A method for extracting extracellular vesicles of white peony root and its application

Abstract

The present invention relates to an extraction method and application of extracellular vesicles of white peony. Specifically, it relates to an extraction method of extracellular vesicles of white peony, the extraction method comprising: (1) taking fresh peony, pre-treating, and obtaining white peony; (2) soaking and breaking the white peony obtained in step (1) to obtain white peony juice; (3) centrifuging the white peony juice obtained in step (2) to obtain extracellular vesicles of white peony; wherein the pre-treatment step comprises: placing the fresh peony in water, heating, cooling, and drying. The extracellular vesicles of white peony obtained by the extraction method of the invention have excellent anti-wrinkle, whitening, and repairing effects.

Description

Technical Field

[0001] The invention relates to the fields of biotechnology and bioengineering, and in particular to an extraction method and application of extracellular vesicles of white peony root. 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] The traditional Chinese medicine white peony root tastes bitter, sour and slightly cold. Its main components include total glycosides of white peony such as paeoniflorin, paeoniflorin, volatile oil, hydroxypeoniflorin, peony phenol, etc., which play a role in protecting the liver, relieving pain and anti-inflammatory. At present, many studies have reported that white peony or total glycosides of white peony have good anti-inflammatory and immunomodulatory effects. Total glycosides of white peony inhibit abnormally activated immune cells, balance the relationship between immune cells and macrophages, and further regulate the function of immune cells. The results of many studies have shown that total glycosides of white peony can regulate the functions of B lymphocytes, T lymphocytes, macrophages, dendritic cells, endothelial cells, and cancer cells, thereby playing a role in immunomodulation and cell protection.

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

[0005] The purpose of the present invention is to overcome the defects of the above-mentioned prior art and to extract a method for extracting white peony extracellular vesicles that can promote cell regeneration, reduce melanin production and antioxidant repair using peony root as raw material.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In one aspect, the present invention provides a method for extracting extracellular vesicles of white peony root, the extraction method comprising:

[0008] (1) taking fresh peony root and pretreating it to obtain white peony root;

[0009] (2) soaking and breaking the white peony root obtained in step (1) to obtain white peony root juice;

[0010] (3) centrifuging the white peony juice obtained in step (2) to obtain white peony extracellular vesicles;

[0011] The pretreatment step comprises: heating the fresh peony in water, cooling and drying; the heating time is 10 to 30 minutes; controlling the heating temperature to 80 to 100° C.; controlling the drying temperature to 50 to 70° C.; and controlling the drying time to 18 to 36 hours.

[0012] Preferably, the fresh peony is the root of fresh peony.

[0013] Preferably, the mass ratio of the fresh peony to the water is controlled to be 1:1-10.

[0014] Preferably, the soaking time is controlled to be 10 to 36 hours.

[0015] Preferably, the cell wall breaking time is controlled to be 8 to 25 minutes, and the cell wall breaking is intermittent, and the specific steps include: cell wall breaking for 6 to 10 minutes, and intermittently for 8 to 12 minutes.

[0016] Preferably, the paeoniflorin in the white peony root obtained after pretreatment is controlled to be no less than 1.2%.

[0017] Preferably, the centrifugation is density gradient centrifugation or ultracentrifugation.

[0018] Preferably, the centrifugation is density gradient centrifugation, and the specific steps include:

[0019] The white peony juice is centrifuged at 400-800×g for 10-30 min, and the supernatant is collected; then the white peony juice is centrifuged at 1500-3000×g for 10-30 min, and the supernatant is collected; then the white peony juice is centrifuged at 3000-5000×g for 20-40 min, and the supernatant is collected; then the white peony juice is centrifuged at 8000-12000×g for 40-70 min, and the supernatant is collected; then the white peony juice is centrifuged at 100000-120000×g for 60-100 min, and the precipitate is collected, and the precipitate is resuspended in a PBS solution to obtain the extracellular vesicles.

[0020] In a second aspect, the present invention provides extracellular vesicles of white peony root obtained by any of the extraction methods described above.

[0021] In a third aspect, the present invention provides a composition comprising the extracellular vesicles of white peony obtained by any of the extraction methods described above or the extracellular vesicles described above.

[0022] In a fourth aspect, the present invention provides the use of the extraction method according to any one of the above, the extracellular vesicles according to the above, or the composition according to the above in the following:

[0023] (1) Application in the preparation of skin anti-wrinkle and anti-aging products;

[0024] (2) Application in the preparation of skin repair and antioxidant products;

[0025] (3) Application in the preparation of whitening and anti-freckle products.

[0026] Compared with the prior art, the present invention has the following significant advantages and effects:

[0027] The method for extracting extracellular vesicles of white peony provided by the present invention is simple to operate, has mild conditions, short time consumption, low cost and high safety. Meanwhile, the extracellular vesicles of white peony provided by the present invention have few impurities, high purity and high safety. Compared with fresh white peony, the pretreated white peony solves the problem of limited season of materials, and the content of paeoniflorin in the pretreated white peony is higher than that in fresh peony. The extracellular vesicles of white peony extracted by the present invention are applied to the field of skin care, have high active ingredients, whiten and remove spots, improve wrinkles and skin aging, and have effects in skin repair and anti-oxidation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Electron microscopic images of GP-Exo and DP-Exo (red arrows indicate extracellular vesicles derived from white peony root);

[0029] Figure 2 This is the particle size distribution diagram of extracellular vesicles derived from white peony root;

[0030] Figure 3 Mouse melanocytes take up extracellular vesicles derived from white peony root (red: extracellular vesicles derived from white peony root labeled with PKH26; green: phalloidin; blue: DAPI);

[0031] Figure 4 The ex vivo skin of 8-week-old Bama miniature pigs infiltrated with extracellular vesicles derived from white peony root (green arrows: extracellular vesicles derived from white peony root labeled with PKH26; white arrows: DAPI);

[0032] Figure 5 This is a bar graph showing the biochemical inhibition of tyrosinase activity by extracellular vesicles derived from white peony root;

[0033] Figure 6 This is a bar graph showing the inhibition of melanocyte tyrosinase activity by extracellular vesicles derived from white peony root;

[0034] Figure 7 This is a bar graph showing the inhibition of melanin synthesis in melanocytes by extracellular vesicles derived from white peony root;

[0035] Figure 8Human dermal fibroblasts take up extracellular vesicles derived from white peony root (red: extracellular vesicles derived from white peony root labeled with PKH26; green: phalloidin; blue: DAPI);

[0036] Fig. 9 This is a bar graph showing that extracellular vesicles derived from white peony root promote the secretion of type I collagen in dermal fibroblasts;

[0037] Fig.10 This is a bar graph showing the effect of extracellular vesicles derived from white peony on the secretion of extracellular matrix proteases in dermal fibroblasts. Fig.10 The A part in the middle is TIMP1, Fig.10 The middle B part is MMP1;

[0038] Fig.11 Schematic diagram of how extracellular vesicles derived from white peony root affect the senescence of dermal fibroblasts;

[0039] Fig.12 This is a bar graph of the effects of the extracellular vesicle-like particles derived from white peony root and the white peony root extract on the secretion of type I collagen by dermal fibroblasts in Example 2;

[0040] Fig.13 This is a bar graph showing the effect of extracellular vesicle-like particles derived from white peony root on keratinocyte proliferation. Fig.13 Part A is a schematic diagram showing the effect of DP-Exo at different concentrations on the proliferation of keratinocytes. Fig.13 Part B is a schematic diagram of the effects of DP-Exo and GP-Ex on the proliferation of keratinocytes;

[0041] Fig.14 This is a bar graph showing the effect of extracellular vesicle-like particles derived from white peony root on keratinocyte migration;

[0042] Fig.15 This is a bar graph showing the effect of extracellular vesicle-like particles derived from white peony root on the expression level of keratin-related proteins;

[0043] Fig.16 This is a bar graph showing the effect of extracellular vesicles derived from white peony root on the level of reactive oxygen species in keratinocytes;

[0044] Fig.17 Schematic diagram of the whitening effect of the face cream in a human test using a test group containing the extracellular vesicles derived from white peony root in Example 2 and a matrix group not containing the extracellular vesicles;

[0045] Fig.18 Schematic diagram of the anti-wrinkle effect of the face creams of the test group containing the extracellular vesicles derived from white peony root of Example 2 and the matrix group not containing the extracellular vesicles on the 0th day, the 14th day and the 28th day in the human body test;

[0046] Fig.19 Schematic diagram of the anti-wrinkle effect of the facial cream containing the extracellular vesicles derived from white peony root of Example 2 on the 0th day and the 28th day of the human body test on periocular wrinkles;

[0047] Fig. 20 This is a schematic diagram of the skin repair effect of a human trial using a facial cream containing the extracellular vesicles derived from white peony root of Example 2. DETAILED DESCRIPTION

[0048] 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.

[0049] Human skin is the largest organ in the human body, located on the surface of the body and composed of multiple layers of tissue. It has many important functions, including protecting internal organs from damage by the external environment, regulating body temperature, sensing external stimuli and participating in water regulation. The causes of damaged skin barrier are complex; when the skin is damaged, it will appear dry and dehydrated, burning and stinging, redness and inflammation, dullness and loss of elasticity.

[0050] my country has abundant resources of Chinese herbal medicines at low cost. Chinese herbal vesicles are derived from natural plants and have higher safety. The Expert Consensus on the Research and Application of Chinese Herbal Extracellular Vesicles (2023) points out that Chinese herbal vesicles can be used by the human body for skin regeneration, anti-inflammatory, immunomodulation, anti-tumor, freeze-dried beauty, industrialization, etc. Application in the field of medical cosmetology and skin care products: Chinese herbal vesicles carry highly bioactive substances such as proteins and active factors secreted by original cells, activate cell regeneration function, and release nutrients encapsulated in vesicles after penetrating into the skin, continuously nourishing, adjusting the cell microenvironment, and enhancing cell vitality, thereby promoting cell migration, promoting collagen and elastic repair damage, cell regeneration, and restoring the youthful state of the skin. Based on this, the applicant completed the present invention.

[0051] In one aspect, the present invention provides a method for extracting extracellular vesicles of white peony root, the extraction method comprising:

[0052] (1) taking fresh peony root and pretreating it to obtain white peony root;

[0053] (2) soaking and breaking the white peony root obtained in step (1) to obtain white peony root juice;

[0054] (3) centrifuging the white peony juice obtained in step (2) to obtain white peony extracellular vesicles;

[0055] Wherein, the pretreatment step comprises: placing the fresh peony in water for heating, cooling and then drying;

[0056] The heating time is 10-30 min (for example, 12 min, 14 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, etc.); the heating temperature is controlled to be 80-100°C (for example, 82°C, 84°C, 86°C, 88°C, 90°C, 92°C, 94°C, 96°C, 98°C, etc.); the drying temperature is controlled to be 50-70°C (for example, 52°C, 54°C, 56°C, 58°C, 60°C, 62°C, 64°C, 66°C, 68°C, etc.), and the drying time is controlled to be 18-36 hours (for example, 20h, 22h, 24h, 26h, 28h, 30h, 32h, 34h, etc.).

[0057] In certain embodiments, the fresh peony is the root of fresh peony.

[0058] In some embodiments, the mass ratio of the fresh peony to the water is controlled to be 1:1-10, for example, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, etc.

[0059] In certain embodiments, the mass ratio of the fresh peony to the water is controlled to be 1:1-5.

[0060] In certain embodiments, the mass ratio of the fresh peony to the water is controlled to be 1:1-3.

[0061] In some embodiments, the drying temperature is controlled to be 55-65°C.

[0062] In certain embodiments, the soaking time is controlled to be 10 to 36 hours, for example, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 26 h, 28 h, 30 h, 32 h, 34 h, etc.

[0063] In certain embodiments, the soaking time is controlled to be 10 to 15 hours.

[0064] In certain embodiments, the soaking time is controlled to be 12 hours.

[0065] In certain embodiments, the soaking temperature is controlled to be 0-10°C, such as 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, etc.

[0066] In certain embodiments, the soaking temperature is controlled to be 0-6°C.

[0067] In certain embodiments, the time for the wall breaking is controlled to be 8 to 25 min (preferably 9 min, 10 min, 11 min, 12 min, 14 min, 16 min, 18 min, 20 min, etc.), and the wall breaking is intermittent, and the specific steps include: wall breaking for 6 to 10 min (preferably 6 min, 7 min, 8 min, 9 min, 10 min, etc.), and intermittent for 8 to 12 min (preferably 9 min, 10 min, 11 min, etc.).

[0068] In some embodiments, the cell wall breaking time is controlled to be 20 minutes, and the cell wall breaking is intermittent, and the specific steps include: cell wall breaking for 8 minutes and intermittently for 10 minutes. The intermittent cell wall breaking adopted by the present invention avoids the continuous heat generation during the cell wall breaking process from affecting the extracellular vesicle-like particles of white peony.

[0069] In certain embodiments, the paeoniflorin in the white peony root obtained after pretreatment is controlled to be no less than 1.2%.

[0070] In certain embodiments, the centrifugation is density gradient centrifugation or ultracentrifugation.

[0071] In certain embodiments, the centrifugation is density gradient centrifugation, and the specific steps include:

[0072] The white peony juice is centrifuged at 400-800×g for 10-30 min, and the supernatant is collected; then the white peony juice is centrifuged at 1500-3000×g for 10-30 min, and the supernatant is collected; then the white peony juice is centrifuged at 3000-5000×g for 20-40 min, and the supernatant is collected; then the white peony juice is centrifuged at 8000-12000×g for 40-70 min, and the supernatant is collected; then the white peony juice is centrifuged at 100000-120000×g for 60-100 min, and the precipitate is collected, and the precipitate is resuspended in a PBS solution to obtain the extracellular vesicles.

[0073] In certain embodiments, the centrifugation is density gradient centrifugation, and the specific steps include:

[0074] The white peony juice was centrifuged at 500 × g for 10 min, and the supernatant was collected; then centrifuged at 2000 × g for 20 min, and the supernatant was collected; then centrifuged at 4000 × g for 30 min, and the supernatant was collected; then centrifuged at 10000 × g for 60 min, and the supernatant was collected; then centrifuged at 110,000 × g for 70 min, the precipitate was collected, and the precipitate was resuspended in a PBS solution to obtain the extracellular vesicles. The centrifugation of each step before the centrifugation step at 10000 × g provided by the present invention cannot be omitted, so as to remove large particle precipitation and improve the purity of the white peony extracellular vesicles. Compared with directly drying peony or directly breaking the wall and centrifuging peony, the pretreated white peony exosomes show better safety and efficacy.

[0075] In certain embodiments, the diameter of the extracellular vesicles of Paeonia lactiflora obtained in the present invention is 20-500 nm.

[0076] In certain embodiments, the particle concentration of the extracellular vesicles of white peony obtained in the present invention is 10 9 ~10 13 Particles / mL.

[0077] In a second aspect, the present invention provides extracellular vesicles of white peony root obtained by any of the extraction methods described above.

[0078] In a third aspect, the present invention provides a composition comprising the extracellular vesicles of white peony obtained by any of the extraction methods described above or the extracellular vesicles described above.

[0079] In a fourth aspect, the present invention provides the use of the extraction method according to any one of the above, the extracellular vesicles according to the above, or the composition according to the above in the following:

[0080] (1) Application in the preparation of skin anti-wrinkle and anti-aging products;

[0081] (2) Application in the preparation of skin repair and antioxidant products;

[0082] (3) Application in the preparation of whitening and anti-freckle products.

[0083] In certain embodiments, the concentration of the extracellular vesicles is 20 μg / mL.

[0084] The extracellular vesicles obtained by the method provided by the present invention have their own phospholipid bilayers, which are easier to be absorbed by cells and skin, especially to penetrate into the dermis of the skin to exert their effects, and the extracellular vesicle-like particles derived from white peony are natural isolated products with higher safety. At the same time, the extracellular vesicle-like particles derived from white peony carry a large number of functional ingredients related to skin care, including flavonoids, polyphenols, reducing sugars, proteins, DNA, RNA, etc., which can be used to maintain skin aging, increase the secretion of type I collagen and matrix metalloproteinase tissue inhibitor 1 (TIMP1) of skin dermal fibroblasts, reduce the secretion of matrix metalloproteinase 1 (MMP1) in the extracellular matrix, reduce the level of active oxygen and cell aging, etc., thereby improving wrinkles, aging and other problems, and can be used for skin care products for face, neck, feet, hands and body care, such as water, milk, cream, ointment, essence, mask, etc., to obtain firm and young skin and healthy skin appearance. On the other hand, the extracellular vesicle-like particles derived from white peony provided by the present invention have application value in skin repair, anti-oxidation and freckle removal and whitening.

[0085] 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).

[0086] Example 1

[0087] Weigh 100g of fresh peony (peeled), wash it three times with ultrapure water to remove impurities, transfer it to a wall breaking machine, cover it tightly, break the wall for 8-10 min, and ensure that there are no obvious block fragments; transfer the peony juice to a 50ml sterile centrifuge tube, centrifuge it at 500×g at 4℃ low speed centrifuge for 10 min, discard the precipitate, and take the supernatant; centrifuge it at 2000×g at 4℃ low speed centrifuge for 20 min, discard the precipitate, and take the supernatant; centrifuge it at 4000×g at 4℃ low speed centrifuge for 30 min, discard the precipitate, and take the supernatant; centrifuge it at 10000×g at 4℃ high speed centrifuge for 1 h, discard the precipitate, and take the supernatant; centrifuge it at 110000×g at 4℃ ultracentrifuge for 70 min, discard the supernatant, and resuspend the precipitate with an appropriate amount of PBS to obtain extracellular vesicle-like particles (labeled as GP-Exo).

[0088] Example 2

[0089] Weigh 100g of fresh peony, wash it, remove the head, tail and thin roots, put it into 200mL of 90℃ water, boil it for 10min, remove it, put it in cold water to cool it quickly, remove the skin, slice it and dry it at 60℃ for 18h. The paeoniflorin (C 23 H 28 O 11 ) shall not be less than 1.2%. Wash with ultrapure water for 3 times to remove impurities, place in a clean beaker; add 500 mL of PBS and soak overnight at 4°C; transfer to a wall-breaking machine, cover tightly, break the wall for 15 minutes, and ensure that there are no obvious block fragments; transfer the white peony 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 extracellular vesicles derived from white peony (labeled as DP-Exo).

[0090] Example 3 Characterization of extracellular vesicles from white peony root

[0091] According to the sample conditions, adjust the samples of Examples 1 and 2 to the 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 min. Use filter paper to blot 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 min. If obvious adsorbents are visible on the copper mesh, pure water can be dripped onto the surface, quickly blotted off, and repeatedly washed several times. Use filter paper to blot the exosome sample on the copper mesh. Observe and take pictures, and save the pictures. The results show ( Figure 1 ): Saucer-cup-shaped vesicles were observed in both GP-Exo and DP-Exo, and the DP-Exo vesicles were regular and complete compared with the GP-Exo vesicles.

[0092] After diluting the extracellular vesicles derived from white peony root obtained in Examples 1 and 2 with a particle-removing buffer, 4 μL was taken into the well of the chip (C400) (no bubbles were allowed, and bubbles could be squeezed out by hand), and the chip was inserted into the nCS1 microfluidic pulse nanoparticle size analyzer, and the corresponding Catridge mold ID (chip model ID) and other related information were input for detection. The detection results showed ( Figure 2 ): 90% of the particle diameters of GP-Exo and DP-Exo extracellular vesicles are in the range of 20-500nm.

[0093] Example 4 Mouse melanocytes take up extracellular vesicles derived from white peony root

[0094] 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 the extracellular vesicles from white peony prepared in Example 2, 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 (containing 10% BSA, Sigma-Aldrich, D8537) 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 pellet in 1 ml of serum-free medium for use.

[0095] PKH26-labeled extracellular vesicles from white peony were added to mouse melanocytes at 50 ng / 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 3 ): Extracellular vesicles derived from white peony root are easily taken up into the cells by mouse melanocytes.

[0096] Example 5 Extracellular vesicles derived from white peony root penetrate into ex vivo pig skin

[0097] The back or abdominal skin of 8-week-old Bama miniature pigs was cut into 1 cm × 1 cm pieces for use. Take 1 mg of the extracellular vesicles derived from white peony prepared in Example 2, 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 10% BSA in PBS (Sigma-Aldrich, D8537) 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.

[0098] 1cm×1cm 8-week-old Bama miniature pig skin was placed in a 32℃ environment, and 50μL of 1mg / ml PKH26-labeled white peony-derived extracellular vesicles were added to the 1cm×1cm 8-week-old Bama miniature pig skin, evenly covering the entire epidermis, and incubated for 2 hours. After the incubation, frozen sections were taken with a thickness of 10 μm. After DAPI staining the cell nucleus, the cells were observed and photographed under a fluorescence microscope. The results showed ( Figure 4 ): Extracellular vesicles derived from white peony root can penetrate into the skin of 8-week-old Bama miniature pigs.

[0099] Example 6 Biochemical inhibition of tyrosinase activity by extracellular vesicles derived from white peony root

[0100] The extracellular vesicles derived from white peony root obtained in Example 1 and Example 2 were subjected to tyrosinase biochemical inhibition tests respectively, and the specific operations were as follows:

[0101] The tyrosinase biochemical inhibition experiment is divided into sample wells (T, only sample solution, tyrosinase solution and L-DOPA solution are added), sample background wells (T0, only sample solution and L-DOPA solution are added), enzyme reaction wells (C, only tyrosinase solution and L-DOPA solution are added), and solvent background wells (C0, only L-DOPA solution is added). Three parallel wells need to be set up for each sample tube (T) of each tested concentration of each sample, and three parallel wells need to be set up for the enzyme reaction well (C). 100 μL of sample solution of the same concentration is added to each sample well (T) and sample background (T0), and 100 μL of disodium hydrogen phosphate-citrate buffer is added to each enzyme reaction well (C) and solvent background well (C0). Add 50 μL of tyrosinase solution to each sample well (T) and enzyme reaction well (C), replace the sample background (T0) and solvent background (C0) with 50 μL of sodium dihydrogen phosphate-citrate buffer, mix the sample and tyrosinase thoroughly, and incubate in a 37°C water bath for 10 minutes. Add 200 μL of levodopa solution to each well in turn, control the reaction time to 5 minutes, and immediately transfer the 96-well ELISA plate to the ELISA reader to measure the absorbance at 492 nm. The tyrosinase inhibition rate is calculated as follows: Inhibition rate (%) = [1-(T-T0) / (C-C0)] × 100% Where: T—sample tube absorbance, that is, the absorbance of the solution after the sample reacts with tyrosinase; T0—sample background absorbance; C—the average of three absorbance values ​​of the enzyme reaction tube, that is, the absorbance of the reaction between tyrosinase and DOPA when no sample is added; C0—solvent background absorbance. The results show that ( Figure 5 , where NC is the sample well with a sample concentration of 0): 100μg / ml GP-Exo and DP-Exo increased the biochemical inhibition rate of tyrosinase by 11.79% and 19.35%, respectively. DP-Exo is superior to GP-Exo in the biochemical inhibition effect of tyrosinase.

[0102] Example 7 Extracellular vesicles from white peony root inhibit melanocyte tyrosinase activity

[0103] B16 melanocytes were inoculated into 24-well plates at a density of 100,000 cells / 500 μL / well. The cells were cultured for 24 hours. The cell supernatant was discarded, and 500 μL of negative control group reagent (DMEM complete medium) and experimental group reagent (DMEM complete medium + 50 μg / mL GP-Exo or 50 μg / mL DP-Exo) were added respectively. The culture was continued for 72 hours. During this period, the cell status and medium were observed every day and the medium of the negative control reagent and experimental group reagent was replaced with fresh medium. 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 digested in a CO2 incubator for 2 minutes. 400 μL of PBS containing 10% FBS was added to terminate the digestion and blow the cells, and the cells were collected in a centrifuge tube, centrifuged at 4000 rpm for 5 minutes, and the supernatant was discarded. Add 100 μL 1% Triton-x-100 to each tube to lyse the cells, freeze at -80°C for 30 minutes, place at room temperature for 10 minutes, centrifuge at 4°C, 12000 rpm, centrifuge for 20 minutes to remove cell debris. Take 50 μL of the supernatant, add 150 μL 2 mg / ml L-DOPA solution, incubate at 37°C for 1 hour, and measure the absorbance at 492 nm with an enzyme marker. The results show ( Figure 6 ): The inhibition rates of 100μg / ml GP-Exo and DP-Exo on the tyrosinase activity of melanocytes can reach 45.95% and 60.57% respectively; the inhibitory effect of DP-Exo on the tyrosinase activity of melanocytes is better than that of GP-Exo.

[0104] Example 8 Extracellular vesicles from white peony root inhibit melanin synthesis in melanocytes

[0105] B16 melanocytes were inoculated into 24-well plates at a density of 100,000 cells / 500 μL / well. The cells were cultured for 24 hours. The cell supernatant was discarded, and 500 μL-negative control group reagent (NC: DMEM complete medium) and experimental group reagent (DMEM complete medium + 50 μg / mL GP-Exo or 50 μg / mL DP-Exo) were added respectively, and cultured for 72 hours. During this period, the cell status and medium were observed every day and the medium of the negative control reagent and experimental group reagent was replaced with fresh medium. After removing the culture supernatant, wash once with PBS. Add 200 μL 0.25% trypsin to each well to digest the cells, and digest them in a CO2 incubator for 2 minutes. Add 400 μL PBS containing 10% FBS to terminate the digestion and blow the cells, collect the cells into a centrifuge tube, centrifuge at 4000 rpm for 5 minutes, and discard the supernatant. Add 100 μL of melanin extract to each tube, shake well, and heat in an 80°C water bath for 1 hour. After cooling, centrifuge the tube wall droplets moderately, blow and evenly mix, take 90 μL of solution from each centrifuge tube, transfer to a 96-well plate, and use an ELISA reader to measure the absorbance of each well at a wavelength of 405 nm. The results show ( Figure 7 ): 100μg / ml GP-Exo and DP-Exo can inhibit the melanin production of melanocytes by 21.66% and 28.41% respectively. The comparison of the inhibitory effect on melanin synthesis of melanocytes showed that DP-Exo was higher than GP-Exo.

[0106] Example 9 Human dermal fibroblasts take up extracellular vesicle-like particles derived from white peony root

[0107] The extracellular vesicle-like particles derived from white peony root (from Example 2) were labeled with PKH26 to track the uptake of extracellular vesicle-like particles derived from white peony root by human dermal fibroblasts.

[0108] 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.

[0109] Take 1 mg of extracellular vesicle-like particles from white peony, and use diluent C in the kit (Sigma, PKH26 Red Fluorescent Cell Labeling Kit, MINI26-1KT) to make the volume of the sample 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 containing 10% BSA to quench. 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-like particle pellet in 1 mL of serum-free medium for use.

[0110] PKH26-labeled extracellular vesicle-like particles from white peony were added to human dermal fibroblasts at 50 ng / 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 ( Figure 8 ): Extracellular vesicle-like particles derived from white peony root can be taken up into human dermal fibroblasts.

[0111] Example 10 Extracellular vesicle-like particles derived from white peony root promote the secretion of type I collagen in dermal fibroblasts

[0112] Dermal fibroblasts were seeded into 96-well plates at a density of 5000 cells / 100 μL / well. The cells were cultured for 24 h. The cell supernatant was discarded, and 100 μL of negative control reagent (NC: basal culture medium), positive control reagent (PC: 100 ng / mL TGF-β), and experimental group reagent (20 μg / mL white peony-derived extracellular vesicle-like particles in Examples 1 and 2) 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.

[0113] The results show that ( Fig. 9 ): GP-Exo and DP-Exo can significantly promote the secretion of type I collagen in dermal fibroblasts, among which DP-Exo is more effective than GP-Exo.

[0114] Example 11 Extracellular vesicle-like particles derived from white peony root affect the secretion of extracellular matrix proteases in dermal fibroblasts

[0115] Dermal fibroblasts were inoculated into 96-well plates at a density of 5000 cells / 100 μL / well. The cells were cultured for 24 h. The cells were irradiated with UVA to simulate the changes in matrix metalloenzymes caused by photoaging. The cell supernatant was discarded and 50 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 min of equilibration, and then the cell culture plate was placed under the lamp. The lid was opened and irradiated for 1 h, for a total of 5 J / cm 2Energy. PBS was discarded, and negative control reagent (NC: basal culture medium), positive control reagent (PC: 100 ng / mL TGF-β), experimental group reagent (20 μg / mL white peony-derived extracellular vesicle-like particles in Examples 1 and 2) were added. The basal culture medium (BC) was added to the group not treated with UVA, and cultured for 24 hours. The supernatant was taken, and the secretion of matrix metalloproteinase 1 (MMP1) and matrix metalloproteinase tissue inhibitor 1 (TIMP1) proteins were detected by Lianke human MMP1 ELISA kit and human TIMP1 ELISA kit, respectively.

[0116] The results show that ( Fig.10 ): GP-Exo can inhibit the secretion of MMP1 in dermal fibroblasts induced by UVA, but cannot promote the secretion of TIMP1 in dermal fibroblasts induced by UVA, while DP-Exo can both promote the secretion of TIMP1 in dermal fibroblasts induced by UVA and inhibit the secretion of MMP1 in dermal fibroblasts induced by UVA, indicating that DP-Exo is better than GP-Exo in regulating matrix-related enzymes.

[0117] Example 12 Extracellular vesicle-like particles derived from white peony root affect the senescence of dermal fibroblasts

[0118] Dermal fibroblasts were inoculated into 24-well plates at a density of 20,000 cells / 100 μL / well. The cells were cultured for 24 hours. The cells were aged by UVA irradiation, the cell supernatant was discarded, and 100 μL of pre-cooled PBS was added to each well. An ice pack was placed in the biosafety cabinet, and four layers of kraft paper were placed on the ice pack. 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), positive control reagent (PC: 100 ng / mL TGF-β), and experimental reagent (20 μg / mL white peony-derived extracellular vesicle-like particles in Examples 1 and 2) were added and cultured for 24 h. 5 J / 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 DP-Exo could significantly reduce the senescence of dermal fibroblasts induced by UVA, while GP-Exo was not significant.

[0119] Example 13 Comparison of extracellular vesicle-like particles from white peony root and white peony root extract

[0120] The extracellular vesicle-like particles derived from white peony root and white peony root extract (from Shanghai Jiakai Biotechnology Co., Ltd.) with the same protein concentration were compared, and the secretion of type I collagen by dermal fibroblasts was compared according to the method of Example 10. The results showed that ( Fig.12 ): DP-Exo is better than white peony extract in promoting the secretion of type I collagen in dermal fibroblasts.

[0121] Example 14 Effect of extracellular vesicles of white peony root on keratinocyte proliferation

[0122] The CCK-8 method was used to determine the proliferation effect of extracellular vesicles of white peony on keratinocytes. A blank group (no cells inoculated, basal culture medium), a control group (NC: basal culture medium), a positive control group (PC: basal culture medium + 100 ng / mL EGF) and an experimental group (basal culture medium containing different concentrations of white peony EVs) were set up. The white peony EVs solution was diluted to a mother solution with a protein concentration of 1 mg / mL in a serum-free culture medium. Sample solutions of 0.5 μg / mL, 5 μg / mL, and 20 μg / mL were prepared and recorded as experimental groups 1 to 3, respectively, for later use.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] Cell proliferation rate%=(OD 实验组 -OD 空白 ) / (OD 对照组 -OD 空白 )×100%;

[0127] Fig.13 The results in Figure A show that the addition of 20 μg / mL of white peony extracellular vesicles (Example 2) can significantly promote keratinocyte proliferation, and the keratinocyte proliferation ability is increased by 32.2%.

[0128] Fig.13The results in Figure B show that the addition of 20 μg / mL of the extracellular vesicles of White Peony Root in Example 2 can increase the proliferation ability of keratinocytes by 34.0%, while the addition of 20 μg / mL of the extracellular vesicles of Example 1 can only increase the proliferation ability of keratinocytes by 25.8%, indicating that the promoting effect of DP-Exo on the proliferation ability of keratinocytes is significantly higher than that of GP-Exo.

[0129] Example 15 Effect of extracellular vesicles of white peony root on keratinocyte migration

[0130] A control group (NC: basal culture medium), a positive control group (PC: basal culture medium + 100 ng / mL EGF), and an experimental group (basal culture medium containing different concentrations of white peony EVs) were set up. Take the white peony EVs solution and dilute it with serum-free culture medium to a mother solution with a protein concentration of 1 mg / mL. Prepare a 20 μg / mL sample solution, record it as the experimental group, and set aside.

[0131] Keratinocytes in good growth condition were taken and 2×10 5 The cells were seeded at a density of 100 cells / well in a 24-well plate and cultured for 24 h before use.

[0132] Use a 300 μL pipette tip to scrape each well once (the tip should be vertical, not tilted) to form an artificial wound, wash the cells three times with PBS, and remove the scratched cells. Use a microscope to take pictures of each scratch position and record the scratch area at 0h.

[0133] The positive control group and the experimental group were given 500 μL / well of the sample solution with the above concentration gradient, and the control group was given the same dose of incomplete culture medium (i.e., culture medium without serum) and incubated at 37°C and 5% CO2 for 24 h.

[0134] Use a microscope to take pictures of each scratch position, record the scratch area for 24 h, and use imageJ to count the keratinocyte scratch area at 0 h and 24 h.

[0135] Cell migration rate % = (S 0h -S 24h ) / S 0h ×100%, where S 0h : Scratch area at 0 hours; S 24h :24 hours scratch area.

[0136] Fig.14 The results showed that the addition of 20 μg / mL DP-Exo could significantly promote keratinocyte migration, and the cell migration level was increased by 16.49%, while the addition of 20 μg / mL GP-Exo could significantly inhibit keratinocyte migration, and the cell migration level was decreased by 19.13%.

[0137] Example 16 Effect of extracellular vesicles of white peony root on the expression level of keratin-related proteins

[0138] Set up a control group (NC: complete culture medium) and an experimental group (complete culture medium containing white peony EVs). Take the white peony EVs solution and dilute it with serum-containing culture medium to a mother solution with a protein concentration of 1 mg / mL. Prepare a 20 μg / mL sample solution, record it as the experimental group, and set aside.

[0139] Keratinocytes in good growth condition were taken and 2×10 5 The cells were seeded at a density of 100 cells / well on a 12-well plate and cultured for 24 h before use.

[0140] The experimental group was given 1 mL / well of the sample solution with the above concentration gradient, and the control group and the experimental group were given the same dose of complete culture medium (i.e., culture medium without serum) and incubated at 37°C and 5% CO2 for 24 h.

[0141] The RNA of the incubated keratinocytes was extracted according to the instructions of VeZol total RNA extraction reagent (R411, VeZol Reagent).

[0142] The RNA extracted in the previous step was reverse transcribed according to the instructions of the Takara PrimeScript™ RT reagent (Perfect Real Time) kit.

[0143] According to the instructions of the Bimake 2╳SYBR Green qPCR Master Mix (High ROX) kit, the cDNA obtained by reverse transcription in the previous step was used for qPCR experiment.

[0144] The expression level of the corresponding gene was calculated using the 2^-ΔΔCt method:

[0145] ΔΔCt = (Ct(target gene) - Ct(reference gene)) 实验组 -(Ct(target gene) - Ct(reference gene)) 对照组

[0146] Ct (target gene): target detection gene

[0147] Ct (reference gene): internal reference gene (18s)

[0148] Fold Change = 2 -ΔΔCt

[0149] Among them, FoldChange represents the multiple of the expression level of the target gene relative to the expression level of the reference gene.

[0150] Fig.15 The results showed that the addition of 20 μg / mL DP-Exo could significantly promote the expression of loricrin LOR and involucrin IVL in keratinocytes, up-regulating by 79.5% and 51.4%, respectively, while the addition of 20 μg / mL GP-Exo had no significant promoting effect on the expression of loricrin LOR and involucrin IVL in keratinocytes. The present invention made a preliminary exploration of the repair mechanism of extracellular vesicles of white peony root, proving that it can achieve its repair effect by promoting the expression of LOR and IVL.

[0151] Example 17 Effects of extracellular vesicles of white peony root on the level of reactive oxygen species in keratinocytes

[0152] DCFH-DA fluorescent probe was used to determine the effect of extracellular vesicles of white peony on the level of reactive oxygen species in keratinocytes. A blank control group (BL: basal culture medium without irradiation), a control group (NC: basal culture medium), a bare cell group (wells without cells), a positive control group (PC: basal culture medium + 5 μg / mL RA) and an experimental group (basal culture medium containing different concentrations of white peony EVs) were set up. The white peony EVs solution was diluted to a mother solution with a protein concentration of 1 mg / mL in a serum-free culture medium. Sample solutions of 0.5 μg / mL, 5 μg / mL, and 20 μg / mL were prepared respectively and recorded as experimental groups 1 to 3 for later use.

[0153] 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.

[0154] 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.

[0155] The DPBS was discarded, and the positive control group and the drug-treated group were given 100 μL / well of the sample solution with the above concentration gradient, and the control group was 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 6 hours.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] Fig.16 The results showed that adding 20 μg / mL DP-Exo could significantly inhibit the ROS level in keratinocytes, with the inhibition level reaching 22.3%.

[0161] Example 18 Testing of the Effect of Extracellular Vesicle Cream from White Peony Root on Human Body

[0162] The extracellular vesicle-like particles derived from white peony root extracted in Example 2 were added to the matrix cream (commissioned to Keying Group for third-party production) at an addition amount of 0.3%, and the matrix cream without extracellular vesicle-like particles derived from white peony root (provided by Keying Group) was used as a control, and applied once in the morning and evening, and no other skin care products were used during use. 30 experiencers 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 extracellular vesicle-like particles derived from white peony root of the present invention.

[0163] 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 28 of use. The skin surface texture analysis system VisioscanVC20plus, the skin elasticity tester MPA580, and the skin rapid three-dimensional imaging system PRIMOSCR were used to detect skin conditions on day 0 and day 28 of use. Visia photo testing 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.

[0164] The results showed that after using the cream containing extracellular vesicles from white peony root for 28 days, the participants' skin pigmentation was significantly reduced, and three participants showed significant improvement. Fig.17 The results show that ( Fig.18 ) After using the extracellular vesicle-like particle cream derived from white peony root for 28 days, skin elasticity increased, and skin SEw (skin wrinkles), wrinkle length, wrinkle depth, and wrinkle area ratio all decreased. Skin fast three-dimensional imaging system and Visia camera detection photos ( Fig.19 ) Wrinkles around the eyes are visibly reduced.

[0165] Visia takes photos to detect the photo ( Fig. 20) The results showed that after using the extracellular vesicle-like particle cream derived from white peony root for 14 days, the transepidermal water loss (TEWL) of the skin was reduced, and the skin hemoglobin was reduced.

[0166] 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. A method for extracting extracellular vesicles from white peony root, characterized in that: The extraction method comprises: (1) taking fresh peony root and pretreating it to obtain white peony root; (2) soaking and breaking the white peony root obtained in step (1) to obtain white peony root juice; (3) centrifuging the white peony juice obtained in step (2) to obtain white peony extracellular vesicles; Wherein, the pretreatment step comprises: placing the fresh peony in water for heating, cooling and then drying; The heating time is 10-30 minutes, the heating temperature is 80-100°C; the drying temperature is controlled to be 50-70°C, and the drying time is 18-36 hours; The centrifugation specific steps include: The white peony juice is centrifuged at 400-800×g for 10-30 min, and the supernatant is collected; then the white peony juice is centrifuged at 1500-3000×g for 10-30 min, and the supernatant is collected; then the white peony juice is centrifuged at 3000-5000×g for 20-40 min, and the supernatant is collected; then the white peony juice is centrifuged at 8000-12000×g for 40-70 min, and the supernatant is collected; then the white peony juice is centrifuged at 100000-120000×g for 60-100 min, and the precipitate is collected, and the precipitate is resuspended in a PBS solution to obtain the extracellular vesicles.

2. The extraction method according to claim 1, characterized in that The fresh peony is the root of fresh peony; And / or, the mass ratio of the fresh peony to the water is controlled to be 1:1-10.

3. The method for extraction according to claim 1, characterized in that Control the soaking time to be 10 to 36 hours; And / or, the cell wall breaking time is controlled to be 8-25 minutes, and the cell wall breaking is intermittent cell wall breaking, and the specific steps include: cell wall breaking for 6-10 minutes, and intermittently for 8-12 minutes; And / or, the paeoniflorin in the white peony root obtained after pretreatment is controlled to be no less than 1.2%.

4. The extracellular vesicles of white peony root obtained by the extraction method according to any one of claims 1 to 3.

5. A composition, characterized in that The composition comprises extracellular vesicles of white peony root obtained by the extraction method according to any one of claims 1 to 3.

6. Use of the extracellular vesicles of white peony root obtained by the extraction method according to any one of claims 1 to 3 or the composition according to claim 5 in the following: (1) Application in the preparation of skin anti-wrinkle and anti-aging products; (2) Application in the preparation of skin repair and antioxidant products; (3) Application in the preparation of whitening and anti-freckle products.

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