soluble components such as glycyrrhizic acid and glycyrrhizin, and is combined with extract B containing glabridin to form a clathrate, thereby increasing the water solubility and bioavailability of glabridin. In addition, the flavonoids, isoflavones, saponins, polysaccharides and the like contained in Glycyrrhiza have certain anti-inflammatory and anti-allergic effects, which can reduce the risk of allergy during product application. The present application uses the components of Glycyrrhiza itself to wrap glabridin and form a clathrate, which has excellent solubility and water dispersibility, can effectively and greatly reduce the use of various solubilizers, surfactants and emulsifiers and the like during use, effectively reduces the potential risk of allergy and production cost, and widens the use field of the product.

[0040] In some embodiments of the present application, in step S1, the water extraction treatment specifically adopts a reflux extraction method; in some specific embodiments of the present application, in step S1, the water extraction treatment is specifically as follows: mixing Glycyrrhiza with water, boiling and refluxing for extraction.

[0041] In some embodiments of the present application, in the water extraction treatment of step S1, the solid-liquid ratio of Glycyrrhiza to water is 1: (1-20) by mass; in some specific embodiments of the present application, in the water extraction treatment of step S1, the solid-liquid ratio of Glycyrrhiza to water is 1: (3-15) by mass; in some examples of the present application, in the water extraction treatment of step S1, the solid-liquid ratio of Glycyrrhiza to water is 1: (4-10) by mass; non-limiting specific examples are 1:5, 1:6, 1:7, 1:8 or 1:9.

[0042] In some embodiments of the present application, in the water extraction treatment of step S1, the boiling refluxing time is 0.1-5h; in some specific embodiments of the present application, in the water extraction treatment of step S1, the boiling refluxing time is 0.3-3h; in some examples of the present application, in the water extraction treatment of step S1, the boiling refluxing time is 0.5-2h; non-limiting specific examples are 0.6h, 0.8h, 1h, 1.2h, 1.5h or 1.8h.

[0043] In some embodiments of the present application, the Glycyrrhiza is selected from Glycyrrhiza glabra; in some specific embodiments of the present application, the Glycyrrhiza is selected from Glycyrrhiza glabra root, Glycyrrhiza glabra rhizome or a combination thereof.

[0044] The roots and rhizomes of Glycyrrhiza glabra are rich in flavonoids, isoflavones, triterpenoid saponins, polysaccharides, phytosterols, coumarin and asparagine and the like, and high-content glabridin and other active ingredients can be extracted.

[0045] In some embodiments of the present application, before the water extraction treatment in step S1, the licorice is crushed. In some specific embodiments of the present application, the licorice is crushed to 50-200 mesh. Non-limiting specific examples include 80 mesh, 100 mesh, 120 mesh, or 150 mesh.

[0046] In some embodiments of the present application, in step S2, the precipitate A is obtained by adjusting the filtrate to be acidic. In some specific embodiments of the present application, in step S2, the pH value of the filtrate is adjusted to 1-6. In some more specific embodiments of the present application, in step S2, the pH value of the filtrate is adjusted to 1.5-5. In some examples of the present application, in step S2, the pH value of the filtrate is adjusted to 2-4. Non-limiting specific examples include 2.2, 2.5, 2.8, 3, 3.2, 3.5, or 3.8.

[0047] Adjusting the filtrate to be acidic can precipitate the water-soluble components in the licorice. Further, the pH value of the filtrate can affect the components contained in the precipitate A, the solubility of the precipitate A, and thus the water solubility and bioavailability of the glabridin inclusion compound. Within the specific pH value range of the present application, the inclusion effect of the precipitate A is better, and the glabridin inclusion compound obtained has better water solubility and higher bioavailability.

[0048] In some embodiments of the present application, in step S2, the licorice residue is extracted with an organic solvent. In some specific embodiments of the present application, in step S2, the organic solvent is selected from ester solvents. In some more specific embodiments of the present application, the ester solvent is selected from C2-C10 esters. In some examples of the present application, the ester solvent includes at least one of methyl formate, ethyl formate, methyl acetate, ethyl acetate, or propyl acetate. In some specific examples of the present application, the ester solvent is selected from ethyl acetate.

[0049] In some embodiments of the present application, in step S2, the extraction of the licorice residue is performed as follows: the licorice residue is extracted with an organic solvent to obtain an organic solvent extract, and the organic solvent extract is concentrated to obtain a pre-extract.

[0050] In some embodiments of the present application, in step S2, after the extraction of the licorice residue, the pre-extract is further subjected to separation and purification treatment, which specifically includes: mixing the pre-extract with an ethanol solution to obtain an ethanol extract; subjecting the ethanol extract to adsorption treatment by an imprint resin, and eluting the adsorbed imprint resin to obtain an eluate, and drying the eluate to obtain an extract B containing glabridin.

[0051] In some embodiments of the present application, in the separation and purification treatment of step S2, the ethanol solution is an ethanol aqueous solution.

[0052] In some embodiments of the present application, the ethanol content in the ethanol solution in the separation and purification treatment of step S2 is 30-99.9wt%; in some specific embodiments of the present application, the ethanol content in the ethanol solution in the separation and purification treatment of step S2 is 50-99.9wt%; in some examples of the present application, the ethanol content in the ethanol solution in the separation and purification treatment of step S2 is 70-90wt%; non-limiting specific examples include 72wt%, 75wt%, 78wt%, 80wt%, 82wt%, 85wt% or 88wt%.

[0053] The type of mobile phase used in the imprint resin adsorption treatment will affect the adsorption effect of the imprint resin, and in the embodiments of the present application, ethanol solution is used as the mobile phase, which can promote the adsorption of glabridin by the imprint resin, and thus obtain the extract B with high glabridin content.

[0054] In some embodiments of the present application, the imprint resin in the separation and purification treatment of step S2 is a glabridin molecular imprinting resin.

[0055] The glabridin molecular imprinting resin is a polymer material for purifying glabridin, which is prepared by molecular imprinting technology and has the characteristics of high selectivity and high adsorption capacity for glabridin.

[0056] The glabridin molecular imprinting resin in the present application can be obtained by existing technology, for example, the glabridin molecular imprinting resin can be obtained by the following method: first, glabridin is mixed with a functional monomer (such as trifluoromethacrylic acid, methacrylic acid, 4-vinylpyridine, vinyl imidazole, etc.), a crosslinking agent (such as ethylene glycol dimethyl acrylate, N,N-dimethyl acrylamide, ethylene glycol dimethyl acrylate, etc.), an initiator (such as 2,2'-azobis isobutyronitrile) and a solvent (such as methanol / chloroform, acetonitrile / chloroform, N,N-dimethylformamide / chloroform, etc.) as a template molecule, and then a polymer is formed through polymerization (the polymerization temperature is 1-5℃, and the time is 12-36h), and then the template molecule is removed by elution, leaving a cavity complementary to the structure of glabridin, thereby obtaining a molecularly imprinted polymer with specific recognition ability, which is the glabridin molecular imprinting resin.

[0057] In some embodiments of the present application, the eluent used in the elution in the separation and purification treatment of step S2 includes at least one of ethyl acetate, methanol, ethanol or chloroform; in some specific embodiments of the present application, the eluent used in the elution in the separation and purification treatment of step S2 includes ethyl acetate, methanol or a combination thereof; in some examples of the present application, the eluent used in the elution in the separation and purification treatment of step S2 includes ethyl acetate and methanol.

[0058] In some embodiments of the present application, in the separation and purification process of step S2, the volume ratio of ethyl acetate to methanol in the eluent used for elution is (10-50):1; in some specific embodiments of the present application, in the separation and purification process of step S2, the volume ratio of ethyl acetate to methanol in the eluent used for elution is (15-45):1; in some examples of the present application, in the separation and purification process of step S2, the volume ratio of ethyl acetate to methanol in the eluent used for elution is (20-40):1; non-limiting specific examples include 25:1, 28:1, 30:1, 32:1 or 35:1.

[0059] In some embodiments of the present application, in step S3, the mass ratio of precipitate A to extract B is 1:(0.01-30); in some specific embodiments of the present application, in step S3, the mass ratio of precipitate A to extract B is 1:(0.02-20); in some examples of the present application, in step S3, the mass ratio of precipitate A to extract B is 1:(0.03-10); non-limiting specific examples include 1:0.04, 1:0.05, 1:0.08, 1:0.1, 1:0.3, 1:0.5, 1:1, 1:3, 1:5 or 1:8.

[0060] The mass ratio of precipitate A to extract B affects the inclusion effect, thereby affecting the solubility and dispersibility of the inclusion compound in aqueous solution. The inclusion compound with high solubility and good water dispersibility can be obtained by using the mass ratio within the scope of the present application.

[0061] In some embodiments of the present application, in step S3, the precipitate A solution and the extract B solution are mixed by adding the extract B solution to the precipitate A solution. A specific order of addition can achieve better inclusion effect.

[0062] In some embodiments of the present application, in step S3, the precipitate A solution and the extract B solution are mixed under light shielding conditions.

[0063] In some embodiments of the present application, in step S3, the precipitate A solution and the extract B solution are mixed for 20-60 min; non-limiting examples include 25 min, 30 min, 35 min, 40 min, 45 min, 50 min or 55 min.

[0064] In some embodiments of the present application, in step S3, the drying is performed under vacuum conditions.

[0065] In some embodiments of the present application, in step S3, after drying, a step of crushing is further included.

[0066] In some embodiments of the present application, in step S3, the solvent comprises an alcohol solvent and water. The mixed solvent composed of the alcohol solvent and water has a better dissolving effect on the precipitate A and the extract B, thereby promoting the formation of a better inclusion effect.

[0067] In some embodiments of the present application, in step S3, the alcohol solvent is selected from C1-C10 alcohol; in some specific embodiments of the present application, in step S3, the alcohol solvent comprises at least one of methanol, ethanol, isopropyl alcohol or isobutyl alcohol; in some examples of the present application, in step S3, the alcohol solvent is selected from ethanol.

[0068] The content of the alcohol solvent in the solvent of step S3 can be adjusted according to the amount of glabridin. Specifically, in some embodiments of the present application, in the solvent of step S3, the content of the alcohol solvent is 30-99.9wt%, and the balance is water; in some specific embodiments of the present application, in the solvent of step S3, the content of the alcohol solvent is 50-99.9wt%, and the balance is water; in some examples of the present application, in the solvent of step S3, the content of the alcohol solvent is 70-90wt%, and the balance is water; non-limiting specific examples include an alcohol solvent content of 72wt%, 75wt%, 78wt%, 80wt%, 82wt%, 85wt% or 88wt%.

[0069] The second aspect of the embodiments of the present application provides a glabridin inclusion compound prepared by the preparation method of the first aspect of the embodiments of the present application.

[0070] In the glabridin inclusion compound prepared by the method provided by the first aspect of the embodiments of the present application, the glabridin is surface-included with a specific inclusion layer, so that the glabridin inclusion compound has good water solubility and bioavailability, and the inclusion compound also contains anti-inflammatory, anti-allergic components such as flavones, isoflavones, saponins and polysaccharides, which is conducive to reducing the risk of allergy of the inclusion compound in use.

[0071] In some embodiments of the present application, the solubility of the glabridin inclusion compound is 100-20000μg / mL; in some specific embodiments of the present application, the solubility of the glabridin inclusion compound is 300-15000μg / mL; in some examples of the present application, the solubility of the glabridin inclusion compound is 500-12000μg / mL; non-limiting specific examples include 600μg / mL, 800μg / mL, 1000μg / mL, 3000μg / mL, 5000μg / mL, 8000μg / mL, 10000μg / mL or 11000μg / mL.

[0072] In some embodiments of the present application, the average Zeta potential of the glabridin inclusion complex is 1-5 mV; in some specific embodiments of the present application, the average Zeta potential of the glabridin inclusion complex is 1.5-4 mV; in some examples of the present application, the average Zeta potential of the glabridin inclusion complex is 2-3 mV; non-limiting specific examples are 2.1 mV, 2.2 mV, 2.4 mV, 2.6 mV or 2.8 mV.

[0073] In some embodiments of the present application, the average particle size of the glabridin inclusion complex is 100-400 nm; in some specific embodiments of the present application, the average particle size of the glabridin inclusion complex is 120-300 nm; in some examples of the present application, the average particle size of the glabridin inclusion complex is 150-250 nm; non-limiting specific examples are 160 nm, 180 nm, 200 nm, 220 nm or 240 nm.

[0074] The third aspect of the embodiments of the present application provides a glabridin preparation comprising the glabridin inclusion complex of the second aspect of the embodiments of the present application.

[0075] The glabridin preparation of the present application can fully utilize the biological activity of glabridin, has high bioavailability, high tyrosinase inhibition rate and melanin production inhibition rate of the preparation, has good whitening effect, and does not need to add solubilizers, surfactants and emulsifiers and other auxiliary materials with irritant or allergic properties, thus having the advantages of low potential allergic risk, low production cost and wide application field.

[0076] In some embodiments of the present application, the glabridin preparation is a glabridin liquid preparation; in some specific embodiments of the present application, the solvent in the glabridin liquid preparation comprises water.

[0077] In some embodiments of the present application, when the concentration of glabridin in the glabridin preparation is 30 μmol / L, the tyrosinase inhibition rate of the glabridin preparation is 60-90%; in some specific embodiments of the present application, when the concentration of glabridin in the glabridin preparation is 30 μmol / L, the tyrosinase inhibition rate of the glabridin preparation is 65-88%; in some examples of the present application, when the concentration of glabridin in the glabridin preparation is 30 μmol / L, the tyrosinase inhibition rate of the glabridin preparation is 70-85%; non-limiting specific examples are 72%, 75%, 78%, 80% or 82%.

[0078] In some embodiments of the present application, the tyrosinase inhibition rate of the glabridin preparation is 50-80% when the concentration of glabridin in the glabridin preparation is 15 μmol / L; in some specific embodiments of the present application, the tyrosinase inhibition rate of the glabridin preparation is 55-75% when the concentration of glabridin in the glabridin preparation is 15 μmol / L; in some examples of the present application, the tyrosinase inhibition rate of the glabridin preparation is 58-70% when the concentration of glabridin in the glabridin preparation is 15 μmol / L; non-limiting specific examples include 60%, 62%, 64%, 65% or 68%.

[0079] In some embodiments of the present application, the melanogenesis inhibition rate of the glabridin preparation is 50-70% when the concentration of glabridin in the glabridin preparation is 30 μmol / L; in some specific embodiments of the present application, the melanogenesis inhibition rate of the glabridin preparation is 52-68% when the concentration of glabridin in the glabridin preparation is 30 μmol / L; in some examples of the present application, the melanogenesis inhibition rate of the glabridin preparation is 55-65% when the concentration of glabridin in the glabridin preparation is 30 μmol / L; non-limiting specific examples include 56%, 58%, 60%, 62% or 64%.

[0080] In some embodiments of the present application, the melanogenesis inhibition rate of the glabridin preparation is 40-60% when the concentration of glabridin in the glabridin preparation is 15 μmol / L; in some specific embodiments of the present application, the melanogenesis inhibition rate of the glabridin preparation is 42-58% when the concentration of glabridin in the glabridin preparation is 15 μmol / L; in some examples of the present application, the melanogenesis inhibition rate of the glabridin preparation is 45-55% when the concentration of glabridin in the glabridin preparation is 15 μmol / L; non-limiting specific examples include 46%, 48%, 50%, 52% or 54%.

[0081] The fourth aspect of the embodiments of the present application provides a product comprising the glabridin inclusion complex of the second aspect of the embodiments of the present application or the glabridin preparation of the third aspect of the embodiments of the present application, and the product comprises a cosmetic, a pharmaceutical, a food or a health product.

[0082] The glabridin inclusion complex and the glabridin preparation of the present application have high bioavailability, high tyrosinase inhibition rate and melanogenesis inhibition rate, good whitening effect, low irritation and low risk of allergy, and can be widely applied in the fields of cosmetic, pharmaceutical, food or health product additive, etc.

[0083] In some embodiments of the present application, the glabridin inclusion complex or the glabridin preparation is used as an additive in a product.

[0084] The application will be further described in connection with specific examples and comparative examples.

[0085] Example 1 A preparation method of a glabridin inclusion compound, the specific steps are as follows: 1) Dry and crush licorice, and pass it through a 100-mesh sieve to obtain licorice dry powder; use licorice dry powder and deionized water in a solid-liquid ratio of 1:5 to reflux and boil for 1 h, filter, repeat three times, concentrate to 1 / 2, filter, obtain licorice residue and filtrate, and adjust the filtrate to pH = 4 to obtain precipitate A; 2) Extract the licorice residue with ethyl acetate, filter, obtain ethyl acetate extract, concentrate the ethyl acetate extract, add 60% ethanol water to redissolve, filter, and obtain ethanol water extract; prepare glabridin molecularly imprinted resin, adsorb the ethanol water extract on the imprinted resin, and elute with ethyl acetate and methanol (mixed at 30:1, v / v), collect the eluate, and dry to obtain extract B containing glabridin; The preparation process of the glabridin molecularly imprinted resin is as follows: use glabridin molecules as templates, trifluoromethacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a crosslinking agent, and 2,2'-azobis isobutyronitrile as an initiator, under nitrogen protection, in a chloroform and methanol mixed solvent, react at 3°C for 24 h to obtain glabridin molecularly imprinted resin; 3) Take 1 mass part of precipitate A and 1 mass part of extract B, respectively, and dissolve them in an ethanol water solution to obtain precipitate A ethanol solution and extract B ethanol solution, respectively, slowly drop the extract B ethanol water solution into the precipitate A ethanol water solution, stir in the dark for 30 min, dry the inclusion liquid under vacuum, crush, and obtain a glabridin inclusion compound.

[0086] Example 2 A preparation method of a glabridin inclusion compound, the specific steps are as follows: 1) Dry and crush licorice, and pass it through a 100-mesh sieve to obtain licorice dry powder; use licorice dry powder and deionized water in a solid-liquid ratio of 1:5 to reflux and boil for 1 h, filter, repeat three times, concentrate to 1 / 2, filter, obtain licorice residue and filtrate, and adjust the filtrate to pH = 6 to obtain precipitate A; 2) Extract the licorice residue with ethyl acetate, filter, obtain ethyl acetate extract, concentrate the ethyl acetate extract, add 60% ethanol water to redissolve, filter, and obtain ethanol water extract; prepare glabridin molecularly imprinted resin, adsorb the ethanol water extract on the imprinted resin, and elute with ethyl acetate and methanol (mixed at 30:1, v / v), collect the eluate, and dry to obtain extract B containing glabridin; The preparation process of the glabridin molecularly imprinted resin is as follows: taking glabridin molecule as a template, trifluoromethacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a crosslinking agent, 2,2'-azobis isobutyronitrile as an initiator, under nitrogen protection, mixed solvent of chloroform and methanol, reaction at 3℃ for 24h, to obtain the glabridin molecularly imprinted resin. 3) 1 part by mass of the precipitate A and 20 parts by mass of the extract B are respectively dissolved in an ethanol aqueous solution, to obtain an ethanol solution of the precipitate A and an ethanol solution of the extract B respectively, the ethanol aqueous solution of the extract B is slowly dropped into the ethanol aqueous solution of the precipitate A, and stirring is carried out in the dark for 30 min, the inclusion liquid is dried under vacuum, and after crushing, the glabridin inclusion compound is obtained.

[0087] Example 3 A preparation method of a glabridin inclusion compound, and the specific steps are as follows: 1) The licorice is dried, crushed, and sieved through a 100-mesh sieve to obtain licorice dry powder; the licorice dry powder and deionized water are refluxed and boiled at a solid-liquid ratio of 1:5 for 1h, filtered, repeated three times, concentrated to 1 / 2, filtered, to obtain licorice residue and filtrate, and the filtrate is adjusted to pH=2 to obtain a precipitate A; 2) The licorice residue is extracted with ethyl acetate, filtered, to obtain an ethyl acetate extract, the ethyl acetate extract is concentrated, resuspended with 80% ethanol water, filtered, to obtain an ethanol water extract; the ethanol water extract is adsorbed by a molecularly imprinted resin and eluted with anhydrous ethanol, the eluate is collected and dried to obtain an extract B containing glabridin; The preparation process of the glabridin molecularly imprinted resin is as follows: taking glabridin molecule as a template, trifluoromethacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a crosslinking agent, 2,2'-azobis isobutyronitrile as an initiator, under nitrogen protection, mixed solvent of chloroform and methanol, reaction at 3℃ for 24h, to obtain the glabridin molecularly imprinted resin; 3) 1 part by mass of the precipitate A and 0.05 parts by mass of the extract B are respectively dissolved in an ethanol aqueous solution, to obtain an ethanol solution of the precipitate A and an ethanol solution of the extract B respectively, the ethanol aqueous solution of the extract B is slowly dropped into the ethanol aqueous solution of the precipitate A, and stirring is carried out in the dark for 30 min, to obtain an inclusion liquid. The obtained inclusion liquid is dried under vacuum, and after crushing, the glabridin inclusion compound is obtained.

[0088] Comparative Example 1 A preparation method of a glabridin mixture, which is different from Example 3 in that the step 3) of this example is: 1 part by mass of the precipitate A and 0.05 parts by mass of the extract B are directly mixed, crushed and ground, and sieved through a 100-mesh sieve, to obtain a direct mixture of the two components, i.e. the glabridin mixture of this example. The other steps are the same as those of Example 3.

[0089] Comparative Example 2 A preparation method of a glabridin inclusion compound, which is different from example 3 in that the example does not include the step of preparing precipitate A in step 1); and based on this, the precipitate A in step 3) is replaced with an equal amount of glycyrrhizic acid, and the other steps are the same as example 3, to prepare the glabridin inclusion compound of the example.

[0090] Comparative example 3 A preparation method of a glabridin inclusion compound, which is different from example 3 in that the example does not include the step of preparing precipitate A in step 1); and step 3) is replaced with a mixture of 0.5 parts by mass of glycyrrhizic acid and 0.5 parts by mass of glycyrrhizin polysaccharide, and the other steps are the same as example 3, to prepare the glabridin inclusion compound of the example.

[0091] Performance test (1) Determination of solubility: Under room temperature conditions, the glabridin inclusion compound or glabridin mixture prepared in examples 1-3 and comparative examples 1-3 is prepared into a saturated aqueous solution, which is centrifuged, filtered, and then analyzed by high performance liquid chromatography to test the content of glabridin in the aqueous solution, i.e. the solubility of the glabridin inclusion compound or glabridin mixture; the liquid chromatography analysis conditions are: mobile phase: acetonitrile-water=56:44; 1 mL / min; column temperature: 40°C; chromatographic column: Phenomenex C18 (2) Luna, 4.6x150mm, 5Å; analysis wavelength 282nm.

[0092] (2) Determination of particle size and Zeta potential: The glabridin inclusion compound sample of example 3 is dissolved in deionized water to prepare a 2mg / mL solution, which is left to stand at room temperature for 24h, and then the ZETA potential and PDI of the sample are analyzed by a nanoparticle size and Zeta potential analyzer (ZetasizerNANO ZS).

[0093] (3) XRD test: The glabridin inclusion compound sample of example 3 is adhered to a special adhesive tape paper for X-ray diffraction, and the crystal condition of the sample is analyzed by an X-ray diffractometer.

[0094] (4) SEM characterization: The glabridin inclusion compound sample of example 3 is adhered to the adhesive tape paper of a scanning electron microscope, and the surface and microstructure of the inclusion compound sample are observed by a SEM instrument.

[0095] (5) Mass spectrometry imaging analysis: The extract B and glabridin inclusion compound sample prepared in example 3, and the glabridin mixture sample of comparative example 1 are dispersed in ultrapure water, and then added dropwise into the well plate of a mass spectrometry imaging instrument, dried, and then the positive ion signals are collected by a mass spectrometry imaging instrument to analyze the distribution of the mass-to-charge ratio of 325.274m / z.

[0096] (6) Determination of tyrosinase activity inhibition rate: The glabridin inclusion compound or glabridin mixture prepared by the examples and comparative examples and the glabridin standard sample were dispersed in DMSO to prepare a 0.05 mol / L PBS solution (pH = 6.8), a 10 mmol / L L-dopa solution, a 50 U / mL tyrosinase solution, and a test sample solution of different concentrations (in PBS, the concentration of the test sample solution was 25, 50 and 100 μg / mL, respectively, based on glabridin). The test was divided into four groups, namely the normal group, the normal zero adjustment group, the experimental group, and the experimental zero adjustment group. Among them, the normal group: 50 μL of tyrosinase solution and 50 μL of distilled water were precisely measured in a 96-well plate, mixed well, and incubated at 28°C for 15 min, then 100 μL of L-dopa solution was added, and the reaction was carried out at room temperature for 10 min, and the absorbance was measured at 475 nm, recorded as A1; the normal zero adjustment group: 100 μL of distilled water was precisely measured in a 96-well plate, incubated at 28°C for 15 min, then 100 μL of L-dopa solution was added, and the reaction was carried out at room temperature for 10 min, and the absorbance was measured at 475 nm, recorded as A2; the experimental group: 50 μL of tyrosinase solution and 50 μL of test sample solution were precisely measured in a 96-well plate, mixed well, and incubated at 28°C for 15 min, then 100 μL of L-dopa solution was added, and the reaction was carried out at room temperature for 10 min, and the absorbance was measured at 475 nm, recorded as A3; the experimental zero adjustment group: 50 μL of distilled water and 50 μL of test sample solution were precisely measured in a 96-well plate, mixed well, and incubated at 28°C for 15 min, then 100 μL of L-dopa solution was added, and the reaction was carried out at room temperature for 10 min, and the absorbance was measured at 475 nm, recorded as A4.

[0097] The tyrosinase inhibition rate was calculated according to the absorbance values of each group, and the specific calculation formula was as follows: .

[0098] (7) Determination of melanin production inhibition rate: The glabridin inclusion compound or glabridin mixture of the examples and the comparative examples and the glabridin standard were dispersed in DMSO as test samples. B16-F10 cells (mouse melanoma cells, purchased from the Chinese Academy of Sciences Cell Library) were cultured in DMEM complete medium, 150,000 cells were inoculated in a 6-well plate, and after adhering for 24 h, 15 μM and 30 μM of the test sample (calculated as glabridin) and the glabridin standard were respectively added to the culture medium, and the culture was continued for 48 h; the cells were collected, counted, and 100,000 cells were transferred into a 1.5 mL centrifuge tube, centrifuged at 800 x g for 10 min, and the precipitate was taken out, then 200 μL of 1 μM sodium hydroxide containing 10% DMSO was used to dissolve the precipitate at 80°C water bath for 1 h, after centrifugation to remove the precipitate, 50 μL of the solution was tested on the enzyme label instrument to obtain the absorbance of each group at 490 nm. At the same time, a blank group (containing only DMEM complete medium) and a model group (containing only B16-F10 cells) were set up and the absorbance was measured to obtain the absorbance of the blank group and the control group.

[0099] The melanin production inhibition rate was calculated according to the absorbance of each group, and the specific calculation formula was as follows: .

[0100] Test results (1) The solubility of the inclusion compound or mixture of examples 1-3 and comparative examples 1-3 was analyzed by HPLC, as shown in Table 1. From Table 1, it can be seen that by the inclusion method of examples 1-3 of the present application, the solubility of glabridin in pure water can be significantly improved, and without additional addition of other solubilizers, surfactants and emulsifiers, example 3 can increase the solubility in pure water by at least 100,000 times, while comparative example 2 only uses glycyrrhizic acid as the inclusion layer component, and comparative example 3 only uses glycyrrhizic acid and glycyrrhizin as the inclusion layer component, which are difficult to effectively improve the solubility of glabridin in pure water. Figure 1 is the actual picture of the glabridin inclusion compound powder of example 3 and the dissolution of its aqueous solution, (a) is the glabridin inclusion compound powder, and (b) is the aqueous solution of the glabridin inclusion compound.

[0101] Table 1 Solubility comparison of glabridin inclusion compound or glabridin mixture prepared by examples 1-3 and comparative examples 1-3

[0102] a. The solubility of glabridin is from the literature (DOI: 10.11656 / j.issn.1673-9043.2017.01.13).

[0103] (2) Figure 2Particle size distribution curve of the glabridin inclusion prepared in Example 3 dispersed in aqueous solution. Figure 3 XRD spectrum of the extract B and the glabridin inclusion prepared in Example 3. Figure 4 SEM images of the extract B and the glabridin inclusion prepared in Example 3, wherein (A) is the extract B; and (B) is the glabridin inclusion.

[0104] The glabridin inclusion prepared in Example 3 was subjected to characterization tests, from which Figure 2 It can be seen that the particle size distribution is logarithmically normally distributed, and the glabridin inclusion prepared in Example 3 is well dispersed in aqueous solution, appearing clear and transparent; ZETA potential analysis of the aqueous solution thereof shows that the average potential is 2.09 mV, having certain dispersion stability, the median particle size of dispersion is 169.4 nm, the average particle size is 194.7 nm, and the polydispersity coefficient PDI is 0.341. The inclusion of the glabridin inclusion was studied, from which Figure 3 It can be seen that the XRD crystal peak of the inclusion is basically covered, indicating that the glabridin is basically included therein. Further analysis by SEM shows that Figure 4 It can be seen that the crystal structure of the inclusion is basically disappeared, also proving that the glabridin inclusion is basically completely included. Therefore, when the glabridin inclusion of Example 3 is dispersed in water, a clear and transparent aqueous solution can be formed.

[0105] (3) The extract B and the glabridin inclusion prepared in Example 3, and the glabridin mixture prepared in Comparative Example 1 were analyzed by mass spectrometry imaging instrument, obtaining Figure 5 , Figure 5 Mass spectrometry imaging images of the extract B and the glabridin inclusion prepared in Example 3, and the glabridin mixture of Comparative Example 1, wherein (A) is the extract B prepared in Example 3, (B) is the glabridin inclusion prepared in Example 3, and (C) is the glabridin mixture prepared in Comparative Example 1, the icon below represents the color of different signal intensity at 325.274 m / z, the signal gradually increases from left to right, and the red arrow in (A)~(C) represents the mass spectrum signal with mass-to-charge ratio of 325.274 in different samples. It can be obviously found from the figure that the mass spectrum signal of 325.274 m / z in the extract B is very strong, that in Comparative Example 1 is slightly weaker, and that in Example 3 is the weakest, almost difficult to measure, indicating that after the extract B is treated by inclusion with the precipitate A, the extract B is almost included by the precipitate A.

[0106] (4) In order to explore the whitening effect of the glabridin treated by the specific process, tyrosinase experiment in vitro and cell melanin production experiment were carried out, Figure 6 Tyrosinase inhibition rate of the glabridin inclusion prepared in Example 3, Figure 7The melanin production inhibition rate of the light glycyrrhizin inclusion compound of Example 3, Figure 8 The tyrosinase inhibition rate comparison chart of the light glycyrrhizin inclusion compound of Example 3 and the light glycyrrhizin mixture of Comparative Example 1. From Figures 6-7 It can be seen that, under the same light glycyrrhizin concentration, the ability to inhibit tyrosinase activity and the ability to inhibit cell melanin production are comparable to the un-included light glycyrrhizin standard, that is, after being treated by the process in the embodiment of the application, the inclusion compound has basically no effect on the whitening activity of light glycyrrhizin. From Figure 8 It can be seen that, compared with the sample of Comparative Example 1, Example 3 has stronger tyrosinase inhibition rate in the corresponding filtrate in the aqueous solution test environment of the same amount of sample treatment, indicating that after inclusion, it has more excellent effect than direct single mixing.

[0107] In summary, the application adopts the components of licorice itself to wrap light glycyrrhizin, and the inclusion compound formed has excellent solubility and water dispersibility, without the need to add stimulating auxiliary materials such as solubilizers, can effectively reduce the potential allergic risk of the product and the production cost, and broaden its application field.

Claims

1. A process for the preparation of a glycyrrhizin inclusion complex, characterized in that, The method comprises the following steps: S1, water extraction treatment is performed on liquorice to obtain liquorice residue and filtrate; S2, a precipitate A is obtained in the filtrate; the liquorice residue is extracted to obtain an extract B containing glabridin; S3, the precipitate A and the extract B are mixed with solvents respectively to obtain a solution of the precipitate A and a solution of the extract B; the solution of the precipitate A and the solution of the extract B are mixed, and a glabridin inclusion compound is obtained after drying.

2. The production method according to claim 1, characterized by, In step S2, the precipitate A is obtained in the filtrate by adjusting the filtrate to be acidic; And / or, in step S2, the liquorice residue is extracted by using an organic solvent.

3. The production method according to claim 2, characterized by, The pH value of the filtrate is adjusted to 1-6; And / or, the organic solvent is selected from ester solvents.

4. The method of claim 1, wherein, In step S2, after the liquorice residue is extracted, a pre-extract is obtained, and the pre-extract is further subjected to separation and purification treatment, which specifically comprises: mixing the pre-extract with an ethanol solution to obtain an ethanol extract; the ethanol extract is subjected to adsorption treatment by imprint resin, and the imprint resin after adsorption is subjected to elution to obtain an eluate, and the eluate is dried to obtain the extract B containing glabridin.

5. The production method according to claim 4, characterized by, In the separation and purification treatment, the content of ethanol in the ethanol solution is 30-99.9wt%; And / or, in the separation and purification treatment, the eluent used for elution comprises at least one of ethyl acetate, methanol, ethanol or chloroform.

6. The method of claim 1, wherein, In step S3, the mass ratio of the precipitate A to the extract B is 1:(0.01-30); And / or, in step S3, the solvent comprises an alcohol solvent and water.

7. A glabridin inclusion compound prepared by the preparation method in any one of claims 1-6.

8. The photo-epi-gallocatechin inclusion complex according to claim 7, characterized in that, The solubility of the glabridin inclusion compound is 100-20000 μg / mL; And / or, the average Zeta potential of the glabridin inclusion compound is 1-5 mV; And / or, the average particle size of the glabridin inclusion compound is 100-400 nm.

9. A formulation of glabrene, characterized in that, The product comprises cosmetics, pharmaceuticals, food or health products.

10. A product characterized by, The product comprises cosmetics, pharmaceuticals, food or health products. ​

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