A glycyrrhizin-based composite of bosine and collagen nanoparticles, its preparation method and application

Abstract

This invention belongs to the field of cosmetic technology, specifically a glycyrrhizin-based composite nanoparticle containing sclerotherapy, BPOXY, and collagen, along with its preparation method and applications. The invention first combines glycyrrhizin and glycyrrhizic acid via non-covalent bonds to obtain a glycyrrhizin-glycyrrhizic acid eutectic structure that overcomes the solubility problem of glycyrrhizin. Then, a BPOXY-collagen mixture is used to coat the glycyrrhizin-glycyrrhizic acid eutectic structure, yielding glycyrrhizin-based composite nanoparticles containing sclerotherapy, BPOXY, and collagen. The glycyrrhizin-based composite nanoparticles containing sclerotherapy, BPOXY, and collagen obtained using this method exhibit good water solubility of glycyrrhizin, a simple composition, high stability, and minimal risk of allergic reactions. Furthermore, it enhances the skin permeability of glycyrrhizin, collagen, and BPOXY, resulting in a synergistic effect and improved bioavailability.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically a glycyrrhizin composite boswellicin & collagen nanoparticle, its preparation method and application. Background Technology

[0002] Aging, prolonged exposure to ultraviolet radiation, irregular sleep patterns, and malnutrition are all lifestyle factors that contribute to skin aging, typically accompanied by noticeable signs and symptoms such as sagging skin, wrinkles and fine lines, pigmentation, dryness, and enlarged pores. Adding the natural and highly effective whitening factor glycyrrhizin can enhance the whitening effect by working in conjunction with the skin's natural repair process. However, the water-insoluble nature of glycyrrhizin limits its application.

[0003] Existing technologies generally employ encapsulation to address the water solubility issue of glycyrrhizin, with encapsulation materials primarily consisting of liposomes, cyclodextrins, or exosomes. For example, patent CN113893191A discloses a whitening essence based on phospholipid-encapsulated glycyrrhizin nanoemulsions and its preparation method. In this method, phospholipids and glycyrrhizin are dissolved in an organic solvent to obtain a glycyrrhizin-phospholipid complex, which is then homogenized with an emulsifier to form a nanoemulsion. This method uses a large amount of emulsifiers and stabilizers, posing a certain risk of allergic reactions.

[0004] A patent, CN108815012A, discloses a multi-layer encapsulated solid dosage form of glycyrrhizin and its preparation method. In this method, glycyrrhizin is first encapsulated with cyclodextrin, and then a secondary encapsulation is performed using glycyrrhizic acid and glycyrrhizic acid polysaccharide. However, the glycyrrhizin content encapsulated by this method is limited, and the encapsulating carrier does not have any therapeutic effect.

[0005] In summary, none of these existing technologies have solved the fundamental requirements of licorice extract as a cosmetic ingredient: simplicity, stability, risk-free operation, and high water solubility.

[0006] Both Pro-Xylane and collagen are effective anti-aging ingredients. Collagen has the characteristics of large molecular weight and stable molecular structure, which makes it difficult for large collagen molecules to penetrate the tight structure of the stratum corneum into the deep layers of the skin. In addition, the amino acid composition of collagen contains a large number of hydrophilic amino acids, which makes it easy for collagen to form a hydration layer on the skin surface, making it difficult to penetrate the skin barrier into deep tissues, resulting in poor skin permeability. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides glycyrrhizin-based composite β-xylene and collagen nanoparticles, their preparation method, and applications. This invention first combines glycyrrhizin and glycyrrhizic acid via non-covalent bonds to obtain a glycyrrhizin-glycyrrhizic acid eutectic structure that overcomes the solubility problem of glycyrrhizin. Then, a β-xylene-collagen mixture is used to coat the glycyrrhizin-glycyrrhizic acid eutectic structure to obtain glycyrrhizin-based composite β-xylene and collagen nanoparticles. The glycyrrhizin-based composite β-xylene and collagen nanoparticles obtained using this method exhibit good water solubility of glycyrrhizin, simple composition, high stability, and minimal risk of allergic reactions. Furthermore, it can enhance the skin permeability of collagen, resulting in a synergistic effect among the three components and improved utilization.

[0008] This invention is achieved by adopting the following technical solution:

[0009] This invention protects a method for preparing glycyrrhizin-based composite bosine and collagen nanoparticles, comprising the following steps:

[0010] The raw materials are weighed according to the following weight percentages: collagen 0.1%–15%, glycyrrhizin 1%–15%, glycyrrhizic acid 2.5%–40%, dipotassium glycyrrhizate 5%–40%, and the remainder is bosine. The sum of the weight percentages of each raw material is 100%. The collagen is selected from recombinant collagen, elastin or mussel protein.

[0011] After mixing glycyrrhizin and glycyrrhizic acid, the mixture is ball-milled. The glycyrrhizin and glycyrrhizic acid are bonded through non-covalent bonds to obtain a glycyrrhizin-glycyrrhizic acid eutectic structure. Glycyrrhizic acid gives glycyrrhizin good water solubility. In this invention, the formation of a water-soluble eutectic structure by glycyrrhizin and glycyrrhizic acid significantly improves the water solubility and dispersibility of glycyrrhizin, increasing its solubility in water to ≥20 mg / mL (the initial water solubility of glycyrrhizin is ≤0.1 mg / mL), and also enhances the stability of glycyrrhizin.

[0012] A pH-adjusting solution of dipotassium glycyrrhizate was mixed with glycyrrhizin-glycyrrhizic acid to adjust the pH to 4.0-4.5, resulting in a clear and transparent glycyrrhizin-glycyrrhizic acid mixture. The purpose of adjusting the pH was to make it suitable for skin application.

[0013] A recombinant collagen aqueous solution was added to a Bosein aqueous solution. Bosein and collagen were combined through positive and negative charge interactions to obtain a Bosein-collagen mixture.

[0014] A mixture of glycyrrhizin and glycyrrhizic acid was added to a mixture of bosine and collagen. After mixing, the eutectic structure of glycyrrhizin and glycyrrhizic acid and bosine and collagen assembled into nanoparticles through intermolecular forces (mainly π-π bonds). At this time, bosine and collagen coated the surface of the eutectic structure of glycyrrhizin and glycyrrhizic acid, resulting in a glycyrrhizin-bosine and collagen nanoparticle emulsion. The glycyrrhizin-bosine and collagen nanoparticle emulsion was freeze-dried to obtain glycyrrhizin-bosine and collagen nanoparticles.

[0015] Preferably, the raw materials are weighed according to the following weight percentages: collagen 0.1%–15%, glycyrrhizin 1%–15%, glycyrrhizic acid 2.5%–40%, dipotassium glycyrrhizate 5%–40%, and the remainder is β-xylene. The sum of the weight percentages of each raw material is 100%. Under this weight percentage, the glycyrrhizin-β-xylene & collagen nanoparticles have better performance.

[0016] Preferably, the mass ratio of glycyrrhizin to glycyrrhizic acid is 1-1.5:1-5. Studies have shown that when the mass ratio of glycyrrhizin to glycyrrhizic acid is 1:2.5, the obtained glycyrrhizin-glycyrrhizic acid eutectic structure has the best water solubility.

[0017] Preferably, the ball milling conditions are: ball milling at 30℃~50℃ for 10min~50min.

[0018] Preferably, the mixing time of the recombinant collagen aqueous solution and the BOXER aqueous solution is 10 min to 30 min.

[0019] Preferably, the mixing time of the glycyrrhizin-glycyrrhizic acid mixture and the bosine-collagen mixture is 10 min to 30 min.

[0020] Preferably, in the aqueous solution of recombinant collagen, the amount of deionized water is 10 to 50 times that of the recombinant collagen.

[0021] Preferably, in the aqueous solution of bosine, the amount of deionized water is 5 to 10 times that of bosine.

[0022] This invention also protects the glycyrrhizin-based composite bosine and collagen nanoparticles prepared by the above method.

[0023] Preferably, the glycyrrhizin-glycoside composite β-xylene & collagen nanoparticles have a β-xylene-collagen-coated glycyrrhizin-glycyrrhizic acid eutectic structure.

[0024] This invention also protects the application of glycyrrhizin-based composite bosine and collagen nanoparticles in the preparation of cosmetics.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] 1. This invention first combines glycyrrhizin and glycyrrhizic acid through non-covalent bonding to form a stable structure, resulting in a glycyrrhizin-glycyrrhizic acid eutectic structure, which makes glycyrrhizin water-soluble. Then, the glycyrrhizin-glycyrrhizic acid eutectic structure is dissolved in a pH-adjusting dipotassium glycyrrhizate aqueous solution to obtain a glycyrrhizin-glycyrrhizic acid mixture, and the pH is adjusted to a skin-acceptable range. Next, a collagen aqueous solution and a BOXER aqueous solution are mixed to form a BOXER-collagen mixture through electrostatic interaction. Finally, using BOXER-collagen as the shell and the glycyrrhizin-glycyrrhizic acid eutectic structure as the core, the intermolecular forces of glycyrrhizin-glycyrrhizic acid, collagen, and BOXER are further assembled to form a BOXER-collagen-coated glycyrrhizin-glycyrrhizic acid eutectic structure, resulting in glycyrrhizin composite BOXER & collagen nanoparticles.

[0027] 2. Considering the poor transdermal permeability of collagen and Proxylane when added to cosmetics, the glycyrrhizin-based composite Proxylane & collagen nanoparticles of this invention contain glycyrrhizin-based composite Proxylane & collagen nanoparticles as the solute. The nanoparticle structure exhibits good transdermal permeability and high utilization rate, solving the problems of poor transdermal permeability and bioavailability of collagen and Proxylane. Furthermore, compared to conventional Proxylane and collagen, the glycyrrhizin-based composite Proxylane & collagen nanoparticles prepared in this invention have a more significant skin whitening effect. This is because the nanoparticles have good transdermal permeability and high utilization rate, allowing them to better exert their whitening effects. In addition, the combination of the anti-aging effects of Proxylane and collagen enables wider applications in the skincare industry.

[0028] 3. In this invention, glycyrrhizic acid is used to achieve non-covalent bonding with glycyrrhizin, and collagen-Bosine is used to coat the glycyrrhizin-glycyrrhizic acid eutectic structure, which solves the problem of poor water solubility and instability of glycyrrhizin.

[0029] 4. In terms of efficacy, the glycyrrhizin-based composite nanoparticles of the present invention, which contain sclerosing agents, have anti-wrinkle effects, and collagen can efficiently replenish collagen in the skin, achieve synergistic effects of collagen, sclerosing agents, and glycyrrhizin in the preparation of glycyrrhizin-based composite nanoparticles. Collagen and sclerosing agents exert anti-aging effects, while glycyrrhizin exerts whitening effects, thereby better solving skin problems.

[0030] 5. The glycyrrhizin-based composite bosine and collagen nanoparticles provided by this invention are stable and have good water solubility. At the same time, the raw materials are readily available, the preparation method is simple, the safety risk is low, the stability is high, and the transdermal properties are good, which makes them extremely promising for application in the field of skin care raw materials. Attached Figure Description

[0031] Figure 1 This is a TEM image of the glycyrrhizin-based composite bosine and collagen nanoparticles from Example 1.

[0032] Figure 2 The image shows the cell safety assessment results of the glycyrrhizin-based composite bosine and collagen nanoparticles in Example 1 based on mouse melanoma cells.

[0033] Figure 3 The image shows the results of the glycyrrhizin-based composite bosine and collagen nanoparticles in Example 1 based on the melanin content of mouse melanoma cells.

[0034] Figure 4 The image shows the tyrosinase activity results of the glycyrrhizin-based composite bosine and collagen nanoparticles in Example 1.

[0035] Figure 5 The figures shown are experimental results of the cumulative penetration of glycyrrhizin-based composite bosine and collagen nanoparticles and collagen in Example 1. In Figure (a), the figure is a confocal Raman image, and in Figure (b), the figure is a total integral intensity image.

[0036] Figure 6 Comparison of water solubility of glycyrrhizin (a) and glycyrrhizin-glycyrrhizic acid eutectic structure (b) in Example 1. Detailed Implementation

[0037] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0038] Considering that existing technologies using liposomes, cyclodextrins, or exosomes to encapsulate glycyrrhizin require the addition of emulsifiers or other complex components, and the potential risks associated with adding numerous components, this invention first proposes a non-covalent bond between glycyrrhizin and glycyrrhizic acid. The use of glycyrrhizic acid ensures excellent water solubility of glycyrrhizin, overcoming the defects caused by the addition of complex components during the encapsulation process. It also overcomes the problem of glycyrrhizin being insoluble or poorly soluble in water. Glycyrrhizic acid has antioxidant and whitening properties and is often added to skincare products to reduce skin inflammation, promote skin repair, improve uneven skin tone, or reduce pigmentation. Then, a glycoside-collagen co-crystal structure of glycyrrhizin and glycyrrhizic acid is encapsulated in nanoparticles. Due to the small volume effect, the nanoparticles enhance the transdermal permeability of glycoside and collagen. Furthermore, the encapsulation of the glycyrrhizin-glycyrrhizic acid co-crystal structure within the glycoside-collagen enhances the stability of glycyrrhizin.

[0039] The technical solution of the present invention will be further studied using the following embodiments. The specific research methods and results are shown below:

[0040] Example 1

[0041] A method for preparing glycyrrhizin-based composite bosine and collagen nanoparticles includes the following steps:

[0042] S1. Weigh the raw materials according to the following weight percentages: 10% glycyrrhizin, 25% glycyrrhizic acid, 10% dipotassium glycyrrhizate, 45% bosine, and 10% recombinant collagen.

[0043] S2. Use a vortex mixer to mix glycyrrhizin and glycyrrhizic acid evenly, and then use a ball mill to further refine the particles. The grinding temperature is 50℃ and the grinding time is 30min to obtain the glycyrrhizin-glycyrrhizic acid eutectic structure.

[0044] S3. Dissolve dipotassium glycyrrhizate in deionized water to obtain an aqueous solution of dipotassium glycyrrhizate. Mix the aqueous solution of dipotassium glycyrrhizate with the eutectic structure of glycyrrhizin-glycyrrhizic acid and dissolve until clear and transparent to obtain a mixed solution of glycyrrhizin-glycyrrhizic acid.

[0045] S4. Dissolve BOXER and recombinant collagen in water to obtain BOXER aqueous solution and recombinant collagen aqueous solution. Add the recombinant collagen aqueous solution to the BOXER aqueous solution and stir for 10 min to obtain BOXER-collagen mixture.

[0046] S5. Add the glycyrrhizin-glycyrrhizic acid mixture to the bisphenol A-collagen mixture and stir for 30 minutes to obtain a glycyrrhizin-bisphenol A-collagen nanoparticle emulsion. Freeze-dry the emulsion to obtain glycyrrhizin-bisphenol A-collagen nanoparticles.

[0047] Example 2

[0048] A method for preparing glycyrrhizin-based composite bosine and collagen nanoparticles includes the following steps:

[0049] S1. Weigh the raw materials according to the following weight percentages: 5% glycyrrhizin, 25% glycyrrhizic acid, 10% dipotassium glycyrrhizate, 50% bosine, and 10% recombinant collagen, and set aside.

[0050] S2. Use a vortex mixer to mix glycyrrhizin and glycyrrhizic acid evenly, and then use a ball mill to further refine the particles. The grinding temperature is 50℃ and the grinding time is 30min to obtain the glycyrrhizin-glycyrrhizic acid eutectic structure.

[0051] S3. Dissolve dipotassium glycyrrhizate in deionized water to obtain an aqueous solution of dipotassium glycyrrhizate. Mix the aqueous solution of dipotassium glycyrrhizate with the eutectic structure of glycyrrhizin-glycyrrhizic acid and dissolve until clear and transparent to obtain a mixed solution of glycyrrhizin-glycyrrhizic acid.

[0052] S4. Dissolve BOXER and recombinant collagen in water to obtain BOXER aqueous solution and recombinant collagen aqueous solution. Add the recombinant collagen aqueous solution to the BOXER aqueous solution and stir for 10 min to obtain BOXER-collagen mixture.

[0053] S5. Add the glycyrrhizin-glycyrrhizic acid mixture to the bisphenol A-collagen mixture and stir for 30 minutes to obtain a glycyrrhizin-bisphenol A-collagen nanoparticle emulsion. Freeze-dry the emulsion to obtain glycyrrhizin-bisphenol A-collagen nanoparticles.

[0054] Example 3

[0055] A method for preparing glycyrrhizin-based composite bosine and collagen nanoparticles includes the following steps:

[0056] S1. Weigh the raw materials according to the following weight percentages: 10% glycyrrhizin, 25% glycyrrhizic acid, 30% dipotassium glycyrrhizate, 25% bosine, and 10% recombinant collagen.

[0057] S2. Use a vortex mixer to mix glycyrrhizin and glycyrrhizic acid evenly, and then use a ball mill to further refine the particles. The grinding temperature is 50℃ and the grinding time is 30min to obtain the glycyrrhizin-glycyrrhizic acid eutectic structure.

[0058] S3. Dissolve dipotassium glycyrrhizate in deionized water to obtain an aqueous solution of dipotassium glycyrrhizate. Mix the aqueous solution of dipotassium glycyrrhizate with the eutectic structure of glycyrrhizin-glycyrrhizic acid and dissolve until clear and transparent to obtain a mixed solution of glycyrrhizin-glycyrrhizic acid.

[0059] S4. Dissolve BOXER and recombinant collagen in water to obtain BOXER aqueous solution and recombinant collagen aqueous solution. Add the recombinant collagen aqueous solution to the BOXER aqueous solution and stir for 10 min to obtain BOXER-collagen mixture.

[0060] S5. Add the glycyrrhizin-glycyrrhizic acid mixture to the bisphenol A-collagen mixture and stir for 30 minutes to obtain a glycyrrhizin-bisphenol A-collagen nanoparticle emulsion. Freeze-dry the emulsion to obtain glycyrrhizin-bisphenol A-collagen nanoparticles.

[0061] Example 4

[0062] A method for preparing glycyrrhizin-based composite bosine and collagen nanoparticles includes the following steps:

[0063] S1. Weigh the raw materials according to the following weight percentages: 15% glycyrrhizin, 10% glycyrrhizic acid, 5% dipotassium glycyrrhizate, 65% bosine, and 5% recombinant collagen.

[0064] S2. Use a vortex mixer to mix glycyrrhizin and glycyrrhizic acid evenly, and then use a ball mill to further refine the particles. The grinding temperature is 50℃ and the grinding time is 30min to obtain the glycyrrhizin-glycyrrhizic acid eutectic structure.

[0065] S3. Dissolve dipotassium glycyrrhizate in deionized water to obtain an aqueous solution of dipotassium glycyrrhizate. Mix the aqueous solution of dipotassium glycyrrhizate with the eutectic structure of glycyrrhizin-glycyrrhizic acid and dissolve until clear and transparent to obtain a mixed solution of glycyrrhizin-glycyrrhizic acid.

[0066] S4. Dissolve BOXER and recombinant collagen in water to obtain BOXER aqueous solution and recombinant collagen aqueous solution. Add the recombinant collagen aqueous solution to the BOXER aqueous solution and stir for 20 minutes to obtain BOXER-collagen mixture.

[0067] S5. Add the glycyrrhizin-glycyrrhizic acid mixture to the bisphenol A-collagen mixture and stir for 20 minutes to obtain a glycyrrhizin-bisphenol A-collagen nanoparticle emulsion. Freeze-dry the emulsion to obtain glycyrrhizin-bisphenol A-collagen nanoparticles.

[0068] Example 5

[0069] A method for preparing glycyrrhizin-based composite bosine and collagen nanoparticles includes the following steps:

[0070] S1. Weigh the raw materials according to the following weight percentages: 1% glycyrrhizin, 2.5% glycyrrhizic acid, 5% dipotassium glycyrrhizate, 76.5% bosine, and 15% recombinant collagen, and set aside.

[0071] S2. Use a vortex mixer to mix glycyrrhizin and glycyrrhizic acid evenly, and then use a ball mill to further refine the particles. The grinding temperature is 30℃ and the grinding time is 50min to obtain the glycyrrhizin-glycyrrhizic acid eutectic structure.

[0072] S3. Dissolve dipotassium glycyrrhizate in deionized water to obtain an aqueous solution of dipotassium glycyrrhizate. Mix the aqueous solution of dipotassium glycyrrhizate with the eutectic structure of glycyrrhizin-glycyrrhizic acid and dissolve until clear and transparent to obtain a mixed solution of glycyrrhizin-glycyrrhizic acid.

[0073] S4. Dissolve BOXER and recombinant collagen in water to obtain BOXER aqueous solution and recombinant collagen aqueous solution. Add the recombinant collagen aqueous solution to the BOXER aqueous solution and stir for 30 minutes to obtain BOXER-collagen mixture.

[0074] S5. Add the glycyrrhizin-glycyrrhizic acid mixture to the bisphenol A-collagen mixture and stir for 30 minutes to obtain a glycyrrhizin-bisphenol A-collagen nanoparticle emulsion. Freeze-dry the emulsion to obtain glycyrrhizin-bisphenol A-collagen nanoparticles.

[0075] Example 6

[0076] A method for preparing glycyrrhizin-based composite bosine and collagen nanoparticles includes the following steps:

[0077] S1. Weigh the raw materials according to the following weight percentages: 15% glycyrrhizin, 40% glycyrrhizic acid, 40% dipotassium glycyrrhizate, 9.9% bosine, and 0.1% recombinant collagen, and set aside.

[0078] S2. Use a vortex mixer to mix glycyrrhizin and glycyrrhizic acid evenly, and then use a ball mill to further refine the particles. The grinding temperature is 40℃ and the grinding time is 10min to obtain the glycyrrhizin-glycyrrhizic acid eutectic structure.

[0079] S3. Dissolve dipotassium glycyrrhizate in deionized water to obtain an aqueous solution of dipotassium glycyrrhizate. Mix the aqueous solution of dipotassium glycyrrhizate with the eutectic structure of glycyrrhizin-glycyrrhizic acid and dissolve until clear and transparent to obtain a mixed solution of glycyrrhizin-glycyrrhizic acid.

[0080] S4. Dissolve BOXER and recombinant collagen in water to obtain BOXER aqueous solution and recombinant collagen aqueous solution. Add the recombinant collagen aqueous solution to the BOXER aqueous solution and stir for 30 minutes to obtain BOXER-collagen mixture.

[0081] S5. Add the glycyrrhizin-glycyrrhizic acid mixture to the bisphenol A-collagen mixture and stir for 30 minutes to obtain a glycyrrhizin-bisphenol A-collagen nanoparticle emulsion. Freeze-dry the emulsion to obtain glycyrrhizin-bisphenol A-collagen nanoparticles.

[0082] Examples 1 to 6 of this invention all yielded glycyrrhizin-based composite boswelliae and collagen nanoparticles suitable for cosmetic applications. The following study uses the glycyrrhizin-based composite boswelliae and collagen nanoparticles from Example 1 as an example. Specific research methods and results are shown below:

[0083] Figure 1 The image shows a TEM image of the glycyrrhizin-glycyrrhizic acid eutectic structure coated with β-xylene and collagen nanoparticles in Example 1. The results show that the β-xylene-collagen coated glycyrrhizin-glycyrrhizic acid eutectic structure is composed of nanoparticles.

[0084] The whitening and permeability tests of the glycyrrhizin-based composite boswellicin and collagen nanoparticles of this invention were conducted, specifically:

[0085] Whitening Test: Based on mouse melanoma cells, changes in melanin content and tyrosinase activity were detected to confirm that glycyrrhizin combined with styrosinase and collagen nanoparticles has whitening effects.

[0086] Permeability test: Based on the pig skin-Franz cell system, this test is based on the ex vivo skin model-Franz cell system. After the test sample is applied to the skin for 1 hour, the residual amount of the test sample in the skin is detected by confocal Raman spectroscopy to evaluate the permeability behavior of the sample in the skin.

[0087] The detailed steps for verifying efficacy are as follows:

[0088] 1. Cell safety testing:

[0089] Based on mouse melanoma cells, cell safety tests were conducted to determine the safe dosage range of glycyrrhizin combined with bosine and collagen nanoparticles in mouse melanoma cells.

[0090] 1.1 Experimental Reagents:

[0091] DMEM culture medium (Gibco), fetal bovine serum (Sijiqing), PBS (Boster Biological), CCK-8 detection solution (Diyi), and pancreatic enzyme (Gibco).

[0092] 1.2 Experimental Methods:

[0093] (1) Cell resuscitation: Mouse melanoma cells were taken from a liquid nitrogen tank, resuscitated and inoculated into culture flasks, and cultured in a 37°C, 5% CO2 incubator.

[0094] (2) Cell passage: When the cells reach 70%-80% growth, digest and passage them. After digestion, resuspend the cells in culture medium and count them for later use.

[0095] (3) Cell seeding: Seed cells into 96-well plates at a seeding density of 5000 cells / well and incubate overnight in an incubator (37°C, 5% CO2).

[0096] (4) Experimental grouping: The experiment set up a control group and a sample group; in the sample group, each sample was set with 8 concentration gradients, and 3 replicate wells were set under each concentration gradient.

[0097] (5) Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table (Table 1).

[0098] (6) Drug administration: Drug administration was performed when the cell deposition rate in the 96-well plate reached 40%–60%. 200 μL of the special culture medium was added to each well of the control group, and 200 μL of the corresponding concentration of culture medium was added to each well of the sample group. After drug administration, the 96-well plate was placed in an incubator (37℃, 5% CO2) for culture.

[0099] (7) Detection: After culturing the cells for 24 hours, add 100 μL of 10% CCK-8 detection solution and incubate at 37°C in the dark for 2 hours. After incubation, aspirate 90 μL and read the OD value at 450 nm.

[0100] (8) Calculation of relative cell viability: Calculated according to the formula.

[0101] Table 1 Test Concentration Setting Table

[0102]

[0103]

[0104] Security test results as follows Figure 2 As shown, the sample group demonstrated no cytotoxicity to B16 cells at a concentration of 0.15%, indicating the safety of the glycyrrhizin-based composite bosine and collagen nanoparticles of the present invention.

[0105] 2. Whitening Test:

[0106] 2.1 Melanin content assay based on mouse melanoma cells:

[0107] Based on mouse melanoma cells, an experiment was conducted to detect melanin content and determine whether the sample group had a whitening effect.

[0108] 2.1.1 Experimental reagents:

[0109] 1640 medium (Gibco), DMEM medium (Gibco), fetal bovine serum (Lanzhou Rongye), PBS (Boster), α-melanocyte-stimulating hormone (a-MSH APEX), L-DOPA (Solepro), Triton X-100 (Solepro).

[0110] 2.1.2 Experimental Methods:

[0111] (1) Cell resuscitation: Frozen mouse melanoma cells were taken out of the liquid nitrogen tank and revived. When the cells grew to 70%-80%, they were digested and counted.

[0112] (2) Cell seeding and grouping: Cells were divided into a blank group, a negative control group, and an experimental group, with three replicate wells in each group. 8 × 10⁸ cells were seeded per well. 4Cells were seeded at a density of 100 cells per well in 12-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0113] (3) Drug administration:

[0114] Control group: 1 mL of DMEM culture medium.

[0115] Model group: 1 mL of DMEM medium containing 200 nmol / L α-MSH.

[0116] VC group: 1 mL of DMEM medium containing 200 nmol / L α-MSH and VC (500 ug / mL).

[0117] Sample group: 1 mL of DMEM medium containing 200 nmol / L α-MSH and 0.15% of the sample group.

[0118] Continue culturing for 48 hours in a 37℃, 5% CO2 incubator.

[0119] (4) Melanin content detection:

[0120] After aspirating the supernatant from each well, wash the cells three times with PBS, then fix them with 4% paraformaldehyde for 10 min, wash them three times with ultrapure water, and finally stain them with 500 μL of 0.1% (w / v) L-DOPA solution for 3 h. Observe the cells and take pictures under a 10x microscope using an inverted microscope.

[0121] Melanin content results as follows Figure 3 As shown, the enzyme activity in the model group was significantly increased, indicating that the modeling was successful. Compared with the model group, the enzyme activity in the positive control (VC) group was significantly reduced, indicating that the experiment was effective. Compared with the model group, the enzyme activity in the sample group decreased by 35.82%, which was better than that in the positive control (VC) group, indicating that the combination of glycyrrhizin and styraxin & collagen nanoparticles has a whitening effect.

[0122] 2.2 Tyrosinase activity assay based on mouse melanoma cells:

[0123] Based on mouse melanoma cells, a tyrosinase activity detection experiment was conducted to determine whether the sample group had a whitening effect.

[0124] 2.2.1 Experimental reagents:

[0125] 1640 medium (Gibco), DMEM medium (Gibco), fetal bovine serum (Lanzhou Rongye), PBS (Boster), α-melanocyte-stimulating hormone (a-MSHAPEX), L-DOPA (Solepro), Triton X-100 (Solepro).

[0126] 2.2.2 Experimental Methods:

[0127] (1) Cell resuscitation: Frozen mouse melanoma cells were taken out of the liquid nitrogen tank for resuscitation. When the cells grew to 70%-80%, they were digested and counted.

[0128] (2) Cell seeding and grouping: Cells were divided into a blank group, a negative control group, and an experimental group, with three replicate wells in each group. 8 × 10⁸ cells were seeded per well. 4 Cells were seeded at a density of 100 cells per well in 12-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0129] (3) Drug administration:

[0130] Control group: 1 mL of DMEM culture medium.

[0131] Model group: 1 mL of DMEM medium containing 200 nmol / L α-MSH.

[0132] VC group: 1 mL of DMEM medium containing 200 nmol / L α-MSH and VC (500 ug / mL).

[0133] Sample group: 1 mL of DMEM medium containing 200 nmol / L α-MSH and 0.15% of the sample group.

[0134] Continue culturing for 48 hours in a 37℃, 5% CO2 incubator.

[0135] (4) Tyrosinase activity detection:

[0136] After aspirating the supernatant from each well, wash once with PBS. Add 400 μL of cell lysis buffer containing 1% (V%) Triton X-100 to each well, freeze-lyse at -80℃ for 30 min, then remove and collect the cell lysis buffer from each well. Centrifuge, transfer 100 μL of supernatant to a 96-well plate, add 100 μL of 0.1% (W / V) L-DOPA solution, and incubate at 37℃ for 2 h. Measure the absorbance (A) of each well at 495 nm using a microplate reader and calculate the enzyme activity.

[0137] Enzyme activity results as follows Figure 4 As shown, Figure 4 The results showed that, compared with the control group, melanin production in the model group was significantly increased under α-MSH stimulation, indicating that the model was successfully constructed. Compared with the model group, the cells in the positive control (VC) treatment group were lighter in color, indicating that VC could significantly inhibit melanin production in B16 cells. Compared with the model group, the vast majority of cells in the sample treatment group were significantly lighter in color, indicating that the sample group could inhibit melanin production in B16 cells.

[0138] 3. Transdermal absorption test:

[0139] 3.1 Experimental Methods:

[0140] (1) First, the ex vivo skin is fixed between the supply chamber and the receiving chamber of the Franz cell diffusion cell, with the stratum corneum facing the supply chamber and the dermis facing the receiving chamber.

[0141] (2) Add 4.0 mL of receiving solution to the receiving chamber. After tightening and fixing the excised skin, add 1 mL of receiving solution (PBS) to the receiving chamber through the sampler, remove the air, and make the dermal layer of the skin in close contact with the receiving solution.

[0142] (3) Sample loading: The sample is added to the skin surface in the supply chamber. The sample is selected from collagen or glycyrrhizin-collagen composite nanoparticles of Example 1 of this invention, with an effective penetration area S of approximately 0.785 cm². 2 The sample was added to the surface of the ex vivo skin and spread evenly from the center of the skin to the edge. To meet the statistical requirements of biological samples, each experiment was set up with 3 replicates.

[0143] (4) Infiltration: Turn on the electromagnetic stirrer and stir at a speed of 300 rpm, maintain a constant temperature water bath of 32±1℃, and ensure that there are no air bubbles in the water bath jacket.

[0144] (5) Collect skin samples at 1 hour, clean the skin surface 5 times, wipe the residual liquid on the surface with cotton swabs, cut the skin in a circular motion with a blade, freeze and slice, and set aside for use.

[0145] 3.2 Raman spectroscopy detection experiment:

[0146] (1) The confocal Raman spectrometer was calibrated using a silicon wafer before testing.

[0147] (2) Place the gold-plated quartz slide with frozen sections on the sample stage, and select a part with no separation of the epidermis, no damage to the skin surface, no wrinkles, and clear structure under a microscope to measure its single spectrum.

[0148] (3) Select a range centered on the point where the measured characteristic peak is prominent and the noise is relatively small, start scanning the sample, and obtain the Raman spectrum dataset.

[0149] Figure 5 In the study, the control group consisted of collagen (red in Figure (b)) and the sample group consisted of glycyrrhizin-collagen nanoparticles combined with β-hydroxyl and collagen nanoparticles from Example 1 (blue in Figure (b)). Confocal Raman spectroscopy results showed that the total integrated intensity of both the sample group and the control group increased significantly after 1 hour of skin application, and the permeability of the sample group was better than that of the control group.

[0150] Equal amounts of glycyrrhizin and the glycyrrhizin-glycyrrhizic acid eutectic structure from Example 1 were dissolved in equal amounts of deionized water, and the dissolution was observed. The results are as follows: Figure 6 As shown, Figure 6 The results showed that glycyrrhizin was insoluble in water, while the glycyrrhizin-glycyrrhizic acid eutectic structure was completely soluble in water.

[0151] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from the spirit and scope of the invention. Therefore, it is intended to include any modifications and variations of this invention that fall within the scope of the claims and their equivalents.

Claims

1. A method for preparing glycyrrhizin-composite bosine & collagen nanoparticles, characterized in that, Includes the following steps: Weigh the raw materials according to the following weight percentages: collagen 0.1%~15%, glycyrrhizin 1%~15%, glycyrrhizic acid 2.5%~40%, dipotassium glycyrrhizate 5%~40%, and the remainder is bosine. The sum of the weight percentages of all raw materials is 100%. After mixing glycyrrhizin and glycyrrhizic acid, the mixture was ball-milled. The glycyrrhizin and glycyrrhizic acid were bonded together through non-covalent bonds to obtain a glycyrrhizin-glycyrrhizic acid eutectic structure. A pH adjuster, dipotassium glycyrrhizate aqueous solution, was mixed with a glycyrrhizin-glycyrrhizic acid eutectic structure to adjust the pH to 4.0-4.5, and a glycyrrhizin-glycyrrhizic acid mixture was obtained. Collagen aqueous solution is added to BOXER aqueous solution and mixed. BOXER and collagen bind through positive and negative charge interactions to obtain a BOXER-collagen mixture. A mixture of glycyrrhizin and glycyrrhizic acid was added to a mixture of bosine and collagen. After mixing, the eutectic structure of glycyrrhizin and glycyrrhizic acid was bonded to bosine and collagen through π-π bonds by intermolecular forces. The eutectic structure of glycyrrhizin and glycyrrhizic acid was then coated with the bosine and collagen mixture to obtain a glycyrrhizin composite bosine & collagen nanoparticle emulsion. After freeze-drying, glycyrrhizin composite bosine & collagen nanoparticles were obtained.

2. The preparation method of glycyrrhizin-composite bosine & collagen nanoparticles according to claim 1, characterized in that, Weigh the raw materials according to the following weight percentages: collagen 5%~10%, glycyrrhizin 5%~10%, glycyrrhizic acid 10%~25%, dipotassium glycyrrhizate 5%~10%, and the remainder is bosine. The sum of the weight percentages of each raw material is 100%.

3. The preparation method of the glycyrrhizin-composite bosine & collagen nanoparticles according to claim 1, characterized in that, The mass ratio of glycyrrhizin to glycyrrhizic acid is 1~1.5:1~5.

4. The preparation method of the glycyrrhizin-composite bosine & collagen nanoparticles according to claim 1, characterized in that, The ball milling conditions are: ball milling at 30℃~50℃ for 10min~50min.

5. The preparation method of glycyrrhizin-composite bosine & collagen nanoparticles according to claim 1, characterized in that, The mixing time between the collagen aqueous solution and the BOXER aqueous solution is 10 min to 30 min.

6. The preparation method of glycyrrhizin composite bosine & collagen nanoparticles according to claim 1, characterized in that, The mixing time for the glycyrrhizin-glycyrrhizic acid mixture and the bosine-collagen mixture is 10 min to 30 min.

7. A glycyrrhizin-based composite bosine and collagen nanoparticle prepared by the preparation method according to any one of claims 1 to 6.

8. The glycyrrhizin-composite bosine & collagen nanoparticles according to claim 7, characterized in that, The glycyrrhizin-glycoside composite β-xylene & collagen nanoparticles have a β-xylene-collagen-coated glycyrrhizin-glycyrrhizic acid eutectic structure.

9. The application of the glycyrrhizin-composite bosine & collagen nanoparticles as described in claim 7 in the preparation of cosmetics, characterized in that, The glycyrrhizin-based composite of bosine and collagen nanoparticles is used to improve the stability and solubility of glycyrrhizin; enhance the permeability of collagen; reduce melanin production; and have anti-aging effects.

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