Use of Extracellular Vesicles Derived from Coffee Beans

Abstract

The present invention relates to the use of extracellular vesicles derived from coffee beans, specifically their use in skin care products. More specifically, the present invention provides the application of extracellular vesicles derived from coffee beans in the preparation of products capable of inhibiting tyrosinase activity and / or reducing melanin production, promoting cell regeneration and / or having antioxidant, repair, and anti-aging effects, and products with whitening and freckle-removing effects.

Description

Technical Field

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

[0002] Extracellular vesicles (EVs) are nanoscale (30 - 150 nm) membranous vesicles released from cells into the extracellular matrix, which participate in processes such as 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 various diseases. Moreover, plant EVs, especially those from traditional Chinese herbs (medicinal plants), are considered an important emerging field in recent years.

[0003] Coffee beans belong to the Rubiaceae family and are mainly produced in places such as Brazil, Indonesia, and Colombia. In China, they are mainly produced in places such as Yunnan, Hainan, and Guangdong. The seeds are dark green or dark brown, with a specific aroma and a slightly bitter and astringent taste. Coffee beans are not only widely used as a beverage but also have certain medicinal value. It is recorded in the Guangxi Pharmacopoeia: "Coffee beans have the effects of exciting diuresis, and roasted coffee beans can help digestion." Modern research shows that coffee beans have the effects of exciting the central nervous system, lowering blood pressure, antioxidant, and anti-tumor.

[0004] Currently, there are few reports on the related content of extracellular vesicles derived from coffee beans. Therefore, there is an urgent need for a method for extracting extracellular vesicles of coffee beans using coffee beans as raw materials and applying them to aspects such as reducing melanogenesis, promoting skin cell regeneration, and antioxidant repair and anti-aging. Summary of the Invention

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

[0006] Solutions for Solving the Problems:

[0007] The present invention provides the application of extracellular vesicles derived from coffee beans in the preparation of products capable of inhibiting tyrosinase activity and / or reducing melanogenesis.

[0008] The present invention provides the application of extracellular vesicles derived from coffee beans in the preparation of products with the effects of promoting cell regeneration and / or antioxidant, repair, and anti-aging.

[0009] The present invention provides the application of extracellular vesicles derived from coffee beans in the preparation of products with the effect of whitening and freckle removal.

[0010] Preferably, the coffee beans are fresh coffee beans.

[0011] Preferably, the extracellular vesicles derived from coffee beans are prepared by a preparation method including the following steps:

[0012] The coffee beans are soaked in sterile water containing penicillin, streptomycin, and amphotericin B and then broken to obtain coffee bean juice. The coffee bean juice is centrifuged, and the supernatant is collected. The filtrate is ultracentrifuged, and the precipitate is collected. The precipitate is resuspended to obtain the extracellular vesicles of coffee beans. The extraction method of the extracellular vesicles derived from coffee beans is not limited to the ultracentrifugation process mentioned in this patent, but also includes common extracellular vesicle extraction processes on the market, such as tangential flow, fractional filtration, precipitation method, etc.

[0013] Preferably, 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.

[0014] Preferably, the soaking time is controlled to be 12 - 18 hours.

[0015] Preferably, the time for breaking the wall is controlled to be 5 - 10 min.

[0016] Preferably, the centrifugation is carried out at 0 - 8°C at 200 - 1000 g for 5 - 15 min, the supernatant is collected, then it is carried out at 1000 - 5000 g for 10 - 30 min, the supernatant is collected, and then it is carried out at 3000 - 6000 g for 20 - 40 min.

[0017] Preferably, the ultracentrifugation is carried out at 0 - 8°C at 8000 - 12000 g for 40 - 70 min, the supernatant is collected, and then it is carried out at 100000 - 120000 g for 60 - 100 min.

[0018] Preferably, the product includes cosmetics or pharmaceuticals.

[0019] Preferably, the product is cosmetics, and its dosage forms include emulsions, aqueous solutions, oils, or gels.

[0020] Preferably, the product is pharmaceuticals, and its dosage forms include oils, emulsions, ointments, pastes, film-forming agents, gels, aerosols, sprays, solutions, liniments.

[0021] Effects of the invention:

[0022] The present invention uses coffee beans as raw materials to obtain extracellular vesicles of coffee beans through an extraction method with simple operation, mild conditions, short time consumption, low cost and high safety, and applies them to the skin care field. They have high active ingredients and have effects in reducing melanin production, inhibiting tyrosinase activity, whitening and removing spots, improving wrinkles and skin aging, as well as skin repair and antioxidant aspects. Description of the Drawings

[0023] Figure 1 Appearance of extracellular vesicles derived from coffee beans;

[0024] Figure 2 Electron microscopy image of CB-Exo (arrows indicate extracellular vesicles derived from coffee beans);

[0025] Figure 3 Particle size distribution diagram of extracellular vesicles derived from coffee beans;

[0026] Figure 4 SDS-PAGE diagram of extracellular vesicles derived from coffee beans. Among them, lane 1 represents the protein molecular weight standard, and lane 2 represents exosomes derived from coffee beans;

[0027] Figure 5 Line graph of cytotoxicity of extracellular vesicles derived from coffee beans against human dermal fibroblasts HDF, human keratinocytes HaCat, and mouse melanoma cells B16;

[0028] Figure 6 Schematic diagram of mouse melanocytes ingesting extracellular vesicles derived from coffee beans (red: extracellular vesicles derived from coffee beans labeled with PKH26; green: phalloidin; blue: DAPI);

[0029] Figure 7 Schematic diagram of the penetration of extracellular vesicles derived from coffee into the ex vivo skin of 8-week-old Bama minipigs and statistical chart of single penetration amount (extracellular vesicles derived from coffee beans labeled with PKH26, 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 indicates the exosomes labeled on the surface of the ex vivo skin). Among them, A is a schematic diagram of the penetration of extracellular vesicles derived from coffee into the ex vivo skin at 0 h and 6 h, and B is a statistical chart of the single penetration amount of extracellular vesicles derived from coffee into the ex vivo skin;

[0030] Figure 8 Histogram of the inhibition of tyrosinase activity in melanocytes by extracellular vesicles derived from coffee beans;

[0031] Figure 9Schematic diagram of the effect of extracellular vesicles derived from coffee beans on melanin precipitation in melanocytes and bar graph of the inhibition of melanin synthesis. Among them, A is the bar graph of the inhibition of melanin synthesis by extracellular vesicles derived from coffee beans, and B is the schematic diagram of the effect of extracellular vesicles derived from coffee beans on melanin precipitation in melanocytes;

[0032] Figure 10 Schematic diagram of human dermal fibroblasts ingesting extracellular vesicles derived from coffee beans (Red: extracellular vesicles derived from coffee beans labeled with PKH26; Green: phalloidin; Blue: DAPI);

[0033] Figure 11 Schematic diagram of the effect of extracellular vesicles derived from coffee beans on the senescence of human dermal fibroblasts;

[0034] Figure 12 Bar graph of the effect of extracellular vesicle-like particles derived from coffee beans on the proliferation of human dermal fibroblasts;

[0035] Figure 13 Bar graph of the promotion of type I collagen secretion in human dermal fibroblasts by extracellular vesicles derived from coffee beans;

[0036] Figure 14 Bar graph of the effect of extracellular vesicles derived from coffee beans on the reactive oxygen species level in human dermal fibroblasts;

[0037] Figure 15 Bar graph of the effect of extracellular vesicles derived from coffee beans on the superoxide dismutase level in keratinocytes;

[0038] Figure 16 Bar graph of the effect of extracellular vesicles derived from coffee beans on the reactive oxygen species level in keratinocytes;

[0039] Figure 17 Bar graph of the effect of extracellular vesicles derived from coffee beans on the antioxidant capacity of keratinocytes;

[0040] Figure 18 Schematic diagram of the whitening effect of a human trial using a facial cream containing extracellular vesicles derived from coffee beans in Example 1;

[0041] Figure 19 Schematic diagram of the anti-wrinkle effect on nasolabial folds and wrinkles on the face on the 0th day and the 14th day of a human trial using a facial cream containing extracellular vesicles derived from coffee beans in Example 1. Detailed implementation methods

[0042] To make the technical solutions and beneficial effects of the present invention more obvious and understandable, the following will be described in detail by listing specific embodiments. Among them, the attached drawings are not necessarily drawn to scale, and local features can 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 and scientific fields to which this application belongs.

[0043] Through extensive and in-depth research, the inventors unexpectedly found that exosomes derived from coffee beans have new uses in skin care products. Specifically, extracellular vesicles derived from coffee beans are used in the preparation of products that can inhibit tyrosinase activity and / or reduce melanin production, products with effects of promoting cell regeneration and / or antioxidant, repair, and anti-aging, and products with whitening and freckle-removing effects. Based on this, the technical solutions of this application are proposed.

[0044] The present invention provides the use of extracellular vesicles derived from coffee beans in the preparation of products that can inhibit tyrosinase activity and / or reduce melanin production.

[0045] The present invention provides the use of extracellular vesicles derived from coffee beans in the preparation of products with effects of promoting cell regeneration and / or antioxidant, repair, and anti-aging.

[0046] The present invention provides the use of extracellular vesicles derived from coffee beans in the preparation of products with whitening and freckle-removing effects.

[0047] In some embodiments, the coffee beans are from Santos coffee beans in Brazil.

[0048] In some embodiments, the extracellular vesicles derived from coffee beans are prepared by a preparation method including the following steps:

[0049] The coffee beans are soaked in sterile water containing penicillin, streptomycin, and amphotericin B and then 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 extracellular vesicles of coffee beans.

[0050] In some embodiments, the penicillin concentration is 50-150 U / mL, such as 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.

[0051] In some embodiments, the penicillin concentration is 80-120 U / mL.

[0052] In some embodiments, the penicillin concentration is 100 U / mL.

[0053] In some embodiments, the streptomycin concentration is 0.05 - 0.5 mg / mL, such as 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.

[0054] In some embodiments, the streptomycin concentration is 0.05 - 0.2 mg / mL.

[0055] In some embodiments, the streptomycin concentration is 0.1 mg / mL.

[0056] In some embodiments, the amphotericin B concentration is 0.1 - 0.5 μg / mL, such as 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.

[0057] In some embodiments, the amphotericin B concentration is 0.1 - 0.3 μg / mL.

[0058] In some embodiments, the amphotericin B concentration is 0.25 μg / mL.

[0059] In some embodiments, the soaking time is controlled to be 12 - 18 hours, such as 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, etc.

[0060] In some embodiments, the time for cell wall breaking is controlled to be 5 - 10 min, such as 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.

[0061] In some embodiments, the cell wall breaking is intermittent cell wall breaking, and the specific steps include: cell wall breaking for 5 - 10 min and intermittent for 5 - 10 min.

[0062] In some embodiments, the centrifugation is carried out at 0 - 8 °C (such as 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, etc.) at 200 - 1000 g (such as 200 g, 300 g, 400 g, 500 g, 600 g, 700 g, 800 g, 900 g, 1000 g, etc.) for 5 - 15 min (such as 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 - 5000 g (such as 1000 g, 1500 g, 2000 g, 2500 g, 3000 g, 3500 g, 4000 g, 4500 g, 5000 g, etc.) for 10 - 30 min (such as 10 min, 15 min, 20 min, 25 min, 30 min, etc.). The supernatant is collected again, and then centrifuged at 3000 - 6000 g (such as 3000 g, 3500 g, 4000 g, 4500 g, 5000 g, 5500 g, 6000 g, etc.) for 20 - 40 min (such as 20 min, 25 min, 30 min, 35 min, 40 min, etc.).

[0063] In some embodiments, the ultra - centrifugation is carried out at 0 - 8 °C (such as 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, etc.) at 8000 - 12000 g (such as 8000 g, 8500 g, 9000 g, 9500 g, 10000 g, 10500 g, 11000 g, 11500 g, etc.) for 40 - 70 min (such as 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, etc.). The supernatant is collected, and then centrifuged at 100000 - 120000 g (such as 100000 g, 105000 g, 110000 g, 115000 g, 120000 g, etc.) for 60 - 100 min (such as 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, 95 min, etc.).

[0064] In some embodiments, the product includes cosmetics or pharmaceuticals.

[0065] In some embodiments, the product is a cosmetic, and its dosage forms include emulsion, aqueous solution, oil - based or gel - based.

[0066] In some embodiments, the product is a pharmaceutical, and its dosage forms include oil - based, emulsion, ointment, paste, film - forming agent, gel - based, aerosol, spray, solution, liniment.

[0067] 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

[0068] 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.

[0069] Example 2 Characterization of coffee bean-derived extracellular vesicles

[0070] 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.

[0071] After diluting the coffee bean-derived extracellular vesicles obtained in Example 1 with a particle-removing buffer, 4 μL was taken and placed in the wells of a chip (C400) (no air bubbles allowed, air bubbles can be removed by hand-squeezing). The chip was inserted into an nCS1 microfluidic pulse nanosizer, and relevant information such as the chip model ID (Catridge mold ID) corresponding to the chip was input for detection. The detection results showed ( Figure 3 ): 90% of the particle diameters of CB-Exo extracellular vesicles were in the range of 20 - 500 nm.

[0072] After mixing the coffee bean-derived extracellular vesicles obtained in Example 1 with 5× loading buffer in a volume ratio of 4:1, it was boiled in a thermostatic metal bath at 100 °C for 5 - 10 min to completely denature the proteins (8 μL of exosomes was taken and 2 μL of 5× loading buffer was added; a 10% or 12% precast gel was selected. After assembling the electrophoresis apparatus, electrophoresis buffer was added to ensure there was no leakage, and the comb was removed; 5 μL of protein molecular weight standard (Marker) and 5 μL of coffee bean exosome sample were successively added to the gel wells with a pipette; the electrophoresis conditions were set and electrophoresis was started. Generally, a voltage of 100 - 120 V was set and the time was set to 2 h; after electrophoresis, the gel was transferred to a clean glass or plastic container, and Coomassie Brilliant Blue staining solution was added, and it was incubated overnight on a shaker at room temperature; then it was rinsed with deionized water until there was no blue color in the container, and new deionized water was added, and it was incubated on a shaker for 2 h for decolorization. After decolorization, the SDS-PAGE gel was photographed with a camera. The results showed ( Figure 4 ): The molecular weights of CB-Exo were approximately at the positions of 60 KD, 35 KD, and 25 KD.

[0073] Example 3 Cytotoxicity of coffee bean-derived extracellular vesicles on human dermal fibroblasts HDF, human keratinocytes HaCat, and mouse melanoma cells B16

[0074] 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 specific steps were as follows:

[0075] The 3 types of cells were respectively inoculated into 96-well plates at a density of 1×10 4 cells / well and cultured for 12 h. Subsequently, the culture medium was discarded, and 100 μL of different concentrations of free coffee bean-derived extracellular vesicles at 0, 0.5, 1, 5, 10, 20, 50, and 100 μg / mL were added to the 3 types of cells respectively and co-cultured for 24 h. Then 10 μL of CCK8 solution was added to each well and co-incubated with the 3 types of cells for 2 - 4 h, and the absorbance was measured at a wavelength of 450 nm with an enzyme-linked immunosorbent assay reader. The results showed (Figure 5 ): The extracellular vesicles derived from coffee beans have a cell viability of greater than 90% for human dermal fibroblasts HDF, human keratinocytes HaCaT, and mouse melanoma cells (B16), and do not show cytotoxicity.

[0076] Example 4: Uptake of extracellular vesicles derived from coffee beans by mouse melanocytes

[0077] Mouse melanocytes were added to a 3.5 cm confocal dish at 100,000 cells / well and cultured for 24 h for later use. Take 1 mg of the extracellular vesicles derived from coffee beans prepared in Example 1, and the diluent C in the kit (Sigma, PKH26 Red Fluorescent Cell Labeling Kit, MINI26-1KT) was used to make up the volume of the sample to 1 mL. Take 6 μL of the PKH26 dye in the kit and add it to a test tube containing 1 mL of diluent C. Gently pipette and mix continuously for 30 seconds. Let it stand at room temperature for 5 minutes. Add 2 mL of PBS containing 10% BSA (Sigma-Aldrich, D8537) as the solvent for quenching. Make up the volume to 30 mL with serum-free medium. Centrifuge at 110,000 g at 2 - 8 °C for 2 hours. Gently pipette to resuspend the extracellular vesicle pellet in 1 mL of serum-free medium for later use.

[0078] The extracellular vesicles derived from coffee beans labeled with PKH26 were added to mouse melanocytes at 50 μg / mL and incubated for 24 h. After incubation, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min. After washing with PBS, the cell skeleton was stained with phalloidin for 30 min, and then DAPI was added to stain the cell nucleus, and then observed and photographed with a confocal microscope. The results showed ( Figure 6 ): The extracellular vesicles derived from coffee beans are easily taken up by mouse melanocytes into the cells.

[0079] Example 5: Penetration of extracellular vesicles derived from coffee beans into ex vivo porcine skin

[0080] The dorsal or abdominal ex vivo skin of 8-week-old Bama mini-pigs was cut into 1 cm × 1 cm pieces for later use. Take 1 mg of the extracellular vesicles derived from coffee beans prepared in Example 1, and use Diluent C in the kit (Sigma, PKH26 Red Fluorescent Cell Labeling Kit, MINI26-1KT) to make up the volume of the sample to 1 mL. Take 6 μL of the PKH26 dye in the kit and add it to a test tube containing 1 mL of Diluent C. Gently pipette and mix continuously for 30 seconds. Let it stand at room temperature for 5 minutes. Add 2 mL of PBS containing 10% BSA (Sigma-Aldrich, D8537) as the solvent for quenching. Make up the volume to 30 mL with serum-free medium. Centrifuge at 110,000 g for 2 hours at 2-8 °C. Gently pipette to resuspend the extracellular vesicle precipitate in 1 ml of serum-free medium for later use.

[0081] Place the 1 cm × 1 cm ex vivo skin of 8-week-old Bama mini-pigs in a 32 °C environment. Take 100 μL of the PKH26-labeled extracellular vesicles derived from coffee beans at a concentration of 1 mg / ml and drop them onto the skin of 8-week-old Bama mini-pigs with a size of 1 cm × 1 cm, evenly covering the entire epidermis, and incubate for 0 h and 6 h. After the incubation, perform cryosectioning with a section thickness of 10 μm. After staining the cell nuclei with DAPI, observe and take pictures with a fluorescence microscope. The results show that ( Figure 7 ): The extracellular vesicles derived from coffee beans can penetrate into the ex vivo skin of 8-week-old Bama mini-pigs; and the single penetration amount within 6 h reaches 34.13%.

[0082] Example 6 Inhibition of the tyrosinase activity of melanocytes by extracellular vesicles derived from coffee beans

[0083] B16 melanocytes were seeded into 24-well plates at a density of 100,000 cells / 500 μL / well and cultured for 24 h. The cell supernatant was discarded, and 500 μL of the negative control group 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 h. During this period, the cell state and the medium were observed every day, and the fresh negative control reagent, CB-Exo reagent, CB-T reagent, CAF reagent, and NAM reagent medium were replaced. After removing the culture supernatant, the cells were washed once with PBS. 200 μL of 0.25% trypsin was added to each well to digest the cells, and the cells were placed in a CO2 incubator for 2 min. 400 μL of PBS containing 10% FBS was added to terminate the digestion and pipette the cells. The cells were collected into a centrifuge tube, centrifuged at 4000 rpm for 5 min, and the supernatant was discarded. 100 μL of 1% Triton-x-100 was added to each tube to lyse the cells, and the cells were frozen at -80 °C for 30 min, then placed at room temperature for 10 min, and centrifuged at 12,000 rpm for 20 min in a 4 °C centrifuge to remove cell debris. 50 μL of the supernatant was taken, 150 μL of 2 mg / ml L-DOPA solution was added, and incubated at 37 °C for 1 h. The absorbance at 492 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The results showed ( Figure 8 ) that the inhibition rate of 50 μg / ml CB-Exo on the tyrosinase activity of melanocytes could reach 54.91%, and the inhibition rate of tyrosinase activity was significantly higher than that of CB-T, CAF, and NAM.

[0084] Example 7 Inhibition of Melanin Synthesis in Melanocytes by Extracellular Vesicles Derived from Coffee Beans

[0085] B16 melanocytes were seeded into a 24-well plate at a density of 100,000 cells / 500 μL / well and cultured for 24 h. The cell supernatant was discarded, and 500 μL of the negative control group 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), and NAM (nicotinamide) reagent (DMEM complete medium + 50 μg / mL NAM) were added respectively. The cells were cultured for 72 h, and the cell status and the medium were observed every day during this period, and the fresh negative control reagent, CB-Exo reagent, CB-T reagent, CAF reagent, and NAM reagent media were replaced. After removing the culture supernatant, the cells were washed once with PBS. 200 μL of 0.25% trypsin was added to each well to digest the cells, and the cells were placed in a CO2 incubator for 2 min. 400 μL of PBS containing 10% FBS was added to terminate the digestion and pipette the cells. The cells were collected into a centrifuge tube and centrifuged at 4000 rpm for 5 min, and the supernatant was discarded. 100 μL of melanin extraction solution was added to each tube, shaken well, and heated in a water bath at 80 °C for 1 hour. After cooling, the liquid droplets on the tube wall were centrifuged moderately, pipetted evenly, 90 μL of the solution in each centrifuge tube was aspirated and transferred into a 96-well plate, and the absorbance of each well was measured at a wavelength of 405 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The results showed ( Figure 9 ) that the blackness of the centrifugal precipitate of melanocytes in the 50 μg / ml CB-Exo group decreased significantly; the inhibition rate of melanin synthesis was statistically analyzed, and the inhibition rate of melanin production by 50 μg / ml CB-Exo on melanocytes could reach 45.42%, and the melanin synthesis inhibition rate was significantly higher than that of CB-T, CAF, and NAM.

[0086] Example 8 Human dermal fibroblasts ingest coffee bean-derived extracellular vesicle-like particles

[0087] 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.

[0088] Human dermal fibroblasts were added to a 3.5 cm confocal dish at 200,000 cells / well and cultured for 24 h for standby.

[0089] Take 1 mg of extracellular vesicle-like particles derived from coffee beans and use diluent C in the kit (Sigma, PKH26 Red Fluorescent Cell Labeling Kit, MINI26-1KT) to make up 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. Gently pipette and mix continuously for 30 seconds. Let it stand at room temperature for 5 minutes. Add 2 mL of PBS containing 10% BSA as a quenching agent. Make up the volume to 30 mL using serum-free medium. Centrifuge at 110,000×g at 2-8 °C for 2 hours. Gently pipette and resuspend the precipitate of extracellular vesicle-like particles in 1 mL of serum-free medium for later use.

[0090] The PKH26-labeled extracellular vesicle-like particles derived from coffee beans were added to human dermal fibroblasts at a concentration of 50 μg / mL and incubated for 24 h. After incubation, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min. After washing with PBS, the cell skeleton was stained with phalloidin for 30 min, and then DAPI was added to stain the cell nucleus. Then, it was observed and photographed under a confocal microscope. The results showed ( Figure 10 ): The extracellular vesicle-like particles derived from coffee beans could be taken up by human dermal fibroblasts into the cells.

[0091] Example 9 Effect of extracellular vesicle-like particles derived from coffee beans on the senescence of human dermal fibroblasts

[0092] Human dermal fibroblasts were seeded into 24-well plates at a density of 20,000 cells / 100 μL / well and cultured for 24 h. The cells were senesced by UVA irradiation. The specific operation was as follows: Discard the cell supernatant, and add 100 μL of pre-cooled PBS to each well. Place an ice pack in the biosafety cabinet, lay four layers of kraft paper on the ice pack, and then place the cell culture plate on the kraft paper. Adjust the position of the UVA lamp tube so that the distance from the lamp tube to the cells is 5 cm. Remove the cell culture plate, turn on the lamp tube and balance for 5 min, then place the cell culture plate under the lamp tube, open the lid, and irradiate for 1 h, with a total energy of 5 J / cm 2 . Discard the PBS, add negative control reagent (NC: basal medium), experimental group reagents (10 μg / mL, 20 μg / mL, 50 μg / mL extracellular vesicle-like particles derived from coffee beans in Example 1), and culture for 24 h. At the same time, a blank group (BC group, seeded with cells, added basal medium, not irradiated) was set. Irradiate the cells again with UVA with an energy of 5 J / cm 2 . After incubating with the drugs again for 24 h, stain the senescent cells with the β-galactosidase staining kit from Shanghai Shangbao Biotechnology Co., Ltd. ( Figure 11 ). The results showed that CB-Exo could significantly reduce the senescence of human dermal fibroblasts induced by UVA.

[0093] Example 10: Effect of Coffee Bean Extracellular Vesicles on the Proliferation of Human Dermal Fibroblasts

[0094] The CCK-8 method was used to determine the proliferative effect of coffee bean extracellular vesicles on human dermal fibroblasts. A blank group (without cell inoculation, basal medium), a control group (NC: basal medium), and an experimental group (basal medium containing different concentrations of coffee bean EVs) were set up. The coffee bean EVs solution was taken and diluted with serum-free medium to a stock 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 standby.

[0095] Except for the blank group, cells in good growth state were inoculated on 96-well plates at a density of 1×10 4 cells / well and cultured for 24 h for standby.

[0096] The experimental groups were respectively given 100 μL / well of the above concentration gradient sample solutions, and the blank group and the normal group were given the same dose of incomplete medium (i.e., serum-free medium). After incubation at 37 °C and 5% CO2 for 24 h, the medium was discarded.

[0097] The proliferative activity of the cells was detected according to the instructions of the CCK-8 kit. Each group was measured in parallel for 3 times, and the cell survival rate and cell proliferation rate were calculated according to the following formula.

[0098] Cell proliferation rate % = (OD 实验组 - OD 空白 ) / (OD 对照组 - OD 空白 ) × 100%;

[0099] Figure 12 The results showed that the addition of 10 μg / mL and 20 μg / mL of coffee bean extracellular vesicles (Example 1) could significantly promote the proliferation of human dermal fibroblasts, and the proliferation ability of human dermal fibroblasts was up-regulated by 12.16% and 15.59% respectively with 10 μg / mL and 20 μg / mL of coffee bean extracellular vesicles.

[0100] Example 11: Coffee Bean-derived Extracellular Vesicle-like Particles Promote the Secretion of Type I Collagen by Human Dermal Fibroblasts

[0101] Human dermal fibroblasts were seeded into 96-well plates at a density of 5000 cells / 100 μL / well, and the cells were cultured for 24 h. The cell supernatant was discarded, and 100 μL of negative control reagent (NC: basal medium) and experimental group reagent (10 μg / mL and 20 μg / mL coffee bean-derived extracellular vesicle-like particles of Example 1) were added respectively, and cultured for 24 h. The supernatant was taken, and the secretion of type I collagen was detected by Lianke type I collagen detection kit.

[0102] The results showed ( Figure 13 ): CB-Exo could significantly promote the secretion of type I collagen by human dermal fibroblasts. The coffee bean extracellular vesicles at 10 μg / mL and 20 μg / mL up-regulated the secretion of type I collagen by human dermal fibroblasts by 15.39% and 18.07% respectively.

[0103] Example 12 Effect of coffee bean extracellular vesicles on the reactive oxygen species level of human dermal fibroblasts

[0104] The DCFH-DA fluorescent probe was used to determine the effect of coffee bean extracellular vesicles on the reactive oxygen species level of human dermal fibroblasts. A blank control group (BC: T4 basal medium, without irradiation), a control group (NC: T4 basal medium), a naked cell group (wells without seeded cells), and an experimental group (basal medium containing different concentrations of coffee bean Evs) were set up. The coffee bean EVs solution was taken and diluted with serum-free medium to a stock solution with a protein concentration of 1 mg / mL. A sample solution of 20 μg / mL was prepared and denoted as experimental group 1 for standby.

[0105] Cells in good growth state were seeded on 24-well plates at a density of 5×10 4 cells / well, and the cells were cultured for 24 h for standby.

[0106] The cell culture supernatant was discarded. The blank control group was given 500 μL / well of T4 basal medium, the control group was given 500 μL / well of T4 basal medium, the naked cell group was given 500 μL / well of T4 basal medium, and the experimental group was given 500 μL / well of the above 20 μg / ml concentration of coffee bean EVs sample solution. After incubation at 37 °C and 5% CO2 for 24 h, the medium was discarded.

[0107] 200 μL of PBS was added to each well, and placed at a distance of 5 cm below the UVA ultraviolet lamp tube and irradiated for 60 min (except for the blank control group).

[0108] Discard the PBS, and add 500 μL of blank group 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) respectively, and culture for 3 - 5 h.

[0109] Prepare the DCFH-DA working solution according to a dilution ratio of 1:1000, and dilute DCFH-DA with T4 basal medium to a final concentration of 10 μmol / L.

[0110] Discard the culture medium in the remaining wells, wash 3 times with PBS, then add 200 μL of DCFH-DA working solution to each well, and incubate in a CO2 incubator for 30 min.

[0111] After the incubation, wash the cells in each well 3 times with the basal medium, place them on the detection platform of a fluorescence microplate reader, set the incident light wavelength to 529 nm and the excitation light wavelength to 504 nm, and take readings.

[0112] ROS level (%) = (T - C0) / (C - C0) × 100%, where T is the average of 3 measurements of the fluorescence intensity of the test sample; C is the average of 3 measurements of the fluorescence intensity of the control group; C0 is the average of 3 measurements of the fluorescence intensity of the naked cell group.

[0113] Figure 14 The results showed that adding 20 μg / mL of CB-Exo could significantly inhibit the ROS level in human dermal fibroblasts, and the inhibition level reached 19.26%.

[0114] Example 13 Effects of coffee bean-derived extracellular vesicle-like particles on the superoxide dismutase level of keratinocytes

[0115] Seed keratinocytes into 24-well plates at a density of 200,000 cells / 100 μL / well. Culture the cells for 24 h. Discard the culture medium, add blank control reagent (BC: basal medium), negative control reagent (NC: basal medium), and experimental group reagent (50 μg / mL coffee bean-derived extracellular vesicle-like particles of Example 1), and culture for 24 h. Reduce the SOD level in keratinocytes by UVB irradiation (the blank control group BC is not irradiated). Discard the cell supernatant, and add 200 μL of pre-cooled PBS to each well. In a biosafety cabinet, adjust the position of the UVA lamp tube so that the distance from the lamp tube to the cells is 15 cm, and irradiate for 16 min. Aspirate the PBS, add 500 μL of basal medium to each well, and culture for 24 h. Detect the superoxide dismutase level of the cells using the total SOD activity detection kit from Beyotime Biotechnology Co., Ltd. Figure 15 The results showed that CB-Exo could significantly promote the SOD level in keratinocytes.

[0116] Example 14 Effects of coffee bean-derived extracellular vesicles on the reactive oxygen species level in keratinocytes

[0117] Keratinocytes were seeded into 96-well plates at a density of 20,000 cells / 100 μL / well and cultured for 24 h. The culture medium was discarded, and reagents for the blank control group (BC: basal medium), control group (NC: basal medium), naked cell group (wells without seeded cells), and experimental group (50 μg / mL coffee bean-derived extracellular vesicle-like particles of Example 1) were added, followed by culturing for 24 h. The content of reactive oxygen species in keratinocytes was increased by UVB irradiation (the BC group of the blank control 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 tube was adjusted so that the distance from the lamp tube to the cells was 15 cm, and irradiation was performed for 16 min. The PBS was aspirated, and the DCFH-DA working solution was prepared by diluting DCFH-DA in a 1:1000 dilution ratio with basal medium to a final concentration of 10 μmol / L. After discarding the culture medium in the remaining wells and washing 3 times with PBS, 100 μL of DCFH-DA working solution was added to each well, and incubation was carried out in a CO2 incubator for 30 min. After the incubation, the cells in each well were washed 3 times with PBS, placed on the detection platform of a fluorescence microplate reader, and the incident light wavelength was set to 529 nm, the excitation light wavelength was set to 504 nm, and readings were taken.

[0118] ROS level (%) = (T - C0) / (C - C0) × 100%, where T is the average of 3 measurements of the fluorescence intensity of the test sample; C is the average of 3 measurements of the fluorescence intensity of the control group; C0 is the average of 3 measurements of the fluorescence intensity of the naked cell group.

[0119] Figure 16 The results showed that the addition of 50 μg / mL of CB-Exo could significantly inhibit the ROS level in keratinocytes, and the inhibition level reached 25.8%.

[0120] Example 15 Effects of coffee bean extracellular vesicles on the antioxidant capacity of keratinocytes

[0121] The DCFH-DA fluorescent probe was used to determine the effect of coffee bean extracellular vesicles on the reactive oxygen species level in keratinocytes. A blank control group (BC: basal medium, without irradiation), a control group (NC: basal medium), a naked cell group (wells without seeded cells), and an experimental group (basal medium containing different concentrations of coffee bean Evs) were set up. The coffee bean EVs solution was taken and diluted with serum-free medium to a stock solution with a protein concentration of 1 mg / mL. A sample solution of 50 μg / mL was prepared and denoted as the experimental group for standby.

[0122] Take cells in good growth condition and inoculate them on a 96-well plate at a density of 1×10 4 cells / well, and incubate the cells for 24 h for standby.

[0123] Aspirate and discard the cell culture supernatant, add 50 μL of DPBS to each well, place it at a distance of 15 cm below the ultraviolet lamp tube, and irradiate for 16 min.

[0124] Discard the DPBS, and add 100 μL of blank group 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) respectively. After incubating for 6 h at 37°C and 5% CO2, discard the medium.

[0125] Prepare the DCFH-DA working solution according to a dilution ratio of 1:1000, and dilute DCFH-DA with the basal medium to a final concentration of 10 μmol / L.

[0126] Discard the medium in the remaining wells, wash 3 times with PBS, then add 100 μL of DCFH-DA working solution to each well, and incubate in a CO2 incubator for 30 min.

[0127] After the incubation, wash the cells in each well 3 times with the basal medium, place them on the detection platform of a fluorescence microplate reader, set the incident light wavelength to 529 nm and the excitation light wavelength to 504 nm, and read the values.

[0128] ROS level (%) = (T - C0) / (C - C0) × 100%, where T is the average of 3 measurements of the fluorescence intensity of the test sample; C is the average of 3 measurements of the fluorescence intensity of the control group; C0 is the average of 3 measurements of the fluorescence intensity of the naked cell group.

[0129] Figure 17 The results show that adding 50 μg / mL of CB-Exo can significantly inhibit the ROS level in keratinocytes, and the inhibition level reaches 22.8%, indicating that coffee bean extracellular vesicles can improve the antioxidant capacity of keratinocytes.

[0130] Example 16 Testing the human effect of a coffee bean-derived extracellular vesicle cream

[0131] Add the coffee bean-derived extracellular vesicle-like particles extracted in Example 1 to the matrix cream at an addition amount of 0.3%. At the same time, use the matrix cream without coffee bean-derived extracellular vesicle-like particles as a control, and apply it once in the morning and once in the evening. Do not use other skin care products during the use period. Select 30 experiencers and divide them into a matrix group and an experimental group. The matrix group uses the matrix cream, and the experimental group uses the matrix cream containing the coffee bean-derived extracellular vesicle-like particles of the present invention.

[0132] Using the skin elasticity tester MPA580, connect the pigment test probe (Mexamter MX18) to detect skin pigment deposition at 0 day and 14 days of use respectively; use the skin surface texture analysis system Visioscan VC20plus and the skin rapid three-dimensional imaging system PRIMOS CR to detect the skin condition at 0 day and 14 days of use respectively; use Visia to take pictures to detect the skin condition at 0 day and 14 days of use. All of the above detections are completed by the third-party PONY Testing International Group.

[0133] The results show that after using the face cream containing extracellular vesicles derived from coffee beans for 14 days, the skin pigment deposition of the experiencers is significantly reduced. The effects of three experiencers are shown in Figure 18 , the overall face of the experiencer was dull before the test, and there were visible spots on the face; after the experiencer used it, the overall facial skin color was significantly brightened, and the skin texture became more delicate from rough. After one experiencer used the face cream containing extracellular vesicle-like particles derived from coffee beans for 14 days, the skin rapid three-dimensional imaging system and the Visia taken pictures ( Figure 19 ) showed that the nasolabial folds on the face were significantly lightened.

[0134] It should be understood that the above embodiments are all exemplary and do not cover all possible implementation manners included in the claims. Without departing from the scope of the present disclosure, various deformations and changes can also be made on the basis of the above embodiments. Similarly, the technical features of the above embodiments can also be combined arbitrarily to form additional embodiments of the present invention that may not be clearly described. Therefore, the above embodiments only represent several implementation manners of the present invention and do not limit the protection scope of the present invention patent.

Claims

1. Use of extracellular vesicles derived from coffee beans in the preparation of a product capable of inhibiting tyrosinase activity and / or reducing melanogenesis, wherein the extracellular vesicles derived from coffee beans are prepared by a preparation method comprising the following steps: Soak coffee beans in sterile water containing penicillin, streptomycin, and amphotericin B and then break the cell walls to obtain coffee bean juice. Centrifuge the coffee bean juice, collect the supernatant, ultracentrifuge the filtrate, collect the precipitate, and resuspend the precipitate to obtain the extracellular vesicles of coffee beans. 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.

2. Use of extracellular vesicles derived from coffee beans in the preparation of a product with the efficacy of promoting cell regeneration and / or antioxidant, repair, and anti - aging effects, wherein the extracellular vesicles derived from coffee beans are prepared by a preparation method comprising the following steps: Soak coffee beans in sterile water containing penicillin, streptomycin, and amphotericin B and then break the cell walls to obtain coffee bean juice. Centrifuge the coffee bean juice, collect the supernatant, ultracentrifuge the filtrate, collect the precipitate, and resuspend the precipitate to obtain the extracellular vesicles of coffee beans. 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.

3. Use of extracellular vesicles derived from coffee beans in the preparation of a product with the efficacy of whitening and removing freckles, wherein the extracellular vesicles derived from coffee beans are prepared by a preparation method comprising the following steps: Soak coffee beans in sterile water containing penicillin, streptomycin, and amphotericin B and then break the cell walls to obtain coffee bean juice. Centrifuge the coffee bean juice, collect the supernatant, ultracentrifuge the filtrate, collect the precipitate, and resuspend the precipitate to obtain the extracellular vesicles of coffee beans. 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.

4. The application according to any one of claims 1 to 3, characterized in that Control the soaking time to be 12 - 18 hours; and / or, control the cell wall - breaking time to be 5 - 10 min.

5. The application according to any one of claims 1 to 3, characterized in that The centrifugation is carried out at 0 - 8°C at 200 - 1000 g for 5 - 15 min to collect the supernatant, then at 1000 - 5000 g for 10 - 30 min to collect the supernatant, and then at 3000 - 6000 g for 20 - 40 min.

6. The application according to any one of claims 1 to 3, characterized in that The ultracentrifugation is carried out at 0 - 8°C at 8000 - 12000 g for 40 - 70 min to collect the supernatant, and then at 100000 - 120000 g for 60 - 100 min.

7. The application according to any one of claims 1 to 3, characterized in that The product includes cosmetics or pharmaceuticals.

8. The application according to claim 7, wherein The product is a cosmetic, and its dosage form includes emulsion, aqueous solution, oil, or gel; or, the product is a pharmaceutical, and its dosage form includes oil, emulsion, ointment, paste, film - forming agent, gel, aerosol, spray, solution, liniment.

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