Glabridin nano-vesicle based on glycyrrhizic acid wrapping, preparation method of glabridin nano-vesicle and application of glabridin nano-vesicle in preparation of whitening skincare cosmetics

Abstract

The invention provides glabridin nano-vesicles based on glycyrrhizic acid wrapping, a preparation method of the glabridin nano-vesicles and application of the glabridin nano-vesicles in preparation of whitening and skin care cosmetics, and relates to the technical field of cosmetics. According to the glabridin nano-vesicle based on glycyrrhizic acid wrapping, glabridin is carried by glycyrrhizic acid, glycyrrhizic acid serves as a carrier, the solubility of glabridin can be improved, the skin transmittance of glabridin can be increased, the anti-inflammatory effect of glabridin can be achieved, then the effect of one stone for three birds is achieved, the characteristic and advantage of integration of medicine and auxiliary materials are achieved, and the glabridin nano-vesicle based on glycyrrhizic acid wrapping has good application prospects. The tyrosinase inhibition activity of the glabridin is greatly improved. Meanwhile, the glabridin nano-vesicles wrapped based on glycyrrhizic acid have the advantages of high encapsulation efficiency, large drug loading capacity, good stability and high safety, can effectively promote the whitening factor glabridin to penetrate through the skin cuticle to enter the skin deep tissue, can stay in the skin at a high concentration for a long time, and can continuously play a whitening role.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetics, and in particular to a glabridin nanovesicle based on glycyrrhizic acid encapsulation, a preparation method thereof, and an application thereof in the preparation of whitening and skin care cosmetics. Background Art

[0003] Glabridin is an isoflavone compound with outstanding whitening effect, mainly derived from the root of Glycyrrhiza glabra L. In the current numerous biological activity studies, glabridin has a variety of biological activities, such as whitening, anti-inflammatory, antioxidant, phytoestrogen activity, etc. The reason why glabridin is known as "whitening gold" is mainly because it is difficult to purify, has a low extraction rate, and the price of raw materials is comparable to gold. This ingredient has an outstanding whitening effect, which is 140 times that of niacinamide. Glabridin can not only prevent darkening, but also help whiten the skin. It can be said to be both internal and external among whitening ingredients, which is rare.

[0004] However, the application of glabridin in cosmetics has the following defects: (1) glabridin has poor water solubility, which limits its further development and application; (2) glabridin has poor stability and is easily oxidized and deteriorated; (3) the glabridin content in whitening products currently on the market is low, which affects the skin care effect.

[0005] In recent years, some solutions for the application of glabridin in cosmetics have been given in the prior art, but they all have certain defects. For example: Chinese patent application number CN202311866730.5 discloses a tremella nanovesicle loaded with glabridin delivery system, which uses fresh tremella as raw material to prepare tremella nanovesicles with uniform particle size by differential centrifugation and sucrose density gradient centrifugation, and then loads glabridin into the interior of the tremella nanovesicles by intermittent ultrasonic incubation to prepare a tremella nanovesicle loaded with glabridin delivery system. However, the average particle size of the prepared nanovesicles is 200-300nm, the particle size is large, the skin permeability of glabridin is poor, and its preparation process is complicated, which is not conducive to industrial production.

[0006] Chinese patent application application number CN202410566769.3 discloses a method for embedding glabridin emulsion based on lipopeptide sodium, which comprises: (1) preparing oil phase: taking solid lipid, liquid lipid, glabridin, lipopeptide sodium and Tween 80 and stirring to obtain oil phase; (2) preparing water phase: taking polyol and adding it into water and stirring to obtain water phase; (3) preparing micron-scale dispersion: adding the stirred water phase into the stirred oil phase and stirring evenly to fully emulsify it to obtain colostrum; (4) preparing nanocomposite: high-pressure homogenization of colostrum to obtain glabridin nanoemulsion. However, the average particle size of the prepared nanoemulsion is 100-300nm, the particle size is relatively large, and the skin permeability is poor.

[0007] Chinese patent application number CN202111338459.9 discloses a whitening essence based on phospholipid-encapsulated glabridin nanoemulsion and its preparation method. The preparation process contains the organic solvent acetone, which may cause the toxic substance acetone to remain in the finished product, increasing the safety risk of using the glabridin nanoemulsion essence.

[0008] Chinese patent application number CN202110351039.8 discloses a method for preparing a double-layer glabridin nano-microcapsule freeze-dried powder. The obtained microcapsules are in the nanoparticle range after dissolution and can be dissolved in aqueous formulations. However, its preparation process is cumbersome and not suitable for industrial production. In addition, the microcapsule particle size is large and the skin permeability is relatively poor.

[0009] Chinese patent application number CN201611209981.6 discloses a glabridin nanosuspension and a preparation method thereof, wherein the formulation contains 0.5-20% glabridin, 0.1-20% stabilizer and the remainder water by weight percentage. The glabridin nanosuspension is prepared by ultrasonic pre-dispersion combined with high-pressure homogenization. The average particle size of the nanosuspension is 776nm, which is too large and is not conducive to skin absorption.

[0010] Chinese invention patent CN106691888A discloses a high skin retention glabridin nanocomposition and its preparation method and use, wherein the glabridin nanocomposition is prepared by dropping an oil phase prepared by glabridin, solid lipid, liquid lipid and octyldodecanol into an aqueous phase prepared by vitamin E succinate polyethylene glycol ester, polyol and purified water, and then processed by high-speed shear emulsification, high-pressure homogenization or high-pressure microfluidization equipment to obtain. However, this method still belongs to a thermodynamically unstable system of solid-liquid mixing, and it is difficult to ensure storage stability and dilution stability, and it cannot well meet the raw material requirements such as simple composition and good compatibility required by the cosmetic system. Moreover, emulsifiers and stabilizers are used in large quantities, and there is also a certain risk of allergies.

[0011] Therefore, it is necessary and urgent to study and develop a new type of glabridin delivery system for use in the preparation of whitening cosmetics in order to improve the solubility, water dispersibility and stability of existing glabridin and to improve the transdermal delivery efficiency of glabridin.

[0012] In view of this, the present invention is proposed. Summary of the invention

[0013] The first purpose of the present invention is to provide a glycyrrhizin nanocapsule encapsulated with glycyrrhizic acid, which can significantly increase the skin penetration and skin retention of glycyrrhizin, better exert the skin care effect, and effectively alleviate the problems of existing glycyrrhizin such as poor water solubility, poor stability, low content, low skin penetration rate and low retention rate.

[0014] The second object of the present invention is to provide a method for preparing glabridin nanovesicles encapsulated by glycyrrhizic acid.

[0015] The third object of the present invention is to provide an application of glabridin nanocapsule encapsulated by glycyrrhizic acid.

[0016] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0017] The present invention provides a glabridin nanovesicle based on glycyrrhizic acid encapsulation, wherein the glabridin is encapsulated in glycyrrhizic acid to form a nanovesicle;

[0018] The particle size of the nanovesicles is 5 to 50 nm, and the polydispersity index PDI is 0.203 to 0.217;

[0019] The nanovesicles are prepared from glabridin, glycyrrhizic acid and water;

[0020] The nanovesicles do not contain organic solvents.

[0021] Furthermore, the raw materials for preparing the nanovesicles include, by weight: 1 to 5 parts of glabridin, 2 to 28.07 parts of glycyrrhizic acid and 600 to 5120 parts of water;

[0022] Preferably, the water is deionized water.

[0023] Furthermore, the raw materials for preparing the nanovesicles include, by weight: 1 part of glabridin, 6 parts of glycyrrhizic acid and 1000 parts of water;

[0024] And / or, in parts by mass, the raw materials for preparing the nanovesicles include: 5 parts of glabridin, 28.07 parts of glycyrrhizic acid and 5120 parts of water.

[0025] Furthermore, the particle size of the nanovesicles is 8 to 20 nm, and the polydispersity index (PDI) is 0.203 to 0.206.

[0026] The present invention provides a method for preparing the above-mentioned glycyrrhizic acid-encapsulated glabridin nanovesicles, the preparation method comprising:

[0027] S1: Weigh glabridin and glycyrrhizic acid according to the mass ratio and dissolve them in a solvent to obtain a mixed solution;

[0028] S2: After evaporating and removing the solvent in the mixed solution, a thin film is obtained, which is dried, and then water is added to the thin film for ultrasonic hydration to obtain a hydrated solution;

[0029] S3: The hydrated solution is filtered using a 0.22 μm microporous filter membrane to obtain glycyrrhizic acid-encapsulated glabridin nanovesicles.

[0030] Furthermore, the step S1 comprises any one of 5% ethanol solution, 95% ethanol solution and anhydrous ethanol, preferably anhydrous ethanol.

[0031] Furthermore, the evaporation in step S2 is to remove the solvent in the mixed solution by rotary evaporation under vacuum conditions;

[0032] Further, the parameters of the rotary evaporation include: the rotary evaporation temperature is 40-50°C, the rotation speed is 35-50rpm / min, the vacuum degree is 70-90Pa, and the time is 15-25min;

[0033] Furthermore, the rotary evaporation temperature is 45° C., the rotation speed is 40 rpm / min, the vacuum degree is 80 Pa, and the time is 20 min.

[0034] Furthermore, the drying in step S2 is performed under reduced pressure at 37 to 45° C. for 6 to 10 hours;

[0035] Preferably, the drying is carried out under reduced pressure at 40° C. for 8 h.

[0036] Furthermore, the process parameters of the ultrasonic hydration in step S2 include: the power of the ultrasonic treatment is 100-600W, the time of the ultrasonic treatment is 20-40min, and the temperature of the ultrasonic treatment is 25-40°C.

[0037] The present invention provides an application of the glycyrrhizin nanovesicles encapsulated by glycyrrhizic acid in the preparation of whitening skin care cosmetics.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] In view of the problems of poor water solubility, poor stability, low content, low skin permeability and low retention rate of existing glabridin, the present invention uses traditional Chinese medicine nano-assembly technology to provide a glabridin nano-vesicle based on glycyrrhizic acid encapsulation, wherein the nano-vesicle is equipped with glabridin by glycyrrhizic acid, and glycyrrhizic acid can be used as a carrier to improve the solubility of glabridin, increase the skin permeability of glabridin, and exert its own anti-inflammatory effect, thereby achieving the effect of "killing three birds with one stone", exerting the characteristics and advantages of "combining medicine and adjuvant", and greatly improving the activity of glabridin in inhibiting tyrosinase. At the same time, the glabridin nano-vesicle based on glycyrrhizic acid encapsulation in this application has the advantages of high encapsulation rate, large drug loading, good stability and high safety, which can effectively promote the whitening factor glabridin to penetrate the skin stratum corneum into the deep tissue of the skin, and stay in the skin for a long time at a high concentration and can continuously exert a whitening effect.

[0040] The present invention provides a method for preparing glabridin nanovesicles based on glycyrrhizic acid-encapsulated glycyrrhizic acid. The method first prepares a mixed solution of glabridin and glycyrrhizic acid, then evaporates the solvent to obtain a film, and then performs ultrasonic hydration treatment on the dried film, and then filters the hydrated solution with a microporous filter membrane to obtain nanovesicles. The above-mentioned preparation method has the advantages of simple and easy control of processing technology and easy large-scale industrial implementation.

[0041] The glabridin nanocapsule based on glycyrrhizic acid encapsulation provided by the present invention can be widely used in the preparation process of whitening skin care cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figure 1a The contour diagram of the effect of glycyrrhizic acid dosage and hydration volume on the encapsulation efficiency of glycyrrhizic acid-glabridin;

[0044] Figure 1b The contour map of the effect of glycyrrhizic acid dosage and hydration time on the encapsulation efficiency of glycyrrhizic acid-glabridin;

[0045] Figure 1c The contour diagram of the effect of hydration volume and hydration time on the encapsulation efficiency of glycyrrhizic acid-glabridin;

[0046] Figure 1d The response surface diagram of the effect of glycyrrhizic acid dosage and hydration volume on the encapsulation efficiency of glycyrrhizic acid-glabridin;

[0047] Figure 1e The response surface diagram of the effect of glycyrrhizic acid dosage and hydration time on the encapsulation efficiency of glycyrrhizic acid-glabridin;

[0048] Figure 1f The response surface diagram of the effect of hydration volume and hydration time on the encapsulation efficiency of glycyrrhizic acid-glabridin;

[0049] Figure 2 This is a sample of glabridin nanovesicles encapsulated with glycyrrhizic acid prepared in Example 19 of the present invention;

[0050] Figure 3 This is a particle size distribution diagram of glycyrrhizin nanovesicles encapsulated with glycyrrhizic acid prepared in Example 19 of the present invention;

[0051] Figure 4 This is a graph of the tyrosinase inhibition rates of Example 19 and Comparative Examples 1 to 3 provided in Test Example 5 of the present invention. DETAILED DESCRIPTION

[0052] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] According to one aspect of the present invention, a nanovesicle of glabridin encapsulated by glycyrrhizic acid is provided, wherein the glabridin is encapsulated in glycyrrhizic acid to form a nanovesicle;

[0054] The particle size of the nanovesicles is 5 to 50 nm, and the polydispersity index PDI is 0.203 to 0.217;

[0055] The nanovesicles are prepared from glabridin, glycyrrhizic acid and water;

[0056] The nanovesicles do not contain organic solvents.

[0057] In view of the problems of poor water solubility, poor stability, low content, low skin permeability and low retention rate of existing glabridin, the present invention uses traditional Chinese medicine nano-assembly technology to provide a glabridin nano-vesicle based on glycyrrhizic acid encapsulation, wherein the nano-vesicle is equipped with glabridin by glycyrrhizic acid, and glycyrrhizic acid can be used as a carrier to improve the solubility of glabridin, increase the skin permeability of glabridin, and exert its own anti-inflammatory effect, thereby achieving the effect of "killing three birds with one stone", exerting the characteristics and advantages of "combining medicine and adjuvant", and greatly improving the activity of glabridin in inhibiting tyrosinase. At the same time, the glabridin nano-vesicle based on glycyrrhizic acid encapsulation in this application has the advantages of high encapsulation rate, large drug loading, good stability and high safety, which can effectively promote the whitening factor glabridin to penetrate the skin stratum corneum into the deep tissue of the skin, and stay in the skin for a long time at a high concentration and can continuously exert a whitening effect.

[0058] At the same time, it should be noted that the carrier glycyrrhizic acid in the present application is an amphiphilic active ingredient derived from the root of the Chinese medicinal liquorice, containing two molecules of glucuronic acid and one molecule of glycyrrhetinic acid, which is water-soluble and can self-assemble to form nano micelles, which are also called nano vesicles. In addition, studies have confirmed that glycyrrhizic acid is one of the most important active ingredients in licorice, and has antiviral, anti-inflammatory, antioxidant, immunomodulatory, and liver-protecting effects.

[0059] Therefore, the nanovesicles in this application can also be understood as nanomicelles formed by self-assembly of glycyrrhizic acid and glabridin in water. The glabridin nanovesicles based on glycyrrhizic acid encapsulation in this application can also be called "glabridin nanomicelles based on glycyrrhizic acid encapsulation".

[0060] In a preferred embodiment of the present invention, the raw materials for preparing the nanovesicles include, by weight: 1 to 5 parts of glabridin, 2 to 28.07 parts of glycyrrhizic acid and 600 to 5120 parts of water;

[0061] In the above preferred embodiment, the water is deionized water.

[0062] In the above preferred embodiment, the raw materials for preparing the nanovesicles include, by weight: 1 part of glabridin, 6 parts of glycyrrhizic acid and 1000 parts of water;

[0063] In the above preferred embodiment, the raw materials for preparing the nanovesicles include, by weight: 5 parts of glabridin, 28.07 parts of glycyrrhizic acid and 5120 parts of water.

[0064] In a preferred embodiment of the present invention, the particle size of the nanovesicles is 8 to 20 nm, and the polydispersity index PDI is 0.203 to 0.206.

[0065] According to one aspect of the present invention, a method for preparing the above-mentioned glabridin nanovesicles encapsulated by glycyrrhizic acid comprises:

[0066] S1: Weigh glabridin and glycyrrhizic acid according to the mass ratio and dissolve them in a solvent to obtain a mixed solution;

[0067] S2: After evaporating and removing the solvent in the mixed solution, a thin film is obtained, which is dried, and then water is added to the thin film for ultrasonic hydration to obtain a hydrated solution;

[0068] S3: Filter the hydrated solution using a microporous filter membrane to obtain glycyrrhizic acid-encapsulated glabridin nanovesicles.

[0069] The present invention provides a method for preparing glabridin nanovesicles based on glycyrrhizic acid-encapsulated glycyrrhizic acid. The method first prepares a mixed solution of glabridin and glycyrrhizic acid, then evaporates the solvent to obtain a film, and then performs ultrasonic hydration treatment on the dried film, and then filters the hydrated solution with a microporous filter membrane to obtain nanovesicles. The above-mentioned preparation method has the advantages of simple and easy control of processing technology and easy large-scale industrial implementation.

[0070] In a preferred embodiment of the present invention, the step S1 comprises any one of 5% ethanol solution, 95% ethanol solution and anhydrous ethanol, preferably anhydrous ethanol.

[0071] In a preferred embodiment of the present invention, the evaporation in step S2 is to remove the solvent in the mixed solution by rotary evaporation under vacuum conditions;

[0072] In a preferred embodiment of the present invention, the parameters of the rotary evaporation include:

[0073] The temperature of rotary evaporation is 40-50°C, the speed is 35-50rpm / min, the vacuum degree is 70-90Pa, and the time is 15-25min;

[0074] In the above preferred embodiment, the rotary evaporation temperature is 45° C., the rotation speed is 40 rpm / min, the vacuum degree is 80 Pa, and the time is 20 min.

[0075] In a preferred embodiment of the present invention, the drying in step S2 is performed under reduced pressure at 37 to 45° C. for 6 to 10 hours;

[0076] Preferably, the drying is carried out under reduced pressure at 40° C. for 8 h.

[0077] In a preferred embodiment of the present invention, the process parameters of the ultrasonic hydration in step S2 include:

[0078] The power of the ultrasonic treatment is 100-600W, the time of the ultrasonic treatment is 20-40min, and the temperature of the ultrasonic treatment is 25-40°C.

[0079] According to one aspect of the present invention, a use of the above-mentioned glycyrrhizic acid-encapsulated glabridin nanovesicles in the preparation of whitening skin care cosmetics.

[0080] The glabridin nanocapsule based on glycyrrhizic acid encapsulation provided by the present invention can be widely used in the preparation process of whitening skin care cosmetics.

[0081] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0082] Example 1

[0083] A method for preparing glabridin nanovesicles coated with glycyrrhizic acid, the preparation method comprising:

[0084] 1. Accurately weigh glycyrrhizic acid and glabridin and place them in an eggplant-shaped bottle; then add an appropriate amount of anhydrous ethanol to ultrasonically dissolve glycyrrhizic acid and glabridin to obtain a mixed solution;

[0085] The glycyrrhizic acid is 6 parts and the glabridin is 1 part.

[0086] 2. Remove the anhydrous ethanol added to the mixed solution by rotary evaporation in a 45°C water bath at a speed of 40 rpm / min and a vacuum degree of 80 Pa. Rotate for 20 minutes to form a uniform transparent film at the bottom;

[0087] 3. Dry under reduced pressure at 40°C for 8 h to remove traces of anhydrous ethanol in the film of step 2;

[0088] 4. Add deionized water, hydrate and dissolve by ultrasonic treatment for 40 minutes at a temperature of 50°C to obtain a hydrated solution;

[0089] The deionized water is 1000 parts;

[0090] 5. Filter the hydrated solution with a 0.22 m microporous filter membrane to obtain nanovesicles.

[0091] Embodiments 2 to 5

[0092] The difference between Examples 2 to 6 and Example 1 is that the added mass ratio of glycyrrhizic acid is different, and the rest is the same as Example 1;

[0093] The added amounts of glycyrrhizic acid, glabridin and deionized water in Examples 2 to 6 are shown in Table 1:

[0094] Table 1:

[0095] Group Glycyrrhizic acid (part) Glabridin (unit) Deionized water (parts) Example 2 2 1 1000 Example 3 4 1 1000 Example 4 8 1 1000 Example 5 10 1 1000 Example 6 12 1 1000

[0096] Test Example 1

[0097] The above-mentioned Examples 1 to 6 are examples of the present application for testing the performance of the glabridin nanovesicles based on glycyrrhizic acid encapsulation by the amount of glycyrrhizic acid. Further, the present invention uses high performance liquid chromatography to measure and calculate the encapsulation efficiency of each example, and uses a nanoparticle size analyzer to measure the particle size, Zeta potential and polydispersity index (PDI). The specific test results are shown in Table 2:

[0098] Table 2 Effect of glycyrrhizic acid dosage on nanovesicle particle size, Zeta potential, polydispersity index (PDI) and encapsulation efficiency:

[0099]

[0100] It can be seen from the above table that when the dosage of glycyrrhizic acid is 6 parts, the encapsulation efficiency is the highest at 89.68%, the particle size is as small as 10.73 nm, the Zeta potential and PDI are small, the Zeta potential is -22.64 mV, and the PDI is 0.203. Therefore, 6 parts of glycyrrhizic acid (Example 1) are finally selected for subsequent experiments.

[0101] Embodiments 7 to 12

[0102] The difference between Examples 7 to 12 and Example 1 is that the mass ratio of deionized water added is different, and the rest is the same as Example 1;

[0103] The amounts of glycyrrhizic acid, glabridin and deionized water added in Examples 7 to 12 are shown in Table 3:

[0104] Table 3:

[0105] Formula Glycyrrhizic acid (part) Glabridin (unit) Deionized water (parts) Example 7 6 1 600 Example 8 6 1 800 Example 9 6 1 1000 Example 10 6 1 1200 Embodiment 11 6 1 1400 Example 12 6 1 1600

[0106] Test Example 2

[0107] The above-mentioned Examples 7 to 12 are examples of the present application for testing the performance of the glabridin nanovesicles based on glycyrrhizic acid encapsulation by the amount of deionized water. Further, the present invention uses high performance liquid chromatography to measure and calculate the encapsulation efficiency of each example, and uses a nanoparticle size analyzer to measure the particle size, Zeta potential and polydispersity index (PDI). The specific test results are shown in Table 4:

[0108] Table 4 Effect of deionized water dosage on nanovesicle particle size, Zeta potential, polydispersity index (PDI) and encapsulation efficiency:

[0109]

[0110] It can be seen from the above table that when the amount of deionized water is 1000 parts, the Zeta potential is as low as -25.91mV, and the encapsulation efficiency is as high as 90.98%. The particle size is small and the PDI is also the smallest, with a particle size of 9.37nm and a PDI of 0.167. Therefore, when the amount of deionized water is 1000 parts, the nanovesicles are relatively stable.

[0111] Examples 13 to 18

[0112] The difference between Examples 13 to 18 and Example 1 is only that the time of hydration ultrasound in step 4 is different, and the rest is the same as Example 1;

[0113] The addition amounts of glycyrrhizic acid, glabridin and deionized water and the hydration ultrasonic time in Examples 13 to 18 are shown in Table 5:

[0114] Table 5:

[0115]

[0116]

[0117] Test Example 3

[0118] The above-mentioned Examples 13 to 18 are examples of the present application for testing the performance of the glabridin nanovesicles based on glycyrrhizic acid encapsulation by hydration ultrasonic time. Further, the present invention uses high performance liquid chromatography to measure and calculate the encapsulation efficiency of each embodiment, and uses a nanoparticle size analyzer to measure the particle size, Zeta potential and polydispersity index (PDI). The specific test results are shown in Table 4:

[0119] Table 6 Effect of hydration ultrasound time on nanovesicle particle size, Zeta potential, polydispersity index (PDI) and encapsulation efficiency:

[0120]

[0121] It can be seen from the above table that when the hydration ultrasound time is 45 min, the encapsulation efficiency of the nanovesicles is the highest at 91.54%, the PDI is the smallest at 0.151, and the particle size and Zeta potential are also small, with a particle size of 9.96 nm and a Zeta potential of -27.22 mV.

[0122] Embodiment 19

[0123] A method for preparing glabridin nanovesicles coated with glycyrrhizic acid, the preparation method comprising:

[0124] 1. Accurately weigh glycyrrhizic acid and glabridin and place them in an eggplant-shaped bottle; then add an appropriate amount of anhydrous ethanol to ultrasonically dissolve glycyrrhizic acid and glabridin to obtain a mixed solution;

[0125] 5 parts of glabridin and 28.07 parts of glycyrrhizic acid;

[0126] 2. Remove the anhydrous ethanol added to the mixed solution by rotary evaporation in a 45°C water bath at a speed of 40 rpm / min and a vacuum degree of 80 Pa. Rotate for 20 minutes to form a uniform transparent film at the bottom;

[0127] 3. Dry under reduced pressure at 40°C for 8 h to remove traces of anhydrous ethanol in the film of step 2;

[0128] 4. Add deionized water, hydrate and dissolve by ultrasonic treatment for 45.48 minutes, the hydration temperature is 50°C, and a hydration solution is obtained; the amount of deionized water used is 5120 parts;

[0129] 5. Filter the hydrated solution with a 0.22 m microporous filter membrane to obtain nanovesicles.

[0130] The process parameters of this embodiment are selected based on the results of the above-mentioned test examples 1 to 3. The selected factors are the amount of glycyrrhizic acid (X1), the amount of deionized water (X2) and the hydration time (X3). The encapsulation efficiency (Y) is used as the investigation index, the amount of glabridin is fixed at 5 parts, the range of each investigation factor is determined, and the Box-Benhken design-response surface method experiment is used to optimize the preparation process of nanovesicles (Table 7).

[0131] Table 7 Box-Behnken response surface factors and level design table:

[0132]

[0133] The preparation method is the same as that in Example 1.

[0134] The encapsulation efficiency was calculated by high performance liquid chromatography, and the particle size, Zeta potential and polydispersity index (PDI) were determined by nanoparticle size analyzer. The factors investigated, experimental design and results are shown in Table 8, and the variance analysis is shown in Table 9.

[0135] Table 8 Box-Benhken response surface method experimental design and results:

[0136]

[0137] Table 9 ANOVA results:

[0138]

[0139]

[0140] Input the encapsulation efficiency data in Table 8 into Design-Expert 8.0.6.1 software, and obtain the fitting equation Y=92.99-1.75X1+0.74X2+0.70X3-1.00X1X2-1.58X1X3-2.33X2X3-1.00X1 2 -1.58X2 2 -2.33X3 2 (r=0.9360,P<0.05).

[0141] Through the significance test, we can see that X1 in the fitting model 2 、X3 2 The Y value was significantly affected (P < 0.05), and the lack of fit term was not significant (P ≥ 0.05), indicating that the regression model has good predictive power and the regression equation has a good fit in the entire regression region. The order of the influence of each factor on the encapsulation efficiency is X1 > X3, that is, the amount of glycyrrhizic acid > the amount of deionized water. The effect surface diagram and contour map between the factors were drawn using Design-Expert 8.0.6.1 software. The results are shown in the attached Figure 1a to Figure 1f .

[0142] Figure 1a The contour diagram of the effect of glycyrrhizic acid dosage and hydration volume on the encapsulation efficiency of glycyrrhizic acid-glabridin;

[0143] Figure 1b The contour map of the effect of glycyrrhizic acid dosage and hydration time on the encapsulation efficiency of glycyrrhizic acid-glabridin;

[0144] Figure 1c The contour diagram of the effect of hydration volume and hydration time on the encapsulation efficiency of glycyrrhizic acid-glabridin;

[0145] Figure 1d The response surface diagram of the effect of glycyrrhizic acid dosage and hydration volume on the encapsulation efficiency of glycyrrhizic acid-glabridin;

[0146] Figure 1e The response surface diagram of the effect of glycyrrhizic acid dosage and hydration time on the encapsulation efficiency of glycyrrhizic acid-glabridin;

[0147] Figure 1f Response surface diagram of the effect of hydration volume and hydration time on the encapsulation efficiency of glycyrrhizic acid-glabridin.

[0148] According to the analysis results, the predicted optimal process conditions for nanovesicles were obtained by Design-Expert 8.6.0.1 as follows:

[0149] 5 parts of glabridin, 28.07 parts of glycyrrhizic acid, 5.12 parts of deionized water / 10 -3 The hydration time was 45.48 min and the encapsulation efficiency was 93.22%.

[0150] Three batches of nanovesicles were prepared according to the prescription and process conditions, and the encapsulation efficiencies of the three batches of nanovesicles were 92.71%, 91.54% and 93.34%, respectively. The test results were compared with the predicted values ​​of the fitting equation, and the relative deviation was calculated according to the formula [relative deviation = (predicted value - measured value) / predicted value]. The results showed that the relative deviation between the measured value and the predicted value was less than 5%, indicating that the preferred preparation process was stable and reliable.

[0151] Figure 2 This is a sample of glycyrrhizin nanovesicles encapsulated with glycyrrhizic acid prepared in Example 19.

[0152] Figure 3 This is the diameter distribution diagram of the glycyrrhizin nanovesicles encapsulated with glycyrrhizic acid prepared in Example 19.

[0153] In addition, the nanovesicles are stable in an aqueous environment and are colorless and transparent ( Figure 2 ), particle size distribution as shown in the attached Figure 3 As shown, the average diameter is (10.93±2.21) nm, the Zeta potential is (-25.42±1.03) mV, and the PDI is (0.157±0.035).

[0154] Comparative Example 1

[0155] A method for preparing nanovesicles, comprising:

[0156] 1. Accurately weigh glabridin and place it in an eggplant-shaped bottle; then add an appropriate amount of anhydrous ethanol and ultrasonically dissolve it to obtain a glabridin solution;

[0157] 2. Remove the anhydrous ethanol added to the glabridin solution by rotary evaporation in a 45°C water bath at a speed of 40 rpm / min and a vacuum degree of 80 Pa. Rotate for 20 minutes to form a uniform transparent film at the bottom;

[0158] 3. Dry under reduced pressure at 40°C for 8 h to remove traces of anhydrous ethanol in the film of step 2;

[0159] 4. Add deionized water, hydrate and dissolve by ultrasonic treatment for 40 minutes, the hydration temperature is 50° C., and obtain a hydrated solution; the mass ratio of glabridin to deionized water is 1:1000;

[0160] 5. Filter the hydrated solution with a 0.22 m microporous filter membrane to obtain nanovesicles.

[0161] This comparative example differs from Example 1 in that glycyrrhizic acid is not included.

[0162] Comparative Example 2

[0163] A method for preparing nanovesicles, comprising:

[0164] 1. Accurately weigh glycyrrhizic acid and place it in an eggplant-shaped bottle; then add an appropriate amount of anhydrous ethanol and ultrasonically dissolve it to obtain a glycyrrhizic acid solution;

[0165] 2. Remove the anhydrous ethanol added to the glycyrrhizic acid solution by rotary evaporation in a 45°C water bath, with a rotation speed of 40 rpm / min and a vacuum degree of 80 Pa. Rotate for 20 minutes to form a uniform transparent film at the bottom;

[0166] 3. Dry under reduced pressure at 40°C for 8 h to remove traces of anhydrous ethanol in the film of step 2;

[0167] 4. Add deionized water, hydrate and dissolve by ultrasonic treatment for 40 minutes, the hydration temperature is 50°C, and obtain a hydrated solution; the mass ratio of the glycyrrhizic acid to deionized water is 1:1000;

[0168] 5. Filter the hydrated solution with a 0.22 m microporous filter membrane to obtain nanovesicles.

[0169] This comparative example differs from Example 1 in that glabridin is not included.

[0170] Comparative Example 3

[0171] A method for preparing a mixed solution of glabridin and glycyrrhizic acid, the preparation method comprising:

[0172] 1. Accurately weigh glycyrrhizic acid and glabridin and place them in an eggplant-shaped bottle; then add an appropriate amount of anhydrous ethanol to ultrasonically dissolve glycyrrhizic acid and glabridin to obtain a mixed solution;

[0173] The mass ratio of glycyrrhizic acid to glabridin is 6:1.

[0174] 2. Add deionized water and mix well to obtain a mixed solution of glabridin and glycyrrhizic acid.

[0175] The mass ratio of the glycyrrhizic acid, glabridin and deionized water is 6:1:1000.

[0176] Experimental Example 4: Stability Study of Glycyrrhizin Nanocapsules Encapsulated with Glycyrrhizic Acid

[0177] The nanovesicles prepared in Example 19 were stored at 4°C (cold-resistant), 25°C (normal temperature), and 45°C (heat-resistant), and then taken out at 1, 2, 4, 8, 14, and 21 days, respectively, and the appearance was observed, and the particle size, Zeta potential, PDI, and encapsulation efficiency of the nanovesicles were determined.

[0178] The results of stability investigation showed that the average particle size, Zeta potential, PDI and encapsulation efficiency of the nanovesicles did not change much when placed in an environment at 4°C (cold-resistant), 25°C (normal temperature) and 45°C (heat-resistant) for 21 days (Tables 10 to 12), indicating that the nanovesicles had good stability when placed in an environment at 4°C (cold-resistant), 25°C (normal temperature) and 45°C (heat-resistant) for 21 days.

[0179] Table 10 Stability of nanovesicles at 4°C (cold resistance):

[0180]

[0181] Table 11 Stability of nanovesicles at 25°C (normal temperature):

[0182]

[0183]

[0184] Table 12 Stability of nanovesicles at 45°C (heat resistant)

[0185]

[0186] Experimental Example 5 Tyrosinase Inhibitory Activity Test Based on Glycyrrhizic Acid-Encapsulated Glabridin Nanocapsules

[0187] (1) Preparation of solution:

[0188] Preparation of PBS buffer: Weigh 1.55 g of pH 6.8 phosphate buffer solid powder, dilute to 200 mL with distilled water, and set aside.

[0189] Preparation of 0.1mol / L hydrochloric acid: Accurately measure 0.431mL of hydrochloric acid with a mass fraction of 36-38%, and make up to 50mL with distilled water.

[0190] Preparation of 0.05% L-tyrosine solution: weigh 0.05 g L-tyrosine and dissolve it in 35 mL 0.1 mol / L hydrochloric acid, then add 65 mL PBS buffer with pH = 6.8.

[0191] Take Example 19 and Comparative Examples 1-3 and dilute them with PBS buffer to the required mass fraction:

[0192] (1) Comparative Example 1 Glabridin solution: 0.5 μg / mL (low concentration), 1 μg / mL (medium concentration), 5 μg / mL (high concentration);

[0193] (2) Comparative Example 2 Glycyrrhizic acid solution: 3 μg / mL (low concentration), 6 μg / mL (medium concentration), 30 μg / mL (high concentration);

[0194] (3) Comparative Example 3: Mixture of glycyrrhizic acid and glabridin (quantitated by glabridin): 0.5 μg / mL (low concentration), 1 μg / mL (medium concentration), 5 μg / mL (high concentration);

[0195] (4) Nanovesicles of Example 19 (quantified by glabridin): 0.5 μg / mL (low concentration), 1 μg / mL (medium concentration), 5 μg / mL (high concentration).

[0196] Preparation of tyrosinase solution: accurately weigh 3 mg of tyrosinase with an enzyme activity of 25 KU and dissolve it in 15 mL of pure water to make a tyrosinase solution with an enzyme activity of 100 U / mL. Divide it into 1.5 mL EP tubes, fill each tube with 1 mL and store it in a -40°C refrigerator.

[0197] (II) Tyrosinase activity inhibition experiment:

[0198] As shown in Table 13, L-tyrosine, sample solution, and PBS buffer were added to the tubes numbered A1, A2, B1, and B2 in turn and mixed; tyrosinase was added to the tubes of groups A1 and B1 in turn, shaken, and spotted on 96-well plates in turn. After incubation in a 37°C incubator for 30 minutes to fully react, the time was accurately set, the plates were taken out, and the absorbance at a wavelength of 450nm was measured with an enzyme marker and the inhibition rate was calculated. The inhibition rate calculation formula is as follows:

[0199] Inhibition rate (%) = (A1-A2)-(B1-B2) / (A1-A2)*100.

[0200] In the formula, A1 is the absorbance of the blank sample with enzyme system; A2 is the absorbance of the blank sample without enzyme system; B1 is the absorbance of the sample group with enzyme system; B2 is the absorbance of the sample group without enzyme system. In the experiment, the negative control was replaced by PBS buffer, and the positive control was selected as ascorbic acid solution. Each group was measured in parallel 6 times.

[0201] Table 13 Reaction solution composition:

[0202]

[0203] The tyrosinase inhibition rates obtained in Example 19 and Comparative Examples 1 to 3 are shown in Table 14 and Attached Figure 4 shown.

[0204] Figure 4 This is a graph showing the tyrosinase inhibition rate of Example 19 and Comparative Examples 1 to 3 provided in this test example.

[0205] Table 14:

[0206]

[0207] From the above, it can be seen that after being coated with glycyrrhizic acid, the activity of glabridin in inhibiting tyrosinase has been greatly improved. At low concentrations, the inhibition rate of glabridin solution is 18.03%, the inhibition rate of glycyrrhizic acid solution is 13.49%, the inhibition rate of glycyrrhizic acid + glabridin mixed solution is 18.26%, and the inhibition rate of nanovesicles is 96.50%, which greatly improves the whitening effect. The results also show that the inhibitory effect of glycyrrhizic acid-coated glabridin nanovesicles on tyrosinase is not a simple "1+1=2" effect after mixing glycyrrhizic acid and glabridin, but the result of "drug-adjuvant combination" synergistic enhancement.

[0208] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanocapsule of glabridin encapsulated by glycyrrhizic acid, characterized in that: The glabridin is encapsulated in glycyrrhizic acid to form nanovesicles; The particle size of the nanovesicles is 5 to 50 nm, and the polydispersity index PDI is 0.203 to 0.217; The nanovesicles are prepared from glabridin, glycyrrhizic acid and water; The nanovesicles do not contain organic solvents.

2. The nanovesicle according to claim 1, characterized in that The raw materials for preparing the nanovesicles include, by weight: 1 to 5 parts of glabridin, 2 to 28.07 parts of glycyrrhizic acid and 600 to 5120 parts of water; Preferably, the water is deionized water.

3. The nanovesicle according to claim 1, characterized in that The raw materials for preparing the nanovesicles include, by weight: 1 part of glabridin, 6 parts of glycyrrhizic acid and 1000 parts of water; And / or, in parts by mass, the raw materials for preparing the nanovesicles include: 5 parts of glabridin, 28.07 parts of glycyrrhizic acid and 5120 parts of water.

4. The nanovesicle according to claim 1, characterized in that The particle size of the nanovesicles is 8-20 nm, and the polydispersity index PDI is 0.203-0.

206.

5. A method for preparing glabridin nanovesicles based on glycyrrhizic acid encapsulation according to any one of claims 1 to 4, characterized in that: The preparation method comprises: S1: Weigh glabridin and glycyrrhizic acid according to the mass ratio and dissolve them in a solvent to obtain a mixed solution; S2: After evaporating and removing the solvent in the mixed solution, a thin film is obtained, which is dried, and then water is added to the thin film for ultrasonic hydration to obtain a hydrated solution; S3: The hydrated solution is filtered using a 0.22 μm microporous filter membrane to obtain glycyrrhizic acid-encapsulated glabridin nanovesicles.

6. The method for preparing nanovesicles according to claim 5, characterized in that: The step S1 comprises any one of 5% ethanol solution, 95% ethanol solution and anhydrous ethanol, preferably anhydrous ethanol.

7. The method for preparing nanovesicles according to claim 5, characterized in that: The evaporation in step S2 is to remove the solvent in the mixed solution by rotary evaporation under vacuum conditions; Preferably, the parameters of the rotary evaporation include: the rotary evaporation temperature is 40-50°C, the rotation speed is 35-50rpm / min, the vacuum degree is 70-90Pa, and the time is 15-25min; More preferably, the rotary evaporation temperature is 45° C., the rotation speed is 40 rpm / min, the vacuum degree is 80 Pa, and the time is 20 min.

8. The method for preparing nanovesicles according to claim 5, characterized in that: The drying in step S2 is performed under reduced pressure at 37-45° C. for 6-10 hours; Preferably, the drying is carried out under reduced pressure at 40° C. for 8 h.

9. The method for preparing nanovesicles according to claim 5, characterized in that: The process parameters of the ultrasonic hydration in step S2 include: the power of the ultrasonic treatment is 100-600W, the time of the ultrasonic treatment is 20-40min, and the temperature of the ultrasonic treatment is 25-40°C.

10. Use of the glycyrrhizic acid-encapsulated glabridin nanovesicles according to any one of claims 1 to 4 in the preparation of whitening skin care cosmetics.

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