Preparation of grape exosome or composition thereof and application of grape exosome or composition thereof in skin medical treatment and beauty and health care

Abstract

The invention particularly relates to preparation of grape exosomes or a composition thereof and application of the grape exosomes or the composition in skin medical treatment and beauty and health care. Skin cells Hacat are used as a model, and it is verified that the grape exosome can significantly reduce the active oxygen level in the skin cells Hacat, inhibit expression of inflammatory factors TNF-alpha and IL-1beta, significantly promote expression of Collagen1, Elastin, FLG and LOR in the skin cells Hacat, inhibit inflammatory factors TNF-alpha and IL-1beta, inhibit inflammatory factors TNF-alpha and IL-1beta, inhibit inflammatory factors TNF-alpha and IL-1beta, inhibit inflammatory factors TNF-alpha and IL-1beta and inhibit inflammatory factors TNF-alpha and IL-1beta from inflammatory factors TNF It is proved that when the grape exosome acts on skin cells, the effects of resisting inflammation, resisting oxidation, promoting skin scar healing and tightening and resisting wrinkles are expected to be achieved at the same time, and the application prospect in the aspect of medical cosmetology is huge.

Description

Technical Field

[0001] The present invention relates to the technical field of plant exosomes, and in particular to application of plant exosomes derived from grapes in the preparation of skin care products. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the 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 a person skilled in the art.

[0003] Plant exosome-like extracellular vesicles are nano-sized vesicles secreted by plant cells, which have important biological functions and broad prospects for medical and cosmetic applications. Natural ingredients, especially plant ingredients, are becoming increasingly popular in cosmetic and medical beauty applications, and plant exosomes, as natural active nanovesicles, have become a hot topic of research due to their high efficiency and low immunogenicity, and have great application potential.

[0004] Grapes, as the best beauty fruit, have received extensive attention in the medical beauty field. Grape seed extract has antioxidant, antiviral, and anti-tumor effects. The peel and pulp are rich in polyphenolic compounds such as flavonoids, resveratrol, phenolic acid, anthocyanin, acid, and tannin. Studies have found that these ingredients have strong free radical scavenging capabilities such as DPPH, and can play a significant anti-aging role.

[0005] In view of the above research status, the inventors believe that the current research on grape exosomes in this field is mainly focused on improving gastroenteritis and promoting stem cell proliferation. There are no reports on applying grape-derived exosome-like extracellular vesicles to skin cells to explore their skin care effects. Summary of the invention

[0006] In response to the above-mentioned gaps in the prior art, the present invention uses human skin cells Hacat as a research model and verifies that grape exosomes have highly effective anti-inflammatory, antioxidant, skin scar healing and firming and anti-wrinkle effects. They are expected to be added to beauty products as a skin care ingredient, and have broad application prospects in the field of medical cosmetology.

[0007] Based on the above technical effects, the present invention provides the following technical solutions: In a first aspect, a grape exosome is provided, which is an extracellular vesicle secreted by grape pulp tissue and has a particle size of 95-187 nm.

[0008] The above exosomes are derived from the peeled and pitted grape pulp tissue, and feasible grape varieties include but are not limited to Kyoho, Summer Black, Blueberry, Jingya, Hutai No. 8, Moldova, Crystal, Red Globe, Clenson, Rose Fragrance, Jasmine Fragrance, Ruby, Beauty Finger, Red Grape, Victoria, Sunshine Rose, Sunny King, etc. The grapes used to prepare exosomes should be fresh and can be sterilized in advance by irradiation.

[0009] The exosomes are obtained by crushing grape pulp tissue and then performing composite enzymolysis, and directional filtering the enzymolysis solution. The present invention verifies that in one embodiment, the preparation of the grape exosomes includes the following steps: selecting fresh grapes, peeling and pitting to obtain grape pulp, adding PBS solution and soaking for 1 to 4 hours; grinding the grape pulp and PBS solution together to extract juice, using the obtained grinding solution to remove solid insoluble matter, and retaining the supernatant; adding 0.1 to 0.3% of the initial grape mass of the composite enzyme to the supernatant for enzymolysis, retaining the enzymolysis solution, freezing and removing impurities, passing through an 80 to 100 mesh sieve to obtain a filtrate, and tangentially filtering and concentrating it to obtain the grape exosomes.

[0010] Furthermore, the grinding and juicing can be performed by a food processor, etc., with a preferred rotation speed of 1000-3000 rpm / min.

[0011] Furthermore, the grinding liquid can achieve the removal of solid insoluble matter by step-by-step filtration and the like. The present invention provides a specific implementation as follows: the grinding liquid is coarsely filtered through gauze, and the obtained coarse filtrate is centrifuged to remove impurities, the centrifugal speed is 800-2000rpm, the centrifugal time is 5-30min, and the supernatant is retained.

[0012] Furthermore, the complex enzyme is composed of pectinase, cellulase and β-1,3-glucanase, and the preferred mass ratio is 5:2~4:1~3.

[0013] In a second aspect, a composition is provided, comprising the grape exosomes described in the first aspect.

[0014] In the above composition, the grape exosomes should be in an active dose and also include other active ingredients; the "active dose" referred to here means: based on the administration method of the composition, it can be applied to subjects to slow down skin aging, anti-oxidation, anti-inflammation, fade skin scars, increase the content of collagen and elastin in the skin, and other technical effects. The specific dosage value belongs to the technical content that can be directly determined according to the conventional research methods in this field.

[0015] The purposes of adding other active ingredients include but are not limited to: (1) Improving and maintaining the physicochemical activity of the grape exosomes described in the first aspect; (2) enhancing the physiological activity of the composition; (3) As a preparation excipient.

[0016] In the above aspect (2), other active ingredients may be other products with skin care effects, including but not limited to autophagy promoters, antioxidants, whitening ingredients or moisturizing ingredients, etc. The above ingredients may be small molecule compounds or natural compounds.

[0017] In the above aspect (3), feasible formulation excipients include but are not limited to solvents, surfactants, lubricants, wetting agents, flavoring agents, emulsifiers, suspending agents, preservatives, penetrants or carriers, etc.; specific examples of the solvents are water, ethanol, propylene glycol, and glycerol; specific examples of the surfactants are fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic acid monoesters, hydroxyethyl sulfonates, imidazoline derivatives, methyl taurates, sarcosinates, fatty acid amide ether sulfates, alkylamide betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

[0018] In a third aspect, a use of the grape exosomes described in the first aspect or the composition described in the second aspect in the preparation of a skin care product is provided.

[0019] In the above application, the care product is any one or more of the following; (1) The care product is an anti-inflammatory product, and further, is a product related to improving, relieving, and treating skin inflammation; (2) The care product is an antioxidant product; (3) The care product is an anti-aging product. Furthermore, the anti-aging product is a product that improves skin aging, delays the formation of wrinkles and eye bags; (4) The care product is a skin repair product. Furthermore, the repair includes skin barrier damage, sensitivity, sunburn or scars.

[0020] In the above applications, the care product mainly acts on the skin surface, including the scalp and body skin, preferably the scalp, face, neck, hands, feet and other parts that may be exposed to the outside and need improvement; the care product can act by directly contacting the skin surface or by staying on the skin surface before being removed. Feasible types include but are not limited to cleansers, facial soaps, toners, lotions, face creams, facial masks, facial masks, sunscreens, essences, foundations, concealers and other products; products that act on the hands or feet can also be soaps, hand creams, body lotions, hand sanitizers and the like.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention uses skin cells Hacat as the research object and confirms that 1.6×10 8Particles / mL grape exosomes can significantly reduce the level of reactive oxygen in skin cells Hacat, indicating that it has the ability to scavenge free radicals and play an antioxidant role; 3.2×10 8 Grape exosomes with a concentration of 1.6×10 8 Particles / mL grape exosomes can significantly promote the expression of Collagen1, Elastin, FLG, and LOR in skin cells Hacat, indicating that it can significantly promote the regeneration of basal collagen, promote skin firming and play an anti-wrinkle role. Therefore, according to the results of the present invention, grape exosomes acting on the skin are expected to simultaneously achieve multiple skin care effects such as anti-inflammatory, anti-oxidation, enhancing collagen activity, and repairing skin barrier function, and are expected to be added to care products as a beauty ingredient.

[0022] 2. The improvement effect of grape exosomes on skin cells has not been reported in the art, especially the improvement effect on collagen, elastin and barrier function-related proteins in skin cells. This is the first report of the present invention, filling the gap in existing research.

[0023] 3. Grape is a common fruit on the market. The exosome preparation process adopted in the present invention adopts a composite enzymatic hydrolysis process, which has simple steps, economical cost, and is easier to achieve large-scale extraction, which is more conducive to the industrialization of grape exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings in the specification, 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.

[0025] Figure 1 This is a process flow chart for preparing grape exosomes according to the present invention; Figure 2 The performance characterization results of grape exosomes prepared in Example 1; in, Figure 2 A is the particle size distribution result described in Example 4; Figure 2 B and 2C are the transmission electron microscopy analysis results described in Example 4; Figure 3 The antioxidant effect test results of grape exosomes described in Example 4; in, Figure 3 A is the level of intracellular reactive oxygen species in skin cells Hacat after treatment with normal saline; Figure 3 B is the level of intracellular reactive oxygen species in skin cells Hacat after being treated with grape juice; Figure 3C is the level of intracellular reactive oxygen species after skin cells Hacat were treated with grape exosomes; Figure 4 The anti-inflammatory effect test results of grape exosomes described in Example 4; in, Figure 4 A is a graph showing the mRNA expression level of inflammatory factor TNF-α after grape exosomes treated microglia HMC3; Figure 4 B is a graph showing the mRNA expression level of the inflammatory factor IL-1β after grape exosomes treated microglia HMC3; Figure 5 The test results of the grape exosomes promoting skin firming and anti-wrinkle in Example 4; in, Figure 5 A is a graph showing the mRNA expression level of Collagen1 in skin cells Hacat after cells were treated with grape exosomes; Figure 5 B is a graph showing the mRNA expression level of Elastin in skin cells Hacat after the cells were treated with grape exosomes; Figure 5 C is a graph showing the mRNA expression level of FLG in skin cells Hacat after the cells were treated with grape exosomes; Figure 5 D is a graph showing the mRNA expression level of LOR in skin cells Hacat after cells were treated with grape exosomes; Figure 6 The results of the skin cell proliferation experiment described in Example 4; Figure 7 The results of the skin cell scratch test in Example 4; in, Figure 7 A shows the cell scratch interface conditions in different experimental groups; Figure 7 B shows the change of scratch distance of cells in different experimental groups. DETAILED DESCRIPTION

[0026] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0027] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0028] 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 in conjunction with specific embodiments.

[0029] Example 1 In this embodiment, a grape exosome and a corresponding extraction method are provided, comprising the following steps: fresh Kyoho grapes are washed and irradiated for sterilization, the peel and grape seeds are removed to obtain grape pulp, and the grape pulp is immersed in PBS for soaking treatment for 2 hours. The PBS solution and the pulp tissue after soaking treatment are transferred to a large-capacity food processor for shearing, grinding and juicing at a speed of 3000rpm / min. The obtained grinding liquid is coarsely filtered through three layers of sterile gauze, and the coarse filtrate is transferred to a 50ml sterile centrifuge tube for centrifugal removal of impurities, the centrifugal speed is 1000rpm, the centrifugal time is 20min, and the supernatant is retained.

[0030] 0.2% of the initial grape weight of composite enzyme was added to the supernatant to perform 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.

[0031] The above enzymatic hydrolysate was transferred to a freezing impurity removal device, and the impurities were removed by freezing centrifugation, the centrifugal speed was 2000 rpm, and the filter screen was 100 mesh. The obtained filtrate was tangentially filtered and concentrated to obtain plant exosome-like vesicles with a filter diameter of 30-600 nm, and then irradiated for sterilization.

[0032] Example 2 In this embodiment, another grape exosome and corresponding extraction method are provided, comprising the following steps: fresh summer black grapes are washed and irradiated for sterilization, the peel and grape seeds are removed to obtain grape pulp, and the grape pulp is immersed in PBS for soaking treatment for 1 hour. The PBS solution and the pulp tissue after soaking treatment are transferred to a capacity food processor for shearing, grinding and juicing at a speed of 1000rpm / min. The obtained grinding liquid is coarsely filtered through three layers of sterile gauze, and the coarse filtrate is transferred to a 50ml sterile centrifuge tube for centrifugal removal of impurities, the centrifugal speed is 800rpm, the centrifugal time is 30min, and the supernatant is retained.

[0033] 0.1% of the initial grape weight of the composite enzyme was added to the supernatant to perform enzymatic hydrolysis to obtain an enzymatic solution. The enzymatic reaction time was 4 h. The composite enzyme consisted of 50% pectinase, 30% cellulase, and 20% β-1,3-glucanase.

[0034] The above enzymatic hydrolysate was transferred to a freezing impurity removal device, and the impurities were removed by freezing centrifugation, the centrifugal speed was 2000 rpm, and the filter screen was 100 mesh. The obtained filtrate was tangentially filtered and concentrated to obtain plant exosome-like vesicles with a filter diameter of 30-600 nm, and then irradiated for sterilization.

[0035] Example 3 In this embodiment, another grape exosome and corresponding extraction method are provided, comprising the following steps: fresh blueberry grapes are washed and irradiated for sterilization, the peel and grape seeds are removed to obtain grape pulp, and the grape pulp is immersed in PBS for soaking treatment for 4 hours. The PBS solution and pulp tissue after soaking treatment are transferred to a capacity food processor for shearing, grinding and juicing at a speed of 3000rpm. The obtained grinding liquid is coarsely filtered through three layers of sterile gauze, and the coarse filtrate is transferred to a 50ml sterile centrifuge tube for centrifugal removal of impurities, the centrifugal speed is 2000rpm, the centrifugal time is 5min, and the supernatant is retained.

[0036] 0.3% of the initial grape weight of the composite enzyme was added to the supernatant to perform enzymatic hydrolysis to obtain an enzymatic solution. The enzymatic reaction time was 4 h. The composite enzyme consisted of 50% pectinase, 30% cellulase, and 20% β-1,3-glucanase.

[0037] The above enzymatic hydrolysate was transferred to a freezing impurity removal device, and the impurities were removed by freezing centrifugation, the centrifugal speed was 2000 rpm, and the filter screen was 100 mesh. The obtained filtrate was tangentially filtered and concentrated to obtain plant exosome-like vesicles with a filter diameter of 30-600 nm, and then irradiated for sterilization.

[0038] Example 4 In this example, the grape exosomes prepared in Example 1 above were used as an example to perform performance characterization and activity studies.

[0039] 1. Performance Characterization 1. Particle size analysis 1 mL of grape exosome suspension was added to the sample pool, and the sample pool was placed in NanoSight (Nano-ZS) for testing and analysis, and the particle size map was drawn. Figure 2 As shown in the particle size distribution diagram A, the average diameter of grape exosomes is 140 nm.

[0040] Table 1 Grape exosome particle size distribution results in Example 1 2. Transmission electron microscopy (TEM) analysis For TEM imaging, 30 μL of grape exosome suspension was added to the discharge treated sample grid, left horizontally to allow exosomes to adsorb, and then the liquid was carefully blotted with filter paper. The sample was stained with 2% phosphotungstic acid dye for 2 min, and the liquid was carefully blotted with filter paper. After drying for 30 min, the sample was sent for analysis and TEM observation and photography were obtained. The transmission electron microscopy micrographs are shown in the figure. Figure 2 As shown in B-2C.

[0041] 2. Activity Study 1. Detection of the antioxidant effect of grape exosomes Hacat cells, a skin cell line in logarithmic growth phase, were inoculated with 8000 cells / well in a 6-well cell culture plate and allowed to adhere overnight for 12 h. The normal saline group, grape juice treatment group, and grape exosome treatment group (1.6×10 8 Particles / mL), the above experimental groups were set up as follows: Normal saline group: 2% normal saline was added to the complete medium; Grape juice group: The peeled and pitted grape pulp was directly put into a food processor to squeeze the juice to obtain grape juice, and 2% of grape juice was added to the complete culture medium for treating cells, which is called the grape juice group; Grape exosome treatment group: The grape exosomes prepared in Example 1 were used as the mother solution, and the mother solution was diluted 50 times to a final concentration of 1.6×10 8 particles / mL, and 2% of the above diluted grape exosomes were added to the complete culture medium for treating cells, which was called the grape exosome treatment group.

[0042] The above experimental groups were cultured at 37°C for 24h. DCFH-DA (2,7-dichlorofluorescein diacetate, 4091-99-0, purchased from Beyotime) was diluted with PBS solution at a ratio of 1:1000 to a final concentration of 10μmol / L. The cell culture medium was removed and 500uL / well of the diluted DCFH-DA was added. Incubate in a 37ºC cell culture incubator for 20 minutes. The cells were washed three times with PBS to fully remove the DCFH-DA that did not enter the cells. The excitation wavelength was set to 488nm and the emission wavelength was set to 525nm. The live cell workstation was used to take pictures to detect the intracellular ROS level. The results are shown below. Figure 3 As shown in the figure, grape exosomes can significantly reduce the level of intracellular ROS, and the effect is significantly better than that of grape juice. This shows that grape exosomes have the function of clearing intracellular ROS and reducing the level of oxidative stress.

[0043] 2. Detection of the anti-inflammatory effect of grape exosomes HMC3 cells, a microglial cell line in the logarithmic growth phase, were inoculated with a cell suspension of 50,000 cells / well in a 6-well cell culture plate and allowed to adhere overnight for 12 h. A physiological saline group, a grape juice treatment group, and a grape exosome treatment group were set up (the above experimental groups were set up in the same way as in "1. Detection of the antioxidant effect of grape exosomes", among which there were four grape exosome groups with a mother liquor concentration of 1.6×10 10 particles / mL, the four groups of dilution factors were 25x, 50x, 100x, and 200x respectively); cultured at 37°C for 24 h.

[0044] Total cell RNA was manually extracted using the Trizol method. Reverse transcription was performed using the BeyoRT™ II cDNA First Strand Synthesis Kit (RNase H-) (Beyotime, reaction conditions 25°C 10min, 42°C 60min) to obtain cDNA. TB Green® Premix Ex Taq™ II FAST qPCR Kit was used for detection on an ABI7500 instrument (Takara, reaction conditions 95°C 3min, (95°C 5s, 58°C 30s, 72°C 40s, 40 cycles), 72°C 5min, 95°C 15s, 60°C 1min, 95°C 15s.) qPCR experiments were performed to detect the relative expression levels of mRNA of inflammatory factors TNF-α and IL-1β. The internal reference for the qPCR reaction was GADPH. The primer sequences used are shown in Table 2 below: Table 2 The results are as follows Figure 4 As shown, based on Figure 4 It can be seen that the mRNA levels of inflammatory factors TNF-α and IL-1β in cells cultured with the addition of grape exosomes were significantly reduced, and the high concentration of exosomes had a more prominent inhibitory effect on the above inflammatory factors, which indicates that grape exosomes can effectively reduce the transcriptional expression of TNF-α and IL-1β in cells and reduce the level of inflammation in the organism.

[0045] 3. Detection of the effect of grape exosomes on promoting skin firming and anti-wrinkle Hacat cells, a skin cell line in logarithmic growth phase, were inoculated with a cell suspension of 50,000 cells / well in a 6-well cell culture plate and allowed to adhere overnight for 12 h. A physiological saline group, a grape juice treatment group, and a grape exosome treatment group were set up (the above experimental groups were set up in the same way as in "1. Detection of the antioxidant effect of grape exosomes", among which there were four grape exosome groups with a mother liquor concentration of 1.6×10 10 particles / mL, the four groups of dilution factors were 25x, 50x, 100x, and 200x respectively); cultured at 37°C for 24 h.

[0046] Total RNA of cells was manually extracted using the Trizol method. Reverse transcription was performed using the BeyoRT™ II cDNA First-Strand Synthesis Kit (RNase H-) (Beyotime, reaction conditions 25°C 10min, 42°C 60min) to obtain cDNA. TB Green® Premix Ex Taq™ II FAST qPCR Kit was used for detection on an ABI7500 instrument (Takara, reaction conditions 95°C 3min, (95°C 5s, 58°C 30s, 72°C 40s, 40 cycles), 72°C 5min, 95°C 15s, 60°C 1min, 95°C 15s.) qPCR experiments were performed to detect the relative expression levels of skin firming and anti-wrinkle related genes Collagen1, Elastin, FLG, and LOR. The internal reference for the qPCR reaction was GADPH. The primer sequences used are shown in Table 3 below: Table 3 Test results such as Figure 5 As shown in the figure, the expression levels of Collagen1, Elastin, FLG, and LOR-related mRNAs in skin cells treated with grape exosomes were all increased. Among them, the expression levels of Collagen1 and Elastin-related mRNAs were positively correlated with the concentration of exosomes. As the concentration of exosomes increased, the expression levels of Collagen1 and Elastin in cells also increased. For the expression of FLG and LOR-related mRNAs, lower concentrations of exosomes can achieve a good expression-promoting effect.

[0047] 4. Detection of the effect of grape exosomes on scar repair (1) Proliferation assay Hacat cells, a skin cell line in logarithmic growth phase, were inoculated with a cell suspension of 6000 cells / well in a 96-well cell culture plate and allowed to adhere overnight for 12 h. A physiological saline group, a grape juice treatment group, and a grape exosome treatment group were set up in duplicate (the above experimental groups were set up in the same way as in “1. Detection of the antioxidant effect of grape exosomes”, where the concentration of the grape exosome mother solution after dilution was 1.6×10 8 CCK8 was detected at 0h, 24h, and 48h. The detection process is as follows: 10μL CCK-8 solution was added to each well. Incubate in the cell culture incubator for 2 hours, and then detect with an enzyme marker to measure the absorbance at 450nm.

[0048] Test results such as Figure 6 As shown, grape exosomes can promote skin cell regeneration and play a role in skin scar repair.

[0049] (2) Migration detection Hacat cells, a skin cell line in logarithmic growth phase, were inoculated with a cell suspension of 50,000 cells / well in a 6-well cell culture plate and allowed to adhere overnight for 12 hours. After starvation treatment for 12 hours, a 200-ul pipette tip was used to vertically scratch the plate, with 3 strips per well. The cells were washed with serum-free culture medium. A physiological saline group, a grape juice treatment group, and a grape exosome treatment group were set up respectively (the above experimental groups were set up in the same way as "1. Detection of the antioxidant effect of grape exosomes", where the concentration of the grape exosome mother solution after dilution was 1.6×10 8 The well plate was placed on the living cell workstation, and the parameters were set, with a shooting interval of 24h.

[0050] Test results such as Figure 7 As shown, Figure 7 A is the scratched cell culture dish interface. It can be seen that the spacing between the central scratches in the grape exosome culture group became significantly narrower after 48 hours of culture, indicating that grape exosomes can enhance the ability of skin cells to migrate and fill the blank parts in the skin tissue, thereby hopefully achieving a repair effect on skin scars.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A grape exosome, characterized in that: The exosomes are extracellular vesicles secreted by grape pulp tissue, and the particle size is 95-187nm.

2. The grape exosomes according to claim 1, characterized in that The exosomes are derived from peeled and pitted grape pulp tissue, and the grape variety is selected from one of Kyoho, Summer Black, Jingya, Moldova, Crystal, Red Globe, Karenson, Rose Fragrance, Jasmine Fragrance, Ruby, Beauty Finger, Red Grape, Victoria, Sunshine Rose, and Qingwang.

3. The grape exosomes according to claim 1, characterized in that The preparation of grape exosomes comprises the following steps: selecting fresh grapes, peeling and pitting to obtain grape pulp, adding PBS solution to soak for 1-4 hours; grinding the grape pulp and PBS solution together to extract juice, removing solid insoluble matter from the obtained grinding liquid, and retaining the supernatant; adding 0.1-0.3% of the initial grape weight of the composite enzyme to the supernatant for enzymolysis, freezing the retained enzymolysis liquid to remove impurities, passing through an 80-100 mesh sieve to obtain a filtrate, tangentially filtering and concentrating the filtrate to obtain the grape exosomes.

4. The grape exosomes according to claim 3, characterized in that The grinding and juicing is performed using a food processor at a rotation speed of 1000-3000 rpm / min; Alternatively, the grinding liquid is filtered step by step to remove the solid insoluble matter, the steps are as follows: the grinding liquid is filtered through gauze, the obtained coarse filtrate is centrifuged to remove impurities, the centrifugal speed is 800-2000rpm, the centrifugal time is 5-30min, and the supernatant is retained; Alternatively, the complex enzyme is composed of pectinase, cellulase, and β-1,3-glucanase in a mass ratio of 5:2-4:1-3.

5. A composition, characterized in that The composition comprises the grape exosomes as described in any one of claims 1 to 4; in the composition, the grape exosomes are in an active dose and also include other active ingredients; the "active dose" referred to herein means that based on the administration method of the composition, the application to a subject can have the technical effects of slowing down skin aging, anti-oxidation, anti-inflammation, fading skin scars, and increasing the content of collagen and elastin in the skin.

6. The composition according to claim 5, characterized in that The purpose of adding other active ingredients include but are not limited to: (1) Improving and maintaining the physicochemical activity of the grape exosomes according to any one of claims 1 to 4; (2) enhancing the physiological activity of the composition; (3) As a preparation excipient.

7. The composition according to claim 6, characterized in that In aspect (2), the other active ingredients are other products with skin care effects, including but not limited to autophagy promoters, antioxidants, whitening ingredients or moisturizing ingredients, selected from small molecule compounds or natural compounds; Or, in aspect (3), the formulation excipients include but are not limited to solvents, surfactants, lubricants, wetting agents, flavoring agents, emulsifiers, suspending agents, preservatives, penetrants or carriers; specific examples of the solvents are water, ethanol, propylene glycol, and glycerol; specific examples of the surfactants are fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinic acid monoesters, hydroxyethyl sulfonates, imidazoline derivatives, methyl taurates, sarcosinates, fatty acid amide ether sulfates, alkylamide betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glyceryl fatty acid esters.

8. Use of the grape exosomes according to any one of claims 1 to 4 or the composition according to any one of claims 5 to 7 in the preparation of a skin care product.

9. The use according to claim 8, characterized in that The care product is any one or more of the following; (1) The care product is an anti-inflammatory product, and further, is a product related to improving, relieving, and treating skin inflammation; (2) The care product is an antioxidant product; (3) The care product is an anti-aging product. Furthermore, the anti-aging product is a product that improves skin aging, delays the formation of wrinkles and eye bags; (4) The care product is a skin repair product. Furthermore, the repair includes skin barrier damage, sensitivity, sunburn or scars.

10. The use according to claim 8, characterized in that The care product mainly acts on the skin surface, including the scalp and body skin, preferably the scalp, face, neck, hands, feet and other parts that may be exposed to the outside and need improvement; the care product can act by directly contacting the skin surface or by staying on the skin surface before being removed. Possible types include but are not limited to cleansers, facial soaps, toners, lotions, face creams, facial masks, facial masks, sunscreens, essences, foundations or concealers; products acting on the hands or feet can also be soaps, hand creams, body lotions or hand sanitizers.

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