Resveratrol-tocoxolam nano-micelle and application thereof in preparation of antioxidant and skin whitening products

Abstract

The invention relates to the technical field of cosmetics and transdermal drug delivery, in particular to a resveratrol-tocoxolam nano-micelle and application of the resveratrol-tocoxolam nano-micelle 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 (the weight percentage is 0.1-2%), and heating and dissolving at a first temperature of 80-100 DEG C to obtain a first solution; adding tocoxolam into the first solution, mixing (the weight percentage is 0.5-20%), and heating and dissolving at a second temperature (80-100 DEG C) to obtain a second solution; adding the second solution into the deionized water at a first rate (1mL / min to 3mL / min), and stirring and dispersing at a third temperature (50 DEG C to 70 DEG C); the nano-micelle is high in stability, excellent in transdermal absorption performance and good in anti-light aging effect, and has good skin whitening and anti-oxidation effects.

Description

Technical Field

[0001] This application relates to the fields of cosmetics and transdermal drug delivery technology, specifically to resveratrol-tocoxelen nanomicelles and their application in the preparation of antioxidant and skin whitening products. Background Technology

[0002] Skin aging is a natural process, but it is also related to some exogenous factors. Sunlight's ultraviolet radiation is one of the main exogenous factors causing photoaging. Photoaging is skin damage caused by long-term ultraviolet (UV) radiation, characterized by oxidative stress, inflammatory responses, and collagen degradation, ultimately leading to wrinkles, pigmentation, and impaired skin barrier function. With increasing attention to skin health and beauty, the development of effective antioxidant 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 antioxidant and anti-inflammatory properties.

[0003] Resveratrol is a plant-derived protective factor produced in response to stimulation. It is a stilbene compound with multiple hydroxyl substituents, systematically named (E)-3,4',5-trihydroxystilbene. It appears as white needle-like crystals, is poorly soluble in water, but readily soluble in organic solvents such as ether, chloroform, methanol, ethanol, acetone, and ethyl acetate. Resveratrol possesses strong free radical scavenging capabilities, effectively inhibiting reactive oxygen species-mediated cell damage. It also exhibits various activities, including regulating melanin-producing enzyme activity, modulating inflammatory responses, and promoting collagen production, demonstrating great potential in the field of anti-photoaging skincare. However, resveratrol is photosensitive, easily oxidized and inactivated, and has poor aqueous solubility, resulting in poor permeability to the skin surface and difficulty in effectively crossing the epidermal barrier to exert its biological functions. These characteristics significantly limit the practical application of resveratrol. Summary of the Invention

[0004] This application has discovered through extensive experiments that resveratrol-tocoxelane nanomicelles prepared using tocoxelane as a carrier and through appropriate methods have small particle size, high stability and excellent transdermal absorption performance, and have a good anti-photoaging effect on the skin, as well as good skin whitening and antioxidant effects.

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

[0006] In a first aspect, this application provides a resveratrol-toxoxelamne nanomicelle, the preparation method of which includes the following steps:

[0007] Resveratrol and / or its derivatives are added to a first organic solvent and mixed, then heated to a first temperature to dissolve, thereby obtaining a first solution; the weight percentage of resveratrol or its derivatives in the first solution is 0.1%-2%; the first temperature is 80℃-100℃.

[0008] Toxoxelam is added to the first solution and mixed, then heated to a second temperature to dissolve, thus obtaining a second solution; the weight percentage of toxoxelam in the second solution is 0.5%-20%; the second temperature is 80℃-100℃.

[0009] The second solution was added to water at a first rate and stirred and dispersed under a third temperature condition to obtain the resveratrol-tocoxelane nanomicelles; the first rate was 1 mL / min-3 mL / min and the third temperature was 50℃-70℃.

[0010] The resveratrol-tocoxelane nanomicelles of this application are constructed based on self-assembly technology. This preparation method is simple to operate and suitable for large-scale industrial production. The nanomicelles have excellent skin permeability, which can significantly enhance the retention and absorption of drugs in the skin. They also have good stability, which can effectively prevent the degradation of resveratrol. At the same time, they have good dispersion characteristics and do not settle or agglomerate in solution.

[0011] In some embodiments, the first organic solvent includes at least one selected from dipropylene glycol, glycerol, 1,3-butanediol, isopropanol, butanol, pentanol, phenoxyethanol, ethanol, propylene glycol, ethyl acetate, isopropyl myristate, polyethylene glycol, and medium-chain triglycerides.

[0012] In some embodiments, the resveratrol derivatives include natural derivatives and / or synthetic derivatives;

[0013] Optionally, the natural derivatives include one or more of resveratrol glycoside, glucosinolate, pterostilbene, lecithin, and tetramers of resveratrol;

[0014] Optionally, the synthetic derivatives include one or more of hydroxylated derivatives, methoxylated derivatives, halogenated derivatives, and acylated derivatives.

[0015] In some embodiments, the tocoxexyl is polyethylene glycol succinate; the polyethylene glycol structural unit in the polyethylene glycol succinate has a molecular weight of 200-6000.

[0016] In some embodiments, the resveratrol or a resveratrol derivative comprises 1%-2% by weight in the first solution; and / or,

[0017] The tococelen in the second solution is 0.5%-5% by weight.

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

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

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

[0021] The resveratrol-tocoxelane nanomicelles of this application have the advantages of good stability, good transdermal absorption, and high bioavailability. Resveratrol or resveratrol derivatives and tocoxelane work synergistically, showing better anti-melanin production, anti-oxidation, anti-inflammation, anti-photoaging and anti-wrinkle effects than resveratrol or resveratrol derivatives, and have good skin whitening and antioxidant effects.

[0022] Secondly, this application provides an application of the resveratrol-tocoxelane nanomicelles described above in the preparation of cosmetics.

[0023] In some embodiments, the cosmetics include cosmetics with anti-photoaging effects.

[0024] The resveratrol-tocoxelane nanomicelles of this application are highly safe for use in the preparation of cosmetics, have no skin irritation, and have good anti-photoaging effects.

[0025] Thirdly, this application provides an application of the resveratrol-tocoxelane nanomicelles described above in the preparation of antioxidant products.

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

[0027] Furthermore, the dosage form of antioxidant products is selected from one of the following: serum, ointment, cream, mask, powder, and microneedle.

[0028] Fourthly, this application provides the application of the resveratrol-tocoxelane nanomicelles described above in the preparation of skin whitening products.

[0029] In some embodiments, skin whitening products are cosmetics, including medical aesthetic products, skin care products, or beauty products.

[0030] Furthermore, the dosage form of skin whitening products is selected from one of the following: serum, ointment, cream, mask, powder, and microneedling. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0032] Figure 1 These are transmission electron microscope images of nanomicelles (R@T-NM) of Tocosolvan and Example 1 of this application;

[0033] Figure 2 These are stability tests and Tyndall effect illustrations of resveratrol, toxoxexole, and nanomicelles of Examples 1 and Comparative Example 1 of this application.

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

[0035] Figure 4 This is a cytotoxicity statistic and cell viability / death staining results of the nanomicelles (R@T-NM) of Example 1 of this application;

[0036] Figure 5 This describes the antioxidant effect of the nanomicelles (R@T-NM) in Example 1 of this application;

[0037] Figure 6 This is the result of the inhibition of inflammatory factors by the nanomicelles (R@T-NM) of Example 1 of this application;

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

[0039] Figure 8 This is the photoaging result of the nanomicelles (R@T-NM) of Example 1 of this application. Detailed Implementation

[0040] The present application is further described below with reference to embodiments and examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the protection scope of the appended claims.

[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 herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0042] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0043] The term "and / or" as used herein includes any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations encompass 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 using at least two conjunctions selected from "and / or," "or / and," or "and / or," it should be understood that, in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0044] In this document, terms such as "preferred," "better," and "more preferred" are merely descriptions of implementation methods or examples that achieve better results, and should be understood as not constituting a limitation on the scope of protection of this application.

[0045] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0046] In this application, the terms "first aspect," "second aspect," "third aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0047] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0048] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, optional numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0049] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5°C, ±4°C, ±3°C, ±2°C, or ±1°C.

[0050] In this application, weight can be a well-known unit of mass in the chemical industry, such as μg, mg, g, or kg.

[0051] In this application, unless otherwise specified, the terms "size", "particle size", and "diameter" generally refer to average values.

[0052] In this application, unless otherwise specified, molecular weight refers to average molecular weight, and further, unless otherwise specified, refers to weight-average molecular weight.

[0053] In a first aspect, this application provides a resveratrol-toxoxelamne nanomicelle, the preparation method of which includes the following steps:

[0054] Resveratrol and / or its derivatives are added to a first organic solvent and mixed, then heated to a first temperature to dissolve, thereby obtaining a first solution; the weight percentage of resveratrol or its derivatives in the first solution is 0.1%-2%; the first temperature is 80℃-100℃.

[0055] Toxoxelam is added to the first solution and mixed, then heated to a second temperature to dissolve, thus obtaining a second solution; the weight percentage of toxoxelam in the second solution is 0.5%-20%; the second temperature is 80℃-100℃.

[0056] The second solution was added to deionized water at a first rate and stirred and dispersed under a third temperature condition to obtain the resveratrol-tocoxelane nanomicelles; the first rate was 1 mL / min-3 mL / min and the third temperature was 50℃-70℃.

[0057] The resveratrol-tocoxelen nanomicelles of this application are constructed based on self-assembly technology. This preparation method is simple to operate, suitable for large-scale industrial production, has excellent skin permeability, can significantly enhance drug retention and absorption in the skin, and has good stability, which can effectively avoid resveratrol degradation. At the same time, it has good dispersion characteristics, and does not settle or agglomerate in the solution environment.

[0058] In some embodiments, the resveratrol-tocoxelane nanomicelles have an average particle size of 16.52 ± 0.02 nm, a zeta potential of -2.473 ± 1.30 mV, and a polydispersity index of 0.09 ± 0.008.

[0059] The resveratrol-tocoxolane nanomicelles of this application exhibit enhanced stability compared tocoxolane alone. This enhanced stability may arise through two synergistic mechanisms: (1) electronic modulation mediated by tocoxolane, which alters the electron density distribution of resveratrol through potential covalent or non-covalent interactions (e.g., hydrogen bonding and π-π stacking), thereby stabilizing the phenol redox center; and (2) steric protection, i.e., the tocoxolane matrix physically shields the active hydroxyl groups of resveratrol through three-dimensional encapsulation, thereby limiting the accessibility of free radicals and oxide species. These dual stabilization pathways jointly contribute to the improved antioxidant properties and chemical stability of the nanomicelles.

[0060] In the preparation method of the embodiments of this application, resveratrol is heated in a first solvent at a first temperature to fully dissolve it at a temperature that will not denature, resulting in a homogeneous solution (first solution) of appropriate concentration. Toxoxelam is added to the first solution and heated at a second temperature to ensure that both the hydrophilic part (polyethylene glycol chain) and the hydrophobic part (vitamin E succinate) are in a relatively relaxed state, which is conducive to the more complete interaction between resveratrol molecules and the hydrophobic part. The resulting second solution is injected into water at an appropriate rate and stirred and dispersed at a third temperature. During this process, when the solvent formulation changes drastically, toxoxelam undergoes self-assembly driven by hydrophobic forces, selectively encapsulating the resveratrol molecules distributed near the hydrophobic region to form spherical nanomicelles of a certain particle size.

[0061] In some embodiments, the weight percentage of resveratrol or a resveratrol derivative in the first solution is 0.1%-2% (e.g., 0.1%, 0.2%, 0.5%, 0.8%, 1%, 1.2%, 1.5%, 2% or any two thereof), preferably 1%-2%.

[0062] In some embodiments, the weight percentage of toxoxelam in the second solution is 0.5%-20% (e.g., 0.5%, 1%, 5%, 10%, 15%, 20% or any two thereof), preferably 0.5%-5%.

[0063] In some embodiments, the first organic solvent includes at least one selected from dipropylene glycol, glycerol, 1,3-butanediol, isopropanol, butanol, pentanol, phenoxyethanol, ethanol, propylene glycol, ethyl acetate, isopropyl myristate, polyethylene glycol, and medium-chain triglycerides.

[0064] In some embodiments, the resveratrol derivatives include natural derivatives and / or synthetic derivatives.

[0065] Optionally, the natural derivative includes one or more of resveratrol glycoside, glucosinolate, pterostilbene, paclitaxel, and tetramers of resveratrol.

[0066] Optionally, the artificially synthesized derivatives include one or more of the following: hydroxylated derivatives (e.g., resveratrol-4'-methyl ether, resveratrol-4'-O-glucoside, dihydroxystyrene compounds, etc.), methoxylated derivatives (e.g., p-hydroxyacetophenone resveratrol, trimethoxyacetophenone resveratrol, tetramethoxyacetophenone resveratrol, etc.), halogenated derivatives (e.g., oxidized resveratrol, brominated resveratrol, etc.), and acylated derivatives (e.g., triacetyl resveratrol, etc.).

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

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

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

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

[0071] The resveratrol-tocoxelane nanomicelles prepared by the above method have the advantages of good stability, good transdermal absorption, and high bioavailability. Resveratrol or resveratrol derivatives and tocoxelane exert a synergistic effect, showing better anti-melanin production, anti-oxidation, anti-inflammation, anti-photoaging and anti-wrinkle effects than resveratrol or resveratrol derivatives.

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

[0073] Secondly, this application provides an application of the resveratrol-tocoxelane nanomicelles described above in the preparation of cosmetics.

[0074] In some embodiments, the cosmetics include cosmetics with anti-photoaging effects.

[0075] In some embodiments, the cosmetic has at least one of the following effects: inhibiting melanin production, anti-oxidation, anti-inflammation, anti-photoaging, and anti-wrinkle. It is understood that since anti-photoaging is defined as skin damage caused by long-term ultraviolet (UV) radiation, including melanin deposition, oxidation, inflammation, and wrinkle formation, the cosmetic efficacy of this application includes anti-photoaging damage such as melanin deposition, oxidation, inflammation, and wrinkle formation, as well as skin problems such as melanin deposition, oxidation, inflammation, and wrinkles that do not exist due to ultraviolet (UV) radiation, such as those caused by aging.

[0076] The resveratrol-tocoxelen nanomicelles of this application have the advantages of high safety, no skin irritation, and good anti-melanin production, anti-oxidation, anti-inflammation, anti-photoaging and anti-wrinkle effects when used to prepare cosmetics. They can be used to prepare functional skin care products with one or more functions such as anti-aging, anti-oxidation, whitening, anti-inflammation, moisturizing and skin barrier repair.

[0077] For example, applications in cosmetics include the development of anti-aging cosmetics that promote collagen synthesis, inhibit matrix metalloproteinase (MMP) activity, and reduce wrinkle formation; antioxidant skincare products that scavenge free radicals and reduce UV-induced oxidative damage; skin brightening and whitening products that inhibit tyrosinase activity and reduce melanin production; anti-inflammatory and soothing agents that reduce the expression of pro-inflammatory factors (such as IL-6 and TNF-α) and alleviate skin sensitivity; and repair skincare products that enhance skin barrier function and reduce transepidermal water loss.

[0078] Thirdly, this application provides an application of the resveratrol-tocoxelane nanomicelles described above in the preparation of antioxidant products.

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

[0080] Furthermore, the dosage form of antioxidant products is selected from one of the following: serum, ointment, cream, mask, powder, and microneedle.

[0081] Fourthly, this application provides the application of the resveratrol-tocoxelane nanomicelles described above in the preparation of skin whitening products.

[0082] In some embodiments, skin whitening products are cosmetics, including medical aesthetic products, skin care products, or beauty products.

[0083] Furthermore, the dosage form of the skin whitening product is selected from one of the following: serum, ointment, cream, mask, powder, and microneedling. Some specific examples are provided below.

[0084] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

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

[0086] I. Preparation of resveratrol-toxoxelamne nanomicelles

[0087] Example 1

[0088] This embodiment provides a method for preparing the resveratrol-toxoxalon nanomicelles of this application, including the following steps:

[0089] (1) Add 1.5g of resveratrol to 100ml of dipropylene glycol and mix. Heat at 90℃ to dissolve and obtain the first solution.

[0090] (2) Add 7.5g of tocoxexyl (2500 Da) to the first solution and mix. Heat at 90°C to dissolve and obtain the second solution.

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

[0092] Example 2

[0093] This embodiment provides a method for preparing the resveratrol-toxoxalon nanomicelles of this application, including the following steps:

[0094] (1) Add 1.5g of resveratrol-4'-O-glucoside to 100ml of dipropylene glycol and mix. Heat at 90℃ to dissolve and obtain the first solution.

[0095] (2) Add 7.5g of tocoxexyl (2500 Da) to the first solution and mix. Heat at 90°C to dissolve and obtain the second solution.

[0096] (3) The second solution was added to 7.4L of deionized water at a rate of 2mL / min. During the addition process, the solution was stirred and dispersed at 50℃ to form nano micelles.

[0097] Example 3

[0098] This embodiment provides a method for preparing the resveratrol-toxoxalon nanomicelles of this application, including the following steps:

[0099] (1) Add 1.5g of pterostilbene to 100ml of dipropylene glycol and mix. Heat at 90℃ to dissolve and obtain the first solution.

[0100] (2) Add 7.5g of tocoxexyl (2500 Da) to the first solution and mix. Heat at 90°C to dissolve and obtain the second solution.

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

[0102] Example 4

[0103] This embodiment provides a method for preparing resveratrol-tocoxelane nanomicelles of this application, which is prepared by a method that is basically the same as that in Example 1, except that in step (3), the addition rate of the second solution is 1 mL / min.

[0104] Example 5

[0105] This embodiment provides a method for preparing resveratrol-tocoxelane nanomicelles of this application, which is prepared by a method that is basically the same as that in Example 1, except that tocoxelane 6000 is used in step (2).

[0106] Example 6

[0107] This embodiment provides a method for preparing resveratrol-tocoxelane nanomicelles of this application. The preparation method is basically the same as that in Example 1, except that in step (1), the amount of resveratrol is changed to 0.1g and in step (2), the amount of tocoxelane is changed to 0.5g.

[0108] Example 7

[0109] This embodiment provides a method for preparing resveratrol-tocoxelane nanomicelles of this application. The preparation method is basically the same as that in Example 1, except that in step (1), the amount of resveratrol is changed to 2g, and in step (2), the amount of tocoxelane is changed to 20g.

[0110] Example 8

[0111] This embodiment provides a method for preparing resveratrol-tocoxelane nanomicelles of this application. The preparation method is basically the same as that in Example 1. The difference is that in step (3), the amount of the second solution added is 1 mL, so that the concentration of tocoxelane in the nanomicelle solution is 0.01 wt%.

[0112] Example 9

[0113] This embodiment provides a method for preparing resveratrol-tocoxelane nanomicelles of this application. The preparation method is basically the same as that in Example 1. The difference is that in step (3), the amount of the second solution added is 50 mL, so that the concentration of tocoxelane in the nanomicelle solution is 0.5 wt%.

[0114] Comparative Example 1

[0115] This comparative example provides a method for preparing resveratrol-tocoxelen nanomicelles, which is prepared using a method that is basically the same as that in Example 1, except that the amount of resveratrol used in step (1) is changed to 5g.

[0116] Comparative Example 2

[0117] This comparative example provides a method for preparing resveratrol-tocoxelen nanomicelles, which is prepared using a method that is basically the same as that in Example 1, except that in step (1), the amount of resveratrol is changed to 0.05g.

[0118] Comparative Example 3

[0119] This comparative example provides a method for preparing resveratrol-tocoxelane nanomicelles, which is prepared using a method that is basically the same as that in Example 1, except that in step (2), the amount of tocoxelane is changed to 30g.

[0120] Comparative Example 4

[0121] This comparative example provides a method for preparing resveratrol-tocoxelane nanomicelles, which is prepared using a method that is basically the same as that in Example 1, except that in step (2), the amount of tocoxelane is changed to 0.1g.

[0122] Comparative Example 5

[0123] This comparative example provides a method for preparing resveratrol-tocoxelane nanomicelles, which is basically the same as the method in Example 1, except that the heating temperature in step (1) is 50°C.

[0124] Comparative Example 6

[0125] This comparative example provides a method for preparing resveratrol-tocoxelen nanomicelles, which is basically the same as the method in Example 1, except that the heating temperature in step (1) is 120°C.

[0126] Comparative Example 7

[0127] This comparative example provides a method for preparing resveratrol-tocoxelane nanomicelles, which is basically the same as the method in Example 1, except that the heating temperature in step (2) is 50°C.

[0128] Comparative Example 8

[0129] This comparative example provides a method for preparing resveratrol-tocoxelen nanomicelles, which is basically the same as the method in Example 1, except that the heating temperature in step (2) is 120°C.

[0130] Comparative Example 9

[0131] This comparative example provides a method for preparing resveratrol-tocoxelane nanomicelles, which is basically the same as the method in Example 1. The difference is that steps (1) and (2) are combined, and resveratrol, solvent and tocoxelane are directly dissolved by stirring at 90°C.

[0132] II. Morphology and Particle Size Characterization

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

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

[0135] Table 1. Results of nanomicelle particle size and zeta potential determination for resveratrol, tocoxolelen, and Example 1.

[0136]

[0137] According to Table 1, the average particle size of the nanomicelles in Example 1 was 16.52 nm, and the zeta potential was -2.473 mV, indicating a negative surface charge. This negative surface charge promoted electrostatic repulsion between the R@T-NM nanoparticles, thereby enhancing their dispersibility and contributing to the stability of the system. The polydispersity index of the nanomicelles in Example 1 was much lower than that of resveratrol or tococelen, indicating a significant improvement in molecular distribution uniformity. The surface charge of the nanomicelles in Example 1 was much higher than that of resveratrol or tococelen, indicating a significant reduction in reducibility.

[0138] III. Performance Testing

[0139] (1) Light stability test

[0140] Following the light stability test conditions described in the 2020 edition of the Chinese Pharmacopoeia, "Guidelines for Stability Testing of Raw Materials and Preparations (9001)," the light stability of each example and comparative example was examined. The state of each system after being placed in a light chamber for 0, 7, and 14 days was observed and recorded. The test results of each example and comparative example are summarized in Table 2.

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

[0142]

[0143]

[0144] According to Table 2, the nanomicelles in the examples showed no significant changes after 7 and 14 days of storage, with no crystallization or stratification, exhibiting good surface stability and no other stability issues, indicating that the examples have relatively good photostability. In contrast, the comparative examples showed significant crystallization or color changes after 7 and 14 days of storage, indicating poor stability of the nanomicelles prepared in the comparative examples.

[0145] Example 1 and Comparative Example 1 are used as examples for illustration: Resveratrol and tococelesin were dissolved and prepared into solutions (or dispersions) at the same concentrations as in Example 1; the prepared solutions and the nanomicelle solutions of Example 1 and Comparative Example 1 were subjected to photostability testing according to known principles. Stability test images on day 0 and day 7 are shown below. Figure 2 As shown. According to Figure 2The results on day 0 showed that resveratrol had low solubility, with a large number of suspended particles and some precipitation; toxoxelenol was completely soluble and showed no Tyndall effect, indicating that a solution was formed; the nanomicelle solution of Comparative Example 1 had a large number of suspended particles and some precipitation; the nanomicelle solution of Example 1 was transparent and clear, and the Tyndall effect could be observed when irradiated with a laser beam, confirming that a colloid was formed; the results on day 7 showed that the nanomicelle solution of Example 1 was still transparent and clear, and the Tyndall effect could be observed when irradiated with a laser beam, indicating that it had good photostability.

[0146] The above photostability test results show that Example 1 has the best stability. Therefore, the nanomicelle solution (R@T-NM) of Example 1 was selected for further antioxidant, anti-aging and anti-inflammatory tests.

[0147] (2) Transdermal efficacy test

[0148] Methacrylamide gelatin (GelMA, 15 wt%, 100 μL) supplemented with phenyl-2,4,6-trimethylbenzoyl lithium phosphinate (LAP) photoinitiator (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 onto the hydrogel to generate a cell layer, thereby mimicking the 3D structure of skin with a dense epidermis and relatively loose subcutaneous tissue. After incubation in 24-well plates for 24 hours, the culture medium was removed, and FITC-R@T-NM (1 wt%) with a fluorescent group from Example 1 was added to the upper chamber containing 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 as follows Figure 3 ,according to Figure 3 As shown in Figure A, the GelMA hydrogel has a bilayer structure: the upper dense porous layer corresponds to the epidermis, while the lower relatively loose porous layer simulates the dermis, together forming a skin-like barrier. According to... Figure 3 As seen in image B, a large number of cells are attached to the surface of the hydrogel. According to... Figure 3 As shown in Figure C, strong green fluorescence was observed when the FITC-labeled R@T-NM solution was applied to the hydrogel surface under 488 nm excitation. Figure 3 As can be seen in the middle section, when examined by an inverted fluorescence microscope, vertical sections of the hydrogel showed a large amount of fluorescence signal throughout the matrix, confirming the permeability of FITC-R@T-NM.

[0150] (3) Biocompatibility of the resveratrol-tocoxelen 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 in a humidified incubator at 37°C and 5% CO2 (Thermo Fisher Scientific, USA). The cytotoxicity of R@T-NM prepared in Example 1 to HSF was assessed using the CCK-8 assay and a live / dead staining kit. Different concentrations of R@T-NM were prepared in PBS and sterilized by filtration through a 0.2 μm filter. HSF was cultured at 4 × 10⁻⁶ ppm. 4 Cells were seeded at densities of 200 μl / well in 96-well plates and cultured overnight under optimal conditions to allow attachment. Cells were then treated for 24 hours with complete medium containing different concentrations of R@T-NM. After removing the medium, the prepared CCK-8 reaction solution was added to each well, and the cells were incubated for another 2 hours. Optical density (OD) was quantified by measuring 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) × 100%, where Ab represents the absorbance of the test sample at 450 nm, Ac represents the absorbance of the control sample at 450 nm, and As is the OD value of the CCK-8 reaction solution.

[0152] The results are as follows Figure 4 As shown, the cytotoxicity of R@T-NM was assessed within a concentration range of 5–30 g / mL. Cell viability was found to be ≥100% at concentrations below 10 g / mL, indicating that low concentrations of R@T-NM promoted HSF cell proliferation. At a concentration of 15 g / mL, cell viability remained above 90%. Live / dead cell staining showed that cells in the R@T-NM group exhibited intact morphology with a spindle-shaped elongated appearance, and no cell death was observed. This demonstrates that the R@T-NM system exhibits good biocompatibility even when using organic solvents as solubilizers.

[0153] (4) Evaluation of antioxidant effect

[0154] DPPH radical scavenging assay: 1 mg / mL R@T-NM was added to 0.12 mg / mL DPPH ethanol. After incubation in the dark for a predetermined 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 × 100%.

[0155] Intracellular reactive oxygen species (ROS) scavenging assay: HSF cells were seeded in 24-well plates and cultured until 80-90% confluence. HSF cells were then pretreated with hydrogen peroxide (H2O2) for 30 minutes, followed by incubation in medium containing R@T-NM (10 μg / mL) for 24 hours. After treatment, the R@T-NM-containing medium was removed, and 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. Cells were incubated with DCFH-DA in the dark at 37°C for 30 minutes. The DCFH-DA solution was then aspirated, and cells were washed three times with serum-free medium to remove residual probes. Fluorescence images were acquired using an inverted fluorescence microscope, and fluorescence intensity was quantified using ImageJ software.

[0156] The results are as follows Figure 5 As shown, according to Figure 5 As shown in Figure A, toxoxexlavone, resveratrol, and R@T-NM all exhibited strong DPPH free radical scavenging capabilities, with DPPH scavenging rates of 72.97%, 90.09%, and 88%, respectively, indicating that toxoxexlavone, resveratrol, and R@T-NM possess superior antioxidant abilities. Figure 5 As shown in Figure B, the minimal green fluorescence signal in the untreated control group confirmed a healthy cell state with low basal ROS levels and no apoptotic tendency. Conversely, treatment with 50 μM H2O2 significantly increased fluorescence intensity. However, co-treatment with R@T-NM significantly attenuated this fluorescence signal. Notably, R@T-NM exhibited the weakest fluorescence intensity among all treatment groups, but did not achieve optimal free radical scavenging efficiency in the DPPH assay. This observation suggests that the antioxidant effect of resveratrol may rapidly diminish due to excessive reactivity (e.g., rapid binding with free radicals). In contrast, toxoxelam may prolong the half-life of resveratrol by reducing reactivity, thus achieving a "sustained release" effect. This further validates the protective effect of toxoxelam in preserving 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. The cells were then pretreated with the inflammatory stimulant LPS and incubated for 24 hours with intact DMEM containing R@T-NM from Example 1. Following treatment, the cell culture supernatant was collected, and IL-6 and IL-8 levels were analyzed using a commercial ELISA kit according to the manufacturer's protocol. Absorbance (OD value) was measured at 450 nm using a microplate reader, and cytokine concentrations and inhibition rates were calculated based on a standard curve.

[0159] The results are as follows Figure 6As shown, compared with the blank control group, LPS-stimulated HSF cells exhibited significantly increased secretion of pro-inflammatory cytokines IL-6 and IL-8, indicating successful stimulation of the cellular inflammatory state. However, R@T-NM treatment significantly reduced the expression levels of both cytokines. These results suggest that R@T-NM can alleviate the inflammatory response by downregulating the expression of pro-inflammatory factors.

[0160] (6) Assay on activity against melanin production

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

[0162] The results are as follows Figure 7 As shown, according to Figure 7 In skin tone, color is closely related to melanin produced by melanocytes in the basal layer of the epidermis. Tyrosinase is the rate-limiting enzyme in melanin production, and inhibiting its activity serves as a key molecular target for skin whitening agents. Figure 7 Intracellular tyrosinase activity assays in the B-cell group showed that resveratrol, R@T-NM, and GL exhibited stronger tyrosinase inhibitory effects compared to the control group. Notably, the R@T-NM treatment group showed the lowest tyrosinase activity levels, indicating its effective inhibition of this key enzyme and its potential to promote skin whitening.

[0163] (7) Cell photoaging test: HSF cell anti-aging efficacy test (UVA)

[0164] HSF cells and fibroblasts were seeded into 24-well or 12-well plates and cultured until 50-60% confluence. HSF cells were covered with PBS and inoculated with 5 J / cm² solution each time. 2 The cells were exposed to a total dose of UVA irradiation. After irradiation, the PBS was removed and replaced with intact DMEM containing R@T-NM prepared in Example 1. This irradiation protocol was repeated daily for 5 consecutive days. For aging assessment, cells in 24-well plates were stained using a aging-associated β-galactosidase (SA-β-gal) staining kit according to the manufacturer's instructions. Simultaneously, commercial NAD+ was used according to the manufacturer's instructions. + The assay kit detects NAD in cells of 12-well plates. + The content was quantified.

[0165] Cellular senescence is a marker of aging in the body, representing a state of permanent growth arrest in non-dividing cells. Based on a literature review, the applicant established a method for HSF at 5 J / cm². 2Photoaging model under high-intensity UVA irradiation. For example... Figure 8 As shown in Figure A, 5 J / cm for 5 consecutive days 2 UVA exposure significantly increased the number of SA-β-galactosidase (SA-β-gal)-positive HSF cells. Comparative analysis showed that the SA-β-gal staining intensity was significantly enhanced in UVA-irradiated HSF cells compared to the blank control group. Notably, treatment with resveratrol, R@T-NM, or GL effectively counteracted UVA-induced cellular senescence. Figure 8 As shown in Figure B, among these interventions, R@T-NM exhibited the lowest percentage of SA-β-GAL positive cells, indicating its superior anti-aging activity. Furthermore, metabolomics-based aging studies suggest that NAD... + It plays a crucial regulatory role in many key metabolic pathways closely related to cellular senescence. NAD + It can inhibit oxidative stress, repair nuclear DNA damage, and regulate mitochondrial dysfunction, thereby hindering the cellular aging process. For example... Figure 8 As shown in Figure C, by measuring intracellular NAD... + The effect of R@T-NM on UVA-induced cellular senescence was investigated at different levels. Compared with the blank control group, HSF cells in the UVA group had significantly higher NAD50 levels. + The content was significantly reduced. Compared with the UVA group, the resveratrol group and the R@T-NM group had significantly lower NAD content. + The content was significantly increased. These results indicate that R@T-NM can enhance NAD+ levels. + The expression of this substance has the potential to inhibit cell senescence.

[0166] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0167] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0168] The embodiments described above merely illustrate several implementation methods of this application and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A resveratrol-toxoxelamne nanomicelle, characterized in that, The preparation method of the resveratrol-toxoxelam nanomicelles includes the following steps: Resveratrol and / or its derivatives are added to a first organic solvent and mixed, then heated to a first temperature to dissolve, thereby obtaining a first solution; the weight percentage of resveratrol or its derivatives in the first solution is 0.1%-2%. The first temperature is 80℃-100℃; Toxoxelam is added to the first solution and mixed, then heated to a second temperature to dissolve, thus obtaining a second solution; the weight percentage of toxoxelam in the second solution is 0.5%-20%. The second temperature is 80℃-100℃; The second solution was added to water at a first rate and stirred and dispersed under a third temperature condition to obtain the resveratrol-tocoxelane nanomicelles; the first rate was 1 mL / min-3 mL / min and the third temperature was 50℃-70℃.

2. The resveratrol-tocoxelen nanomicelles according to claim 1, characterized in that, The first organic solvent includes at least one of dipropylene glycol, glycerol, 1,3-butanediol, isopropanol, butanol, pentanol, phenoxyethanol, ethanol, propylene glycol, ethyl acetate, isopropyl myristate, polyethylene glycol, and medium-chain triglycerides.

3. The resveratrol-tocoxelen nanomicelles according to claim 1, characterized in that, The resveratrol derivatives include natural derivatives and / or synthetic derivatives; The natural derivatives include one or more of the following: resveratrol glycoside, glucosinolate, pterostilbene, paclitaxel, and tetramers of resveratrol; The artificially synthesized derivatives include one or more of the following: hydroxylated derivatives, methoxylated derivatives, halogenated derivatives, and acylated derivatives.

4. The resveratrol-tocoxelen nanomicelles according to claim 1, characterized in that, The tocoxelane is polyethylene glycol succinate containing vitamin E; the polyethylene glycol structural unit in the polyethylene glycol succinate containing vitamin E has a molecular weight of 200-6000.

5. The resveratrol-toxoxalam nanomicelles according to any one of claims 1-4, characterized in that, The resveratrol or its derivatives constitute 1%-2% by weight in the first solution; and / or, The tococelen in the second solution is 0.5%-5% by weight.

6. The resveratrol-toxoxelamne nanomicelles according to any one of claims 1-4, characterized in that, The first temperature is 85℃-95℃; and / or, The second temperature is 85℃-90℃.

7. The resveratrol-toxoxelam nanomicelles according to any one of claims 1-4, characterized in that, In the step of adding the second solution to deionized water at a first rate and stirring and dispersing at a third temperature, the stirring speed is 100 rpm to 500 rpm.

8. The use of resveratrol-tocoxelane nanomicelles according to any one of claims 1-7 in the preparation of cosmetics.

9. The use of resveratrol-tocoxelane nanomicelles according to any one of claims 1-7 in the preparation of antioxidant products.

10. The use of resveratrol-tocoxelane nanomicelles according to any one of claims 1-7 in the preparation of skin whitening products.

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