Preparation method and application of glabridin nanocomposite

Abstract

The invention discloses a preparation method and application of a glabridin nanocomposite. The glabridin nanocomposite is based on alcohol body encapsulating glabridin, which greatly improves the stability and solubility of glabridin in a preparation. The glabridin nanocomposite is made into a gel product, which has the advantages of good flexibility, heat dissipation, skin affinity and the like, and the viscosity system of the gel reduces the aggregation of alcohol body, which can increase the stability of alcohol body, and the gel has strong antioxidant activity such as the ability to scavenge ABTS free radicals and DPPH free radicals, and the whitening effect and safety can be guaranteed, thereby promoting the application prospect of glabridin in skin whitening and freckle removal.

Description

Technical Field

[0001] The invention relates to the technical field of glabridin-related cosmetics, in particular to a preparation method and application of a glabridin nanocomposite. Background Art

[0002] In recent years, consumers' demand for whitening products has continued to increase. However, many whitening products on the market contain irritating chemical ingredients, which may cause skin sensitivity or other adverse reactions, and it is difficult to meet the dual requirements of modern consumers for safety and effectiveness. The perfect combination of mildness, non-irritation, safety and high efficiency has become the basic criterion for today's consumers to choose products. Compared with Western medicine, Chinese herbal medicine has the characteristics of safety, mild effect and few side effects. Therefore, the search for whitening active ingredients with high safety and good effects from natural herbal medicines is increasingly favored by whitening cosmetics researchers. It has a good development prospect and has become the development direction of whitening cosmetics.

[0003] Glabridin is a natural isoflavone compound extracted from the root of licorice. It has gradually become an important ingredient in whitening products due to its significant whitening effect and few side effects. Glabridin can not only significantly inhibit tyrosinase activity and reduce melanin production, but also has multiple effects such as antioxidant and anti-inflammatory. In addition, glabridin can exert a powerful whitening effect at extremely low concentrations.

[0004] However, the application of glabridin in whitening products still has problems such as poor stability and low absorption rate, which also weakens the application of glabridin in skin whitening and freckle removal. Therefore, it is of great research significance and application prospect to develop a whitening product with glabridin as the main ingredient to give full play to its whitening effect and ensure the stability and safety of the product. Summary of the invention

[0005] The invention provides a preparation method and application of a glabridin nanocomposite, which solves the defect that glabridin has a weak whitening effect due to low absorption rate and poor stability.

[0006] In view of this, the scheme of the present invention is: The first object of the present invention is to provide a glabridin nanocomposite, which is a mixture of phase A and phase B, wherein: The phase A is composed of lecithin, glabridin and liquid phase 1; the mass volume ratio of lecithin and glabridin to liquid phase 1 is 10-15% and 0.1-0.5%, respectively; the liquid phase 1 is composed of 25-35% ethanol, 5-10% propylene glycol and the balance water by volume; The B phase is composed of sodium hyaluronate, methylparaben, Camus and liquid phase II; the mass volume ratio of sodium hyaluronate, methylparaben and Camus to liquid phase II is 0.1-0.5%, 0.1-0.25% and 0.5-1% respectively; the liquid phase II is composed of 10-15% glycerol, 5-7% butylene glycol and the balance water by volume ratio; The volume ratio of the liquid phase 1 to the liquid phase 2 is 1:2.

[0007] Furthermore, the composition of phase A is: 1.5g of lecithin, 50mg of glabridin, 3mL of ethanol, 1mL of propylene glycol and 6ml of water; the composition of phase B is: 25mg of sodium hyaluronate, 25mg of methylparaben, 125mg of cam, 2.5ml of glycerin, 1.25ml of butylene glycol and 16.25ml of water.

[0008] The second aspect of the present invention is to provide a method for preparing the glabridin nanocomposite described in the first aspect, the steps comprising: Take lecithin, glabridin, ethanol and propylene glycol, heat and stir them evenly, then add water, perform ultrasound to form a dispersion, centrifuge, take the supernatant and filter to obtain phase A; Sodium hyaluronate, methylparaben, glycerin and butylene glycol are added to water and fully dissolved, and carbomer is added to fully swell to obtain phase B; Under stirring, phase A is slowly injected into phase B to fully dissolve, and the pH value is adjusted to 6-7 to obtain the glabridin nanocomposite.

[0009] Furthermore, in the above preparation method, the preparation process of phase A is carried out according to at least one of the following conditions: The dissolution process temperature is 60°C; The dissolution stirring condition is continuous stirring at 1000 rpm for 30 min.

[0010] The ultrasonic condition is 150 W for 10 min; The centrifugation condition is 4000 rpm at 4°C for 10 min; The filtration is performed using a 0.22 μm filter membrane.

[0011] Furthermore, in the above preparation method, during the preparation of phase B, the dissolution process is continuously stirred at 1000 rpm for 30 min.

[0012] Furthermore, a preservative is added before the pH adjustment process.

[0013] The third aspect of the present invention is to propose the use of the glabridin nanocomposite described in the first aspect or the glabridin nanocomposite obtained by the preparation method described in the second aspect in the preparation of skin care products.

[0014] Furthermore, the skin care products include gel, essence, lotion, cream, ointment or mask, and can be made into suitable skin care product types according to needs.

[0015] Furthermore, the skin care product includes at least one of the following uses: a) scavenging ABTS free radicals; b) scavenging DPPH free radicals; c) Reduce melanin deposition.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The glabridin nanocomposite provided by the present invention is based on the encapsulation of glabridin by alcohol bodies, which greatly improves the stability and solubility of glabridin in the preparation, and the encapsulation rate and drug loading meet the requirements, effectively solving the defects of poor stability and low absorption rate of glabridin whitening products in the prior art, and promoting the application prospects of glabridin in skin whitening and freckle removal.

[0017] The glabridin nanocomposite obtained by the preparation method of the present invention belongs to a gel product, has the advantages of good flexibility, heat dissipation, skin affinity, etc., and the viscosity system of the gel reduces the aggregation of alcohol bodies and can increase the stability of alcohol bodies. The glabridin alcohol body mixture is made into a gel product, which has strong antioxidant activity such as the ability to scavenge ABTS free radicals and DPPH free radicals. In vitro and in vivo studies have determined the whitening efficacy and safety of the glabridin nanocomposite. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the appearance of glabridin ethosomes prepared in an embodiment of the present invention.

[0019] Figure 2 This is the standard curve of the glabridin reference substance in Experimental Example 2.

[0020] Figure 3 This is the test result of the scavenging ability of the glabridin complex on ABTS• in Experimental Example 3.

[0021] Figure 4 This is the test result of the scavenging ability of the glabridin complex on DPPH• in Experimental Example 3.

[0022] Figure 5 This is the test result of the irritation of the glabridin complex to rat skin in Experimental Example 3.

[0023] Figure 6 These are the results of the effect of the glabridin complex on UVB-induced guinea pig skin in Experimental Example 3.

[0024] Figure 7 This is the Fontana-Masson staining result of guinea pig skin in Experimental Example 3. DETAILED DESCRIPTION

[0025] The technical solution of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not 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.

[0026] In one embodiment, the root of Glycyrrhiza glabra is selected as a raw material, which is washed, dried and crushed to a certain particle size. Then, an organic solvent extraction method is used to extract the crushed glabridin raw material. Thereafter, the extract is subjected to low-temperature ultrasound and preliminary filtration to remove solid impurities therein. The extract is purified by a macroporous adsorption resin, and the desorbed liquid is concentrated under reduced pressure. The concentrated solution is refined by a crystallization method. Finally, the crystals are dried to remove moisture from the crystals to obtain a final glabridin product.

[0027] Then, an extraction-based glabridin micro-molecule nano whitening gel preparation is provided, which is made of glabridin, butylene glycol, propylene glycol, ethanol, glycerin, sodium hyalate, lecithin, methylparaben, kamut gum and water.

[0028] Specifically, the preparation method of the glabridin nanogel preparation comprises the following steps: 1) Prepare phase A (ethanol body): weigh glabridin and lecithin, add them to the alcohol solution, stir at 60℃ for 30 min under magnetic stirring (1000 rpm) to fully dissolve them. Inject 60℃ ultrapure water into the alcohol solution, ultrasonicate (150 W) for 10 min, then centrifuge at 4000 rpm for 10 min, take the supernatant, and filter to obtain the alcohol body.

[0029] 2) Prepare phase B (aqueous solution). Weigh sodium hyaluronate, methylparaben, kamut gum, glycerin, and butylene glycol and add them to 60°C ultrapure water to prepare an aqueous solution. Then slowly add the alcohol body to the aqueous solution, stir it thoroughly, then add the preservative methylparaben, and finally adjust the pH to a suitable level to finally prepare a gel product.

[0030] The components of phase A, calculated by volume as 100%, are as follows: 10-15% lecithin, 0.1-0.5% glabridin, 25-35% ethanol, 5-10% propylene glycol and the remainder ultrapure water; the components of phase B, calculated by volume as 100%, are as follows: 0.1-0.5% sodium hyaluronate, 0.1-0.25% methylparaben, 0.5-1% cam, 10-15% glycerin, 5-7% butylene glycol and the remainder ultrapure water.

[0031] In the above embodiment, ethosomes are a new type of flexible nanoliposomes, which are vesicle structures with lipid bilayers composed of phospholipids, alcohols, and water, and can encapsulate lipophilic, amphiphilic, and hydrophilic substances. Figure 1 As shown. We selected alcohol bodies to encapsulate glabridin, which greatly improved the stability and solubility of glabridin in the preparation. Then, the particle size, PDI and zeta potential of glabridin alcohol bodies were measured by DLS nanoparticle size analyzer; and the HPLC method for determining the content of glabridin was established to detect its encapsulation rate and drug loading; finally, the optimal formulation scheme was determined.

[0032] In some embodiments, since liquid alcohol bodies are not easy to stay in the skin, they are easy to lose after application, which may affect the efficacy of glabridin. The gel has the advantages of good flexibility, heat dissipation, and skin affinity, and the viscosity system of the gel reduces the aggregation of alcohol bodies and increases the stability of alcohol bodies. Therefore, we choose to make the glabridin alcohol body mixture into a gel product. After that, its antioxidant activity was evaluated by scavenging ABTS free radical ability and scavenging DPPH free radical ability; its skin irritation reaction was evaluated by rat skin irritation; UVB induced guinea pig model to detect its whitening effect; finally, the whitening effect and safety of the glabridin nano-mixture were determined based on in vitro and in vivo related studies.

[0033] In one embodiment, the specific steps of extracting and purifying glabridin are as follows: 1. Raw material pretreatment The root of Glycyrrhiza glabra was selected as the raw material, washed with clean water and dried, and then crushed into 40 meshes with a grinder.

[0034] 2. Extraction 70% methanol was used as the extraction solvent, the extraction temperature was 60°C, the extraction time was 4h, and the solid-liquid ratio was 1:10 (w / v). The extract was then subjected to low-temperature ultrasound (150W, 30min). After the ultrasound, the sample was placed at -20°C for 20min, and then the sample was centrifuged in a low-temperature centrifuge at 4°C, 12000rpm for 10min, the supernatant was collected, and finally the supernatant was filtered to obtain a crude extract.

[0035] 3. Purification AB-8 macroporous adsorption resin was selected to purify the extract. First, a static adsorption experiment was conducted to determine the optimal adsorption conditions: pH 5, adsorption time 4h. Then a dynamic adsorption experiment was conducted, using 70% ethanol solution as the desorbent, the desorption flow rate was 2BV / h, and the desorption volume was 3BV. The desorbed liquid was concentrated under reduced pressure.

[0036] 4. Refining Ethanol was used as the crystallization solvent, and the concentrated solution was slowly stirred and crystallized at 4°C, with a stirring speed of 50 r / min. Finally, the crystals were vacuum dried at a drying temperature of 50°C and a vacuum degree of -0.09 MPa to obtain the glabridin product.

[0037] The following are specific implementation examples. If the manufacturer of the reagents or instruments is not specified in the examples, conventional products can be purchased from the market. If the specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer can be used. Example 1

[0038] Phase A (alcohol body): Weigh 1.5g lecithin, 50mg glabridin, add 3mL ethanol and 1mL propylene glycol, and place in a container with a stirring device and heating function. Heat to 60℃, stir continuously at 1000 rpm for 30 min during heating, cover the container mouth with a layer of plastic wrap during stirring to reduce the evaporation of the alcohol solution, ensure that the solution is heated evenly and no bubbles are generated. Then slowly inject 6ml of ultrapure water at 60℃ into the alcohol solution, and stir continuously to make it a uniform solution. Then use an ultrasonic disruptor with 150w ultrasonication for 10 min to form an alcohol body dispersion. Centrifuge at 4000 rpm for 10 min at 4℃, take the supernatant, and filter with a 0.22μm filter membrane to obtain the alcohol body. Phase B (aqueous solution): Weigh 0.025g sodium hyaluronate, 0.025g methylparaben, 0.125g carbomer, 2.5mL glycerin, and 1.25mL butylene glycol, slowly add to 16.25mL ultrapure water, and stir continuously at 1000 rpm for 30 min under a magnetic stirrer to fully dissolve. Let stand for 2h to allow the carbomer to fully swell to ensure that it forms a uniform gel matrix solution.

[0039] Then, the alcohol body is slowly injected into the aqueous solution, and stirring is performed while adding dropwise to fully stir and dissolve it. Then, 0.1% methylparaben is added as a preservative, and finally, the pH value is adjusted to 6-7 using an acid-base regulator to prepare a glabridin alcohol body gel mixture. Example 2

[0040] Phase A (ethanol body): weigh 1.2g lecithin, 30mg glabridin, add 2.5mL ethanol and 0.8mL propylene glycol (i.e. 8% by volume) and 7.7ml water; Phase B (aqueous solution): weigh 0.05 g sodium hyaluronate, 0.05 g methylparaben, 0.1 g kamut gum, 3 mL glycerin, and 1.4 mL butylene glycol, and slowly add them into 15.6 mL ultrapure water; The preparation methods of phase A, phase B and glabridin nano-mixture are the same as those in Example 1. Example 3

[0041] Phase A (ethanol body): weigh 1g lecithin, 20mg glabridin, add 2.5mL ethanol, 1mL propylene glycol and 6.5ml water; Phase B (aqueous solution): weigh 0.1 g sodium hyaluronate, 0.02 g methylparaben, 0.1 g kamut gum, 2 mL glycerin, and 1 mL butylene glycol, and slowly add them into 17 mL ultrapure water; The preparation method of phase A, phase B and glabridin nano-mixture is the same as that of Example 1.

[0042] Comparative Example 1 The preparation method of the glabridin nano-mixture of this embodiment is different from that of Embodiment 1 only in that the proportion of lecithin in phase A is different: the proportion of lecithin is 1%.

[0043] Comparative Example 2 The preparation method of the glabridin nano-mixture of this embodiment is different from that of Embodiment 1 only in that the ratio of the alcohol solution in phase A is different: ethanol:propylene glycol is 1:3.

[0044] Comparative Example 3 The preparation method of the glabridin nano-mixture of this embodiment is different from that of embodiment 1 only in that the ratio of the alcohol solution in phase A of step is different: ethanol:propylene glycol is 1:1.

[0045] Experimental Example 1 Particle size, PDI and potential results of glabridin ethosomes The specific methods for the determination of particle size, PDI and zeta potential are as follows: The alcohols used to prepare ethosomes include ethanol, propylene glycol, etc. Ethanol can increase the permeability of drugs in the skin, and propylene glycol has high viscosity and hygroscopicity, which can prolong the time for vesicles to pass through the skin and increase the accumulation of drugs in the deep layers of the skin. The mixed use of ethanol and propylene glycol can significantly enhance the skin deposition of drugs and improve the stability of ethosomes. Therefore, this study selected mixed alcohols with different proportions of ethanol and propylene glycol to prepare ethosomes, and compared their characterization. Phospholipids are the main components of the vesicle structure of ethosomes. During transdermal administration, some drugs can achieve penetration effects through the fusion of phospholipids with the lipid bilayer in the stratum corneum of the skin. Therefore, this study selected different proportions of lecithin to prepare ethosomes, compared their characterization, and selected the appropriate proportion.

[0046] Particle size and fineness are important indicators that affect the quality of cosmetics. Generally, the smaller the particle size, the more uniform the liquid distribution and the easier it is to be absorbed by the skin. The most commonly used particle size and fineness detection indicators are particle size, PDI and zeta potential. This study determined the best optimization process by detecting these three indicators of different glabridin alcohol bodies.

[0047] Take an appropriate amount of glabridin nano-alcohol body, dilute it with ultrapure water by a certain multiple so that the Mean CountRate of the sample solution is between 150-300nm, and measure the particle size, PDI and zeta potential in a DLS nanoparticle sizer. The results show that the prepared glabridin alcohol body is a uniform, slightly thick, colloid-like translucent system. The results in Table 1 show that the particle size of glabridin alcohol body is 70-115nm, PDI is 0.2-0.4, and the potential is -25~-35mV. Among them, Example 1 has the best effect, with a measured particle size of 75.34 nm, PDI: 0.22, and zeta potential: -30.2 mV.

[0048] Table 1: Particle size, PDI and potential results of glabridin ethosomes sample Particle size (nm) PDI Potential (mV) Example 1 75.34 0.220 -30.2 Example 2 75.91 0.261 -29.1 Example 3 75.65 0.232 -29.5 Comparative Example 1 88.84 0.238 -32.3 Comparative Example 2 114.70 0.249 -26.8 Comparative Example 3 96.98 0.381 -25.4 Experimental Example 2 Determination of encapsulation efficiency and drug loading of glabridin ethosomes Establishment of HPLC method for detecting glabridin content The content of glabridin in ethosomes was determined by ultra-high performance liquid chromatography / tandem mass spectrometry (Thermo Fisher Orbitrap Exploris 120). Ethosomes were ultrasonically disrupted with methanol, separated by liquid chromatography, and detected by tandem mass spectrometry in multiple reaction monitoring mode (PRM).

[0049] 1) Preparation of working solution Take 1 g of glabridin standard and dissolve it in 1 mL of 50% methanol water. Mix well to obtain a 1 g / mL glabridin stock solution. Take 20 µL of 1 g / mL glabridin stock solution and add 980 µL of methanol. Mix well to obtain a 20 mg / mL working solution, which is the concentration of EP tube No. 9 in Table 2-1.

[0050] Table 2-1: Preparation of standard curve reaction system Sample volume (µL) Add methanol to dilute (µL) EP pipe number Concentration after dilution (µg / mL) Glabridin concentration (µg / mL) EP09 preparation solution 0 9 20000 1000 Take EP09 No. 80 20 8 16000 800 Take EP09 No. 50 50 7 10000 500 Take EP07 No. 40 60 6 4000 200 Take EP06 No. 50 50 5 2000 100 Take EP05 No. 50 50 4 1000 50 Take EP04 No. 20 80 3 200 10 Take EP03 No. 20 80 2 40 2 Take EP02 No. 50 50 1 20 1 Blank ethosome 0 0 0 0 Table 2-2: Preparation of spike recovery system serial number Volume of blank alcohol added (mL) Glabridin spike concentration (µg / mL) Spike volume (mL) Total volume (mL) Final concentration of glabridin (µg / mL) EP10 95 0 5 100 0 EP11 95 100 5 100 5 EP12 95 1600 5 100 80 EP13 95 12000 5 100 600 2) Pre-processing methods for standard points and punctuation points As shown in Table 2-2, take 95 µL of blank alcohol and 5 µL of standard point / specified point, add 1 mL of methanol, ultrasonicate for 10 min, mix, centrifuge at 12000 r / min for 10 min, filter with 0.22 µm filter membrane, take the supernatant into a sample injection bottle, and freeze it in a -80℃ refrigerator until detection.

[0051] 3) Pre-processing method of actual samples Take 100 µL of sample, add 1 mL of methanol, sonicate for 10 min, mix, centrifuge at 12000 r / min for 10 min, filter with 0.22 µm filter membrane, take the supernatant into a sample injection bottle, and freeze it in a -80 °C refrigerator until detection.

[0052] 4) Liquid chromatography conditions Thermo hypersil gold C18 (1.9µm, 2.1×10mm) chromatographic column; column oven temperature 40°C; mobile phase A: water, mobile phase B: acetonitrile; flow rate: 0.3 mL / min; gradient elution program: 0-3 min, 40%-100% B; 3-4 min, 100% B; 4.1-9 min, 100%-40% B; 5) Mass spectrometry conditions Ionization mode: electrospray ionization positive ion mode (ESI-); capillary temperature: 350°C, sheath gas flow rate: 40Arb, auxiliary gas flow rate: 20Arb, mass-to-charge ratio acquisition range 85-1250, resolution: 60000; detection method: PRM acquisition mode, the quantitative ion pair of glycyrrhizin is 323.1289 / 201.0921 (CE=44 eV), and the qualitative ion pair is 323.1289 / 135.0451 (CE=44 eV).

[0053] Standard curve of glabridin reference substance ( Figure 2 ), the concentration X of glabridin is calculated as follows (Y is the peak area): Y = 438726 X + 146540, R 2 =0.9987 The results showed that under the above conditions, the chromatographic peak area and concentration of glabridin had a good linear relationship in the linear range of 1-1000µg / mL, and the precision, stability and method recovery rate all met the requirements of the pharmacopoeia. The encapsulation efficiency and drug loading of glabridin ethosomes can be determined by high performance liquid chromatography.

[0054] Determination of Encapsulation Efficiency and Drug Loading of Glabridin Ethosomes Encapsulation efficiency (EE) refers to the percentage of encapsulated drugs in a preparation to the total amount of drugs in the preparation. Drug loading (DL) refers to the percentage of the amount of drugs in a preparation to the amount of carriers. Encapsulation efficiency and drug loading are important indicators for formula process screening and quality evaluation in the preparation of nanoformulations. Common methods for determining encapsulation efficiency include centrifugation, ultrafiltration centrifugation, microcolumn centrifugation, dialysis and reverse dialysis, protamine coagulation, fluorescence, etc. The present invention uses ultrafiltration centrifugation to determine its encapsulation efficiency and drug loading.

[0055] Take 1mL of alcohol body sample and place it on the top of a 10 kDa centrifugal ultrafiltration tube, centrifuge it at 12000 r / min for 30min at 4℃, and remove the filtrate and the remaining liquid on it. At the same time, take another 1ml of the same alcohol body. Perform liquid phase detection in triplicate. Take 100μL of sample, add 1mL of methanol, ultrasonicate for 10min, mix, centrifuge at 12000r / min for 10min, take 100μL of supernatant, add 900μL of methanol, mix, take 100μL of supernatant, and bottle. Inject into HPLC under the established chromatographic conditions, record the peak area, substitute the obtained peak area into the regression equation, and calculate the drug concentration C. Then use the following formula to calculate its encapsulation efficiency and drug loading.

[0056] Encapsulation efficiency = C × D × V / Wt × 100% Drug loading = C × D × V / Ws × 100% Wherein, D is the dilution multiple; V is the total volume of a batch of preparations; Wt is the dosage of glabridin; and Ws is the total amount of carrier materials and drugs.

[0057] The results showed that the recovery rate of glabridin in the glabridin ethosomes in Example 1 was 92.5%, the encapsulation efficiency was 96%, and the drug loading was 0.3%.

[0058] Experimental Example 3 Evaluation of the Antioxidant Capacity of Glabridin Nanomixture ABTS free radical scavenging ability assay The ABTS method is a method for determining the total antioxidant capacity of plants (or Chinese herbal extracts), beverages and pure substances. The free radical cation ABTS is usually used for antioxidant testing of foods and water-soluble substances. ABTS reacts with potassium persulfate to generate ABTS free radicals (ABTS·) that are blue-green in color. The free radical has a maximum absorption wavelength at 734 nm. When an antioxidant substance is added to the reaction system, it reacts with a hydrogen-donating antioxidant to generate colorless ABTS molecules. The absorbance value of the colorless solution at the maximum absorption wavelength decreases. Therefore, the total antioxidant activity of the substance can be determined and calculated by the change in absorbance. The smaller the absorbance at 734 nm, the stronger the antioxidant capacity.

[0059] Take a 96-well plate, add 100 μL ABTS working solution, and then add 100 μL of the sample solution to be tested. Incubate in dark for 10 min, and use a multifunctional microplate reader to measure the absorbance at 734 nm, which is recorded as A determination. Use ultrapure water instead of sample solution as A control, and calculate the ABTS clearance rate according to the formula: ABTS clearance rate D% = [A blank - (A assay - A control)] ÷ A blank × 100%.

[0060] like Figure 3 As shown, the results showed that compared with the blank gel group, the ABTS· clearance rate of the glabridin nanogel product in Example 1 was significantly increased, reaching 98%, indicating that it has a strong ability to clear ABTS·.

[0061] DPPH free radical scavenging ability assay DPPH free radical (DPPH·) scavenging activity assay is a common in vitro simulation method for determining antioxidant activity. DPPH· is a stable free radical in organic solvents, purple in ethanol, and has a maximum absorption value at 517 nm. When antioxidant substances pair with the lone electron pair of DPPH·, the color of the solution becomes lighter, and the absorption value at 517 nm decreases. The change in its absorbance is linearly related to the degree of free radical scavenging, that is, the lower the absorbance value, the stronger the free radical scavenging ability of the substance. This antioxidant ability is expressed by the DPPH· scavenging rate. The higher the scavenging value, the stronger the antioxidant ability.

[0062] Take a 96-well plate, add 100 μL of DPPH solution, and then add 100 μL of sample solution of different concentrations. Place it on a shaker to react for 30 minutes in the dark, and use an enzyme marker to measure the absorbance at 517 nm, which is recorded as A determination. Use ultrapure water as the A control and calculate the DPPH clearance rate according to the formula: DPPH free radical scavenging rate D% = [[A blank - (A determination - A control)] ÷ A blank] × 100% like Figure 4 As shown, the results showed that compared with the blank gel group, the scavenging rate of DPPH free radicals by the glabridin nanogel product in Example 1 was significantly increased, reaching 42.4%, indicating that it has a strong ability to scavenge DPPH free radicals.

[0063] Experimental Example 4 Safety and whitening efficacy evaluation of glabridin nano-mixture Skin irritation evaluation Three healthy male SD rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at 0.3 mL / 100 g. The backs of the rats were depilated using a depilatory apparatus, with a depilatory area of ​​approximately 4 cm × 2 cm. After resting for 24 hours, 300 μL of glabridin gel product was applied twice a day for 7 consecutive days to observe whether erythema and edema appeared on the skin, and the scores were given according to Table 3, and the irritation intensity was evaluated according to Table 4. Figure 5 As shown, the results showed that no erythema or edema occurred after applying the product every day for 7 days, indicating that the product is non-irritating to the skin.

[0064] Table 3: Skin irritation response scoring criteria Erythema formation integral Edema formation integral No erythema 0 No edema 0 Barely visible 1 Barely visible 1 Moderate erythema 2 Moderate edema (noticeable swelling) 2 Severe erythema 3 Severe edema (1 mm bulging of the skin with clear contours) 3 Purple-red erythema with eschar formation 4 Severe edema (skin swelling greater than 1 mm and expanding) 4 Table 4: Skin irritation evaluation criteria Points evaluate 0-0.49 Non-irritating 0.5-2.99 Slight irritation 3.0-5.99 Moderate stimulation 6.0-8.0 Strong irritation Evaluation of the skin-whitening efficacy of glabridin in animal models 1) UVB-induced pigmentation in guinea pigs and sample processing Nine normal-grade healthy adult female brown guinea pigs weighing (250 ± 20) g were randomly divided into three groups, with three animals in each group, namely control group, model group and experimental group. The rats were anesthetized with 10% chloral hydrate at 0.3 mL / 100 g intraperitoneally, and the backs of the rats were depilated using a depilatory device. The hair on the back of the guinea pigs was removed and irradiated with 310nm UVB ultraviolet light. Irradiation was performed once a day for 30 minutes each time for 3 days. The product was applied to the back skin area (circle the application area first, about 2cm×2cm), once a day for one month. Figure 6 As shown, the results showed that the model group suffered severe skin damage due to ultraviolet irradiation, with purple-red erythema and eschar formation; the experimental group (Example 1) was able to alleviate skin damage and reduce melanin deposition.

[0065] 2) Fontana-Masson staining The dorsal skin after biopsy was fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. The embedded paraffin blocks were cut into a thickness of approximately 4 μm and stained with Fontana-Masson staining solution (IHC WORLD, GA, USA). The stained slides were examined under an optical microscope. Melanogenesis in skin tissue was evaluated by histological observation after Fontana-Masson staining. The results are shown in Figure 2. Figure 7 As shown, it can be clearly observed that the synthesis of melanin in the skin epidermis is increased after UVB irradiation. After applying the glabridin whitening mixed preparation (Example 1), the production of melanin in the skin is reduced.

[0066] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A glabridin nanocomposite, characterized in that: It is a mixture of phase A and phase B, where: The phase A is composed of lecithin, glabridin and liquid phase 1; the mass volume ratio of lecithin and glabridin to liquid phase 1 is 10-15% and 0.1-0.5% respectively, and the liquid phase 1 is composed of 25-35% ethanol, 5-10% propylene glycol and the balance water by volume; The B phase is composed of sodium hyaluronate, methylparaben, Camus and liquid phase II; the mass volume ratio of sodium hyaluronate, methylparaben and Camus to liquid phase II is 0.1-0.5%, 0.1-0.25% and 0.5-1% respectively; the liquid phase II is composed of 10-15% glycerol, 5-7% butylene glycol and the balance water by volume ratio; The volume ratio of the liquid phase 1 to the liquid phase 2 is 1:

2.

2. The glabridin nanocomposite according to claim 1, characterized in that: The composition of phase A is: 1.5g of lecithin, 50mg of glabridin, 3mL of ethanol, 1mL of propylene glycol and 6ml of water; the composition of phase B is: 25mg of sodium hyaluronate, 25mg of methylparaben, 125mg of cam, 2.5ml of glycerin, 1.25ml of butylene glycol and 16.25ml of water.

3. The method for preparing the glabridin nanocomposite according to claim 1 or 2, characterized in that the steps include: Take lecithin, glabridin, ethanol and propylene glycol, heat and stir evenly, add water, ultrasonicate to form a dispersion, centrifuge to take the supernatant and filter to obtain phase A; Sodium hyaluronate, methylparaben, glycerin and butylene glycol are added to water and fully dissolved, and carbomer is added to fully swell to obtain phase B; Under stirring, phase A is slowly injected into phase B to fully dissolve, and the pH value is adjusted to 6-7 to obtain the glabridin nanocomposite.

4. The preparation method according to claim 3, characterized in that: During the preparation of phase A, the dissolution process temperature is 60°C; And / or, the stirring condition is to continue stirring at 1000 rpm for 30 min.

5. The preparation method according to claim 3, characterized in that: During the preparation of phase A, the ultrasonic condition was 150w ultrasonic for 10min; And / or, the centrifugation condition is 4000 rpm at 4°C for 10 min; And / or, the filtration is performed using a 0.22 μm filter membrane.

6. The preparation method according to claim 3, characterized in that: During the preparation of phase B, the dissolution process was continued with stirring at 1000 rpm for 30 min.

7. The preparation method according to claim 3, characterized in that: A preservative is also added before the pH adjustment process.

8. Use of the glabridin nanocomposite according to claim 1 or 2, or the glabridin nanocomposite obtained by the preparation method according to any one of claims 3 to 7 in the preparation of skin care products.

9. The use according to claim 8, characterized in that: The skin care products include gel, essence, lotion, cream, ointment or mask.

10. The use according to claim 8, characterized in that: The skin care product comprises at least one of the following uses: a) Remove ABTS free radicals; b) scavenging DPPH free radicals; c) Reduce melanin deposition.

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