Glabridin composite glabridin collagen nano-particles as well as preparation method and application of glabridin composite glabridin collagen nano-particles

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a glabridin composite glabridin amp. The invention discloses collagen nanoparticles as well as a preparation method and application thereof. The preparation method comprises the following steps: firstly, combining glabridin and glycyrrhizic acid through a non-covalent bond to obtain a glabridin-glycyrrhizic acid eutectic structure capable of overcoming the problem of solubility of glabridin, and then coating the glabridin-glycyrrhizic acid eutectic structure with a glabridin-collagen mixed solution to obtain the glabridin composite glabridin amp. The invention relates to collagen nanoparticles. The glabridin composite glabridin amp obtained by the method disclosed by the invention; the collagen nano-particles and the glabridin are good in water solubility, simple in component, high in stability and free of excessive allergy risks, the skin permeability of the glabridin, the collagen and the glabridin can be improved, the collagen, the glabridin and the glabridin are in synergistic interaction, and the utilization degree is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and specifically relates to a liquiritin complex with hydroxyprolisilane & collagen nanoparticles and its preparation method and application. Background Art

[0002] Aging, long-term ultraviolet irradiation, irregular work and rest, and malnutrition and other lifestyles can all lead to skin aging, usually accompanied by some obvious characteristics and manifestations, such as skin relaxation, wrinkles and fine lines, pigmentation, skin dryness, enlarged pores, etc. Adding the natural and highly effective whitening factor liquiritin can cooperate with the skin's self-repair process and enhance the whitening effect. However, due to the water-insoluble property of liquiritin, the application of liquiritin is affected.

[0003] The prior art generally uses a coating method to solve the water solubility problem of liquiritin. The main encapsulants for liquiritin are liposomes, cyclodextrins, or exosomes. For example, in the patent "A Whitening Essence Based on Phospholipid-Encapsulated Liquiritin Nanomulsion and Its Preparation Method", publication number CN113893191A, in this method, phospholipids and liquiritin are dissolved in an organic solvent to obtain a liquiritin-phospholipid complex, and then an emulsifier is added for homogenization to form a nanomulsion. A large amount of emulsifiers and stabilizers are used in this method, and there is a certain risk of allergy.

[0004] In the patent "A Multiple-Encapsulated Solid Preparation of Liquiritin and Its Preparation Method", publication number CN108815012A, in this method, liquiritin is first encapsulated with cyclodextrin, and then secondary encapsulation is carried out with glycyrrhizic acid and glycyrrhizic acid polysaccharide. The content of liquiritin encapsulated by this method is limited, and the encapsulation carrier also does not have a functional effect.

[0005] In summary, none of these prior arts have solved the basic characteristics of liquiritin preparations as cosmetic raw materials, which are simple, stable, risk-free, and highly water-soluble.

[0006] Both hydroxyprolisilane and collagen are effective anti-aging ingredients. The characteristics of large molecular weight and stable molecular structure of collagen make it difficult for macromolecular collagen to enter the deep layer of the skin through the tight structure of the stratum corneum; in addition, a large number of hydrophilic amino acids are contained in the amino acid composition of collagen, making it easy for collagen to form a hydration layer on the skin surface and difficult to penetrate the skin barrier into the deep tissue, resulting in poor skin permeability. Summary of the Invention

[0007] In view of the deficiencies of the above-mentioned existing technologies, the present invention provides a glabridin complex with hydroxyprolisilane and collagen nanoparticles, a preparation method thereof, and an application thereof. First, glabridin and glycyrrhizic acid are combined by non-covalent bonds to obtain a glabridin-glycyrrhizic acid co-crystal structure that overcomes the solubility problem of glabridin. Then, a hydroxyprolisilane-collagen mixed solution is used to coat the glabridin-glycyrrhizic acid co-crystal structure to obtain glabridin complex with hydroxyprolisilane and collagen nanoparticles. The glabridin complex with hydroxyprolisilane and collagen nanoparticles obtained by the method of the present invention has good water solubility, simple composition, high stability, and no excessive allergy risk. It can also improve the skin permeability of collagen, enabling the three of them to synergistically enhance the utilization rate.

[0008] The present invention is realized by adopting the following technical solutions:

[0009] The present invention protects a preparation method of glabridin complex with hydroxyprolisilane and collagen nanoparticles, comprising the following steps:

[0010] Weigh the raw materials according to the following weight percentages: collagen 0.1% - 15%, glabridin 1% - 15%, glycyrrhizic acid 2.5% - 40%, dipotassium glycyrrhizinate 5% - 40%, and the balance is hydroxyprolisilane. 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 glabridin and glycyrrhizic acid, ball milling is carried out. Glabridin and glycyrrhizic acid are combined by non-covalent bonds to obtain a glabridin-glycyrrhizic acid co-crystal structure. Glycyrrhizic acid makes glabridin have good water solubility; in the present invention, a water-soluble co-crystal of glabridin and glycyrrhizic acid is formed, significantly improving the water solubility and dispersibility of glabridin. Its solubility in water is increased to ≥20mg / mL (the initial water solubility of glabridin ≤0.1mg / mL), and the stability of glabridin can be enhanced.

[0012] Mix an aqueous solution of the pH regulator dipotassium glycyrrhizinate with the glabridin-glycyrrhizic acid mixture, adjust the pH to 4.0 - 4.5, and obtain a clear and transparent glabridin-glycyrrhizic acid mixture; the purpose of adjusting the pH is to be suitable for skin application.

[0013] Add an aqueous solution of recombinant collagen to an aqueous solution of hydroxyprolisilane. Hydroxyprolisilane and collagen are combined by positive and negative charge interaction to obtain a hydroxyprolisilane-collagen mixed solution.

[0014] Add the glabridin-glycyrrhizic acid mixture to the hydroxyprolisilane-collagen mixture, mix them, and utilize the intermolecular forces. The glabridin-glycyrrhizic acid co-crystal structure and hydroxyprolisilane-collagen are assembled into nanoparticles through intermolecular forces (mainly π-π bonds). At this time, hydroxyprolisilane-collagen coats the surface of the glabridin-glycyrrhizic acid co-crystal structure to obtain a glabridin composite hydroxyprolisilane & collagen nanoparticle emulsion. Freeze-dry the glabridin composite hydroxyprolisilane & collagen nanoparticle emulsion to obtain glabridin composite hydroxyprolisilane & collagen nanoparticles.

[0015] Preferably, weigh the raw materials according to the following weight percentages: collagen 0.1% - 15%, glabridin 1% - 15%, glycyrrhizic acid 2.5% - 40%, dipotassium glycyrrhizinate 5% - 40%, and the balance is hydroxyprolisilane. The sum of the weight percentages of each raw material is 100%. Under this mass percentage, the obtained glabridin composite hydroxyprolisilane & collagen nanoparticles have better performance.

[0016] Preferably, the mass ratio of glabridin to glycyrrhizic acid is 1 - 1.5:1 - 5. Research shows that when the mass ratio of glabridin to glycyrrhizic acid is 1:2.5, the obtained glabridin-glycyrrhizic acid co-crystal structure has the best water solubility.

[0017] Preferably, the conditions for ball milling are: ball milling at 30°C - 50°C for 10 min - 50 min.

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

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

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

[0021] Preferably, in the hydroxyprolisilane aqueous solution, deionized water is 5 - 10 times that of hydroxyprolisilane.

[0022] The present invention also protects the glabridin composite hydroxyprolisilane & collagen nanoparticles prepared by the above preparation method.

[0023] Preferably, the glabridin composite hydroxyprolisilane & collagen nanoparticles are: a hydroxyprolisilane-collagen-coated glabridin-glycyrrhizic acid co-crystal structure.

[0024] The present invention also protects the application of the glabridin composite hydroxyprolisilane & 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. First, liquiritin and glycyrrhizic acid are combined through non-covalent bonds to form a stable structure, obtaining a liquiritin-glycyrrhizic acid co-crystal structure, which makes liquiritin become a water-soluble substance. Then, the liquiritin-glycyrrhizic acid co-crystal structure is dissolved in an aqueous solution of dipotassium glycyrrhizinate as a pH regulator to obtain a liquiritin-glycyrrhizic acid mixture, and the pH is adjusted to the application range acceptable to the skin. Next, an aqueous solution of collagen is mixed with an aqueous solution of hydroxyprolisilane to form a hydroxyprolisilane-collagen mixture through electrostatic interaction. Finally, with hydroxyprolisilane-collagen as the shell and the liquiritin-glycyrrhizic acid co-crystal structure as the core, further assembly is carried out by using the intermolecular forces among liquiritin-glycyrrhizic acid, collagen, and hydroxyprolisilane to form a hydroxyprolisilane-collagen-coated liquiritin-glycyrrhizic acid co-crystal structure, obtaining liquiritin composite hydroxyprolisilane & collagen nanoparticles.

[0027] 2. Considering the problem of poor transdermal permeability of adding collagen and hydroxyprolisilane in cosmetics, in the liquiritin composite hydroxyprolisilane & collagen nanoparticles of the present invention, the solute is liquiritin composite hydroxyprolisilane & collagen nanoparticles. The nanoparticle structure has good transdermal permeability and high utilization rate, solving the problems that collagen and hydroxyprolisilane are difficult to penetrate the skin and have poor bioavailability. And compared with conventional hydroxyprolisilane and collagen, the liquiritin composite hydroxyprolisilane & collagen nanoparticles prepared in the present invention have a more obvious skin whitening effect. The reason is that the nanoparticles have good transdermal permeability and high utilization rate, enabling them to better exert the whitening effect, and combining the anti-aging effects of hydroxyprolisilane and collagen, and can have more applications in the field of skin care products.

[0028] 3. In the present invention, on the one hand, glycyrrhizic acid is used to achieve non-covalent binding with liquiritin, and on the other hand, collagen-hydroxyprolisilane is used to coat the liquiritin-glycyrrhizic acid co-crystal structure, solving the problems of poor water solubility and instability of liquiritin.

[0029] 4. In terms of efficacy, the liquiritin composite hydroxyprolisilane & collagen nanoparticles of the present invention, hydroxyprolisilane has an anti-wrinkle effect, and collagen can efficiently supplement collagen in the skin. The liquiritin composite hydroxyprolisilane & collagen nanoparticles prepared with these three as raw materials achieve the synergistic effect of collagen, hydroxyprolisilane, and liquiritin in terms of efficacy. Collagen and hydroxyprolisilane play an anti-aging role, and liquiritin plays a whitening effect, thus better solving skin problems.

[0030] 5. The liquiritin composite hydroxyprolisilane & collagen nanoparticles provided by the present invention are in a stable state and have good water solubility; at the same time, the raw materials for preparation are easily available, the preparation method is simple, the safety risk is small, the stability is high, and the transdermal permeability is good, and it has great application potential in the field of skin care product raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 TEM image of glabridin complexed with hydroxyprolisilane & collagen nanoparticles in Example 1.

[0032] Figure 2 Cell safety evaluation results of glabridin complexed with hydroxyprolisilane & collagen nanoparticles in Example 1 based on mouse melanoma cells.

[0033] Figure 3 Results of melanin content of glabridin complexed with hydroxyprolisilane & collagen nanoparticles in Example 1 based on mouse melanoma cells.

[0034] Figure 4 Results of tyrosinase enzyme activity of glabridin complexed with hydroxyprolisilane & collagen nanoparticles in Example 1.

[0035] Figure 5 Results of cumulative penetration amount experiment of glabridin complexed with hydroxyprolisilane & collagen nanoparticles and collagen in Example 1. Among them, (a) is the confocal Raman image, and (b) is the total integral intensity image.

[0036] Figure 6 Water solubility comparison of glabridin (a) and glabridin-glycyrrhizic acid co-crystal structure (b) in Example 1. Detailed Description of the Invention

[0037] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the protection scope of the present invention is not limited by the specific embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0038] Considering that the existing technology uses liposomes, cyclodextrins or exosomes to coat glabridin, which requires adding emulsifiers or other complex components, and the addition of more components will cause potential risk problems. The present invention first proposes to bind glabridin and glycyrrhizic acid through non-covalent bonds. Glycyrrhizic acid makes glabridin have good water solubility, overcomes the defects caused by doping complex components during the coating process, and at the same time overcomes the problem that glabridin is insoluble or hardly soluble in water. Glycyrrhizic acid has antioxidant and whitening properties and is often added to skin care products, which can reduce skin inflammation, promote skin repair, improve uneven skin tone or reduce age spots. Then, hydroxyprolisilane-collagen is used to coat the glabridin-glycyrrhizic acid co-crystal structure to form nanoparticles. Due to the small volume effect of the nanoparticles, the transdermal permeability of hydroxyprolisilane and collagen is improved. At the same time, since the glabridin-glycyrrhizic acid co-crystal structure is coated inside the hydroxyprolisilane-collagen, the stability of glabridin is improved.

[0039] The technical solution of the present invention is further studied by using the following examples, and the specific research methods and results are as follows:

[0040] Example 1

[0041] A preparation method of glabridin composite with hydroxyprolisilane & collagen nanoparticles comprises the following steps:

[0042] S1. Weigh the raw materials according to the following weight percentages: 10% of glabridin, 25% of glycyrrhizic acid, 10% of dipotassium glycyrrhizinate, 45% of hydroxyprolisilane, and 10% of recombinant collagen, and set aside.

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

[0044] S3. Dissolve dipotassium glycyrrhizinate in deionized water to obtain a dipotassium glycyrrhizinate aqueous solution. Mix the dipotassium glycyrrhizinate aqueous solution with the glabridin-glycyrrhizic acid eutectic structure and dissolve it until it is clear and transparent to obtain a glabridin-glycyrrhizic acid mixture.

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

[0046] S5. Add the glabridin-glycyrrhizic acid mixture to the hydroxyprolisilane-collagen mixture, mix and stir for 30 min to obtain a glabridin composite with hydroxyprolisilane & collagen nanoparticle emulsion, and then freeze-dry it to obtain glabridin composite with hydroxyprolisilane & collagen nanoparticles.

[0047] Example 2

[0048] A preparation method of glabridin composite with hydroxyprolisilane & collagen nanoparticles comprises the following steps:

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

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

[0051] S3. Dissolve dipotassium glycyrrhizinate in deionized water to obtain a dipotassium glycyrrhizinate aqueous solution. Mix the dipotassium glycyrrhizinate aqueous solution with the glabridin-glycyrrhizic acid co-crystal structure and dissolve until clear and transparent to obtain a glabridin-glycyrrhizic acid mixture.

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

[0053] S5. Add the glabridin-glycyrrhizic acid mixture to the hydroxyproline-collagen mixture, mix and stir for 30 min to obtain a glabridin composite hydroxyproline & collagen nanoparticle emulsion, and then freeze-dry to obtain glabridin composite hydroxyproline & collagen nanoparticles.

[0054] Example 3

[0055] A preparation method of glabridin composite hydroxyproline & collagen nanoparticles, comprising the following steps:

[0056] S1. Weigh the raw materials according to the following weight percentages: 10% of glabridin, 25% of glycyrrhizic acid, 30% of dipotassium glycyrrhizinate, 25% of hydroxyproline, and 10% of recombinant collagen, and set aside.

[0057] S2. Use a vortex mixer to mix glabridin and glycyrrhizic acid evenly, and then further refine the particles through a ball mill. The grinding temperature is 50 °C and the grinding time is 30 min to obtain a glabridin-glycyrrhizic acid co-crystal structure.

[0058] S3. Dissolve dipotassium glycyrrhizinate in deionized water to obtain a dipotassium glycyrrhizinate aqueous solution. Mix the dipotassium glycyrrhizinate aqueous solution with the glabridin-glycyrrhizic acid co-crystal structure and dissolve until clear and transparent to obtain a glabridin-glycyrrhizic acid mixture.

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

[0060] S5. Add the glabridin-glycyrrhizic acid mixture to the hydroxyproline-collagen mixture, mix and stir for 30 min to obtain a glabridin composite hydroxyproline & collagen nanoparticle emulsion, and then freeze-dry to obtain glabridin composite hydroxyproline & collagen nanoparticles.

[0061] Example 4

[0062] A preparation method of glabridin composite hydroxyproline & collagen nanoparticles, comprising the following steps:

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

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

[0065] S3. Dissolve dipotassium glycyrrhizate in deionized water to obtain a dipotassium glycyrrhizate aqueous solution. Mix the dipotassium glycyrrhizate aqueous solution with the glabridin-glycyrrhizic acid eutectic structure and dissolve until it is clear and transparent to obtain a glabridin-glycyrrhizic acid mixture.

[0066] S4. Dissolve hydroxyprolisilane and recombinant collagen in water respectively to obtain a hydroxyprolisilane aqueous solution and a recombinant collagen aqueous solution. Add the recombinant collagen aqueous solution to the hydroxyprolisilane aqueous solution and stir for 20 min to obtain a hydroxyprolisilane-collagen mixture.

[0067] S5. Add the glabridin-glycyrrhizic acid mixture to the hydroxyprolisilane-collagen mixture, mix and stir for 20 min to obtain a glabridin composite hydroxyprolisilane & collagen nanoparticle emulsion, and freeze-dry it to obtain glabridin composite hydroxyprolisilane & collagen nanoparticles.

[0068] Example 5

[0069] A preparation method of glabridin composite hydroxyprolisilane & collagen nanoparticles, comprising the following steps:

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

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

[0072] S3. Dissolve dipotassium glycyrrhizate in deionized water to obtain a dipotassium glycyrrhizate aqueous solution. Mix the dipotassium glycyrrhizate aqueous solution with the glabridin-glycyrrhizic acid eutectic structure and dissolve until it is clear and transparent to obtain a glabridin-glycyrrhizic acid mixture.

[0073] S4. Dissolve hydroxyprolisilane and recombinant collagen in water respectively to obtain a hydroxyprolisilane aqueous solution and a recombinant collagen aqueous solution. Add the recombinant collagen aqueous solution to the hydroxyprolisilane aqueous solution and stir for 30 min to obtain a hydroxyprolisilane-collagen mixture.

[0074] S5. Add the glabridin-glycyrrhizic acid mixture to the hydroxyprolisilane-collagen mixture, mix and stir for 30 min to obtain a glabridin composite hydroxyprolisilane & collagen nanoparticle emulsion, and then freeze-dry it to obtain glabridin composite hydroxyprolisilane & collagen nanoparticles.

[0075] Example 6

[0076] A preparation method of glabridin composite hydroxyprolisilane & collagen nanoparticles comprises the following steps:

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

[0078] S2. Use a vortex mixer to mix glabridin and glycyrrhizic acid evenly, and then further refine the particles by a ball mill at a grinding temperature of 40 °C for 10 min to obtain a glabridin-glycyrrhizic acid eutectic structure.

[0079] S3. Dissolve dipotassium glycyrrhizinate in deionized water to obtain a dipotassium glycyrrhizinate aqueous solution, mix the dipotassium glycyrrhizinate aqueous solution with the glabridin-glycyrrhizic acid eutectic structure, and dissolve until it is clear and transparent to obtain a glabridin-glycyrrhizic acid mixture.

[0080] S4. Dissolve hydroxyprolisilane and recombinant collagen in water respectively to obtain a hydroxyprolisilane aqueous solution and a recombinant collagen aqueous solution, add the recombinant collagen aqueous solution to the hydroxyprolisilane aqueous solution, and stir for 30 min to obtain a hydroxyprolisilane-collagen mixture.

[0081] S5. Add the glabridin-glycyrrhizic acid mixture to the hydroxyprolisilane-collagen mixture, mix and stir for 30 min to obtain a glabridin composite hydroxyprolisilane & collagen nanoparticle emulsion, and then freeze-dry it to obtain glabridin composite hydroxyprolisilane & collagen nanoparticles.

[0082] In Examples 1 to 6 of the present invention, glabridin composite hydroxyprolisilane & collagen nanoparticles that can be applied to cosmetics are prepared. Taking the glabridin composite hydroxyprolisilane & collagen nanoparticles of Example 1 as an example for research, the specific research methods and results are as follows:

[0083] Figure 1 is the TEM image of the glabridin composite hydroxyprolisilane & collagen nanoparticles of Example 1. The results show that the glabridin-glycyrrhizic acid eutectic structure coated with hydroxyprolisilane-collagen is nanoparticles.

[0084] The whitening test and permeability test of the glabridin complex hydroxyprolisilane & collagen nanoparticles of the present invention were carried out, specifically as follows:

[0085] Whitening test: Based on mouse melanoma cells, the changes in melanin content and the enzyme activity of tyrosinase were detected to confirm that the glabridin complex hydroxyprolisilane & collagen nanoparticles have whitening effects.

[0086] Permeability test: Based on the suckling pig skin - Franz cell system, this test is based on the in vitro skin model - Franz cell system. After the test sample acts on the skin for 1 h, a confocal Raman spectrometer is used to detect the residual amount of the test sample in the skin to evaluate the penetration behavior of the sample in the skin.

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

[0088] 1. Cell safety test:

[0089] Based on mouse melanoma cells, cell safety detection was carried out to determine the safe dosing concentration range of the glabridin complex hydroxyprolisilane & collagen nanoparticles on mouse melanoma cells.

[0090] 1.1 Experimental reagents:

[0091] DMEM culture medium (Gibco), fetal bovine serum (Sijiqing), PBS (Boshide), CCK-8 detection solution (Diyi), trypsin (Gibco).

[0092] 1.2 Experimental methods:

[0093] (1) Cell resuscitation: Take mouse melanoma cells from the liquid nitrogen tank, resuscitate and inoculate them into a culture flask, and culture them in an incubator at 37°C and 5% CO 2 .

[0094] (2) Cell passage: When the cells grow to 70% - 80%, digest and passage them. The digested cells are resuspended with the culture medium and counted for standby.

[0095] (3) Cell seeding: Seed the cells into a 96-well plate at an inoculation density of 5000 cells / well, and incubate them overnight in an incubator (37°C, 5% CO 2 ).

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

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

[0098] (6) Administration: Administration is carried out when the cell seeding rate in the 96-well plate reaches 40% - 60%. Add 200 μL of the special culture medium to each well in the control group, and add 200 μL of the culture medium containing the corresponding concentration to each well in the sample group. After the administration is completed, place the 96-well plate in an incubator (37 °C, 5% CO 2 ) for incubation.

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

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

[0101] Table 1 Test concentration setting table

[0102]

[0103]

[0104] The safety test results are as Figure 2 shown, proving that the sample group has no cytotoxicity based on B16 cells at a concentration of 0.15%, indicating that the photoglycyrolide complex with hydroxyprolisilane & collagen nanoparticles of the present invention has safety.

[0105] 2. Whitening test:

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

[0107] Based on mouse melanoma cells, conduct an experiment to detect the melanin content to determine whether the samples in the sample group have 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 (Solarbio), TritonX-100 (Solarbio).

[0110] 2.1.2 Experimental method:

[0111] (1) Cell resuscitation: Take out the cryopreserved mouse melanoma cells from the liquid nitrogen tank for resuscitation, and digest and count the cells when they grow to 70% - 80%.

[0112] (2) Cell seeding and grouping: It is divided into a blank group, a negative group and an experimental group, with 3 replicate wells in each group. At 8×10 cells per well 4Cells were seeded in 12-well plates at a density of 2 and cultured in an incubator at 37°C with 5% CO₂ for 24 h.

[0113] (3) Drug administration treatment:

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

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

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

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

[0118] The cells were further cultured in an incubator at 37°C with 5% CO₂ 2 for 48 h.

[0119] (4) Detection of melanin content:

[0120] After aspirating the supernatant of each well, the cells were washed 3 times with PBS, then fixed with 4% paraformaldehyde for 10 min, washed with ultrapure water 3 times, and finally stained with 500 μL of 0.1% (W / V) L-DOPA solution for 3 h. The cells were observed and photographed under an inverted microscope at 10× magnification.

[0121] The results of melanin content were as Figure 3 shown. The enzyme activity in the model group was significantly increased, indicating that the model was successfully established in this experiment; compared with the model group, the enzyme activity in the positive control (VC) group was significantly decreased, indicating the effectiveness of this experiment; compared with the model group, the enzyme activity in the sample group decreased, and the decrease rate was 35.82%, and it was better than the positive control (VC) group, indicating that glabridin complexed with hydroxyprolisilane & collagen nanoparticles has a whitening effect.

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

[0123] Based on mouse melanoma cells, a tyrosinase activity detection experiment was carried out to determine whether the sample in the sample group has 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 (Solarbio), Triton X-100 (Solarbio).

[0126] 2.2.2 Experimental method:

[0127] (1) Cell resuscitation: Take out the cryopreserved mouse melanoma cells from the liquid nitrogen tank for resuscitation. When the cells grow to 70%-80%, digest and count them.

[0128] (2) Cell seeding and grouping: It was divided into a blank group, a negative group and an experimental group, with 3 replicate wells in each group. Seed at a density of 8×10 4 cells per well in a 12-well plate and culture in an incubator at 37°C and 5% CO 2 for 24 h.

[0129] (3) Drug administration treatment:

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

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

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

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

[0134] Continue to culture in an incubator at 37°C and 5% CO 2 for 48 h.

[0135] (4) Tyrosinase activity detection:

[0136] After aspirating the supernatant of each well, wash it once with PBS. Add 400 μL of cell lysate containing 1% (V%) Triton X-100 to each well. Take it out after freezing and lysing in a -80°C refrigerator for 30 min. Centrifuge the cell lysate collected from each well, take 100 μL of the supernatant and transfer it to a 96-well plate, and add 100 μL of 0.1% (W / V) L-DOPA solution. Incubate at 37°C for 2 h, measure the absorbance (A) of each well at a wavelength of 495 nm with an enzyme-linked immunosorbent assay (ELISA) reader and calculate the enzyme activity.

[0137] The enzyme activity results are as Figure 4 shown, Figure 4The results showed that, compared with the control group, the melanogenesis of cells in the model group increased significantly under the stimulation of α-MSH, indicating the successful establishment of the model. Compared with the model group, the cells in the positive control (VC) treatment group were lighter in color, indicating that VC could significantly inhibit the melanogenesis of B16 cells. Compared with the model group, the vast majority of cells in the sample group treatment group were significantly lighter in color, indicating that the sample group could inhibit the melanogenesis of B16 cells.

[0138] 3. Transdermal absorption experiment:

[0139] 3.1 Experimental method:

[0140] (1) First, fix the excised skin between the supply chamber and the receiving chamber of the Franz cell diffusion cell, with the stratum corneum of the skin facing the supply chamber and the dermal layer facing the receiving chamber.

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

[0142] (3) Sample loading: Add the sample to the skin surface in the supply chamber. The sample is selected from collagen or the liquiritin complex with boswellia serrata extract & collagen nanoparticles of Example 1 of the present invention, and the effective penetration area S is about 0.785 cm 2 ; Add the sample to the excised skin surface, and spread it evenly from the central part of the skin radially to the edge. To meet the statistics of biological samples, 3 replicates are set for each group of experiments.

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

[0144] (5) Collect skin samples at the 1 h time point, wash the skin surface 5 times, dry the residual liquid on the surface with a cotton swab, cut the skin with a scalpel, and prepare frozen sections for use.

[0145] 3.2 Raman spectroscopy detection experiment:

[0146] (1) Calibrate the confocal Raman spectrometer with a silicon wafer before detection.

[0147] (2) Place the quartz gold-plated slide with the frozen section on the sample stage, and select a site under the microscope where the epidermis and dermis are not separated, the skin surface is not damaged, there are no wrinkles, and the structure is clear, and measure its single spectrum.

[0148] (3) Select a range centered on the point with prominent measured characteristic peaks and relatively low noise, and start scanning the sample to obtain a Raman spectroscopy data set.

[0149] Figure 5 Among them, the control group was collagen (red in Figure (b)), and the sample group was the glabridin complex with hydroxyprolisilane & collagen nanoparticles of Example 1 (blue in Figure (b)). The confocal Raman results showed that after the sample group and the control group acted on the skin for 1 h, the total integral intensity increased significantly, and the penetration performance of the sample group was better than that of the control group.

[0150] Equal amounts of glabridin and the glabridin-glycyrrhizic acid co-crystal structure of Example 1 were respectively dissolved in equal amounts of deionized water, and the dissolution situation was observed. The results are as Figure 6 shown Figure 6 The results showed that glabridin was insoluble in water, and the glabridin-glycyrrhizic acid co-crystal structure was completely soluble in water.

[0151] Obviously, those skilled in the art can make various changes and deformations to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and deformations of the present invention fall within the scope of the claims of the present invention and its equivalent technologies, these changes and deformations are also intended to be included.

Claims

1. A method for preparing glabridin composite bosine & collagen nanoparticles, characterized in that: The steps include: Weigh the raw materials according to the following weight percentages: collagen 0.1%-15%, glabridin 1%-15%, glycyrrhizic acid 2.5%-40%, dipotassium glycyrrhizinate 5%-40%, and the remainder is bosine. The sum of the weight percentages of the raw materials is 100%; After mixing glabridin and glycyrrhizic acid, ball milling is performed, and glabridin and glycyrrhizic acid are bonded via non-covalent bonds to obtain a glabridin-glycyrrhizic acid eutectic structure; Mixing a pH regulator dipotassium glycyrrhizinate aqueous solution with a glabridin-glycyrrhizic acid eutectic structure, adjusting the pH to 4.0-4.5, and obtaining a glabridin-glycyrrhizic acid mixed solution; Adding the collagen aqueous solution to the bosogen aqueous solution, mixing, bosogen and collagen are combined through the action of positive and negative charges to obtain a bosogen-collagen mixed solution; The glabridin-glycyrrhizic acid mixed solution is added to the bosone-collagen mixed solution, mixed, and the glabridin-glycyrrhizic acid eutectic structure is combined with the bosone-collagen through a π-π bond by utilizing intermolecular forces, and the bosone-collagen mixed solution is used to coat the glabridin-glycyrrhizic acid eutectic structure to obtain a glabridin composite bosone & collagen nanoparticle emulsion, which is freeze-dried to obtain glabridin composite bosone & collagen nanoparticles.

2. The method for preparing glabridin composite bosine & collagen nanoparticles according to claim 1, characterized in that: The raw materials are weighed according to the following weight percentages: 5% to 10% collagen, 5% to 10% glabridin, 10% to 25% glycyrrhizic acid, 5% to 10% dipotassium glycyrrhizinate, and the remainder is bosquinone. The sum of the weight percentages of the raw materials is 100%.

3. The method for preparing glabridin composite bosine & collagen nanoparticles according to claim 1, characterized in that: The mass ratio of glabridin to glycyrrhizic acid is 1-1.5:1-5.

4. The method for preparing glabridin composite bosine & collagen nanoparticles according to claim 1, characterized in that: The ball milling conditions are: ball milling at 30°C to 50°C for 10 min to 50 min.

5. The method for preparing glabridin composite bosine & collagen nanoparticles according to claim 1, characterized in that: The mixing time of the collagen aqueous solution and the bosogenin aqueous solution is 10 minutes to 30 minutes.

6. The method for preparing glabridin composite bosine & collagen nanoparticles according to claim 1, characterized in that: The mixing time of the glabridin-glycyrrhizic acid mixed solution and the bosone-collagen mixed solution is 10 minutes to 30 minutes.

7. A glabridin composite bosogenin & collagen nanoparticle prepared by the preparation method according to any one of claims 1 to 6.

8. The glabridin composite bosine & collagen nanoparticles according to claim 7, characterized in that: The glabridin composite bosera & collagen nanoparticles are: a glabridin-glycyrrhizic acid eutectic structure coated with bosera-collagen.

9. Use of the glabridin combined with bosine & collagen nanoparticles according to claim 7 in the preparation of cosmetics.

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