Nanomicelles of resveratrol-tocophersolan and their use in the preparation of antioxidant and skin whitening products

Abstract

The application relates to the technical field of cosmetics and transdermal drug delivery, in particular to a resveratrol-tocophersolan nanomicelle and application thereof in preparation of antioxidant and skin whitening products. The preparation method comprises the following steps: adding resveratrol or a derivative thereof into a first organic solvent (0.1%-2% in weight percentage) to be heated and dissolved at a first temperature (80-100 DEG C) to obtain a first solution; adding tocophersolan into the first solution to be mixed (0.5%-20% in weight percentage) to be heated and dissolved at a second temperature (80-100 DEG C) to obtain a second solution; and adding the second solution into deionized water at a first speed (1-3 mL / min) to be stirred and dispersed at a third temperature (50-70 DEG C). The nanomicelle has high stability and excellent transdermal absorption performance, good anti-photoaging effect, and good skin whitening and antioxidant effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetics and transdermal drug delivery, in particular to resveratrol-tocophersolan nanomicelles and their use in the preparation of anti-oxidation and skin whitening products. BACKGROUND

[0002] Skin aging is a natural law of the human body, but it is also related to some exogenous factors, and the ultraviolet radiation of the sun is one of the main exogenous factors causing skin photoaging. Skin photoaging is a skin damage caused by long-term ultraviolet (UV) radiation, characterized by oxidative stress, inflammatory response and collagen degradation, ultimately leading to wrinkles, pigmentation and impaired skin barrier function. With people's increasing concern about skin health and beauty, developing effective anti-oxidation and anti-photoaging products has become a research hotspot. In recent years, resveratrol and its derivatives have received widespread attention in the field of anti-photoaging due to their excellent anti-oxidation and anti-inflammatory properties.

[0003] Resveratrol is a self-protective factor produced by plants when stimulated, and is a stilbene compound with multiple hydroxyl substituents, systematically named (E)-3,4',5-trihydroxystilbene. It is a white needle-like crystal, poorly soluble in water, and easily soluble in organic solvents such as diethyl ether, chloroform, methanol, ethanol, acetone, ethyl acetate, etc. Resveratrol has strong free radical scavenging ability and can effectively inhibit active oxygen free radical-mediated cell damage. It also has multiple activities such as regulating melanin production enzyme activity, regulating inflammatory response of the body, and promoting collagen production, and shows great potential in the field of skin anti-photoaging. However, resveratrol is sensitive to light, easily oxidized and deactivated, and has poor solubility in aqueous phase, resulting in poor permeability on the skin surface and difficulty in effectively penetrating the epidermal barrier to exert its biological function. The above characteristics greatly limit the practical application of resveratrol. SUMMARY

[0004] The present application found through a large number of experiments that resveratrol-tocophersolan nanomicelles prepared by using tocophersolan as a carrier and by a suitable method have small particle size, high stability and excellent transdermal absorption performance, and have good anti-photoaging effect on the skin, good skin whitening and anti-oxidation effect.

[0005] The technical solution of the present application includes the following content:

[0006] In a first aspect, the present application provides a resveratrol-tocophersolan nanomicelle, and a preparation method thereof, comprising the following steps:

[0007] adding resveratrol and / or a derivative of resveratrol into a first organic solvent to mix, heating to dissolve at a first temperature to prepare a first solution; the weight percentage of the resveratrol or the derivative of resveratrol in the first solution is 0.1%-2%; the first temperature is 80℃-100℃;

[0008] adding tocophersolan into the first solution to mix, heating to dissolve at a second temperature to prepare a second solution; the weight percentage of the tocophersolan in the second solution is 0.5%-20%; the second temperature is 80℃-100℃;

[0009] adding the second solution into water at a first rate, stirring to disperse under a third temperature condition to prepare the resveratrol-tocophersolan nanomicelle; the first rate is 1mL / min-3mL / min, and the third temperature is 50℃-70℃.

[0010] The preparation method of the resveratrol-tocophersolan nanomicelle of the present application is constructed based on self-assembly technology, which is simple to operate and suitable for large-scale industrial production. The nanomicelle has excellent skin permeability, can significantly enhance the retention and absorption of drugs in the skin, has good stability, can effectively avoid the degradation of resveratrol, and has good dispersion characteristics, does not settle and does not agglomerate in a solution environment.

[0011] In some embodiments, the first organic solvent includes at least one of dipropylene glycol, glycerol, 1,3-butanediol, isopropyl alcohol, butanol, pentanol, phenoxy ethanol, ethanol, propylene glycol, ethyl acetate, isopropyl myristate, polyethylene glycol, and medium-chain triglyceride.

[0012] In some embodiments, the derivative of resveratrol includes a natural derivative and / or an artificially synthesized derivative.

[0013] Optionally, the natural derivative includes one or more of resveratrol glucoside, ampelopsin, pterostilbene, taxifolin, and tetramer of resveratrol.

[0014] Optionally, the artificially synthesized derivative includes one or more of a hydroxylated derivative, a methoxylated derivative, a halogenated derivative, and an acylated derivative.

[0015] In some embodiments, the tocophersolan is vitamin E succinate polyethylene glycol ester; the molecular weight of the polyethylene glycol structural unit in the vitamin E succinate polyethylene glycol ester is 200-6000.

[0016] In some embodiments, the weight percentage of the resveratrol or the derivative of resveratrol in the first solution is 1%-2%; and / or,

[0017] The weight percentage of the tocolosal in the second solution is 0.5%-5%.

[0018] In some embodiments, the first temperature is 85-95℃; and / or,

[0019] The second temperature is 85-90℃.

[0020] In some embodiments, the second solution is added to the deionized water at a first rate, and the stirring speed in the step of stirring and dispersing at a third temperature is 100-500rpm.

[0021] The resveratrol-tocosal nanomicelles of the present application have the advantages of good stability, good transdermal absorption effect, and high bioavailability. Resveratrol or resveratrol derivatives and tocolsal play a synergistic role, showing better anti-melanogenesis, antioxidant, anti-inflammatory, anti-photoaging, and anti-wrinkle effects than resveratrol or resveratrol derivatives, and have good skin whitening and antioxidant effects.

[0022] In a second aspect, the present application provides a use of the resveratrol-tocosal nanomicelles described above in the preparation of a cosmetic product.

[0023] In some embodiments, the cosmetic product comprises a cosmetic product with anti-photoaging efficacy.

[0024] The resveratrol-tocosal nanomicelles of the present application are safe for use in the preparation of cosmetic products, have no skin irritation, and have good anti-photoaging effects.

[0025] In a third aspect, the present application provides a use of the resveratrol-tocosal nanomicelles described above in the preparation of an antioxidant product.

[0026] In some embodiments, the antioxidant product is a cosmetic product, including a medical aesthetic product, a skin care product, or a beauty product.

[0027] Further, the dosage form of the antioxidant product is selected from one of a serum, an ointment, a cream, a mask, a powder, and a microneedle.

[0028] In a fourth aspect, the present application provides a use of the resveratrol-tocosal nanomicelles described above in the preparation of a skin whitening product.

[0029] In some embodiments, the skin whitening product is a cosmetic product, including a medical aesthetic product, a skin care product, or a beauty product.

[0030] Further, the dosage form of the skin whitening product is selected from one of a serum, an ointment, a cream, a mask, a powder, and a microneedle. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the drawings required to be used in the specific embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0032] Figure 1 is a transmission electron micrograph of torcixolene and nanomicelles (R@T-NM) of Example 1 of the present application;

[0033] Figure 2 is a stability test and Tyndall effect display of nanomicelles of resveratrol, torcixolene, and Example 1 and Comparative Example 1 of the present application;

[0034] Figure 3 is a percutaneous penetration verification based on a three-dimensional cell skin model of nanomicelles (R@T-NM) of Example 1 of the present application;

[0035] Figure 4 is a cytotoxicity statistical graph and cell live-dead staining result of nanomicelles (R@T-NM) of Example 1 of the present application;

[0036] Figure 5 is the antioxidant effect of nanomicelles (R@T-NM) of Example 1 of the present application;

[0037] Figure 6 is the result of inhibiting inflammatory factors of nanomicelles (R@T-NM) of Example 1 of the present application;

[0038] Figure 7 is the anti-melanin production result of nanomicelles (R@T-NM) of Example 1 of the present application;

[0039] Figure 8 is the anti-photoaging result of nanomicelles (R@T-NM) of Example 1 of the present application. DETAILED DESCRIPTION

[0040] The present application will be further described below in conjunction with embodiments and examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. In addition, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the content taught by the present application, and these equivalent forms also fall within the protection scope of the claims appended to the present application.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0042] Unless otherwise defined, or as the context requires, the terms used herein have the same meaning as commonly understood one of ordinary skill in the art to which this application belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an overly literal or overly formal sense unless expressly so defined herein.

[0043] The term "and / or", as used in the application herein, is intended to cover the structure of two or more related listed items, and also includes any and all combinations of the related listed items, which includes any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B, and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (i.e., the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C, and D, i.e., includes the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (i.e., the technical solution connected by "logical and").

[0044] In the present application, "preferably", "more preferably", "even more preferably", and the like are only used to describe the embodiments or examples with better effects, and should be understood as not constituting a limitation on the protection scope of the present application.

[0045] In the present application, "further", "even further", "particularly", and the like are used for the purpose of description, and should not be understood as constituting a limitation on the protection scope of the present application.

[0046] In the present application, the terms "first", "second", "third", and the like in "first aspect", "second aspect", "third aspect", and the like are only used for the purpose of description, and should not be understood as indicating or implying relative importance or quantity, and should not be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", and the like only serve the purpose of non-exhaustive enumeration description, and should be understood as not constituting a closed limitation on the quantity.

[0047] In the present application, the technical features described in an open manner include both the closed technical solution consisting of the listed features, and the open technical solution including the listed features.

[0048] In the present application, when referring to a numerical interval (i.e. a numerical range), unless otherwise specified, the optional numerical distribution within the numerical interval is considered to be continuous, and includes both numerical end points (i.e. the minimum and maximum values) of the numerical range, and every numerical value between the two numerical end points. When a numerical interval refers only to integers within the numerical interval, unless otherwise specified, the two end point integers of the numerical range, and every integer between the two end points are included. Furthermore, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, ranges disclosed herein are to be understood to include any and all sub-ranges subsumed therein.

[0049] In the present application, unless otherwise specified, the temperature parameter allows for constant temperature treatment, and also allows for variation within a certain temperature interval. It should be understood that the constant temperature treatment allows for fluctuations within the accuracy range of the instrument control. Fluctuations within a range of, for example, ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0050] In the present application, weight can be μg, mg, g, kg, and other mass units commonly known in the chemical industry.

[0051] In the present application, unless otherwise specified, when referring to size, particle size, diameter, the average value is generally meant.

[0052] In the present application, unless otherwise specified, the molecular weight is the average molecular weight, and further, unless otherwise specified, refers to the weight average molecular weight.

[0053] In a first aspect, the present application provides a resveratrol-tocophersol nanomicelle, and a preparation method thereof, comprising the following steps:

[0054] Resveratrol and / or a derivative of resveratrol is added to a first organic solvent for mixing, and is dissolved by heating at a first temperature to obtain a first solution; the weight percentage of the resveratrol or the derivative of resveratrol in the first solution is 0.1%-2%; the first temperature is 80°C-100°C;

[0055] Tocophersol is added to the first solution for mixing, and is dissolved by heating at a second temperature to obtain a second solution; the weight percentage of the tocophersol in the second solution is 0.5%-20%; the second temperature is 80°C-100°C;

[0056] The second solution is added to deionized water at a first rate, and is dispersed by stirring at a third temperature to obtain the resveratrol-tocophersol nanomicelle; the first rate is 1 mL / min-3 mL / min, and the third temperature is 50°C-70°C.

[0057] The preparation method of the resveratrol-tocophersolan nanomicelle of the present application is based on self-assembly technology. The preparation method is simple to operate, suitable for large-scale industrial production, has excellent skin permeability, can significantly enhance the retention and absorption of drugs in the skin, has good stability, can effectively prevent resveratrol degradation, and has good dispersion characteristics, does not settle and does not agglomerate in a solution environment.

[0058] In some embodiments, the average particle size of the resveratrol-tocophersolan nanomicelle is 16.52±0.02 nm, the zeta potential is -2.473±1.30 mV, and the polydispersity index is 0.09±0.008.

[0059] The resveratrol-tocophersolan nanomicelle of the present application has enhanced stability compared to tocophersolan alone. This enhanced stability can be produced by two synergistic mechanisms: (1) electronic modulation of the electronic density distribution of resveratrol mediated by tocophersolan, which stabilizes the phenolic redox center through potential covalent or non-covalent interactions (such as hydrogen bonds and π-π stacking); and (2) spatial protection, i.e., the tocophersolan matrix physically shields the active hydroxyl group of resveratrol through three-dimensional encapsulation, thereby limiting the accessibility of free radicals and oxidative species. These dual stabilization pathways collectively promote the improvement of the antioxidant properties and chemical stability of the nanomicelle.

[0060] In the preparation method of the embodiments of the present application, resveratrol is heated in a first solvent at a first temperature to fully dissolve it at a temperature that does not denature it, obtaining a uniform solution of suitable concentration (first solution). Tocophersolan is added to the first solution, heated at a second temperature to place its hydrophilic part (polyethylene glycol chain) and hydrophobic part (vitamin E succinate) in a relatively relaxed state, which facilitates more complete interaction between the resveratrol molecules and the hydrophobic part, and the obtained second solution is injected into water at a suitable rate and dispersed by stirring at a third temperature. During this process, tocophersolan undergoes a dramatic change in solvent form, driven by hydrophobic forces, and self-assembles to selectively wrap the resveratrol molecules distributed near the hydrophobic region, forming nanomicelles of a certain particle size in the form of spherical-like.

[0061] In some embodiments, the weight percentage of resveratrol or a derivative of resveratrol in the first solution is 0.1%-2% (for example, 0.1%, 0.2%, 0.5%, 0.8%, 1%, 1.2%, 1.5%, 2%, or any value between any two of these values), preferably 1%-2%.

[0062] In some embodiments, the weight percentage of tocophersolan in the second solution is 0.5%-20% (for example, 0.5%, 1%, 5%, 10%, 15%, 20%, or any value between any two of these values), preferably 0.5%-5%.

[0063] In some embodiments, the first organic solvent comprises at least one of dipropylene glycol, glycerol, 1,3-butanediol, isopropyl alcohol, butanol, pentanol, phenoxyethanol, ethanol, propylene glycol, ethyl acetate, isopropyl myristate, polyethylene glycol, and medium-chain triglyceride.

[0064] In some embodiments, the derivative of resveratrol comprises a natural derivative and / or an artificially synthesized derivative.

[0065] Optionally, the natural derivative comprises one or more of resveratrol glucoside, ampelopsin, pterostilbene, piceatannol, and tetramer of resveratrol.

[0066] Optionally, the artificially synthesized derivative comprises one or more of a hydroxylated derivative (e.g., resveratrol-4'-methyl ether, resveratrol-4'-O-glucoside, dihydroxystilbenes, etc.), a methoxylated derivative (e.g., p-hydroxyacetophenone resveratrol, trimethoxyacetophenone resveratrol, tetramethoxyacetophenone resveratrol, etc.), a halogenated derivative (e.g., oxidized resveratrol, brominated resveratrol, etc.), and an acylated derivative (e.g., triacetyl resveratrol, etc.).

[0067] In some embodiments, the tocophersolan is vitamin E succinate polyethylene glycol ester; the molecular weight of the polyethylene glycol structural unit in the vitamin E succinate polyethylene glycol ester is 200-6000 (e.g., 200, 238, 400, 600, 1000, 2000, 3400, 3500, 4000, 6000, etc.).

[0068] In some embodiments, the first temperature is 85-95℃; and / or,

[0069] The second temperature is 85-90℃.

[0070] In some embodiments, the second solution is added to the deionized water at a first rate, and the stirring speed in the step of stirring and dispersing at a third temperature is 100-500 rpm.

[0071] The resveratrol-tocophersolan nanomicelles prepared by the above preparation method have the advantages of good stability, good transdermal absorption effect, and high bioavailability. Resveratrol or a resveratrol derivative and tocophersolan exert a synergistic effect, and exhibit better anti-melanogenesis, antioxidant, anti-inflammatory, anti-photoaging, and anti-wrinkle effects than resveratrol or a resveratrol derivative.

[0072] In some embodiments, the resveratrol-tocophersolan nanomicelles are spherical, and have an ultra-small particle size (e.g., the particle size can be ≤20 nm).

[0073] In a second aspect, the present application provides use of the resveratrol-tocophersolan nanomicelle as described above in the preparation of a cosmetic product.

[0074] In some embodiments, the cosmetic product comprises a cosmetic product having anti-photoaging efficacy.

[0075] In some embodiments, the cosmetic product has at least one of the efficacies of inhibiting melanin production, anti-oxidation, anti-inflammation, anti-photoaging, and anti-wrinkle. It can be understood that, since the definition of anti-photoaging is skin damage caused by long-term ultraviolet (UV) radiation, which includes melanin deposition, oxidation, inflammation, wrinkle formation, etc., the efficacy of the cosmetic product of the present application includes damage caused by anti-photoaging such as melanin deposition, oxidation, inflammation, wrinkle formation, etc., and also includes skin problems such as aging, etc. that cause the above-mentioned problems.

[0076] The resveratrol-tocophersolan nanomicelle of the present application has the advantages of high safety, no skin irritation, and good effects of anti-melanin production, anti-oxidation, anti-inflammation, anti-photoaging, and anti-wrinkle, and can be used to prepare a functional skin care product having one or more functions of anti-aging, anti-oxidation, whitening, anti-inflammation, moisturizing, and skin barrier repair.

[0077] For example, the use in the preparation of a cosmetic product includes the preparation of an anti-aging cosmetic product having effects of promoting collagen synthesis, inhibiting matrix metalloproteinase (MMP) activity, reducing wrinkle formation, etc.; an anti-oxidation skin care product having effects of scavenging free radicals, reducing ultraviolet (UV) induced oxidative damage, etc.; a whitening and skin lightening product having effects of inhibiting tyrosinase activity, reducing melanin production, etc.; an anti-inflammatory soothing preparation having effects of reducing the expression of pro-inflammatory factors (such as IL-6, TNF-α), relieving skin sensitivity, etc.; and a repair skin care product having effects of enhancing skin barrier function, reducing transdermal water loss, etc.

[0078] In a third aspect, the present application provides use of the resveratrol-tocophersolan nanomicelle as described above in the preparation of an anti-oxidation product.

[0079] In some embodiments, the anti-oxidation product is a cosmetic product, including a medical aesthetic product, a skin care product, or a beauty product.

[0080] Further, the dosage form of the anti-oxidation product is selected from one of a serum, an ointment, a cream, a mask, a powder, and a microneedle.

[0081] In a fourth aspect, the present application provides use of the resveratrol-tocophersolan nanomicelle as described above in the preparation of a skin whitening product.

[0082] In some embodiments, the skin whitening product is a cosmetic product, including a medical cosmetic product, a skin care product, or a beauty product.

[0083] Further, the dosage form of the skin whitening product is selected from one of a serum, a paste, a cream, a mask, a powder, and a microneedle. Some specific embodiments are listed below.

[0084] For experimental parameters not specified in the following specific embodiments, refer to the guidelines provided in this application document, or refer to experimental manuals in the art or other known experimental methods in the art, or refer to the recommended experimental conditions of the manufacturers.

[0085] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art according to known means.

[0086] I. Preparation of Resveratrol-Tocophersolan Nanomicelles

[0087] Example 1

[0088] This example provides a preparation method of the resveratrol-tocophersolan nanomicelles of the present application, including the following steps:

[0089] (1) 1.5 g of resveratrol was added to 100 ml of dipropylene glycol and mixed to dissolve at 90°C to prepare a first solution.

[0090] (2) 7.5 g of tocophersolan (2500 Da) was added to the first solution and mixed to dissolve at 90°C to prepare a second solution.

[0091] (3) The second solution was added to 7.4 L of deionized water at a rate of 2 mL / min, and dispersed by stirring at 60°C during the addition process to form nanomicelles.

[0092] Example 2

[0093] This example provides a preparation method of the resveratrol-tocophersolan nanomicelles of the present application, including the following steps:

[0094] (1) 1.5 g of resveratrol-4’-O-glucoside was added to 100 ml of dipropylene glycol and mixed to dissolve at 90°C to prepare a first solution.

[0095] (2) 7.5 g of tocophersolan (2500 Da) was added to the first solution and mixed to dissolve at 90°C to prepare a second solution.

[0096] (3) The second solution was added to 7.4 L of deionized water at a rate of 2 mL / min, and dispersed by stirring at 60°C during the addition process to form nanomicelles.

[0097] Example 3

[0098] This example provides a preparation method of resveratrol-tocophersolan nanomicelles of the present application, comprising the following steps:

[0099] (1) 1.5 g of pterostilbene was added to 100 ml of dipropylene glycol and mixed to dissolve at 90°C to prepare a first solution.

[0100] (2) 7.5 g of tocophersolan (2500 Da) was added to the first solution and mixed to dissolve at 90°C to prepare a second solution.

[0101] (3) The second solution was added to 7.4 L of deionized water at a rate of 2 mL / min, and dispersed by stirring at 60°C during the addition process to form nanomicelles.

[0102] Example 4

[0103] This example provides a preparation method of resveratrol-tocophersolan nanomicelles of the present application, which is prepared by using the substantially same method as in Example 1, except that in step (3), the second solution is added at a rate of 1 mL / min.

[0104] Example 5

[0105] This example provides a preparation method of resveratrol-tocophersolan nanomicelles of the present application, which is prepared by using the substantially same method as in Example 1, except that in step (2), tocophersolan 6000 is used.

[0106] Example 6

[0107] This example provides a preparation method of resveratrol-tocophersolan nanomicelles of the present application, which is prepared by using the substantially same method as in Example 1, except that in step (1), the amount of resveratrol is changed to 0.1 g, and in step (2), the amount of tocophersolan is changed to 0.5 g.

[0108] Example 7

[0109] This example provides a preparation method of resveratrol-tocophersolan nanomicelles of the present application, which is prepared by using the substantially same method as in Example 1, except that in step (1), the amount of resveratrol is changed to 2 g, and in step (2), the amount of tocophersolan is changed to 20 g.

[0110] Example 8

[0111] This example provides a preparation method of resveratrol-tocofersolan nanomicelles of the present application, which is prepared by using the substantially same method as that of Example 1, with the difference that in step (3), the second solution is added in an amount of 1 mL, so that the concentration of tocophersolan contained in the nanomicelle solution is 0.01 wt%.

[0112] Example 9

[0113] This example provides a preparation method of resveratrol-tocofersolan nanomicelles of the present application, which is prepared by using the substantially same method as that of Example 1, with the difference that in step (3), the second solution is added in an amount of 50 mL, so that the concentration of tocophersolan contained in the nanomicelle solution is 0.5 wt%.

[0114] Comparative Example 1

[0115] This comparative example provides a preparation method of resveratrol-tocofersolan nanomicelles, which is prepared by using the substantially same method as that of Example 1, with the difference that in step (1), the amount of resveratrol is changed to 5 g.

[0116] Comparative Example 2

[0117] This comparative example provides a preparation method of resveratrol-tocofersolan nanomicelles, which is prepared by using the substantially same method as that of Example 1, with the difference that in step (1), the amount of resveratrol is changed to 0.05 g.

[0118] Comparative Example 3

[0119] This comparative example provides a preparation method of resveratrol-tocofersolan nanomicelles, which is prepared by using the substantially same method as that of Example 1, with the difference that in step (2), the amount of tocophersolan is changed to 30 g.

[0120] Comparative Example 4

[0121] This comparative example provides a preparation method of resveratrol-tocofersolan nanomicelles, which is prepared by using the substantially same method as that of Example 1, with the difference that in step (2), the amount of tocophersolan is changed to 0.1 g.

[0122] Comparative Example 5

[0123] This comparative example provides a preparation method of resveratrol-tocofersolan nanomicelles, which is prepared by using the substantially same method as that of Example 1, with the difference that in step (1), the heating temperature is 50°C.

[0124] Comparative Example 6

[0125] The comparative example provides a preparation method of resveratrol-tocophersolan nanomicelles. The preparation is carried out by using the basically same method as in Example 1, with the difference that in step (1), the heating temperature is 120°C.

[0126] Comparative Example 7

[0127] The comparative example provides a preparation method of resveratrol-tocophersolan nanomicelles. The preparation is carried out by using the basically same method as in Example 1, with the difference that in step (2), the heating temperature is 50°C.

[0128] Comparative Example 8

[0129] The comparative example provides a preparation method of resveratrol-tocophersolan nanomicelles. The preparation is carried out by using the basically same method as in Example 1, with the difference that in step (2), the heating temperature is 120°C.

[0130] Comparative Example 9

[0131] The comparative example provides a preparation method of resveratrol-tocophersolan nanomicelles. The preparation is carried out by using the basically same method as in Example 1, with the difference that steps (1) and (2) are combined, and resveratrol, solvent and tocophersolan are directly dissolved at 90°C.

[0132] II. Morphology and particle size characterization

[0133] The tocophersolan and the nanomicelle solution (R@T-NM) of Example 1 were drop-casted onto copper grids. After the copper grids were air-dried at room temperature, the morphology was observed using a transmission electron microscope, and the transmission electron microscope image is shown in Figure 1 As shown in Figure 1 , the nanomicelles of Example 1 exhibit a transparent, uniform and spherical morphology.

[0134] Resveratrol, tocophersolan and the nanomicelle solution (R@T-NM) of Example 1 were injected into a PS particle size cell or a U-shaped capillary sample cell (DTS0012), and dynamic light scattering was used to test the particle size, and the particle size and Zeta potential were determined, and the determination results are shown in Table 1.

[0135] Table 1. Determination results of particle size and Zeta potential of resveratrol, tocophersolan and nanomicelles of Example 1

[0136]

[0137] According to Table 1, the average particle size of the nanomicelles of Example 1 is 16.52 nm, and the zeta potential is -2.473 mV, indicating that the surface is negatively charged. This negative surface charge promotes the electrostatic repulsion between R@T-NM nanoparticles, thereby enhancing their dispersibility and contributing to the stability of the system. The polydispersity index of the nanomicelles of Example 1 is much lower than that of resveratrol or tocolosol, indicating that the uniformity of molecular distribution is significantly improved. The surface charge of the nanomicelles of Example 1 is much higher than that of resveratrol or tocolosol, indicating that the reduction is significantly reduced.

[0138] III. Performance detection

[0139] (1) Light stability detection

[0140] According to the light stability test conditions recorded in the 9001 Raw Materials and Preparation Stability Test Guidelines of the Chinese Pharmacopoeia 2020 edition, the light stability of each example and comparative example was investigated. The state of each system after being placed in the light box for 0, 7, 14 days was observed and recorded. The detection results of each example and comparative example are shown in Table 2.

[0141] Table 2 Stability detection results of Examples 1-9 and Comparative Examples 1-9

[0142]

[0143]

[0144] According to Table 2, the nanomicelles of the examples have no obvious changes after being placed for 7 and 14 days, without crystallization and delamination, with good surface stability, and no other stability problems, indicating that the examples have relatively good light stability. The comparative examples have obvious crystallization or color change after being placed for 7 and 14 days, indicating that the nanomicelles prepared by the comparative examples have poor stability.

[0145] Take Example 1 and Comparative Example 1 as examples for illustration: raw materials resveratrol, tocolosol are dissolved and prepared into solutions (or dispersions) according to the concentration of Example 1, etc.; the prepared solution and the nanomicelle solution of Example 1 and Comparative Example 1 are subjected to light stability determination according to the known principle, and the stability test images of the 0th day and the 7th day are as shown in Figure 2 Figure 2 ​, the results of day 0 show that resveratrol has low solubility, there are a large number of suspended particles and partial precipitation; Tocophersolan can be completely dissolved, no Tyndall effect, indicating that a solution is formed; the nanomicelle solution of Comparative Example 1 has a large number of suspended particles and partial precipitation; the nanomicelle solution of Example 1 is transparent and clear, and the Tyndall effect can be observed under laser beam irradiation, confirming that a colloid is formed; the results of day 7 show that the nanomicelle solution of Example 1 is still transparent and clear, and the Tyndall effect can be observed under laser beam irradiation, indicating that it has good light stability.

[0146] The above light stability detection results show that the stability of Example 1 is the best, so the nanomicelle solution (R@T-NM) of Example 1 is selected for further testing of antioxidant, anti-aging, and anti-inflammatory.

[0147] (2) Transdermal effect detection

[0148] GelMA (15 wt%, 100 μL) supplemented with the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) (0.1 wt%) was added to the Transwell chamber and crosslinked under UA light to form a hydrogel. A sufficient number of cells were seeded on the hydrogel to generate a cell layer, thereby mimicking the 3D structure of skin with a dense epidermis and a relatively loose subcutaneous tissue. After incubation for 24 hours in a 24-well plate, the medium was removed and FITC-R@T-NM (1 wt%) of Example 1 with a fluorescent group was added to the upper chamber with the generated 3D cell skin model. After incubation for 24 hours, the hydrogel was examined by fluorescence microscopy to evaluate the transmittance of FITC-R@T-NM in the 3D cell skin model.

[0149] The results are shown in Figure 3 , according to A of Figure 3 , the GelMA hydrogel has a double-layer structure, the upper dense porous layer corresponds to the epidermis layer, and the lower relatively loose porous layer simulates the dermis layer, together forming a skin-like barrier. According to B of Figure 3 , the surface of the hydrogel shows a large number of cell adhesion. According to C of Figure 3 , after applying the FITC-labeled R@T-NM solution to the surface of the hydrogel, strong green fluorescence was observed under 488 nm excitation. According to D of Figure 3 , when examined by inverted fluorescence microscopy, the vertical section of the hydrogel showed a large amount of fluorescent signal throughout the matrix, confirming the penetration ability of FITC-R@T-NM.

[0150] (3) Biocompatibility of resveratrol-tocophersolan nanotransdermal delivery system

[0151] Human skin fibroblasts (HSF) were cultured in complete DMEM supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin / streptomycin at 37 °C and 5% CO2 in a humidified incubator (Thermo Fisher Scientific, USA) to detect the cytotoxicity of R@T-NM prepared in Example 1 on HSF using CCK-8 method and live / dead staining kit. Different concentrations of R@T-NM were prepared in PBS and sterilized by 0.2-micron filter membrane. HSF were seeded in 96-well plates at a density of 4 x 10 4 Cells / mL and 200 μl / well and incubated overnight under optimal conditions to allow attachment. Cells were then treated with complete medium containing different concentrations of R@T-NM for 24 h. After removing the medium, the prepared CCK-8 reaction solution was added to each well and incubated for another 2 h in the incubator. The optical density (OD) was quantified by measuring the absorbance at 450 nm. Cells cultured in complete DMEM served as a control group. Cell viability was calculated using the following equation: Cell viability (%) = (Ab-as) / (Ac-As) x 100%, where Ab represents the absorbance at 450 nm of the test sample, Ac represents the absorbance at 450 nm of the control sample, and As is the OD value of the CCK-8 reaction solution.

[0152] The results, as shown in Figure 4 , the cytotoxicity of R@T-NM was evaluated in the concentration range of 5-30 g / mL. It was found that the cell viability was ≥ 100% at a concentration below 10 g / mL, indicating that low concentrations of R@T-NM promoted the proliferation of HSF cells. When the concentration of R@T-NM was 15 g / mL, the cell viability remained above 90%. Live / dead cell staining showed that cells in the R@T-NM group exhibited an intact morphology with a spindle-shaped elongated appearance, with no cell death. This indicates that even R@T-NM systems containing organic solvents as solubilizers exhibit good biocompatibility.

[0153] (4) Evaluation of antioxidant effect

[0154] DPPH radical scavenging test: R@T-NM at a concentration of 1 mg / mL was added to 0.12 mg / mL DPPH ethanol. After incubation in the dark for a predetermined period of time, the absorbance of the treated DPPH solution was measured at 517 nm (As) using a UV-Vis spectrometer. The absorbance of the untreated DPPH solution (Ab) was used as a blank control. The DPPH scavenging rate (%) was calculated using the equation DPPH scavenging rate (%) = (Ab-As) / Ab x 100%.

[0155] Intracellular reactive oxygen species scavenging assay: HSF cells were seeded in 24-well plates and cultured until reaching 80-90% confluence. Then, HSF cells were pre-treated with hydrogen peroxide (H2O2) for 30 min, followed by incubation with R@T-NM-containing medium (10 pg / mL) for 24 h. After treatment, the R@T-NM-containing medium was removed and the cells were washed with PBS. DCFH-DA was diluted 1:1000 in serum-free medium and 1 mL of the prepared DCFH-DA reaction solution was added to each well. The cells were incubated with DCFH-DA in the dark at 37 °C for 30 min. Then, the DCFH-DA solution was aspirated and the cells were washed three times with serum-free medium to remove the residual probe. Fluorescent images were acquired using an inverted fluorescence microscope and the fluorescence intensity was quantified using ImageJ software.

[0156] Results are shown in Figure 5 According to A in Figure 5 , it can be seen that tocolsolon, resveratrol and R@T-NM all showed strong DPPH radical scavenging ability, with DPPH scavenging rates of 72.97%, 90.09% and 88%, respectively, indicating that tocolsolon, resveratrol and R@T-NM have superior antioxidant capacity. According to B in Figure 5 , the smallest green fluorescence signal in the untreated control group confirmed the healthy cell state with low basal ROS levels and no apoptotic tendency. In contrast, 50 mM H2O2 treatment significantly increased the fluorescence intensity. However, co-treatment with R@T-NM significantly attenuated this fluorescence signal. Notably, R@T-NM had the weakest fluorescence intensity among all treatment groups, but did not achieve the best radical scavenging efficiency in the DPPH experiment. This observation suggests that the antioxidant effect of resveratrol may rapidly disappear due to overreaction (e.g., rapid binding with free radicals). In contrast, tocolsolon may extend the half-life of resveratrol by reducing its reactivity, thereby achieving a "sustained release" effect. This further validates the protective effect of tocolsolon in protecting the activity of resveratrol.

[0157] (5) Detection of inflammatory cytokine expression

[0158] HSF cells were seeded in 24-well plates and cultured until 80-90% confluence. Then, the cells were pre-treated with the inflammatory stimulant LPS, followed by incubation with complete DMEM containing R@T-NM of Example 1 for 24 h. After treatment, the cell culture supernatant was collected and analyzed for IL-6 and IL-8 levels using commercial ELISA kits according to the manufacturer's protocol. The absorbance (OD value) was measured at 450 nm using a microplate reader, and the cytokine concentration and inhibition rate were calculated according to the standard curve.

[0159] Results are shown in Figure 6As shown, LPS-stimulated HSF cells exhibited significantly elevated secretion of pro-inflammatory cytokines IL-6 and IL-8 compared to the blank control group, indicating successful stimulation of the cells' inflammatory state. However, treatment with R@T-NM significantly reduced the expression levels of both cytokines. These results suggest that R@T-NM can attenuate the inflammatory response by downregulating the expression of pro-inflammatory factors.

[0160] (6) Inhibition of melanogenesis activity assay

[0161] Melanoma cells were seeded in 12-well plates and cultured to 80-90% confluence. Cells were then treated with complete DMEM containing R@T-NM and G@T-NM prepared in Example 1 and Example 2 for 72 hours. Intracellular tyrosinase activity was measured using a commercial tyrosinase activity assay kit according to the manufacturer's protocol.

[0162] Results are shown in Figure 7 Figure 6. As shown in Figure 7 A, skin color is closely related to the melanin produced by melanocytes in the basal layer of the epidermis. Tyrosinase is the rate-limiting enzyme in melanogenesis, and its activity is inhibited as a key molecular target for skin whitening agents. As shown in Figure 7 B, intracellular tyrosinase activity assay showed that resveratrol, R@T-NM and GL showed stronger tyrosinase inhibition compared to the control group. Notably, the R@T-NM treatment group exhibited the lowest level of tyrosinase activity, indicating its effective inhibition of this key enzyme and the consequent potential to promote skin whitening.

[0163] (7) Cell photoaging test HSF cell sunscreen aging efficacy detection (UVA)

[0164] HSF cells and fibroblasts were seeded in 24-well plates or 12-well plates and cultured to 50-60% confluence. HSF cells were overlaid with PBS and exposed to UVA irradiation with a total dose of 5 J / cm 2 per time. After irradiation, the PBS was removed and replaced with complete DMEM containing R@T-NM prepared in Example 1. This irradiation regimen was repeated daily for 5 consecutive days. For senescence evaluation, cells in 24-well plates were stained using a senescence-associated β-galactosidase (SA-β-gal) staining kit according to the manufacturer's instructions. At the same time, the NAD + content of cells in 12-well plates was quantified using a commercial NAD + phosphate assay kit according to the manufacturer's instructions.

[0165] Cellular senescence is a hallmark of organismal aging, representing a state of permanent growth arrest in non-dividing cells. Based on a literature review, the applicant established a HSF cell photoaging model using UVA irradiation at 5 J / cm 2Intensity of photoaging model under UVA irradiation. As Figure 8 shown in Fig. 5A, HSFs were exposed to 5 J / cm2of UVA for 5 consecutive days 2 UVA exposure significantly increased the number of SA-β-gal positive HSF cells. Comparative analysis showed that the intensity of SA-β-gal staining was significantly enhanced in UVA-irradiated HSFs compared to the blank control group. Notably, treatment with resveratrol (Res), R@T-NM or GL effectively counteracted UVA-induced cellular senescence. As Figure 8 shown in Fig. 5B, among these interventions, R@T-NM showed the lowest percentage of SA-β-GAL positive cells, indicating its superior anti-aging activity. Further aging studies based on metabolomics revealed that NAD + plays a crucial regulatory role in multiple key metabolic pathways closely associated with cellular senescence. NAD + can inhibit oxidative stress, repair nuclear DNA damage, and regulate mitochondrial dysfunction, thereby impeding the process of cellular senescence. As Figure 8 shown in Fig. 5C, the effect of R@T-NM on UVA-induced cellular senescence was investigated by measuring intracellular NAD + levels. Compared to the blank control group, NAD + content in UVA group HSF cells was significantly decreased. Compared to the UVA group, NAD + content in resveratrol group and R@T-NM group was significantly increased. These results suggest that R@T-NM can enhance the expression of NAD + , thereby having the potential to inhibit cellular senescence.

[0166] All the documents mentioned in the present application are incorporated by reference in the present application as if each document was individually incorporated by reference. Unless and to the extent that the documents incorporated by reference conflict with the application purpose and / or technical solutions of the present application, the documents incorporated by reference are incorporated by reference in the present application in the entirety and in the entirety purpose. When the present application refers to the documents incorporated by reference, the definition of the relevant technical features, terms, names, phrases, etc. in the documents incorporated by reference are also incorporated by reference. When the present application refers to the documents incorporated by reference, the examples, preferred modes of the relevant technical features incorporated by reference can also be incorporated by reference into the present application, but to the extent that the present application can be implemented. It should be understood that when the incorporated content conflicts with the description in the present application, the present application is used as the reference or is modified according to the description in the present application adaptively.

[0167] The technical features of the above-described embodiments and examples can be combined in any suitable manner. In order to make the description concise, all possible combinations of the technical features in the above-described embodiments and examples are not described, however, as long as the combinations of the technical features do not contradict each other, it should be considered that they are within the scope of the present description.

[0168] The above embodiments only express several implementation ways of the present application, but cannot be understood as limitation to the patent scope. It should be pointed out that, for ordinary skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. In addition, it should be understood that, after reading the above teaching of the present application, the skilled in the art can make various changes or modifications to the present application, and the equivalent forms also fall within the protection scope of the present application. It should also be understood that, the skilled in the art can obtain the technical solutions on the basis of the technical solutions provided by the present application through logical analysis, reasoning or limited test, which all fall within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the description can be used to explain the content of the claims.

Claims

1. A resveratrol-tocophersolan nanomicelle, characterized in that, The preparation method of the resveratrol-tocophersolan nanomicelles comprises the following steps: resveratrol or a derivative of resveratrol is added into a first organic solvent for mixing, and is heated and dissolved at a first temperature to prepare a first solution; the derivative of resveratrol is resveratrol-4'-O-glucoside or pterostilbene; the first organic solvent is dipropylene glycol; the weight percentage of resveratrol or the derivative of resveratrol in the first solution is 0.1%-2%; and the first temperature is 80-100℃; tocophersolan is added into the first solution for mixing, and is heated and dissolved at a second temperature to prepare a second solution; the tocophersolan is vitamin E succinate polyethylene glycol ester; and the weight percentage of the tocophersolan in the second solution is 0.5%-20%; the second temperature is 80-100℃; the second solution is added into water at a first rate, and is dispersed by stirring at a third temperature to prepare the resveratrol-tocophersolan nanomicelles; the first rate is 1-3 mL / min, and the third temperature is 50-70℃.

2. The resveratrol-tocophersol nanomicelle according to claim 1, wherein, the molecular weight of a polyethylene glycol structure unit in the vitamin E succinate polyethylene glycol ester is 200-6000.

3. The resveratrol-tocophersol nanomicelle according to claim 1 or 2, characterized in that, the weight percentage of resveratrol or the derivative of resveratrol in the first solution is 1%-2%; and / or, the weight percentage of the tocophersolan in the second solution is 0.5%-5%.

4. The resveratrol-tocophersol nanomicelle according to claim 1 or 2, characterized in that, the first temperature is 85-95℃; and / or, the second temperature is 85-90℃.

5. The resveratrol-tocophersol nanomicelle as claimed in claim 1 or 2, wherein, in the step of adding the second solution into deionized water at a first rate and dispersing by stirring at a third temperature, the stirring speed is 100-500 rpm.

6. The resveratrol-tocophersolan nanomicelles according to any one of claims 1-5 for use in the preparation of a cosmetic product.

7. The resveratrol-tocophersolan nanomicelles according to any one of claims 1-5 for use in the preparation of an antioxidant cosmetic product.

8. The resveratrol-tocophersolan nanomicelles according to any one of claims 1-5 for use in the preparation of a skin-whitening cosmetic product.

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