Use of coffee bean-derived extracellular vesicles

Abstract

The invention relates to application of coffee bean-derived extracellular vesicles, in particular to application of the coffee bean-derived extracellular vesicles in skin care products, and more particularly relates to application of the coffee bean-derived extracellular vesicles. The invention provides application of coffee bean-sourced extracellular vesicles in preparation of products capable of inhibiting tyrosinase activity and / or reducing melanogenesis, products capable of promoting cell regeneration and / or resisting oxidation, repairing and aging, and products with whitening and freckle removing effects.

Description

Technical Field

[0001] The invention relates to the technical field of extracellular vesicles, in particular to new uses of extracellular vesicles derived from coffee beans. Background Art

[0002] Extracellular vesicles (EVs) are nanoscale (30-150nm) membrane vesicles released by cells into the extracellular matrix. They are involved in cell communication, migration, angiogenesis, and tumor cell growth. They are widely present in various body fluids and cell supernatants, and stably carry important signaling molecules. A large number of studies have shown that extracellular vesicles produced by human cells (especially stem cells) can prevent and treat a variety of diseases. In addition, plant EVs, especially Chinese herbal medicine (medicinal plant) EVs, are considered to be an important emerging field in recent years.

[0003] Coffee beans belong to the Rubiaceae family and are mainly produced in Brazil, Indonesia, Colombia and other places. In China, they are mainly produced in Yunnan, Hainan, Guangdong and other places. The seeds are dark green or dark brown, have a unique aroma, and taste slightly bitter and astringent. Coffee beans are not only widely used as a beverage, but also have certain medicinal value. Guangxi Medicine Records records: "Coffee beans have an excitatory and diuretic effect, and roasted coffee beans can help digestion." Modern research shows that coffee beans have the effects of stimulating the central nervous system, lowering blood pressure, anti-oxidation, and anti-tumor.

[0004] At present, there are few reports on extracellular vesicles derived from coffee beans. Therefore, there is an urgent need for a method for extracting extracellular vesicles from coffee beans using coffee beans as raw materials, and applying it to reduce melanin production, promote skin cell regeneration, and provide antioxidant repair and anti-aging effects. Summary of the invention

[0005] In view of the above-mentioned defects of the prior art, the present invention provides uses of extracellular vesicles derived from coffee beans in promoting cell regeneration, reducing melanin production and antioxidant repair.

[0006] Solutions used to solve the problem: The present invention provides the use of extracellular vesicles derived from coffee beans in preparing a product capable of inhibiting tyrosinase activity and / or reducing melanin production.

[0007] The present invention provides the use of extracellular vesicles derived from coffee beans in the preparation of products having cell regeneration promotion and / or anti-oxidation, repair and anti-aging effects.

[0008] The present invention provides application of coffee bean-derived extracellular vesicles in preparing a product with whitening and freckle-removing effects.

[0009] Preferably, the coffee beans are fresh coffee beans.

[0010] Preferably, the coffee bean-derived extracellular vesicles are prepared by a preparation method comprising the following steps: Coffee beans are soaked in sterile water containing penicillin, streptomycin, and amphotericin B, and then the coffee beans are broken to obtain coffee bean juice, the coffee bean juice is centrifuged, the supernatant is collected, the filtrate is ultracentrifuged, the precipitate is collected, and the precipitate is resuspended to obtain the coffee bean extracellular vesicles. The extraction method of the coffee bean-derived extracellular vesicles is not limited to the ultracentrifugation process mentioned in this patent, but also includes the commonly used extracellular vesicle extraction processes on the market, such as tangential flow, graded filtration, precipitation method, etc.

[0011] Preferably, the concentration of penicillin is 50-150 U / mL, the concentration of streptomycin is 0.05-0.5 mg / mL, and the concentration of amphotericin B is 0.1-0.5 μg / mL.

[0012] Preferably, the soaking time is controlled to be 12 to 18 hours.

[0013] Preferably, the cell wall breaking time is controlled to be 5 to 10 minutes.

[0014] Preferably, the centrifugation is performed at 200-1000 g for 5-15 min at 0-8° C., the supernatant is collected, then the centrifugation is performed at 1000-5000 g for 10-30 min, the supernatant is collected, and then the centrifugation is performed at 3000-6000 g for 20-40 min.

[0015] Preferably, the ultracentrifugation is performed at 8000-12000 g for 40-70 min at 0-8° C., the supernatant is collected, and then the ultracentrifugation is performed at 100000-120000 g for 60-100 min.

[0016] Preferably, the product comprises a cosmetic or a medicine.

[0017] Preferably, the product is a cosmetic, and its dosage form includes emulsion, water, oil or gel.

[0018] Preferably, the product is a medicine, and its dosage forms include oils, emulsions, ointments, pastes, coatings, gels, aerosols, sprays, solutions, and liniments.

[0019] Effects of the invention: The invention uses coffee beans as raw materials to obtain coffee bean extracellular vesicles through an extraction method that is simple to operate, mild in conditions, short in time, low in cost and high in safety, and applies the extracellular vesicles to the field of skin care. The active ingredients are high and the extracellular vesicles are effective in reducing melanin production, inhibiting tyrosinase activity, whitening and removing spots, improving wrinkles and skin aging, and in skin repair and anti-oxidation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The appearance of coffee bean-derived extracellular vesicles; Figure 2 This is an electron micrograph of CB-Exo (arrows indicate extracellular vesicles derived from coffee beans); Figure 3 This is the particle size distribution diagram of extracellular vesicles derived from coffee beans; Figure 4 This is an SDS-PAGE image of extracellular vesicles derived from coffee beans, where lane 1 represents the protein molecular weight standard and lane 2 represents the exosomes derived from coffee beans; Figure 5 Line graph of cytotoxicity of coffee bean-derived extracellular vesicles to human dermal fibroblasts HDF, human keratinocytes HaCat, and mouse melanoma cells B16; Figure 6 Schematic diagram of coffee bean-derived extracellular vesicles ingested by mouse melanocytes (red: PKH26-labeled coffee bean-derived extracellular vesicles; green: phalloidin; blue: DAPI); Figure 7 The graphs show the infiltration of coffee bean-derived extracellular vesicles into the ex vivo skin of 8-week-old Bama miniature pigs and the statistical graph of the single infiltration amount (PKH26-labeled coffee bean-derived extracellular vesicles, as indicated by the yellow arrow in the figure; DAPI, as indicated by the green arrow in the figure; the white arrow in the figure refers to the exosomes labeled on the surface of the ex vivo skin), where A is a schematic diagram of the infiltration of coffee bean-derived exosomes into the ex vivo skin at 0h and 6h, and B is a statistical graph of the single infiltration amount of coffee bean-derived exosomes into the ex vivo skin; Figure 8 This is a bar graph showing the inhibition of melanocyte tyrosinase activity by coffee bean-derived extracellular vesicles; Fig. 9 The diagram is a schematic diagram of the effect of coffee bean-derived extracellular vesicles on melanin deposition in melanocytes and a bar graph of melanin synthesis inhibition, wherein A is a bar graph of the effect of coffee bean-derived extracellular vesicles on melanin synthesis inhibition, and B is a schematic diagram of the effect of coffee bean-derived extracellular vesicles on melanin deposition in melanocytes; Fig.10 Schematic diagram of coffee bean-derived extracellular vesicles ingested by human dermal fibroblasts (red: PKH26-labeled coffee bean-derived extracellular vesicles; green: phalloidin; blue: DAPI); Fig.11 Schematic diagram of the effect of coffee bean-derived extracellular vesicles on the aging of human dermal fibroblasts; Fig.12 This is a bar graph showing the effect of coffee bean-derived extracellular vesicle-like particles on the proliferation of human dermal fibroblasts; Fig.13The bar graph shows that coffee bean-derived extracellular vesicles promote type I collagen secretion in human dermal fibroblasts; Fig.14 This is a bar graph showing the effect of coffee bean-derived extracellular vesicles on the level of reactive oxygen species in human dermal fibroblasts; Fig.15 The bar graph shows the effect of coffee bean-derived extracellular vesicles on superoxide dismutase levels in keratinocytes. Fig.16 This is a bar graph showing the effect of coffee bean-derived extracellular vesicles on the level of reactive oxygen species in keratinocytes; Fig.17 This is a bar graph showing the effect of coffee bean-derived extracellular vesicles on the antioxidant capacity of keratinocytes; Fig.18 This is a schematic diagram of the whitening effect of a facial cream containing the coffee bean-derived extracellular vesicles of Example 1 in a human test; Fig.19 Schematic diagram of the anti-wrinkle effect of using the coffee bean-derived extracellular vesicles cream of Example 1 on the 0th day and the 14th day of the human test on facial nasolabial folds and wrinkles. DETAILED DESCRIPTION

[0021] In order to make the technical solutions and beneficial effects of the present invention more clearly understandable, the following is a detailed description by listing specific embodiments. The drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0022] After extensive and in-depth research, the inventor unexpectedly discovered that coffee bean-derived exosomes have new uses in skin care products. Specifically, coffee bean-derived extracellular vesicles are used in the preparation of products that can inhibit tyrosinase activity and / or reduce melanin production, promote cell regeneration and / or have antioxidant, repair, and anti-aging effects, and whitening and freckle removal products. Based on this, the technical solution of this application is proposed.

[0023] The present invention provides the use of extracellular vesicles derived from coffee beans in preparing a product capable of inhibiting tyrosinase activity and / or reducing melanin production.

[0024] The present invention provides the use of extracellular vesicles derived from coffee beans in the preparation of products having cell regeneration promotion and / or anti-oxidation, repair and anti-aging effects.

[0025] The present invention provides application of coffee bean-derived extracellular vesicles in preparing a product with whitening and freckle-removing effects.

[0026] In certain embodiments, the coffee beans are derived from Brazilian Santos coffee beans.

[0027] In certain embodiments, the coffee bean-derived extracellular vesicles are prepared by a preparation method comprising the following steps: The coffee beans are soaked in sterile water containing penicillin, streptomycin and amphotericin B, and then the coffee beans are broken to obtain coffee bean juice. The coffee bean juice is centrifuged, the supernatant is collected, the filtrate is ultracentrifuged, the precipitate is collected, and the precipitate is resuspended to obtain the coffee bean extracellular vesicles.

[0028] In certain embodiments, the penicillin concentration is 50-150 U / mL, for example, 50 U / mL, 60 U / mL, 70 U / mL, 80 U / mL, 90 U / mL, 100 U / mL, 110 U / mL, 120 U / mL, 130 U / mL, 140 U / mL, 150 U / mL, etc.

[0029] In certain embodiments, the penicillin concentration is 80-120 U / mL.

[0030] In certain embodiments, the penicillin concentration is 100 U / mL.

[0031] In certain embodiments, the concentration of streptomycin is 0.05-0.5 mg / mL, for example, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, etc.

[0032] In certain embodiments, the concentration of streptomycin is 0.05-0.2 mg / mL.

[0033] In certain embodiments, the streptomycin concentration is 0.1 mg / mL.

[0034] In certain embodiments, the concentration of amphotericin B is 0.1-0.5 μg / mL, for example, 0.15 μg / mL, 0.2 μg / mL, 0.25 μg / mL, 0.3 μg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, etc.

[0035] In certain embodiments, the concentration of amphotericin B is 0.1-0.3 μg / mL.

[0036] In certain embodiments, the concentration of amphotericin B is 0.25 μg / mL.

[0037] In certain embodiments, the soaking time is controlled to be 12 to 18 hours, such as 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, etc.

[0038] In certain embodiments, the cell wall breaking time is controlled to be 5 to 10 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, etc.

[0039] In certain embodiments, the cell wall breaking is intermittent cell wall breaking, and the specific steps include: breaking the cell wall for 5 to 10 minutes, and resting for 5 to 10 minutes.

[0040] In certain embodiments, the centrifugation is carried out at 0-8°C (e.g., 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, etc.) at 200-1000g (e.g., 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1000 g, etc.) for 5-15 min (e.g., 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc.), the supernatant is collected, and then centrifuged at 1000-5000g (e.g., 1000 g, 1500 g, 2000 g, 2500 g, 3000 g, 3500 g, 4000 g, 4500 g, 5000 g, etc.) for 10-30 min (e.g., 10 min, 15 min, 20 min, 25 min, 30 min, etc.), collect the supernatant and centrifuge at 3000-6000 g (e.g., 3000 g, 3500 g, 4000 g, 4500 g, 5000 g, 5500 g, 6000 g, etc.) for 20-40 min (e.g., 20 min, 25 min, 30 min, 35 min, 40 min, etc.).

[0041] In certain embodiments, the ultracentrifugation is performed at 0-8°C (e.g., 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, etc.) at 8000-12000g (e.g., 8000 g, 8500 g, 9000 g, 9500 g, 10000 g, 10500 g, 11000 g, 11500 g, etc.) for 40-70 min (e.g., 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, etc.), the supernatant is collected, and then centrifuged at 100000-120000g (e.g., 100000 g, 105000 g, 110000 g, 115000 g, 120000 g, etc.) for 60-100 min (e.g., 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, 95 min, etc.).

[0042] In certain embodiments, the product comprises a cosmetic or a pharmaceutical.

[0043] In certain embodiments, the product is a cosmetic, and its dosage form includes an emulsion, an aqueous solution, an oil, or a gel.

[0044] In certain embodiments, the product is a medicine, and its dosage form includes oil, emulsion, ointment, paste, film coating, gel, aerosol, spray, solution, and liniment.

[0045] The present invention is further described below by specific examples. Unless otherwise specified herein, "%" represents mass percentage. The materials and reagents in the following examples, unless otherwise specified, are all commonly used materials or reagents in the art, and can be obtained from commercial sources or synthesized by known methods. The experimental methods for which the conditions are not specified in the following implementation cases are usually carried out according to conventional experimental conditions or the conditions recommended by the manufacturers of the relevant reagents (kits). Example 1

[0046] Weigh 100g of fresh coffee beans (Santos coffee beans from Brazil), rinse with clean water, and then wash with ultrapure water for 3 times to remove impurities; place in a clean, UV-irradiated beaker; add 500mL of sterile water containing a mixture of penicillin-streptomycin-amphotericin B (penicillin concentration is 100U / mL, streptomycin concentration is 0.1mg / mL, and amphotericin B concentration is 0.25μg / mL), and soak overnight at 4℃; discard the water after soaking, wash with ultrapure water 2-3 times, and add 500mL of sterile PBS. Transfer to a wall breaking machine, cover tightly, break the wall for 8-10 minutes to ensure that there are no obvious chunks and fragments; transfer the coffee bean juice to a 50 ml sterile centrifuge tube, centrifuge at 500 × g at 4 ° C for 10 minutes, discard the precipitate, and take the supernatant; centrifuge at 2000 × g at 4 ° C for 20 minutes, discard the precipitate, and take the supernatant; centrifuge at 4000 × g at 4 ° C for 30 minutes, discard the precipitate, and take the supernatant; centrifuge at 10000 × g at 4 ° C for 1 hour, discard the precipitate, and take the supernatant; centrifuge at 110000 × g at 4 ° C for 70 minutes, discard the supernatant, and resuspend the precipitate with an appropriate amount of PBS to obtain coffee bean extracellular vesicle-like particles (labeled as CB-Exo). The appearance of coffee bean extracellular vesicles is as follows: Figure 1 As shown, coffee bean extracellular vesicles are shown in light grey. Example 2 Characterization of coffee bean-derived extracellular vesicles

[0047] According to the sample situation, adjust the sample of Example 1 to an appropriate concentration or viscosity. Use a pipette to draw about 15 μL of the extracellular vesicle sample onto the copper mesh and let it stand for 1 minute. Use filter paper to dry the extracellular vesicle sample on the copper mesh, and draw about 15 μL of 2% uranyl acetate staining solution and stain at room temperature for 1 minute. If obvious adsorbents are visible on the copper mesh, pure water can be dripped onto the surface, quickly absorbed, and washed repeatedly. Use filter paper to dry the exosome sample on the copper mesh. Observe and take pictures, and save the pictures. The results show ( Figure 2 ): CB-Exo can observe saucer-cup-shaped vesicles and the vesicles are regular and complete.

[0048] After diluting the coffee bean-derived extracellular vesicles obtained in Example 1 with a particle-removing buffer, take 4 μL into the chip (C400) hole (no bubbles, which can be squeezed out by hand), insert the chip into the nCS1 microfluidic pulse nanoparticle size analyzer, input the chip model ID (Catridge mold ID) and other related information corresponding to the chip, and perform the test. The test results show ( Figure 3 ): 90% of the particle diameters of CB-Exo extracellular vesicles are in the range of 20-500nm.

[0049] The coffee bean-derived extracellular vesicles obtained in Example 1 were mixed with 5× loading buffer in a volume ratio of 4:1, and then boiled in a constant temperature metal bath at 100°C for 5-10 min to completely denature the protein (8 μL of exosomes were added with 2 μL of 5× loading buffer); a 10% or 12% precast gel was selected, and after the electrophoresis instrument was assembled, the electrophoresis buffer was added to ensure that it would not leak, and the comb was pulled out; 5 μL of protein molecular weight standard (Marker) and 5 μL of coffee bean exosome sample were added to the gel wells in sequence with a pipette; the electrophoresis conditions were set and the electrophoresis was started. Generally, the voltage was set to 100-120 V and the time was set to 2 h; after the electrophoresis was completed, the gel was transferred to a clean glass or plastic container, Coomassie brilliant blue staining solution was added, and the gel was incubated overnight on a shaker at room temperature; then the gel was rinsed with deionized water until there was no blue in the container, new deionized water was added, and the gel was incubated on a shaker for 2 h. h for destaining. After destaining, use a camera to take a picture of the SDS-PAGE gel. The results show ( Figure 4 ): The molecular weight of CB-Exo is approximately at 60KD, 35KD, and 25KD. Example 3 Cytotoxicity of coffee bean-derived extracellular vesicles on human dermal fibroblasts HDF, human keratinocytes HaCat, and mouse melanoma cells B16

[0050] The CCK8 method was used to detect the cytotoxicity of coffee bean-derived extracellular vesicles on human dermal fibroblasts HDF, human keratinocytes HaCaT, and mouse melanoma cells (B16). The details are as follows: The three types of cells were cultured at 1×10 4 The cells / well were inoculated in a 96-well plate and cultured for 12 h. The culture medium was then discarded, and 100 μL of free coffee bean-derived extracellular vesicles at different concentrations of 0, 0.5, 1, 5, 10, 20, 50, and 100 μg / mL were added to the three types of cells and co-cultured for 24 h. Then 10 μL of CCK8 solution was added to each well and incubated with the three types of cells for 2-4 h, and the absorbance was measured at a wavelength of 450 nm using an enzyme reader. The results showed ( Figure 5 ): The cell viability of coffee bean-derived extracellular vesicles on human dermal fibroblasts HDF, human keratinocytes HaCaT, and mouse melanoma cells (B16) was greater than 90%, and no cytotoxicity was shown. Example 4 Uptake of coffee bean-derived extracellular vesicles by mouse melanocytes

[0051] Mouse melanocytes were added to a 3.5 cm confocal dish at 100,000 cells / well and cultured for 24 hours. Take 1 mg of coffee bean-derived extracellular vesicles prepared in Example 1, and diluent C in the kit (Sigma, PKH26 red fluorescent cell labeling kit, MINI26-1KT) was used to adjust the volume of the sample to 1 mL. Take 6 μL of PKH26 dye in the kit and add it to a test tube containing 1 mL of diluent C. Use a pipette to blow gently and mix continuously for 30 seconds. Let stand at room temperature for 5 minutes. Add 2 mL of PBS (Sigma-Aldrich, D8537) containing 10% BSA to quench. Use serum-free medium to adjust the volume to 30 mL. Centrifuge at 110,000 g for 2 hours at 2-8°C. Use a pipette to gently blow and resuspend the extracellular vesicle pellet in 1 mL of serum-free medium for use.

[0052] PKH26-labeled coffee bean-derived extracellular vesicles were added to mouse melanocytes at 50 μg / mL and incubated for 24 hours. After incubation, the cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature, washed with PBS, and the cytoskeleton was stained with phalloidin for 30 minutes. DAPI was then added to stain the cell nucleus, and the cells were observed and photographed using a confocal microscope. The results showed ( Figure 6 ): Coffee bean-derived extracellular vesicles are easily taken up into the cells by mouse melanocytes. Example 5: Coffee bean-derived extracellular vesicles penetrate into ex vivo pig skin

[0053] The ex vivo skin of the back or abdomen of an 8-week-old Bama miniature pig was cut into 1 cm × 1 cm pieces for use. Take 1 mg of the coffee bean-derived extracellular vesicles prepared in Example 1, and dilute the sample to 1 mL with diluent C in the kit (Sigma, PKH26 red fluorescent cell labeling kit, MINI26-1KT). Take 6 μL of PKH26 dye in the kit and add it to a test tube containing 1 mL of diluent C. Use a pipette to blow gently and mix continuously for 30 seconds. Let stand at room temperature for 5 minutes. Add 2 mL of PBS (Sigma-Aldrich, D8537) containing 10% BSA for quenching. Use serum-free medium to make the volume 30 mL. Centrifuge at 110,000 g for 2 hours at 2-8°C. Use a pipette to blow gently and resuspend the extracellular vesicle pellet in 1 ml of serum-free medium for use.

[0054] 1cm×1cm ex vivo skin of 8-week-old Bama miniature pig was placed in a 32℃ environment for later use. 100μL of 1mg / mlPKH26-labeled coffee bean-derived extracellular vesicles were added to the 1cm×1cm skin of 8-week-old Bama miniature pig, evenly covering the entire epidermis, and incubated for 0h and 6h. After the incubation, frozen sections were taken with a thickness of 10 μm. After DAPI staining of the cell nucleus, the cells were observed and photographed under a fluorescence microscope. The results showed ( Figure 7 ): Coffee bean-derived extracellular vesicles can penetrate into the ex vivo skin of 8-week-old Bama miniature pigs; and the single penetration amount within 6 hours reaches 34.13%. Example 6 Coffee bean-derived extracellular vesicles inhibit melanocyte tyrosinase activity

[0055] B16 melanocytes were inoculated into 24-well plates at a density of 100,000 cells / 500 μL / well and cultured for 24 hours. The cell supernatant was discarded, and 500 μL of negative control reagent (DMEM complete medium), CB-Exo reagent (DMEM complete medium + 50 μg / mL CB-Exo), CB-T (coffee bean extract) reagent (DMEM complete medium + 50 μg / mL CB-T), CAF (caffeine) reagent (DMEM complete medium + 50 μg / mL CAF), and NAM (nicotinamide) reagent (DMEM complete medium + 50 μg / mL NAM) were added respectively and cultured for 72 hours. During this period, the cell status and culture medium were observed every day and the culture medium of the negative control reagent, CB-Exo reagent, CB-T reagent, CAF reagent, and NAM reagent were replaced with fresh culture medium. After removing the culture supernatant, wash once with PBS. Add 200 μL of 0.25% trypsin to each well to digest the cells and place them in a CO2 incubator for digestion for 2 minutes. Add 400 μL of PBS containing 10% FBS to stop digestion and blow the cells, collect the cells into a centrifuge tube, centrifuge at 4000rpm for 5 minutes, and discard the supernatant. Add 100 μL of 1% Triton-x-100 to each tube to lyse the cells, freeze at -80℃ for 30 minutes, place at room temperature for 10 minutes, centrifuge at 4℃, and centrifuge at 12000rpm for 20 minutes to remove cell debris. Take 50 μL of the supernatant, add 150 μL of 2mg / ml L-DOPA solution, incubate at 37℃ for 1 hour, and measure the absorbance at 492nm with an enzyme reader. The results show ( Figure 8 ): The inhibition rate of 50μg / ml CB-Exo on the tyrosinase activity of melanocytes can reach 54.91%, which is significantly higher than that of CB-T, CAF, and NAM. Example 7 Coffee bean-derived extracellular vesicles inhibit melanin synthesis in melanocytes

[0056] B16 melanocytes were inoculated into 24-well plates at a density of 100,000 cells / 500 μL / well, and the cells were cultured for 24 hours. The cell supernatant was discarded, and 500 μL-negative control reagent (NC: DMEM complete medium), CB-Exo reagent (DMEM complete medium + 50 μg / mL CB-Exo), CB-T (coffee bean extract) reagent (DMEM complete medium + 50 μg / mL CB-T), CAF (caffeine) reagent (DMEM complete medium + 50 μg / mL CAF), NAM (nicotinamide) reagent (DMEM complete medium + 50 μg / mL NAM) were added respectively, and cultured for 72 hours. During this period, the cell status and culture medium were observed every day and the culture medium of the negative control reagent, CB-Exo reagent, CB-T reagent, CAF reagent, and NAM reagent were replaced with fresh culture medium. After removing the culture supernatant, wash once with PBS. Add 200 μL 0.25% trypsin to each well to digest the cells and place them in a CO2 incubator for digestion for 2 minutes. Add 400 μL of PBS containing 10% FBS to stop digestion and blow the cells, collect the cells into a centrifuge tube, centrifuge at 4000rpm for 5 minutes, and discard the supernatant. Add 100 μL of melanin extract to each tube, shake well, and heat in an 80℃ water bath for 1 hour. After cooling, centrifuge the droplets on the tube wall moderately, blow evenly, aspirate 90 μL of solution in each centrifuge tube, transfer to a 96-well plate, and use an enzyme reader to measure the absorbance of each well at a wavelength of 405nm. The results show ( Fig. 9 ): The blackness of the centrifugal sediment of melanocytes in the 50μg / ml CB-Exo group was significantly decreased; the inhibition rate of melanin synthesis was statistically analyzed. The inhibition rate of melanin production by 50μg / ml CB-Exo on melanocytes can reach 45.42%, and the melanin synthesis inhibition rate is significantly higher than that of CB-T, CAF, and NAM. Example 8 Uptake of coffee bean-derived extracellular vesicle-like particles by human dermal fibroblasts

[0057] Coffee bean-derived extracellular vesicle-like particles (from Example 1) were labeled with PKH26 to track the uptake of coffee bean-derived extracellular vesicle-like particles by human dermal fibroblasts.

[0058] Human dermal fibroblasts were added to a 3.5 cm confocal dish at a density of 200,000 cells / well and cultured for 24 hours.

[0059] Take 1 mg of coffee bean-derived extracellular vesicle-like particles and adjust the volume of the sample to 1 mL with diluent C in the kit (Sigma, PKH26 Red Fluorescent Cell Labeling Kit, MINI26-1KT). Take 6 μL of PKH26 dye in the kit and add it to a test tube containing 1 mL of diluent C. Use a pipette to gently blow and mix continuously for 30 seconds. Let stand at room temperature for 5 minutes. Add 2 mL of PBS containing 10% BSA for quenching. Use serum-free medium to adjust the volume to 30 mL. Centrifuge at 110,000 × g for 2 hours at 2-8°C. Use a pipette to gently blow and resuspend the extracellular vesicle-like particle pellet in 1 mL of serum-free medium for use.

[0060] Coffee bean-derived extracellular vesicle-like particles labeled with PKH26 were added to human dermal fibroblasts at 50 μg / mL and incubated for 24 hours. After incubation, the cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature, washed with PBS, stained with phalloidin for 30 minutes, and then DAPI was added to stain the cell nucleus, and then observed and photographed with a confocal microscope. The results showed ( Fig.10 ): Extracellular vesicle-like particles derived from coffee beans can be taken up into human dermal fibroblasts. Example 9: Coffee bean-derived extracellular vesicle-like particles affect the senescence of human dermal fibroblasts

[0061] Human dermal fibroblasts were inoculated into 24-well plates at a density of 20,000 cells / 100 μL / well, and the cells were cultured for 24 hours. Cells were aged by UVA irradiation. The specific operation was as follows: the cell supernatant was discarded and 100 μL of pre-cooled PBS was added to each well. An ice bag was placed in the biosafety cabinet, and four layers of kraft paper were placed on the ice bag. The cell culture plate was then placed on the kraft paper. The position of the UVA lamp was adjusted so that the distance from the lamp to the cells was 5 cm. The cell culture plate was removed, the lamp was turned on for 5 minutes of equilibration, and then the cell culture plate was placed under the lamp. The lid was opened and irradiated for 1 hour, for a total of 5 J / cm 2 PBS was discarded, and negative control reagent (NC: basic culture medium) and experimental group reagent (10μg / mL, 20μg / mL, 50μg / mL coffee bean-derived extracellular vesicle-like particles in Example 1) were added and cultured for 24h. At the same time, a blank group (BC group, inoculated cells, added basic culture medium, no irradiation) was set up. 5J / cm 2 The cells were irradiated with UVA of high energy, and then treated with drugs again for 24 hours. Senescent cells were stained with the β-galactosidase staining kit of Shanghai Shangbao Biotechnology Co., Ltd. ( Fig.11 ). The results showed that CB-Exo can significantly reduce the senescence of human dermal fibroblasts induced by UVA. Example 10 Effect of coffee bean extracellular vesicles on proliferation of human dermal fibroblasts

[0062] The CCK-8 method was used to determine the proliferation effect of coffee bean extracellular vesicles on human dermal fibroblasts. A blank group (no cells inoculated, basal culture medium), a control group (NC: basal culture medium), and an experimental group (basal culture medium containing different concentrations of coffee bean EVs) were set up. The coffee bean EVs solution was diluted with serum-free culture medium to a mother solution with a protein concentration of 1 mg / mL. Sample solutions of 10 μg / mL and 20 μg / mL were prepared and recorded as experimental groups 1~2 respectively for later use.

[0063] In addition to the blank group, cells with good growth status were taken and 1×10 4 The cells were seeded at a density of 100 cells / well in a 96-well plate and cultured for 24 h before use.

[0064] The experimental groups were given 100 μL / well of the sample solutions with the above concentration gradients, and the blank and normal groups were given the same dose of incomplete culture medium (i.e., culture medium without serum). The culture medium was discarded after incubation at 37°C and 5% CO2 for 24 h.

[0065] The cell proliferation activity was detected according to the instructions of the CCK-8 kit. Each group was tested in triplicate, and the cell survival rate and cell proliferation rate were calculated according to the following formula.

[0066] Cell proliferation rate%=(OD 实验组 -OD 空白 ) / (OD 对照组 -OD 空白 )×100%; Fig.12 The results showed that the addition of 10 μg / mL and 20 μg / mL of coffee bean extracellular vesicles (Example 1) can significantly promote the proliferation of human dermal fibroblasts. 10 μg / mL and 20 μg / mL of coffee bean extracellular vesicles increased the proliferation ability of dermal fibroblasts by 12.16% and 15.59%, respectively. Example 11 Coffee bean-derived extracellular vesicle-like particles promote the secretion of type I collagen in human dermal fibroblasts

[0067] Human dermal fibroblasts were seeded into 96-well plates at a density of 5000 cells / 100 μL / well and cultured for 24 h. The cell supernatant was discarded, and 100 μL of negative control reagent (NC: basic culture medium) and experimental group reagent (10 μg / mL and 20 μg / mL coffee bean-derived extracellular vesicle-like particles in Example 1) were added and cultured for 24 h. The supernatant was taken and type I collagen secretion was detected using the Lianke type I collagen detection kit.

[0068] The results show that ( Fig.13 ): CB-Exo can significantly promote the secretion of type I collagen in human dermal fibroblasts. 10 μg / mL and 20 μg / mL of coffee bean extracellular vesicles upregulated the secretion of type I collagen in human dermal fibroblasts by 15.39% and 18.07%, respectively. Example 12 Effect of coffee bean extracellular vesicles on reactive oxygen species levels in human dermal fibroblasts

[0069] The DCFH-DA fluorescent probe was used to determine the effect of coffee bean extracellular vesicles on the level of reactive oxygen species in human dermal fibroblasts. A blank control group (BC: T4 basal medium, no irradiation), a control group (NC: T4 basal medium), a naked cell group (wells without cells), and an experimental group (basal medium containing different concentrations of coffee bean EVs) were set up. The coffee bean EVs solution was diluted with serum-free medium to a mother solution with a protein concentration of 1 mg / mL. A sample solution of 20 μg / mL was prepared and recorded as experimental group 1 for later use.

[0070] Take cells in good growth state and add 5×10 4 The cells were seeded at a density of 100 cells / well in a 24-well plate and cultured for 24 h before use.

[0071] The cell culture supernatant was discarded, and the blank control group was given 500 μL / well T4 basal culture medium, the control group was given 500 μL / well T4 basal culture medium, the naked cell group was given 500 μL / well T4 basal culture medium, and the experimental group was given 500 μL / well of the above-mentioned 20 μg / ml concentration of coffee bean EVs sample solution. The culture medium was discarded after incubation at 37°C and 5% CO2 for 24 h.

[0072] Add 200 μL PBS to each well, place it 5 cm below the UVA lamp, and irradiate for 60 min (except the blank control group).

[0073] The PBS was discarded, and 500 μL of blank control reagent (T4 basal medium), 500 μL of control group reagent (T4 basal medium), 500 μL of naked cell group reagent (T4 basal medium), and 500 μL of experimental group reagent (T4 basal medium containing coffee bean EVs) were added respectively and cultured for 3-5 h.

[0074] The DCFH-DA working solution was prepared at a dilution ratio of 1:1000, and DCFH-DA was diluted with T4 basal medium to a final concentration of 10 μmol / L.

[0075] The culture medium in the remaining wells was discarded, and the wells were washed three times with PBS, and 200 μL DCFH-DA working solution was added to each well, and the wells were incubated in a CO2 incubator for 30 min.

[0076] After the incubation, the cells in each well were washed three times with basal medium, placed on the detection platform of the fluorescence microplate reader, and the incident light wavelength was set to 529 nm, the excitation light wavelength was set to 504 nm, and the reading was taken.

[0077] ROS level (%) = (T-C0) / (C-C0) × 100%, where T is the average of three fluorescence intensity of the test sample; C is the average of three fluorescence intensity of the control group; C0 is the average of three fluorescence intensity of the naked cell group.

[0078] Fig.14 The results showed that adding 20 μg / mL CB-Exo could significantly inhibit the ROS level of human dermal fibroblasts, and the inhibition level reached 19.26%. Example 13: Coffee bean-derived extracellular vesicle-like particles affect the level of superoxide dismutase in keratinocytes

[0079] Keratinocytes were inoculated into 24-well plates at a density of 200,000 cells / 100 μL / well. The cells were cultured for 24 h. The culture medium was discarded, and blank control reagent (BC: basal culture medium), negative control reagent (NC: basal culture medium), and experimental group reagent (50 μg / mL coffee bean-derived extracellular vesicle-like particles in Example 1) were added and cultured for 24 h. The keratinocyte SOD level was reduced by UVB irradiation (the blank control group BC group was not irradiated), the cell supernatant was discarded, and 200 μL of pre-cooled PBS was added to each well. In the biosafety cabinet, the position of the UVA lamp was adjusted so that the distance from the lamp to the cells was 15 cm, and irradiation was performed for 16 minutes. The PBS was aspirated, and 500 μL of basal culture medium was added to each well and cultured for 24 h. The cell superoxide dismutase level was detected using the total SOD activity detection kit of Biyuntian Biotechnology Co., Ltd. ( Fig.15 ). The results showed that CB-Exo could significantly promote the SOD level of keratinocytes. Example 14: Coffee bean-derived extracellular vesicles affect the level of reactive oxygen species in keratinocytes

[0080] Keratinocytes were inoculated into 96-well plates at a density of 20,000 cells / 100 μL / well, and the cells were cultured for 24 h. The culture medium was discarded, and the blank control group reagent (BC: basal culture medium), control reagent (NC: basal culture medium), naked cell group (wells without cells), and experimental group reagent (50 μg / mL coffee bean-derived extracellular vesicle-like particles in Example 1) were added and cultured for 24 h. The reactive oxygen content of keratinocytes was increased by UVB irradiation (the blank control group BC group was not irradiated), the cell supernatant was discarded, and 200 μL of pre-cooled PBS was added to each well. In the biosafety cabinet, the position of the UVA lamp was adjusted so that the distance from the lamp to the cells was 15 cm, and the irradiation was performed for 16 min. The PBS was aspirated, and the DCFH-DA working solution was prepared at a dilution ratio of 1:1000, and DCFH-DA was diluted with basal culture medium to a final concentration of 10 μmol / L. The culture medium in the remaining wells was discarded, and the cells were washed three times with PBS, and 100 μL of DCFH-DA working solution was added to each well, and the cells were incubated in a CO2 incubator for 30 min. After the incubation, the cells in each well were washed three times with PBS, placed on the fluorescence microplate reader, and the incident light wavelength was set to 529 nm, the excitation light wavelength was set to 504 nm, and the reading was taken.

[0081] ROS level (%) = (T-C0) / (C-C0) × 100%, where T is the average of three fluorescence intensity of the test sample; C is the average of three fluorescence intensity of the control group; C0 is the average of three fluorescence intensity of the naked cell group.

[0082] Fig.16 The results showed that adding 50 μg / mL CB-Exo could significantly inhibit the ROS level in keratinocytes, with the inhibition level reaching 25.8%. Example 15 Effect of coffee bean extracellular vesicles on the antioxidant capacity of keratinocytes

[0083] The DCFH-DA fluorescent probe was used to determine the effect of coffee bean extracellular vesicles on the level of reactive oxygen species in keratinocytes. A blank control group (BC: basal culture medium, without irradiation), a control group (NC: basal culture medium), a naked cell group (wells without cells inoculated), and an experimental group (basal culture medium containing different concentrations of coffee bean EVs) were set up. The coffee bean EVs solution was diluted with serum-free culture medium to a mother solution with a protein concentration of 1 mg / mL. A sample solution of 50 μg / mL was prepared and recorded as the experimental group for later use.

[0084] Take cells in good growth state and add 1×10 4 The cells were seeded at a density of 100 cells / well in a 96-well plate and cultured for 24 h before use.

[0085] The cell culture supernatant was discarded, 50 μL DPBS was added to each well, and the well was placed 15 cm below the UV lamp for 16 min.

[0086] The DPBS was discarded, and 100 μL of blank control reagent (T4 basal medium), 100 μL of control group reagent (T4 basal medium), 100 μL of naked cell group reagent (T4 basal medium), and 100 μL of experimental group reagent (T4 basal medium containing coffee bean EVs) were added respectively. The culture medium was discarded after incubation at 37°C and 5% CO2 for 6 h.

[0087] The DCFH-DA working solution was prepared at a dilution ratio of 1:1000, and DCFH-DA was diluted with basal culture medium to a final concentration of 10 μmol / L.

[0088] The culture medium in the remaining wells was discarded, and the wells were washed three times with PBS, and 100 μL DCFH-DA working solution was added to each well, and the wells were incubated in a CO2 incubator for 30 min.

[0089] After the incubation, the cells in each well were washed three times with basal medium, placed on the detection platform of the fluorescence microplate reader, and the incident light wavelength was set to 529 nm, the excitation light wavelength was set to 504 nm, and the reading was taken.

[0090] ROS level (%) = (T-C0) / (C-C0) × 100%, where T is the average of three fluorescence intensity of the test sample; C is the average of three fluorescence intensity of the control group; C0 is the average of three fluorescence intensity of the naked cell group.

[0091] Fig.17 The results showed that adding 50 μg / mL CB-Exo could significantly inhibit the ROS level of keratinocytes, with the inhibition level reaching 22.8%, indicating that coffee bean extracellular vesicles can improve the antioxidant capacity of keratinocytes. Example 16 Testing of the Effect of Coffee Bean-derived Extracellular Vesicle Cream on Human Body

[0092] The coffee bean-derived extracellular vesicle-like particles extracted in Example 1 were added to the matrix cream at an addition amount of 0.3%, and a matrix cream without coffee bean-derived extracellular vesicle-like particles was used as a control, and applied once in the morning and evening, and no other skin care products were used during use. 30 testers were selected and divided into a matrix group and an experimental group, wherein the matrix group used the matrix cream, and the experimental group used the matrix cream containing the coffee bean-derived extracellular vesicle-like particles of the present invention.

[0093] The skin elasticity tester MPA580 was used in conjunction with a pigment test probe (Mexamter MX18) to detect skin pigmentation on day 0 and day 14 of use. The skin surface texture analysis system VisioscanVC20plus and the skin rapid three-dimensional imaging system PRIMOSCR were used to detect skin conditions on day 0 and day 14 of use. Visia photo detection was used to detect skin conditions on day 0 and day 14 of use. All of the above tests were completed by a third-party company, PONY Testing.

[0094] The results showed that after using the cream containing coffee bean-derived extracellular vesicles for 14 days, the participants' skin pigmentation was significantly reduced, with three participants achieving the same effect. Fig.18 Before the test, the overall face of the experiencer was dull and there were visible spots on the face; after using it, the overall facial skin tone was significantly brightened, and the skin texture changed from rough to delicate. After one of the experiencers used the coffee bean-derived extracellular vesicle-like particle cream for 14 days, the skin rapid three-dimensional imaging system and Visia camera detection photos ( Fig.19 ) The facial nasolabial lines are significantly faded.

[0095] It should be understood that the above embodiments are exemplary and are not intended to include all possible implementations included in the claims. Various modifications and changes may be made on the basis of the above embodiments without departing from the scope of the present disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only express several implementations of the present invention and do not limit the scope of protection of the patent of the present invention.

Claims

1. Application of extracellular vesicles derived from coffee beans in the preparation of products capable of inhibiting tyrosinase activity and / or reducing melanin production.

2. Application of coffee bean-derived extracellular vesicles in the preparation of products that promote cell regeneration and / or have antioxidant, repair and anti-aging effects.

3. Application of extracellular vesicles derived from coffee beans in the preparation of products with whitening and freckle removal effects.

4. The use according to any one of claims 1 to 3, characterized in that: The coffee bean-derived extracellular vesicles are prepared by a preparation method comprising the following steps: The coffee beans are soaked in sterile water containing penicillin, streptomycin and amphotericin B, and then the coffee beans are broken to obtain coffee bean juice. The coffee bean juice is centrifuged, the supernatant is collected, the filtrate is ultracentrifuged, the precipitate is collected, and the precipitate is resuspended to obtain the coffee bean extracellular vesicles.

5. The use according to claim 4, characterized in that: The penicillin concentration is 50-150 U / mL, the streptomycin concentration is 0.05-0.5 mg / mL, and the amphotericin B concentration is 0.1-0.5 μg / mL; And / or, controlling the soaking time to be 12 to 18 hours; And / or, the cell wall breaking time is controlled to be 5 to 10 minutes.

6. The use according to claim 4, characterized in that: The centrifugation is performed at 0-8°C and 200-1000g for 5-15 min, the supernatant is collected, and then the centrifugation is performed at 1000-5000g for 10-30 min, the supernatant is collected, and then the centrifugation is performed at 3000-6000g for 20-40 min.

7. The use according to claim 4, characterized in that: The ultracentrifugation is performed at 8000-12000 g for 40-70 min at 0-8° C., the supernatant is collected, and then the ultracentrifugation is performed at 100000-120000 g for 60-100 min.

8. The use according to any one of claims 1 to 3, characterized in that: The product includes a cosmetic or a medicine.

9. The use according to claim 8, characterized in that: The product is a cosmetic, and its dosage form includes emulsion, water, oil or gel; And / or, the product is a medicine, and its dosage forms include oils, emulsions, ointments, pastes, coatings, gels, aerosols, sprays, solutions, and liniments.

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