Glabridin liposome, whitening product, preparation method and application

Abstract

The invention discloses glabridin liposome, a whitening product, a preparation method and application, and relates to the technical field of cosmetics. The glabridin liposome is prepared from the following components in percentage by mass: 0.5%-2.5% of glabridin, 1%-8% of phospholipid, 0%-1% of cholesterol, 10%-15% of liquid grease, 0.5%-1.5% of an emulsifier, 0.5%-1.5% of an antioxidant, 0.2%-1% of trehalose, 10%-30% of glycerol and the balance of water. Through innovation of the process and formula, the problems of instability of glabridin, non-uniformity of lipidosome and the like are solved, the encapsulation efficiency can be improved, the transdermal absorption capacity and stability can be improved, and the inhibitory activity on cell tyrosinase and melanin can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of cosmetics, and in particular to glabridin liposome, a whitening product, and a preparation method and application thereof. Background Art

[0002] Glabridin is a unique isoflavone component of Glycyrrhiza glabra, accounting for about 11% of the total flavonoid components of Glycyrrhiza glabra. Glabridin has an excellent whitening effect, can penetrate deep into the skin and maintain high activity, effectively inhibit the activity of multiple enzymes in the process of melanin production, especially inhibiting the activity of tyrosinase, and also has the effect of preventing skin roughness and anti-inflammatory and antibacterial. Glabridin also has good antioxidant effect, showing a strong anti-free radical oxidation effect, which can significantly inhibit the free radicals produced during the metabolism process in the body, and avoid the oxidation-sensitive biological macromolecules and cell walls from being damaged by free radical oxidation, thereby delaying cell aging, so it is widely used in skin care products. However, glabridin has defects such as low water solubility (solubility is only 0.10 mg / L at 25°C), strong fat solubility (logP=4.85), easy oxidation and degradation, and poor stability, which makes it easy to lose activity in preparations. In addition, glabridin is difficult to penetrate the skin barrier, and its transdermal absorption rate is low, which limits its efficacy in skin care products.

[0003] Existing technologies usually use means such as adding penetration enhancers or new nanoformulation technologies (such as nanoemulsions, hydrogels, liposomes, nanocrystals, cyclodextrin inclusion complexes) to improve the water solubility, stability or transdermal absorption rate of glabridin, but these technologies still have certain defects. For example, penetration enhancers can irritate the skin, especially when used at high concentrations or on sensitive skin, which may cause redness, stinging or allergic reactions; nanoemulsions require high concentrations of emulsifiers, which irritate the skin and are not resistant to dilution. If there is too much water phase, it will affect the physical and chemical properties of the nanoemulsion; hydrogels have relatively low mechanical strength and are easily damaged, with low drug loading rates and unstable release rates; nanocrystals have poor physical stability and may experience aggregation, sedimentation, and crystal transformation; cyclodextrins have a large molecular weight and a hydrophilic surface, making them difficult to penetrate the stratum corneum composed of lipids; the particle size and structure of conventional liposomes may be uneven, with a low encapsulation rate, and they need to be stored at low temperatures and away from light, otherwise they are prone to degradation or stratification.

[0004] Chinese patent CN118662374A discloses a melanocyte-targeted glabridin liposome and its preparation method and application. The liposome is mainly made of the following components in mass percentage: 1.0%-5.0% phospholipids, 0.05%-0.25% targeted phospholipids, 1.0%-2.0% glabridin, 1.0%-6.0% anhydrous ethanol, 0.1%-0.2% cholesterol, 1.0%-5.0% emulsifier, 0%-20.0% co-emulsifier, and 61.55%-95.85% water. Among them, the emulsifier includes behenyl alcohol polyether-25; the targeted phospholipid includes DSPE-PEG-Nonapeptide-1. The liposome has a small particle size, high uniformity, high stability, is mild to the human body and has a sustained release effect. The liposome carries a targeting phospholipid with a targeting molecule through covalent modification and encapsulates glabridin, which can specifically promote the absorption and effect of glabridin.

[0005] Because liposomes have good structure and biocompatibility, they are easier to penetrate the skin barrier, and the double-layer membrane can protect the active ingredients contained therein from damage by light, oxygen or enzymatic hydrolysis, and can release drugs slowly, allowing the active ingredients to continue to act for a longer period of time, which has relatively good advantages. At present, research on the preparation of liposomes for glycyrrhizin is still very scarce. Summary of the invention

[0006] In view of this, the present invention provides a glabridin liposome, a whitening product, a preparation method and an application, which solves the problems of instability of glabridin and unevenness of liposome through innovation of process and formula, and can improve the encapsulation rate, increase the transdermal absorption capacity and stability, and improve the inhibitory activity on cell tyrosinase and melanin.

[0007] To achieve the above-mentioned purpose of the invention, the technical solution of the present invention is as follows: In one aspect, the present invention provides a glabridin liposome, which is made of the following components in percentage by mass: 0.5%-2.5% glabridin, 1%-8% phospholipids, 0%-1% cholesterol, 10%-15% liquid oil, 0.5%-1.5% emulsifier, 0.5%-1.5% antioxidant, 0.2%-1% trehalose, 10%-30% glycerol and the balance water. in: The liquid oil is selected from at least one of caprylic / capric glyceride, polydimethylsiloxane, and rice bran oil; The emulsifier is selected from at least one of sorbitan sesquioleate, polyethylene glycol monolauryl ether, and polyoxyethylene stearate; The mass ratio of the glabridin, liquid oil, emulsifier, antioxidant and trehalose is 0.5-2.5:10:1:0.5-1.5:0.5-1.

[0008] Preferably, the liquid oil is caprylic / capric glyceride.

[0009] Preferably, the emulsifier is sorbitan sesquioleate.

[0010] Preferably, the antioxidant is selected from at least one of ascorbyl tetraisopalmitate and tocopherol acetate, and more preferably ascorbyl tetraisopalmitate.

[0011] Preferably, the phospholipid is selected from at least one of hydrogenated lecithin, hydrogenated phosphatidylcholine, soybean lecithin, and egg yolk lecithin, more preferably at least one of hydrogenated lecithin and hydrogenated phosphatidylcholine, and most preferably hydrogenated lecithin.

[0012] Preferably, the mass ratio of glabridin, liquid oil, emulsifier, antioxidant and trehalose is 1.1:10:1:0.5:0.5.

[0013] Further preferably, as the best embodiment of the present invention, the mass ratio of glabridin, caprylic decanoic acid glyceride, sorbitan sesquioleate, ascorbyl tetraisopalmitate and trehalose is 1.1:10:1:0.5:0.5.

[0014] Preferably, the glabridin liposome is made of the following components in percentage by mass: 1.1% glabridin, 4.5% phospholipids, 1% cholesterol, 10% liquid oil, 1% emulsifier, 0.5% antioxidant, 0.5% trehalose, 20% glycerol and the remainder water.

[0015] Most preferably, the glabridin liposomes are made of the following components in percentage by mass: 1.1% glabridin, 4.5% hydrogenated lecithin, 1% cholesterol, 10% caprylic acid glyceride, 1% sorbitan sesquioleate, 0.5% ascorbyl tetraisopalmitate, 0.5% trehalose, 20% glycerol and the remainder water.

[0016] In another aspect, the present invention provides a method for preparing the above-mentioned glabridin liposome, comprising the following steps: (1) Preparing the oil phase: mixing and dissolving the formulated amounts of glabridin, phospholipids, cholesterol, liquid oil, emulsifier, and antioxidant to obtain the oil phase; (2) Preparing an aqueous phase: mixing the formulated amounts of trehalose, glycerol and water to obtain an aqueous phase; (3) The oil phase and the water phase are mixed to obtain a primary emulsion, and the primary emulsion is subjected to high pressure homogenization to obtain glabridin liposomes.

[0017] Preferably, in step (1), the temperature of the mixed dissolution is 50-85°C, more preferably 80°C.

[0018] As a specific example of the present invention, the mixed dissolution is carried out by stirring in a water bath.

[0019] Preferably, in step (2), the mixing temperature is 50-85°C, more preferably 80°C.

[0020] As a specific example of the present invention, the mixing is performed by stirring in a water bath.

[0021] Preferably, in step (3), the mixing is specifically: adding the oil phase to the water phase, stirring while adding, and after mixing evenly, continuing to stir for 10-30 minutes at a speed of 1000-3000 rpm to obtain a primary emulsion. As a specific example of the present invention, the mixing is specifically: adding the oil phase to the water phase, stirring while adding, and after mixing evenly, continuing to stir for 15 minutes at a speed of 2000 rpm to obtain a primary emulsion.

[0022] Preferably, in step (3), the conditions of the high-pressure homogenization are: pressure 400-600 bar, number of times 4-12 times. As a specific example of the present invention, the conditions of the high-pressure homogenization are: pressure 600 bar, number of times 8 times.

[0023] In yet another aspect, the present invention provides a whitening product, comprising the glabridin liposomes described above or the glabridin liposomes prepared by the above preparation method.

[0024] In another aspect, the present invention provides use of the glabridin liposome, the glabridin liposome prepared by the preparation method, or the whitening product in the preparation of cosmetics.

[0025] Preferably, the cosmetics include eye cream, face cream, primer, essence, lotion, toner, body lotion, and facial mask liquid.

[0026] The beneficial effects of the present invention are: (1) The present invention aims at the ingredient glabridin and solves the problems of instability of glabridin and unevenness of liposomes through innovation of process and formula, which can not only improve the encapsulation rate, but also increase the transdermal absorption capacity and stability, and enhance the inhibitory effect on cell tyrosinase activity and melanin production.

[0027] (2) The present invention selects glycyrrhizin, specific liquid oil, specific emulsifier, antioxidant and trehalose for compatibility, and at a specific ratio, has a synergistic effect, which can significantly improve the encapsulation rate, transdermal absorption capacity, stability and inhibitory activity against cell tyrosinase and melanin.

[0028] (3) The present invention found that when the mass ratio of glabridin, caprylic acid glyceride, sorbitan sesquioleate, ascorbyl tetraisopalmitate and trehalose was selected to be 1.1:10:1:0.5:0.5, the obtained liposome effect was the best, which was significantly better than other ingredients and ratios. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The figure is a comparison of the particle size distribution of Example 1 and commercially available glabridin liposomes.

[0030] Figure 2 This is a morphology diagram of the glabridin liposomes prepared in Example 1.

[0031] Figure 3 The in vitro release curves of glabridin and three types of glabridin liposomes.

[0032] Figure 4 The in vitro release curves of Example 1, Comparative Example 2, Comparative Example 4 and Comparative Example 6 are shown.

[0033] Figure 5 Schematic diagram of the working process of a vertical diffusion cell.

[0034] Figure 6 The cumulative permeation curves of glabridin and three types of glabridin liposomes.

[0035] Figure 7 This is a graph showing the changes in particle size, potential, encapsulation efficiency, and PDI of the glabridin liposome in Example 1.

[0036] Figure 8 This is a graph showing the relative activity of cellular tyrosinase of glabridin and the liposomes of Example 1; In the figure, Indicates significant difference compared with the blank control group. P <0.01.

[0037] Fig. 9 This is a result diagram of the relative content of cellular melanin in glabridin and the liposomes of Example 1; In the figure, Indicates significant difference compared with the blank control group. P <0.01. DETAILED DESCRIPTION

[0038] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present application.

[0039] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0040] In the following embodiments, the brand of commercially available glabridin liposome 1 is Si Ke Xi, and the brand of commercially available glabridin liposome 1 is Feng Tai.

[0041] In the following examples, the sources of raw materials are shown in Table 1: Table 1

[0042] Example 1-Example 2, Comparative Example 1 The formulas of Example 1-Example 2 and Comparative Example 1 are shown in Table 2: Table 2

[0043] Preparation method: (1) preparing the oil phase: mixing the formulated amounts of caprylic decanoic acid glyceride, emulsifier, antioxidant, glabridin, phospholipid and cholesterol, stirring and dissolving in a water bath at 80° C. to obtain the oil phase; (2) Preparation of aqueous phase: Dissolve the formulated amount of trehalose, glycerol and water in a water bath at 80°C with stirring to obtain an aqueous phase; (3) Add the oil phase to the water phase while stirring. After the mixture is evenly mixed, continue stirring for 15 min at a speed of 2000 rpm to obtain a primary emulsion. The primary emulsion is then treated with a high-pressure homogenizer and circulated for 8 times at a pressure of 600 bar. The total high-pressure homogenization time is 5 min to obtain glycyrrhizin liposomes.

[0044] Example 3 The difference from Example 1 is that the liquid oil is polydimethylsiloxane, and the rest is the same.

[0045] Example 4 The difference from Example 1 is that the liquid oil is rice bran oil, and the rest is the same.

[0046] Example 5 The difference from Example 1 is that the emulsifier is polyethylene glycol monododecyl ether, and the rest is the same.

[0047] Example 6 The difference from Example 1 is that the emulsifier is polyoxyethylene stearate, and the rest is the same.

[0048] Example 7 The difference from Example 1 is that the antioxidant is tocopherol acetate, and the rest are the same.

[0049] Example 8 The difference from Example 1 is that the phospholipid is hydrogenated phosphatidylcholine, and the rest is the same.

[0050] Example 9 The difference from Example 1 is that the amount of trehalose added is 0.2%, and the rest are the same.

[0051] Example 10 The difference from Example 1 is that the added amount of trehalose is 1%, and the rest are the same.

[0052] Comparative Example 2 The difference from Example 1 is that the raw material formula is: 1.1% glabridin, 4.5% hydrogenated lecithin, 1% cholesterol, 20% caprylic acid glyceride, 0.5% sorbitan sesquioleate, 0.5% ascorbyl tetraisopalmitate, 0.5% trehalose, 20% glycerol and the balance water, and the rest are the same.

[0053] Comparative Example 3 The difference from Example 1 is that trehalose is not added, and the rest is the same.

[0054] Comparative Example 4 The difference from Example 1 is that the liquid oil is glyceryl behenate, and the rest are the same.

[0055] Comparative Example 5 The difference from Example 1 is that the liquid oil is glyceryl monostearate, and the rest is the same.

[0056] Comparative Example 6 The difference from Example 1 is that the emulsifier is PEG-40 hydrogenated castor oil, and the rest are the same.

[0057] Comparative Example 7 The difference from Example 1 is that the emulsifier is beheneth-25, and the rest are the same.

[0058] Results test: 1. Particle size, zeta potential and PDI determination The particle size, potential, and PDI (polydispersity index) of the liposomes were measured by nanoparticle size and zeta potential analyzer. The results are shown in Table 3: Table 3

[0059] The particle size distribution of the glabridin liposomes prepared in Example 1 and the commercially available glabridin liposomes is shown in the following example: Figure 1As shown. The particle size, potential and PDI of the glabridin liposomes prepared in Examples 1 to 10 are better than those in Comparative Examples 1 to 7, among which Example 1 has the smallest particle size of 93.8 nm and the smallest PDI of 0.151, indicating that its particle size is more uniform. At the same time, the glabridin liposomes prepared by the present invention are significantly better than the commercially available glabridin liposomes.

[0060] 2. Encapsulation efficiency and drug loading The microcolumn centrifugation method was used to determine the liposome encapsulation rate of glabridin. Preparation of microgel column: Weigh a certain amount of Sephadex G-50, swell it with distilled water for 12 hours, and then put it into a 3mL syringe with the inner stopper removed (filled with 3-5mm glass fiber at the bottom). After eliminating bubbles, balance 2-3 column volumes with distilled water, centrifuge at 1000r / min for 2min, and the volume of the gel column after centrifugation is about 2mL. Accurately aspirate 0.2g of liposome and drug solution (dissolved in methanol to make up the volume), slowly add to the top of the column (be careful not to drip into the edge of the column bed when adding the sample), centrifuge at 1000r / min for 1min, so that the sample to be tested enters the microcolumn. Continue to add 1mL of distilled water from the top of the column, repeat the elution, and collect 6mL; dilute the eluate with methanol, detect the content of glabridin, and calculate the encapsulation rate.

[0061] Encapsulation efficiency = drug amount in liposomes / total drug amount × 100%.

[0062] Drug loading = drug amount in liposomes / sample mass × 100%.

[0063] The results are shown in Table 4: Table 4

[0064] The liposomes prepared in the embodiment of the present invention are milky white liquid; the commercially available glabridin liposome 1 is a yellow transparent liquid; and the commercially available glabridin liposome 2 is a light yellow paste. The encapsulation efficiency, drug content, particle size, potential and PDI of the liposomes prepared in the embodiment of the present invention are superior to those of the commercially available liposomes.

[0065] The encapsulation efficiency of the glabridin liposomes prepared in Examples 1-10 is ≥80%, which complies with the guidelines for microparticle preparations in Part 4, 9014, of the 2020 edition of the Chinese Pharmacopoeia. However, the encapsulation efficiency of the glabridin liposomes prepared in Comparative Examples 1-7 did not meet the requirements. Since the proportion of glabridin in Example 2 is relatively high, the drug loading is also relatively high, but the encapsulation efficiency is 82.4%, which meets the requirements but is lower than Example 1.

[0066] Meanwhile, the encapsulation efficiency and drug loading of the glabridin liposomes prepared in Examples 1 to 10 are better than those of the commercially available glabridin liposomes.

[0067] 3. Morphological investigation The liposome morphology was observed by transmission electron microscopy and negative staining technique. 10 μL of liposomes was added to the copper mesh for precipitation for 1 min, and the floating liquid was removed by filter paper. 10 μL of phosphotungstic acid staining solution was added to the copper mesh for precipitation for 1 min, and the floating liquid was removed by filter paper. Dry at room temperature for several minutes, perform electron microscopy detection and imaging at 80-120 kV, observe under a transmission electron microscope, and collect images for analysis.

[0068] The liposomes prepared in Example 1 are as follows Figure 2 The glabridin liposomes were round in shape, double-layered, with rounded edges and good dispersibility.

[0069] 4. In vitro release capacity determination The dialysis bag method was used to investigate and compare the in vitro release behaviors of the liposome raw materials prepared by the optimal process and the commercial raw materials of the reference substance. The specific operation is as follows: a cellulose semipermeable membrane was selected as the dialysis membrane, and 30% ethanol-normal saline was used as the release medium. 1 g of each test sample was placed in a dialysis bag, sealed at both ends and placed in a conical flask containing 50 mL of release medium, and released by shaking at (37±0.5)℃ and 100 r / min in a shaker. 1 mL of each sample was taken at 1 h, 2 h, 4 h, 8 h, and 24 h, and 1 mL of isothermal fresh release medium was quickly added. The samples obtained were analyzed by HPLC and the cumulative release of glabridin at different times was calculated.

[0070] The release curves of glabridin, glabridin liposomes prepared in Example 1, and commercially available glabridin liposomes are as follows: Figure 3 As shown. The release rates of the three glabridin liposomes were all less than 40% at 0.5h, and no burst release occurred. The bare drug was released rapidly in the first 4h, with a release rate of 79.3%. The release rate then slowed down until it was basically stable after 24h, with a final release rate of 84.3%. In the first 4h, at the initial stage of glabridin liposome release, the drug attached to its surface was released rapidly, forming a slight burst release effect. Fengtai glabridin liposomes released faster due to their low encapsulation rate. In the later stage, the three liposomes all released the drug slowly, and the drug release rates after 24h were 64.6%, 56.3% and 52.0%, respectively. Compared with the glabridin group, the liposome group had a lower release rate and a longer sustained release time. This shows that making glabridin into liposomes is conducive to the sustained release of glabridin and increases the duration of action of glabridin.

[0071] The release curves of the glabridin liposomes prepared in Example 1, Comparative Example 2, Comparative Example 4 and Comparative Example 6 are as follows: Figure 4As shown, the results show that the release rates of the four glabridin liposomes at 0.5h are all less than 40%, and no sudden release occurs; however, the glabridin liposomes prepared in Comparative Examples 2, 4 and 6 release faster in the early stage and tend to release more slowly in the later stage; the release rate of the liposomes prepared in Example 1 is relatively uniform, the sustained-release effect lasts for a long time, and is more conducive to the action time of the drug.

[0072] 5. Determination of transdermal absorption capacity The in vitro transdermal performance of liposomes was determined using the vertical Franz diffusion cell method (the schematic diagram of the vertical diffusion cell is shown in Figure 2). Figure 5 ) were evaluated and the in vitro transdermal performance of the liposome raw materials prepared by the optimal process was compared with that of the reference commercial raw materials.

[0073] Bama Xiang pig skin was selected for the experiment. The receiving solution was 30% ethanol-normal saline. The sample amount was 1 g. Sampling was carried out at 1 h, 2 h, 4 h, 8 h, and 24 h. 0.5 mL of the receiving solution was aspirated with an injection needle at different time points, and the same volume of isothermal fresh receiving solution was added in time. The receiving solution sample was taken and centrifuged at 12000 r / min for 10 min. The supernatant was taken for analysis.

[0074] (1) Cumulative permeability Q (μg / cm -2 ): .

[0075] C n is the drug concentration in the receiving solution measured at the nth sampling point, C i is the drug concentration in the receiving solution measured at the i-th (≤n-1) sampling point, V is the total volume of the receiving solution, V i is the volume of each sampling, and A is the effective diffusion area.

[0076] The release curves of glabridin, glabridin liposomes prepared in Example 1, and commercially available glabridin liposomes are shown in FIG. Figure 6 The results show that the cumulative transdermal amount of each group of samples increases with time. After 24 hours of treatment, the cumulative transdermal amount of the glabridin liposome prepared in Example 1 of the present invention is the highest.

[0077] (2) Skin retention (μg / cm -2 ): At the end of the transdermal absorption test, the ex vivo skin was completely removed, the skin was rinsed with warm saline, dried at room temperature, and then cut into pieces and homogenized, and 10 mL of methanol was added. Ultrasonic extraction was performed for 30 minutes, and the skin was filtered through a 0.45 μm microporous membrane. The skin retention amount was determined by HPLC. The results are shown in Table 5: Table 5

[0078] The cumulative permeation amount and skin retention amount of the glabridin liposomes prepared in Examples 1 to 10 are higher than those in Comparative Examples 1 to 7, indicating that the glabridin liposomes prepared in Examples 1 to 10 have stronger transdermal absorption ability. At the same time, the encapsulation efficiency and drug loading of the glabridin liposomes prepared in Examples 1 to 10 are better than those of the commercially available glabridin liposomes.

[0079] The cumulative penetration amounts of Example 1, commercially available glabridin liposome 1, and commercially available glabridin liposome 2 are 2.94 times, 2.33 times, and 1.61 times of free glabridin, respectively; and the skin retention amounts are 1.98 times, 1.71 times, and 1.37 times of free glabridin. The above results show that glabridin can be significantly improved in its transdermal effect after being encapsulated in nanoliposomes, and can increase the skin penetration and skin retention of glabridin; among them, the liposomes prepared in the embodiment of the present invention have better effects.

[0080] 6. Stability inspection The liposomes prepared in Example 1 were stored at room temperature and protected from light. The particle size, potential, PDI and encapsulation efficiency (leakage rate) were tested at 0 months (i.e., just after preparation), 1 month, and 2 months. Figure 7 shown.

[0081] At the same time, the stability in the cosmetic matrix was investigated, and 5% by mass of glabridin liposomes were added to the cosmetic emulsion. The glabridin content and liposome encapsulation efficiency were measured at 0 month (i.e., just after preparation) and 2 months under room temperature and light-proof conditions. The results are shown in Table 6: Table 6

[0082] After the liposomes were added to the cosmetic emulsion matrix for 2 months, the encapsulation efficiency of the glabridin liposomes prepared in Examples 1 to 10 decreased significantly less than that in Comparative Examples 1 to 7, and the stability of glabridin was protected and the loss of components was reduced.

[0083] 7. Whitening efficacy test 1 Reagents and instruments 1.1 Reagents Mouse melanoma cells (B16-F10 cells), fetal bovine serum, DMEM culture medium, etc.

[0084] 1.2 Instruments ELISA reader, water bath, magnetic stirrer, electronic balance, etc.

[0085] 2 Experimental methods 2.2 Cellular tyrosinase activity Cells were inoculated into 6-well plates and divided into a normal cell control group and different concentration sample groups (including samples of different concentrations). DMEM medium containing 10% fetal bovine serum was added to the cell control group, and medium and samples of different concentrations were added to the sample group. The cells were collected, washed twice with PBS, centrifuged and the supernatant was discarded, lysed with 1mL 1% Triton X-100, quickly placed in a -80℃ refrigerator for 30min, thawed at room temperature for about 20min, mixed and allowed to stand at 4℃ for 20min, centrifuged and supernatant was taken, 100μL of cell extract was taken in a 96-well plate, 100μL of L-dopa solution was added and mixed, incubated at 37℃ for 1h, OD475 was measured, and the total protein P content (mg) of each well was measured with a BCA kit. The experiment was repeated 3 times, and the melanin production inhibition rate was calculated as follows: .

[0086] 2.3 Cellular melanin production The cells were inoculated into 6-well plates and divided into a normal cell control group and different concentration sample groups (including samples of different concentrations). DMEM medium containing 10% fetal bovine serum was added to the cell control group, and medium and samples of different concentrations were added to the sample group. The cells were collected, washed twice with PBS, centrifuged and the supernatant was discarded, 1 mL of NaOH (1 mol / L) solution containing 10% DMSO was added, and the solution was incubated at 80°C for 1 hour. 200 μL of the solution was added to a 96-well plate, and A400nm was measured. The experiment was repeated 3 times, and the relative content of melanin production was calculated as follows: .

[0087] 3 Results and analysis 3.1 Cell safety results The safe concentrations of free drug and liposomes for B16-F10 cells are shown in Table 7.

[0088] Table 7

[0089] 3.3 Cellular tyrosinase activity results The results of the inhibition of tyrosinase activity by glabridin and the liposomes in Example 1 are shown in Figure 8 The results showed that glabridin and the liposomes of Example 1 both had significant inhibitory effects on cell tyrosinase activity.

[0090] 3.4 Results of cellular melanin production The results of the inhibition of cellular melanin production by glabridin and the liposomes in Example 1 are shown in Fig. 9 The results showed that the glabridin raw material (purity 90%) and its liposomes can significantly reduce the amount of melanin produced.

[0091] When liposomes were converted into free drugs (about 100 times), the results showed that at the same concentration, liposomes had a higher ability to inhibit tyrosinase activity and melanin production than glabridin, because the structure of liposomes is similar to that of cell membranes and can more easily pass through cell membranes and enter cells to exert their whitening effects.

[0092] The results of the determination of glabridin liposomes prepared in Example 1 and Comparative Examples 2 to 7 at a dosage of 25 μg / mL are shown in Table 8: Table 8

[0093] The results show that the glabridin liposomes prepared in Example 1 have the strongest ability to inhibit the relative activity of cell tyrosinase and also have the strongest effect in reducing the amount of cell melanin production, indicating that the whitening effect is stronger than that of Comparative Examples 2 to 7.

Claims

1. A glabridin liposome, characterized in that: The invention is made of the following components in percentage by mass: 0.5%-2.5% glabridin, 1%-8% phospholipids, 0%-1% cholesterol, 10%-15% liquid oil, 0.5%-1.5% emulsifier, 0.5%-1.5% antioxidant, 0.2%-1% trehalose, 10%-30% glycerol and the balance water. in: The liquid oil is selected from at least one of caprylic / capric glyceride, polydimethylsiloxane, and rice bran oil; The emulsifier is selected from at least one of sorbitan sesquioleate, polyethylene glycol monolauryl ether, and polyoxyethylene stearate; The mass ratio of the glabridin, liquid oil, emulsifier, antioxidant and trehalose is 0.5-2.5:10:1:0.5-1.5:0.5-1.

2. The glabridin liposome according to claim 1, characterized in that The liquid oil is caprylic / capric glyceride.

3. The glabridin liposome according to claim 1, characterized in that The emulsifier is sorbitan sesquioleate.

4. The glabridin liposome according to claim 1, characterized in that The antioxidant is selected from at least one of ascorbyl tetraisopalmitate and tocopherol acetate.

5. The glabridin liposome according to claim 4, characterized in that The antioxidant is ascorbyl tetraisopalmitate.

6. The glabridin liposome according to claim 1, characterized in that The phospholipid is selected from at least one of hydrogenated lecithin, hydrogenated phosphatidylcholine, soybean lecithin and egg yolk lecithin.

7. The glabridin liposome according to claim 6, characterized in that The phospholipid is hydrogenated lecithin.

8. The glabridin liposome according to any one of claims 1 to 7, characterized in that: The mass ratio of the glabridin, liquid oil, emulsifier, antioxidant and trehalose is 1.1:10:1:0.5:0.

5.

9. The glabridin liposome according to claim 1, characterized in that The invention is prepared from the following components in percentage by mass: 1.1% of glabridin, 4.5% of phospholipids, 1% of cholesterol, 10% of liquid oil, 1% of emulsifier, 0.5% of antioxidant, 0.5% of trehalose, 20% of glycerol and the balance of water.

10. The glabridin liposome according to claim 9, characterized in that The glabridin liposome is made of the following components in percentage by mass: 1.1% glabridin, 4.5% hydrogenated lecithin, 1% cholesterol, 10% caprylic acid glyceride, 1% sorbitan sesquioleate, 0.5% ascorbyl tetraisopalmitate, 0.5% trehalose, 20% glycerol and the balance water.

11. The method for preparing the glabridin liposome according to any one of claims 1 to 10, characterized in that: The following steps are involved: (1) Preparing an oil phase: mixing and dissolving the formulated amounts of glabridin, phospholipids, cholesterol, liquid oil, emulsifier, and antioxidant to obtain an oil phase; (2) Preparing an aqueous phase: mixing the formulated amounts of trehalose, glycerol and water to obtain an aqueous phase; (3) The oil phase and the water phase are mixed to obtain a primary emulsion, and the primary emulsion is subjected to high pressure homogenization to obtain glabridin liposomes.

12. The preparation method according to claim 11, characterized in that: In step (3), the mixing is specifically as follows: adding the oil phase to the water phase, stirring while adding, and after mixing evenly, continuously stirring for 10-30 minutes at a rotation speed of 1000-3000 rpm to obtain a primary emulsion.

13. The preparation method according to claim 11, characterized in that: In step (3), the conditions of high pressure homogenization are: pressure 400-600 bar, number of times 4-12 times.

14. A whitening product, characterized in that: The invention comprises the glabridin liposome according to any one of claims 1 to 10, or the glabridin liposome prepared by the preparation method according to any one of claims 11 to 13.

15. Use of the glabridin liposome according to any one of claims 1 to 10, or the glabridin liposome prepared by the preparation method according to any one of claims 11 to 13, or the whitening product according to claim 14 in the preparation of cosmetics.

16. The use according to claim 15, characterized in that The cosmetics include eye cream, face cream, primer, essence, lotion, toner, body lotion, and facial mask liquid.

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