Application of grape exosome in preparation of beauty care products

Abstract

The invention particularly relates to application of grape exosomes in preparation of beauty care products. The grape exosome is rich in proteins, nucleic acids (such as mRNA and miRNA) and bioactive substances, and shows multiple application potentials in the fields of medical treatment, beauty and agriculture. According to the invention, zebra fish is taken as a research model, melanin, ROS (reactive oxygen species), neutrophil and fishtail regeneration detection proves that the grape exosome has melanin inhibition, anti-inflammation, antioxidant and tissue repair effects, and the application of the grape exosome in preparation of beauty products is provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant exosomes, and in particular relates to the application of grape exosomes in the preparation of beauty care products. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Grape exosome-like nanoparticles (GELNs) are nanoscale extracellular vesicles extracted from grape berries, approximately 30-300 nm in diameter and constructed with a phospholipid bilayer structure. Similar to animal exosomes, these plant-derived exosomes can carry bioactive substances such as proteins, nucleic acids (such as miRNAs), lipids, and antioxidants (such as resveratrol and vitamin C). In 2013, researchers first isolated these vesicles from grapes. Protein abundance comparison revealed that grape exosomes are rich in enzymes, GTPases, and membrane- and vesicle-associated proteins, containing as many as 96 protein types. According to a study published in Molecular Therapy on July 21, 2013, grape exosomes, when consumed by mice, evaded digestive enzymes, ultimately reaching the intestine, where they promoted intestinal epithelial proliferation and protected mice from dextran sulfate sodium (DSS)-induced colitis. Grape exosomes have been shown to possess a variety of medical activities. According to a study published in the International Journal of Pharmaceutics on January 17, 2024, oral administration of grape-derived nanovesicles effectively prevented LPS / D-GalN-induced acute liver failure. Grape exosomes not only demonstrate excellent liver protection, but their natural biocompatibility and low immunogenicity make them ideal carriers for cross-species applications, offering new possibilities for breast cancer treatment (according to the Journal of Drug Delivery Science and Technology, December 30, 2023).

[0004] Grape seeds are rich in proanthocyanidins and are known as "natural skincare products." They offer antioxidant, anti-allergic, anti-aging benefits, and can inhibit platelet aggregation and atherosclerosis, making them an important health and beauty ingredient. However, prior research on the cosmetic benefits of grape exosomes is relatively lacking. The present invention proposes that grape pulp, with its much higher yield than grape seeds, and grape exosomes possess excellent biocompatibility, making them safer as a cosmetic ingredient. Furthermore, the preparation process for grape exosomes is relatively simple. Expanding the application of grape exosomes in cosmetic applications could also help increase the economic value of grapes and promote the development of the agricultural industry. Summary of the Invention

[0005] In patent CN119955709A, the inventors used Hacat cells, a skin cell line, as a research model to explore the application of grape exosomes in skin care and cosmetic health. Using zebrafish as a research model, the inventors demonstrated at the biological level that grape exosomes, after ingestion, can still exert anti-inflammatory, antioxidant, and tissue repair effects. Furthermore, the inventors confirmed that grape exosomes inhibit melanin synthesis, suggesting their potential as an active ingredient in skin-whitening products.

[0006] Based on the above research results, the present invention provides the following technical solutions: In a first aspect, the present invention provides the use of grape exosomes in preparing beauty care products.

[0007] Preferably, the grape exosomes are prepared from fresh grapes by mechanical crushing to obtain a filtrate, which can be prepared by differential centrifugation, density gradient centrifugation or ultrafiltration. In one embodiment verified by the present invention, the grape exosomes can be prepared by the method described in patent CN119955709A.

[0008] The beauty care products include external products and internal products. The external products include but are not limited to cleaning products, smear products and dressing products; the cleaning products include facial and body cleaning products, specific examples of which are facial cleansers, makeup remover water / oil, scrubs, etc.; the smear products include water-based solutions, emulsions, creams, oils, gels, ointments or suspensions, specific examples of which are essences, lotions, facial creams, body lotions, sunscreens, lip balms, etc.; the dressing products include but are not limited to patch masks, mud / paste masks or medical dressings, etc.

[0009] The oral products include but are not limited to capsules, tablets, oral liquids, drinks, powders, granules, gels, etc.

[0010] In one embodiment provided by the present invention, use of grape exosomes in the preparation of an anti-inflammatory product is provided.

[0011] In another embodiment, there is provided a use of grape exosomes in preparing an antioxidant product.

[0012] In yet another embodiment, there is provided use of grape exosomes in preparing a tissue repair product.

[0013] In an embodiment with better effects, the present invention also provides the use of grape exosomes in the preparation of whitening products.

[0014] In a second aspect, a skin care product having the effect of inhibiting melanin is provided, wherein the skin care product contains an active dose of grape exosomes.

[0015] In one embodiment, the skin care product is a facial mask, which is further a facial mask composed of facial mask paper and facial mask essence. The ingredients and weight parts of the essence are as follows: 0-20 parts of grape exosomes (not 0), 1-10 parts of butylene glycol, 1-5 parts of 1,2-pentanediol, 1-8 parts of glycerol, 1-5 parts of PEG / PPG-17 / 6 copolymer, and 1-5 parts of bis-PEG-18 methyl ether dimethyl silane. , tocopherol (vitamin E) 0.001-0.1 parts, p-hydroxyacetophenone 0.1-1 parts, sodium hyaluronate 0.05-0.5 parts, polyacrylate crosspolymer-60.05-1 parts, xanthan gum 0.05-0.5 parts, hydroxyphenylpropionamide benzoic acid 0.001-0.5 parts, ascorbyl palmitate 0.001-0.5 parts, panthenol 0.1-2 parts, trehalose 0.1-2 parts, add water to 100 parts.

[0016] Compared with the prior art, the present invention has the following beneficial effects: Previous studies of the present invention have confirmed at the cellular level that grape exosomes have anti-inflammatory and repairing activities. To further investigate the feasibility of grape exosomes as a cosmetic active ingredient, this study used zebrafish as the research subject to confirm at the organismal level that the intake of grape exosomes can indeed achieve anti-inflammatory and repairing effects. In addition, this study also clarified the efficacy of grape exosomes in inhibiting melanin synthesis, providing a promising application for their application in whitening products, which has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0018] Figure 1 This is the test result of the inhibitory effect of grape exosomes on zebrafish melanin in Example 2; in, Figure 1 Middle A is the phenotypic diagram of pigment inhibition of the control group, competitor group 1, competitor group 2, and sample group; Figure 1 Middle B is the statistical analysis result of the pigment area of ​​the control group, competitor group 1, competitor group 2 and sample group; Figure 2 This is the test result of the antioxidant effect of grape exosomes on zebrafish in Example 2; in, Figure 2 Middle A is the phenotype of ROS staining in the control group, model group, competitor group 1, competitor group 2 and sample group; Figure 2 Middle B is the statistical analysis results of ROS fluorescence intensity of the control group, model group, competitor group 1, competitor group 2 and sample group; Figure 3 This is the test result of the anti-inflammatory effect of grape exosomes on zebrafish in Example 2; in, Figure 3 A in the middle is the fluorescence phenotype of neutrophils in the control group, model group, competitor group 1, competitor group 2 and sample group; Figure 3 Figure B shows the statistical results of the number of neutrophils in the control group, model group, competitor group 1, competitor group 2, and sample group; Figure 4 This is the test result of the grape exosomes on zebrafish tissue regeneration in Example 2; in, Figure 4 Middle A is the phenotypic diagram of fish tail regeneration in the control group, model group, competitor group 1, competitor group 2 and sample group; Figure 4 Middle B is the statistical results of the regenerated fish tail area of ​​the control group, model group, competitor group 1, competitor group 2 and sample group; above Figure 1-Figure 4 Data are expressed as mean ± SD, n = 10; one-way ANOVA and Tukey's multiple comparison test were used for statistical analysis, with p < 0.05, p < 0.01, and p < 0.001 compared with the model group. DETAILED DESCRIPTION

[0019] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0020] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0021] In the context of this specification, the word "comprising" is understood to mean "including especially". It should not be interpreted as "consisting only of. . . . "

[0022] As described in the background, existing research on the application of grape exosomes in the field of beauty care is relatively blank. To further verify the cosmetic benefits of grape exosomes on organisms, this paper specifically verifies the whitening, antioxidant, anti-inflammatory, and tissue repair effects of grape exosomes, and provides the application of grape exosomes in the preparation of beauty care products.

[0023] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0024] Example 1 In this embodiment, a method for preparing grape exosomes is provided, with reference to patent CN119955709A, and the specific steps are as follows: Fresh Kyoho grapes were washed and sterilized by irradiation. The skin and seeds were removed to obtain the grape pulp, which was then immersed in PBS for 2 hours. The PBS solution and the pulp were transferred to a large-capacity food processor for shear grinding and juicing at 3000 rpm / min. The resulting grinding solution was coarsely filtered through three layers of sterile gauze. The filtrate was transferred to a 50 mL sterile centrifuge tube and centrifuged at 1000 rpm for 20 minutes to remove impurities. The supernatant was retained.

[0025] A composite enzyme of 0.2% of the initial grape weight was added to the supernatant for enzymatic hydrolysis to obtain an enzymatic solution. The enzymatic hydrolysis reaction time was 4 h. The composite enzyme consisted of 50% pectinase, 30% cellulase, and 20% β-1,3-glucanase.

[0026] The enzymatic hydrolysate was transferred to a freezer for impurity removal and centrifuged at 2000 rpm using a 100-mesh filter. The filtrate was concentrated by tangential flow filtration to obtain plant exosome-like vesicles with a filter diameter of 30-600 nm, which were then sterilized by irradiation.

[0027] Example 2 In this example, the whitening, antioxidant, anti-inflammatory and tissue repair promoting properties of the grape exosomes in Example 1 are provided, and the research method is as follows: 1. Experimental Models and Consumables 1. Test system Wild-type AB strain zebrafish; Tg(mpx:GFP) transgenic zebrafish.

[0028] Zebrafish age: 6 h post fertilization (6 hpf).

[0029] 2. Experimental instruments Artificial climate incubator (RGX-70ES), high-speed refrigerated centrifuge (LC-LX-HR165A), and microplate reader (PL-9602G).

[0030] 3. Experimental consumables 90mm culture dishes, 60mm culture dishes, six-well plates, 3mL plastic droppers, 1.5mL EP tubes, 15mL EP tubes, 50mL EP tubes, etc.

[0031] 4. Experimental reagents 60% E3 solution (NaCl 17.4 g / L; CaCl2•2H2O 2.9 g / L; KCl 0.8 g / L; MgCl2•6H2O 4.89 g / L), methylene blue (CAS: 7220-79-3), glucose, alloxan, and ELISA kit.

[0032] 5. Incubation medium containing the sample (grape exosomes in Example 1).

[0033] Control group: raised under normal conditions without adding any additional active ingredients; Competitive Product 1: The product name of Competitive Product 1 is Arbutin Whitening and Spot Removal Mask, and it claims that the whitening active ingredient is Glycyrrhiza glabra. The incubation solution of Competitive Product 1 contains 1% ( v / v ) Competitor 1 facial mask essence.

[0034] Competitive Product 2: The product name of Competitive Product 2 is Pure Vitamin C Brightening Mask, which claims that the whitening active ingredient is a VC derivative. The incubation solution of Competitive Product 2 contains 1% ( v / v ) Competitor 2 facial mask essence.

[0035] Sample group: Grape exosome concentration was 50 billion particles / mL.

[0036] 2. Experimental Research Methods 1. Whitening efficacy test steps (melanin production inhibition) In the evaluation of whitening efficacy, observing the production of melanin on the surface of zebrafish can directly reflect the inhibitory ability of the candidate on pigment synthesis. In the experiment, zebrafish are usually treated in the early stage of embryonic development, and the changes in the amount or area of ​​melanin are recorded through microscopic imaging to determine the whitening effect. The steps are as follows: a. Take zebrafish embryos that have developed for 24 hours and remove the egg shells; b. The embryos were divided into groups (control group, competitor group 1, competitor group 2, and sample group), with 10 embryos per group, and placed in a 6-well plate; c. Add incubation medium containing the sample and continue culturing until 72 hpf; d. Photograph and record melanin deposition under a microscope, and measure melanin area or pixel density using software such as ImageJ.

[0037] 2. Antioxidant efficacy detection experimental steps (ROS staining) Antioxidant activity testing relies on ROS staining. After zebrafish are exposed to oxidative stressors, the level of reactive oxygen species (ROS) in the body is labeled using a fluorescent probe (e.g., DCFH-DA). Changes in fluorescence intensity reflect the antioxidant's ability to scavenge free radicals. The steps are as follows: a. 72 hpf zebrafish larvae were collected, with 10 fish per group, and divided into treatment groups (control group, model group, competitor group 1, competitor group 2, and sample group); b. H2O2 (200 μM) was added to the model group and sample group to induce ROS generation, and the samples were treated at the same time; c. After 24 hours of treatment, zebrafish were incubated with DCFH-DA probe (10 μM) for 30 minutes; d. After washing, observe and photograph under a fluorescence microscope; e. Quantification of green fluorescence intensity, reflecting the ROS level.

[0038] 3. Anti-inflammatory efficacy test steps (neutrophil count) The evaluation of anti-inflammatory effects is based on the accumulation of neutrophils at the site of inflammation. Using the Tg(mpx:GFP) transgenic zebrafish model, an inflammatory response is induced and the number of GFP-labeled neutrophils throughout the body is counted to determine the anti-inflammatory effect. The steps are as follows: a. Experiments were started at 3 dpf using Tg(mpx:GFP) neutrophil fluorescent transgenic zebrafish. b. Induce inflammation model with LPS; c. Group treatment (control group, model group, competitor group 1, competitor group 2, and sample group), 10 cells per group; Competitor group 1, competitor group 2, and sample group were cultured as an inflammation model. After 6–12 hours of treatment, whole-body neutrophil counts were imaged under a fluorescence microscope. d. Count the number of GFP cells to evaluate the inhibitory effect on inflammation.

[0039] 4. Experimental steps for testing tissue repair efficacy (fish tail regeneration) Tissue repair capacity was assessed through a tail regeneration experiment. After standardized shearing of the zebrafish tail fin, its regeneration was continuously observed. The regenerated length of the tail fin was compared between different treatment groups to verify the sample's ability to promote tissue repair. The steps are as follows: a. Select 3 dpf zebrafish larvae and cut the tail fin at the standard position using microscissors; b. The fish were randomly divided into the tail-docking group + PBS group, the tail-docking group + sample 1 group, the tail-docking group + competitor 2 group, and the tail-docking group + sample group, with 10 fish in each group; c. Cultivate in E3 medium for 24 hours; d. 24 hours later, photograph the regenerated caudal fin area and record the regenerated length; e. Use ImageJ to measure the length or area of ​​the regenerated area and evaluate the repair capacity.

[0040] 3. Experimental Research Results 1. Detection of the melanin-inhibiting effect of grape exosomes The results are as follows Figure 1 Figure A shows the melanin deposition in zebrafish embryos from different treatment groups, based on statistical analysis of the above test data. Compared to the control group, Competitor 1 exhibited a weaker pigmentation inhibition effect, with melanin deposition similar to that of the control group. Competitor 2, however, exhibited a significant reduction in melanin, indicating a moderate inhibitory effect. Notably, the sample group exhibited the least melanin deposition, exhibiting a significant reduction in pigmentation, indicating the strongest inhibitory effect on melanin production.

[0041] Figure B shows the quantitative statistical results of melanin area in each zebrafish group. The statistical data showed no significant difference between Competitor 1 and the control group (p>0.05), suggesting limited inhibitory effect. Competitor 2 showed significantly lower melanin area than the control group (p<0.01), demonstrating its strong whitening potential. The sample group had the smallest melanin area, significantly greater than Competitor 2 (p<0.001), further validating its excellent pigment inhibition ability. These data demonstrate that grape exosomes have a significant whitening effect.

[0042] 2. Antioxidant efficacy testing of grape exosomes The results are as follows Figure 2Figure A shows the ROS fluorescence phenotype of zebrafish treated with different treatment groups under hydrogen peroxide induction. Compared with the control group, the model group showed significantly increased whole-body green fluorescence after H2O2 treatment, indicating a significant increase in ROS levels. The fluorescence intensity of Competitor 1 decreased, indicating some antioxidant activity. Competitor 2 showed strong fluorescence, close to that of the model group, indicating limited ROS scavenging ability. The fluorescence of the sample group was the weakest, almost returning to the level of the control group, demonstrating its effective scavenging of excess ROS and significant antioxidant activity. Figure B shows the quantitative statistical results of ROS fluorescence intensity in zebrafish from each group. The fluorescence intensity of zebrafish in the model group was significantly higher than that in the control group (p<0.001), confirming that H2O2 successfully induced oxidative stress. Competitor 1 showed a significant decrease in ROS levels (p<0.01), demonstrating some scavenging activity. Competitor 2 showed no significant difference from the model group (p>0.05), indicating weak antioxidant capacity. The fluorescence intensity of the sample group was the lowest, significantly lower than that of all other groups (p<0.001), indicating that the sample has excellent antioxidant capacity and can effectively alleviate H2O2-induced oxidative damage. These data show that the sample has significant antioxidant efficacy.

[0043] 3. Anti-inflammatory efficacy testing of grape exosomes The results are as follows Figure 3 Figure 1 shows the fluorescence phenotype of neutrophils in zebrafish treated with different treatment groups after tail injury. In the model group, a large number of GFP-positive neutrophils accumulated at the injury site, indicating a pronounced acute inflammatory response. In Competitor 1 and Competitor 2 groups, neutrophil accumulation remained prominent, similar to that in the model group, and the anti-inflammatory effect was not significant. In contrast, the sample group showed a significant decrease in neutrophil counts at the injury site, and the fluorescence signal was significantly weakened, indicating significant anti-inflammatory activity. Figure 1 shows the statistical results of the neutrophil counts in the injury area of ​​each group. The neutrophil count in the model group was significantly higher than that in the control group (p<0.001), confirming the successful establishment of the inflammatory model. Although the neutrophil counts in Competitor 1 and Competitor 2 groups decreased slightly, the difference was not statistically significant compared to the model group (p>0.05), indicating limited anti-inflammatory effects. The neutrophil count in the sample group was significantly lower than that in the model group and the two competitor groups (p<0.001), approaching the level of the control group, indicating that the sample has a highly significant anti-inflammatory effect and can effectively inhibit the excessive recruitment of inflammatory cells. These data indicate that the sample has significant anti-inflammatory effects. 4. Detection of tissue regeneration efficacy of grape exosomes The results are as follows Figure 4As shown, Panel A shows the regenerative phenotypes of zebrafish treated with different treatments 72 hours after tail amputation. The model group showed slow tail regeneration, with significantly insufficient regenerated tissue at the amputation site. Competitors 1 and 2 showed similar levels of regeneration as the model group, with limited recovery of the caudal fin defect area, indicating a weak effect on tissue repair. In contrast, the sample group showed significantly increased regenerated tissue and significantly improved caudal fin structural integrity, demonstrating a strong regeneration-promoting effect. Panel B shows the quantitative statistical results of the caudal fin regeneration area of ​​each zebrafish group. The regeneration area in the model group was significantly lower than that in the control group (p<0.001). The regeneration areas in Competitors 1 and 2 showed no significant difference from those in the model group (p>0.05), indicating a weaker ability to promote tissue regeneration. The sample group showed a significant increase in regeneration area, significantly exceeding that in the model group and both competitor groups (p<0.001), demonstrating that the sample has a significant effect on promoting tissue repair and can effectively accelerate the regeneration of the zebrafish caudal fin. These data demonstrate that the sample has a significant effect on promoting tissue repair.

[0044] Example 3 In this embodiment, a facial mask containing the grape exosomes of Example 1 is provided. The components and weight proportions of the facial mask essence are as follows: Grape exosomes 0-20 parts (not 0), butylene glycol 1-10 parts, 1,2-pentanediol 1-5 parts, glycerin 1-8 parts, PEG / PPG-17 / 6 copolymer 1-5 parts, bis-PEG-18 methyl ether dimethyl silane 1-5 parts, tocopherol (vitamin E) 0.001-0.1 parts, p-hydroxyacetophenone 0.1-1 parts, sodium hyaluronate 0.05-0.5 parts, polyacrylate crosspolymer-60.05-1 parts, xanthan gum 0.05-0.5 parts, hydroxyphenylpropionamidobenzoic acid 0.001-0.5 parts, ascorbyl palmitate 0.001-0.5 parts, panthenol 0.1-2 parts, trehalose 0.1-2 parts, add water to 100 parts.

[0045] The preparation method is as follows: mix the above ingredients evenly, let them stand, and then fill them together with the disinfected facial mask cloth after passing the inspection.

[0046] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Application of grape exosomes in the preparation of beauty care products.

2. The use according to claim 1, characterized in that The beauty care products include external use products and internal use products; the external use products include but are not limited to cleaning products, smear products and dressing products; the internal use products include but are not limited to capsules, tablets, oral liquids, drinks, powders, granules or gels.

3. The use according to claim 2, characterized in that The cleaning products include facial and body cleaning products, and are further selected from facial cleansers, makeup removers / oils, or scrubs; The smearable product includes an aqueous solution, an emulsion, a cream, an oil, a gel, an ointment or a suspension; further, it is selected from an essence, a lotion, a face cream, a body lotion, a sunscreen or a lip balm; The dressing products include but are not limited to patch masks, mud / cream masks or medical dressings.

4. The use according to claim 1, wherein The beauty care product is an anti-inflammatory product.

5. The use according to claim 1, characterized in that The beauty care product is an antioxidant product.

6. The use according to claim 1, wherein The beauty care product is a tissue repair product.

7. The use according to claim 1, wherein The beauty care product is a whitening product.

8. A skin care product with the effect of inhibiting melanin, characterized in that: The skin care product contains an active dose of grape exosomes.

9. The skin care product according to claim 8, wherein The skin care product is a facial mask.

10. The skin care product according to claim 9, wherein The facial mask is a facial mask, including facial mask paper and facial mask essence. The weight parts of each component in the facial mask essence are as follows: 0-20 parts of grape exosomes (not 0), 1-10 parts of butylene glycol, 1-5 parts of 1,2-pentanediol, 1-8 parts of glycerin, 1-5 parts of PEG / PPG-17 / 6 copolymer, 1-5 parts of bis-PEG-18 methyl ether dimethyl silane, 0.001-0.1 parts of tocopherol, 0.1-1 parts of p-hydroxyacetophenone, 0.05-0.5 parts of sodium hyaluronate, 0.05-1 parts of polyacrylate crosspolymer-6, 0.05-0.5 parts of xanthan gum, 0.001-0.5 parts of hydroxyphenylpropionamidobenzoic acid, 0.001-0.5 parts of ascorbyl palmitate, 0.1-2 parts of panthenol, and 0.1-2 parts of trehalose, and water is added to 100 parts.

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