Application of pholidota chinensis exosome-like nano-vesicles in preparation of products with skin care function

Abstract

The invention relates to application of pholidota chinensis exosome-like nano-vesicles in preparation of a product with a skin care function, and belongs to the technical field of plant biology. The pholidota chinensis exosome-like nano-vesicle is rich in bioactive substances, has excellent biocompatibility and transmembrane delivery potential due to the bimolecular phospholipid layer structure, and has various skin care effects. Tests show that the compound has no toxicity to RBL-2H3 and RAW264.7 cells within the concentration of 6.25-100 [mu] g / mL, has remarkable proliferation promoting capacity to HaCaT cells within the concentration of 50-100 [mu] g / mL, and meets the safety requirements of cosmetics. In addition, the vesicles can effectively remove ABTS < + > and DPPH free radicals, the inhibiting capacity of the vesicles on DPPH is remarkably higher than that of aqueous extracts and close to that of VC, and the vesicles show an excellent antioxidant effect. Meanwhile, NO generation can be remarkably inhibited, and the effect of resisting skin inflammation is achieved.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to the application of exosome-like nanovesicles from *Prunus cerasifera* in the preparation of products with skin care functions. Background Technology

[0002] Allergic dermatitis is one of the most common and prevalent conditions in dermatology. It refers to an inflammatory reaction of the skin triggered by various allergens and falls under the category of allergic skin diseases. However, its etiology and pathogenesis are quite complex. Depending on whether it is acute or chronic, allergic dermatitis mainly manifests as skin redness, edema, papules, itching, burning sensation, dryness, and desquamation. In severe cases, it can even lead to blisters and erosions.

[0003] Currently, the clinical treatment for allergic dermatitis primarily relies on chemical anti-allergy medications, often combined with corticosteroids and immunomodulators. While these approaches can alleviate symptoms, they are prone to causing numerous adverse reactions, leading to recurring symptoms or more serious complications. In contrast, traditional Chinese medicine demonstrates significant advantages in relieving itching and reducing inflammation, not only improving the condition and prolonging remission periods but also exhibiting fewer adverse reactions. However, in-depth reports on the specific mechanisms of action and systemic clinical applications of novel active ingredients from natural medicinal plants suitable for treating allergic dermatitis are still lacking. Summary of the Invention

[0004] The purpose of this invention is to address the current situation where the treatment of allergic dermatitis relies on chemical drugs, has significant side effects, and is prone to recurrence, by providing a solution based on the active ingredients of natural medicinal plants, which offers significant efficacy and low side effects. To this end, this invention provides the application of *Prunus persica* exosome-like nanovesicles in the preparation of products with skin care functions.

[0005] This invention provides an application of *Prunus cerasifera* exosome-like nanovesicles in the preparation of products with skin care functions.

[0006] Preferably, the skin care function includes any one of the following: ① to ③ ① Anti-skin oxidation; ② Inhibit skin inflammation; ③ Improves allergic dermatitis; ④ Promotes skin cell proliferation and repair.

[0007] Preferably, the anti-skin oxidation includes scavenging free radicals ABTS. + And / or free radical DPPH.

[0008] Preferably, the inhibition of skin inflammation includes inhibiting the production of the inflammatory mediator NO.

[0009] Preferably, the improvement of allergic dermatitis includes inhibiting the release of β-aminohexosidase and / or inhibiting the degranulation process of at least one of mast cells and basophils.

[0010] Preferably, the promotion of skin cell proliferation and repair includes promoting the proliferation of human keratinocytes.

[0011] Preferably, the product includes cosmetics and / or pharmaceuticals.

[0012] Preferably, the method for preparing the *Prunus cerasifera* exosome-like nanovesicles includes the following steps: ① Crush fresh rock peaches, mix them with buffer solution, and homogenize to obtain a slurry; ② Filter the slurry, perform gradient centrifugation on the obtained filtrate, collect the supernatant and perform membrane filtration to obtain a clear filtrate; ③ Mix the clarified filtrate and polyethylene glycol solution, let stand, and then centrifuge at ultra-high speed to collect the precipitate; ④ The precipitate was resuspended and dialyzed. The dialysate was then purified by ultrafiltration to obtain *Prunus cerasifera* exosome-like nanovesicles.

[0013] Preferably, the buffer solution in step ① is a phosphate buffer solution; The gradient centrifugation in step ② includes centrifugation at 200~400g, 1000~3000g and 10000~15000g in sequence; the membrane filtration uses a 0.22~0.45 μm filter membrane.

[0014] Preferably, the polyethylene glycol solution in step ③ has a mass-volume concentration of 10%, and the standing operation is performed by standing overnight at 4°C for 12 hours. The dialysis described in step ④ is performed at 4°C using phosphate buffer.

[0015] Beneficial effects: This invention provides the application of *Prunus cerasifera* exosome-like nanovesicles in the preparation of products with skin care functions. The *Prunus cerasifera* exosome-like nanovesicles of this invention are rich in various bioactive substances, including proteins, microRNAs, and lipids; their unique bilayer phospholipid structure endows them with excellent biocompatibility and transmembrane delivery potential, thus enabling them to exert skin care effects through multiple pathways. Verification through examples shows that the *Prunus cerasifera* exosome-like nanovesicles of this invention have no cytotoxicity to RBL-2H3 and RAW264.7 cells within the tested concentration range of 6.25~100 μg / mL, and exhibit significant pro-proliferative ability against HaCaT cells within the concentration range of 50~100 μg / mL, meeting the safety requirements for application in cosmetics. Furthermore, the vesicles of this invention can effectively scavenge free radicals ABTS. +The vesicles described in this invention exhibit significantly stronger inhibitory effects on DPPH free radicals than the water extract and are close to those of ascorbic acid (VC) within the concentration range of 80–200 μg / mL, demonstrating excellent anti-oxidative effects on the skin. Simultaneously, the vesicles described in this invention can significantly inhibit the production of the inflammatory mediator NO within the concentration range of 6.25–100 μg / mL, thus possessing anti-inflammatory effects on the skin.

[0016] Furthermore, the vesicles described in this invention can also inhibit the release of β-aminohexosidase and inhibit the degranulation of RBL-2H3 cells. Their regulatory ability is comparable to that of the positive control drug ketotifen fumarate, exhibiting significant anti-allergic effects and can be used to improve symptoms of allergic dermatitis. The method for preparing *Dendrobium nobile* exosome-like nanovesicles provided by this invention is simple, environmentally friendly, and inexpensive, meeting the requirements of large-scale industrial production. Its biosafety and efficacy are superior to *Dendrobium nobile* water extract, making it an excellent natural raw material for cosmetics with significant practicality and economic benefits. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 Transmission electron microscopy (TEM) image of exosome-like nanovesicles from *Prunus cerasifera*. Figure 2 A schematic diagram showing the particle size distribution of nanovesicles resembling exosomes from *Prunus cerasifera*. Figure 3 A schematic diagram of the ZETA potential of exosome-like nanovesicles from *Prunus cerasifera*. Figure 4 Schematic diagram of the cytotoxicity of HaCaT cells, Raw264.7 cells, and RBL-2H3 cells to *Dendrobium nobile* exosome-like nanovesicles and aqueous extracts of *Dendrobium nobile*. Figure 5 A schematic diagram illustrating the antioxidant activity of *Dendrobium nobile* exosome-like nanovesicles and *Dendrobium nobile* aqueous extract; among which, Figure 5 In the figure, A represents the results of DPPH free radical scavenging rate detection under different drug treatments. Figure 5 In this context, B represents ABTS treated with different drugs. + Free radical scavenging rate test results; Figure 6 A schematic diagram illustrating the anti-inflammatory activity of *Dendrobium nobile* exosome-like nanovesicles and *Dendrobium nobile* aqueous extract; Figure 7 A schematic diagram showing the inhibition rate of β-aminohexosidase release by exosome-like nanovesicles from *Prunus cerasifera*. Figure 8 This is an electron micrograph of exosome-like nanovesicles from *Prunus cerasifera* at the 100-nanometer scale. Detailed Implementation

[0019] This invention provides the application of *Prunus cerasifera* exosome-like nanovesicles in the preparation of products with skin care functions. The *Prunus cerasifera* exosome-like nanovesicles of this invention are rich in various bioactive substances and can exert skin care effects through multiple pathways. As one embodiment, an electron micrograph of the *Prunus cerasifera* exosome-like nanovesicles at the 100 nanometer scale is shown below. Figure 8 As shown. In one embodiment, the *Pyrrosia lingua* exosome-like nanovesicles described in this invention showed no cytotoxicity to RBL-2H3 and RAW264.7 cells within a test concentration range of 6.25~100 μg / mL, meeting the safety requirements for application in cosmetics.

[0020] As one implementation method, the skin care function of the present invention includes any one of the following: ① to ③ ① Anti-skin oxidation; ② Inhibit skin inflammation; ③ Improves allergic dermatitis; ④ Promotes skin cell proliferation and repair.

[0021] As one embodiment, the anti-skin oxidation method of the present invention includes scavenging free radicals ABTS. + And / or free radical DPPH. As one embodiment, the *Dendrobium nobile* exosome-like nanovesicles of the present invention exhibit significantly stronger inhibitory ability against DPPH free radicals than the water extract and approaching the effect of ascorbic acid (VC) in the concentration range of 80~200 μg / mL, demonstrating excellent anti-skin antioxidant efficacy.

[0022] In one embodiment, the inhibition of skin inflammation described in this invention includes inhibiting the production of the inflammatory mediator NO. In another embodiment, the *Pyrrosia lingua* exosome-like nanovesicles described in this invention can significantly inhibit the production of the inflammatory mediator NO in the concentration range of 6.25~100 μg / mL.

[0023] In one embodiment, the improvement of allergic dermatitis according to the present invention includes inhibiting the release of β-aminohexosidase and / or inhibiting the degranulation process of at least one of mast cells and basophils. In one embodiment, the inhibition of mast cells and basophils according to the present invention is manifested in the ability to inhibit RBL-2H3 cell degranulation. In one embodiment, the anti-allergic effect of the improvement of allergic dermatitis according to the present invention is comparable to that of the commercial drug ketotifen fumarate.

[0024] In one embodiment, the promotion of skin cell proliferation and repair described in this invention includes promoting the proliferation of human keratinocytes. In another embodiment, the human keratinocytes described in this invention include promoting HaCaT cells. In yet another embodiment, the *Pyrrosia lingua* exosome-like nanovesicles described in this invention exhibit significant proliferative capacity against HaCaT cells at a concentration range of 50–100 μg / mL.

[0025] As one implementation method, the products described in this invention include cosmetics and / or pharmaceuticals.

[0026] As one embodiment, the preparation method of the *Prunus cerasifera* exosome-like nanovesicles of the present invention includes the following steps: ① Crush fresh rock peaches, mix them with buffer solution, and homogenize to obtain a slurry; ② Filter the slurry, perform gradient centrifugation on the obtained filtrate, collect the supernatant and perform membrane filtration to obtain a clear filtrate; ③ Mix the clarified filtrate and polyethylene glycol solution, let stand, and then centrifuge at ultra-high speed to collect the precipitate; ④ The precipitate was resuspended and dialyzed. The dialysate was then purified by ultrafiltration to obtain *Prunus cerasifera* exosome-like nanovesicles.

[0027] This invention involves crushing fresh *Prunus cerasifera* (a type of peach), mixing it with a buffer solution, and homogenizing the mixture to obtain a slurry. In one embodiment, the buffer solution in step ① is a phosphate buffer solution. In another embodiment, the fresh *Prunus cerasifera* is washed, dried, and then crushed in a juicer. In yet another embodiment, the crushing process is repeated three times over 15 minutes.

[0028] After obtaining the slurry, the present invention filters the slurry, performs gradient centrifugation on the resulting filtrate, collects the supernatant, and performs membrane filtration to obtain a clear filtrate. In one embodiment, the gradient centrifugation in step ② includes sequential centrifugation at 200-400g, 1000-3000g, and 10000-15000g; the membrane filtration uses a 0.22-0.45 μm filter membrane. In another embodiment, the gradient centrifugation in step ② includes sequential centrifugation at 300g, 2000g, and 10000g; the membrane filtration uses a 0.45 μm filter membrane. In one embodiment, the filtration operation of the present invention includes filtering the slurry using a 400-mesh filter cloth to remove plant residue and collecting the filtrate for later use. In one embodiment, the gradient centrifugation of the present invention is performed at 4°C. In one embodiment, the sequential centrifugation times of 300g, 2000g, and 10000g are all 30 minutes.

[0029] After obtaining the clarified filtrate, the present invention mixes the clarified filtrate with a polyethylene glycol solution, allows it to stand, and then performs ultra-high speed centrifugation to collect the precipitate. As one embodiment, the polyethylene glycol solution in step ③ of the present invention has a mass-volume concentration of 10%, and the standing operation is performed overnight at 4°C for 12 hours. As one embodiment, the volume ratio of the clarified filtrate to the polyethylene glycol solution is 1:1. As one embodiment, the ultra-high speed centrifugation operation of the present invention is performed at 4°C, centrifuged at 10000 g for 30 minutes.

[0030] After collecting the precipitate, the present invention resuspends the precipitate, dialyzes it, and then purifies the dialysate by ultrafiltration to obtain *Prunus cerasifera* exosome-like nanovesicles. In one embodiment, the resuspension operation of the present invention uses PBS pre-cooled to 4°C to resuspend the precipitate. In another embodiment, the dialysis operation of the present invention involves transferring the resuspension into a dialysis bag and dialyzing on a shaker at 4°C for 12 h. In another embodiment, the dialysate used in the dialysis operation of the present invention is PBS. In another embodiment, the dialysis in step ④ of the present invention is performed using phosphate buffer at 4°C. In another embodiment, the dialysate is changed every 4 h during the dialysis operation of the present invention. As one embodiment, the method for preparing *Prunus cerasifera* exosome-like nanovesicles provided by the present invention is simple, environmentally friendly, and inexpensive, meeting the requirements of large-scale industrial production. Its biosafety and efficacy are superior to *Prunus cerasifera* water extract, making it an excellent natural raw material for cosmetics with significant practicality and economic benefits.

[0031] To further illustrate the present invention, the application of the *Prunus cerasifera* exosome-like nanovesicles provided by the present invention in the preparation of products with skin care functions is described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1 Extraction, purification and characterization of exosome-like nanovesicles from *Dendrobium nobile* 1. Extraction and purification of exosome-like nanovesicles from *Dendrobium nobile* After washing fresh *Dendrobium nobile* (a type of peach), pat dry and place in a juicer. Add an appropriate amount of PBS and juice thoroughly for 15 minutes, repeating this process three times. Pour out the juice and filter through a 400-mesh filter cloth to remove plant residue. Collect the filtrate for later use. Centrifuge the filtrate sequentially: ① Centrifuge at 300 g, 4℃ for 30 minutes, and collect the supernatant; ② Centrifuge the supernatant again at 2000 g, 4℃ for 30 minutes, and collect the supernatant; ③ Then centrifuge the supernatant at 10000 g, 4℃ for 30 minutes, collect the supernatant, and filter through a 0.45 μm filter membrane for later use. Prepare a 10% (w / v) PEG solution and mix it thoroughly with the supernatant at a 1:1 (v / v) ratio. Incubate overnight at 4℃. Centrifuge the mixture at 10000 g, 4℃ for 30 minutes, discard the supernatant, and retain the precipitate. The precipitate was resuspended in an appropriate amount of freshly pre-cooled (4°C) PBS. The resuspended solution was transferred to a dialysis bag and dialyzed on a shaker at 4°C for 12 h (the dialysate was PBS, which was changed every 4 h). After ultrafiltration to remove impurities, the liquid obtained was Pyrrosia lingua exosome-like nanovesicles, which were stored at -80°C for subsequent analysis.

[0033] 2. Morphological identification by transmission electron microscopy (TEM) The prepared *Pyrrosia lingua* exosome-like nanovesicle suspension was pipetted 20 μL onto a copper grid and allowed to adsorb naturally for 5–10 min. Excess droplets were then removed with filter paper, and the sample was allowed to air dry slightly. Next, 20 μL of 2% phosphotungstic acid solution was pipetted onto the copper grid and allowed to stand for 3–5 min. Excess droplets were removed with filter paper, and the sample was allowed to air dry under an incandescent lamp. The sample was then observed and photographed under a transmission electron microscope. The *Pyrrosia lingua* exosome-like nanovesicles extracted in Example 1 exhibited a spherical structure without aggregation. The results are shown in the figure below. Figure 1 .

[0034] 3. Nanoparticle tracking analysis (NTA) detection After powering on the instrument, dilute the 100 nm polystyrene microsphere standard solution 250,000 times with ultrapure water. Take 1 ml of the diluted standard solution for automatic instrument calibration. After automatic calibration, clean the sample cell with 1X PBS buffer. Dilute the exosome-like nanovesicle sample to a suitable concentration with clean PBS solution and inject it for testing. Enter the sample dilution factor in the software interface and observe whether the number of particles displayed at each detection location is close. After confirming that the number of particles displayed at each detection location is very close, click Measurement and Run Video Acquisition in sequence on the software interface. In the pop-up interface, set the sample name and data save path, select the appropriate SOP, and finally click OK to start the test. The instrument automatically completes the test process, automatically analyzes the data, and automatically generates a test report, providing information such as sample particle size, particle concentration, and ZETA potential. The exosome-like nanovesicles extracted in Example 1 had a particle size concentrated in the range of 30–200 nm, with a peak at 100 nm and a zeta potential of -30.12 ± 0.26 mV, consistent with the particle size characteristics of exosome-like nanovesicles. The results are shown in [Figure number missing]. Figure 2 and Figure 3 .

[0035] Comparative Example 1 Take the same weight of washed fresh rock peaches, add distilled water at a ratio of 1:3, and juice them three times for 15 minutes each time using a juicer. Pour out the juice and filter it through a 400-mesh filter cloth to remove plant residues and obtain the filtrate for later use. After freeze-drying the filtrate, you will get the water extract of rock peaches.

[0036] Example 2 Cytotoxicity test on HaCaT, RBL-2H3, and RAW264.7 cells Experimental methods: HaCaT, RBL-2H3, and RAW264.7 cells (1 x 10) were used. 5 Cells (number per well) were seeded into 96-well plates and incubated for 24 h. Different concentrations of *Pyrrosia lingua* exosome-like nanovesicles and water extract were added to the cells, and incubated for 24 h each. Each concentration was used in a group of 5 parallel wells. 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37 ℃ for 1 h. The absorbance was measured at 450 nm using a microplate reader. The control group consisted of untreated cells.

[0037] Table 1. Effects of *Dendrobium nobile* exosomes and aqueous extracts on the proliferation rate of three cell types.

[0038] Note: Data are expressed as mean ± standard deviation.

[0039] The results of detecting the cytotoxicity of *Dendrobium nobile* exosome-like nanovesicles and *Dendrobium nobile* aqueous extract in HaCaT cells, Raw264.7 cells, and RBL-2H3 cells are shown in Table 1. Figure 4 As shown in Table 1 and... Figure 4 The results showed that, for HaCaT cells, *Pyrrosia lingua* exosome-like nanovesicles exhibited proliferative activity within the tested concentration range of 50–100 μg / mL (p<0.01), while the aqueous extract showed some cytotoxicity at 100 μg / mL (p<0.01). For RBL-2H3 cells, neither *Pyrrosia lingua* exosome-like nanovesicles nor the aqueous extract had a significant effect on cell growth within the tested concentration range of 6.25–100 μg / mL. For RAW264.7 cells, *Pyrrosia lingua* exosome-like nanovesicles had no significant effect on cell growth within the tested concentration range of 6.25–100 μg / mL, while the aqueous extract inhibited cell growth (p<0.001). Therefore, the cytotoxicity of *Pyrrosia lingua* exosome-like nanovesicles was relatively weaker than that of the aqueous extract, demonstrating superior biocompatibility.

[0040] Example 3 Antioxidant activity of *Dendrobium nobile* exosome-like nanovesicles and *Dendrobium nobile* water extract 1. Determination of DPPH· free radical scavenging rate Weigh 10 mM DPPH powder (purchased from Shanghai Yuanye Biotechnology Co., Ltd., CAS: 1898-66-4, 250 mg) and dissolve it in anhydrous ethanol solution. After sonicating for 15 min until the DPPH solution is evenly mixed, dilute the solution to 0.1 mM to obtain DPPH stock solution. Before use, dilute it 20 times to obtain working solution and store it away from light.

[0041] Take appropriate amounts of Example 1 (Dendrobium nobile exosome-like nanovesicles), Comparative Example 1 (Dendrobium nobile water extract), and ascorbic acid (VC) (purchased from Shanghai Titan Technology Co., Ltd., Adamas#CAS:50-81-7, 25g) to prepare a solution of 2mg / mL.

[0042] The experimental group consisted of solutions prepared in Example 1 and Comparative Example 1, with ascorbic acid (VC) as a positive control. 800 μL of DPPH working solution was added to a 5 mL test tube, followed by 0, 10, 20, 40, 60, 80, and 100 μL of the analyte, respectively. The remainder was brought to a final volume of 1 mL with PBS. The mixture was thoroughly mixed and incubated in the dark for 30 min. The absorbance was measured at 517 nm. The free radical scavenging capacity of DPPH was calculated using Equation I: DPPH free radical scavenging rate Formula I; A1: Add different volumes of the analyte and 800 μL of DPPH working solution; A2: Add different volumes of the analyte and 800 μL of PBS solution; A3: Add 200 μL PBS solution and 800 μL DPPH working solution; A4: Add 200 μL of PBS solution.

[0043] The results of DPPH free radical scavenging rate detection of *Dendrobium nobile* exosome-like nanovesicles and *Dendrobium nobile* water extract are as follows: Figure 5 As shown in A and Table 2. According to... Figure 5 As shown in Table 2, within the concentration range of 20–40 μg / mL, both *Pyrrosia lingua* exosome-like nanovesicles and the aqueous extract could inhibit the generation of DPPH free radicals to a certain extent. Within the concentration range of 80–200 μg / mL, the inhibitory ability of *Pyrrosia lingua* exosome-like nanovesicles on DPPH free radicals was significantly stronger than that of the aqueous extract, and approached the effect of the control group (VC). This indicates that both *Pyrrosia lingua* exosome-like nanovesicles and the aqueous extract possess good antioxidant capabilities, with the exosome-like nanovesicles showing superior effects compared to the aqueous extract, demonstrating significant potential for cosmetic applications.

[0044] 2. ABTS + Free radical scavenging rate determination Weigh out an appropriate amount of ABTS (purchased from Shanghai Titan Technology Co., Ltd., Adamas#CAS:30931-67-0, 1g) and potassium persulfate powder (purchased from Shanghai Titan Technology Co., Ltd., Adamas#CAS:7727-21-1, 100g), and prepare 10mL each of 7mM and 2.45mM solutions with anhydrous ethanol. Mix the two solutions thoroughly and store them in the dark for 12 hours to obtain the ABTS stock solution. Before use, dilute it 30 times to obtain the working solution and store it in the dark.

[0045] Take an appropriate amount of Example 1 (Dendrobium nobile exosome-like nanovesicles), Comparative Example 1 (Dendrobium nobile water extract), and ascorbic acid (VC) to prepare a solution of 2 mg / mL.

[0046] The experimental group consisted of solutions prepared in Example 1 and Comparative Example 1, with ascorbic acid (VC) as a positive control. 800 μL of ABTS was added to a 5 mL test tube. + Prepare the working solution, then add 0, 10, 20, 40, 60, 80, and 100 μL of the analyte sequentially, and bring the remaining amount to 1 mL with PBS. Mix well, incubate in the dark for 30 min, and measure the absorbance at 734 nm. ABTS + Free radical scavenging ability is calculated using Equation II: ABTS + Free radical scavenging rate Formula II; A1: Add different volumes of the analyte and 800 μL ABTS. + Working fluid; A2: Add different volumes of the analyte and 800 μL of PBS solution; A3: Add 200 μL PBS solution and 800 μL ABTS + Working fluid; A4: Add 200 μL of PBS solution.

[0047] ABTS was obtained from exosome-like nanovesicles of *Dendrobium nobile* and aqueous extracts of *Dendrobium nobile*. + Free radical scavenging rate test results are as follows Figure 5 As shown in B and Table 2. According to Figure 5 As shown in Table 2, within the concentration range of 20–200 μg / mL, both the exosome-like nanovesicles and the water extract of *Dendrobium nobile* could inhibit ABTS. + The inhibitory effect of *Dendrobium nobile* exosome-like nanovesicles on free radical generation was significantly stronger than that of the water extract, and approached the effect of the control group (VC). This indicates that both *Dendrobium nobile* exosome-like nanovesicles and the water extract possess good antioxidant capabilities, with the exosome-like nanovesicles showing superior efficacy and demonstrating significant potential for cosmetic applications.

[0048] Table 2 Antioxidant Capacity Test

[0049] Note: Data are expressed as mean ± standard deviation; the control drug is vitamin C.

[0050] Example 4 Anti-inflammatory activity assay of exosome-like nanovesicles from *Dendrobium nobile* 1. Experimental Methods RAW264.7 cells were grown at a rate of 8 × 10⁻⁶. 4Cells were seeded at a density of 1 / 2 well in 96-well plates, with Pirelli cell suspension added to each well. After culturing in a CO2 incubator for 24 h, the following groups were established: a blank group (PBS); an LPS group (PBS + LPS); and experimental groups (PBS + LPS + different concentrations of the analyte). Each group had four replicates. The final LPS concentration was 2 μg / mL. After culturing at 37℃ in a CO2 incubator for 24 h, the supernatant was transferred to a microplate, and an equal volume of Griess reagent was added. The reaction was carried out at room temperature for 10 min, and then the absorbance was measured at 540 nm. NaNO2 standard (1M) was diluted to nine different concentrations (0, 1, 2, 5, 10, 20, 40, 60, and 100 µM) using complete DMEM medium. 50 µL of each concentration was added to each well of a clean 96-well microplate, with three replicates per concentration, avoiding air bubbles. 50 µL of Griess I reagent was added to each well sequentially, and the mixture was incubated in the dark with shaking for 10 min. Then, Griess II reagent was added, and the mixture was incubated in the dark with shaking for another 10 min. The OD value was measured at 540 nm, and a standard curve was plotted. The NO content in the cell culture supernatant was calculated based on the NaNO2 standard curve. 2- The concentration of [agent] and its inhibition rate of NO release are shown in Formula III.

[0051] NO release inhibition rate (%) = ([NO 2 ] LPS刺激组 -[NO 2 ] 实验组 ) / ([NO 2 ] LPS刺激组 - [NO 2 ] 空白组 ) ×100%, Formula III.

[0052] 2. Experimental Results The anti-inflammatory activity results of the water extract and the exosome-like nanovesicles of *Dendrobium nobile* are shown in Table 3 and 4, respectively. Figure 6 As shown in A and B in Table 3. Figure 6 The results showed that, within the concentration range of 6.25–100 μg / mL, both *Pyrrosia lingua* exosome-like nanovesicles and their aqueous extract significantly inhibited NO production (P<0.001), indicating that both *Pyrrosia lingua* exosome-like nanovesicles and their aqueous extract possess significant anti-inflammatory activity potential. These experimental results clearly demonstrate that both *Pyrrosia lingua* exosome-like nanovesicles and their aqueous extract can repair damaged skin barriers and alleviate skin discomfort symptoms, exhibiting significant potential for cosmetic applications.

[0053] Table 3 NO generation inhibition rate (%)

[0054] Note: Data are expressed as mean ± standard deviation.

[0055] Example 5 Effects on the release of β-aminohexosidase (β-Hex) from RBL-2H3 cells 1. Experimental Methods When the RBL-2H3 cell growth density reached approximately 80%, it was further increased to 3.5 × 10⁻⁶ cells. 5 The culture medium was seeded at a density of 1 / 2 well in 6-well plates and incubated in a CO2 incubator for 24 h before drug administration. The following groups were established: a blank control (culture medium), a model group (C48 / 80) (C48 / 80 purchased from Shanghai Maclean Biotechnology Co., Ltd., C911503-10mg), a *Pseudomonas aeruginosa* exosome-like nanovesicle group (C48 / 80 + different concentrations of exosomes), a positive control group (C48 / 80 + 2 μg / mL Keto) (ketotifen fumarate Keto purchased from Shanghai Maclean Biotechnology Co., Ltd., K838241-100mg), and a negative control group (different concentrations of exosomes or Keto). 10 μL of drug-containing culture medium was added to the *Pseudomonas aeruginosa* exosome group, positive control group, and negative control group, while an equal volume of drug-free culture medium was added to the blank control and model group. After 24 h, the plates were washed three times with PBS. The model group, *Pyrrosia lingua* exosome-like nanovesicle group, and positive control group were added to 10 μL of complete culture medium containing C48 / 80, with a final C48 / 80 concentration of 20 μg / mL. The remaining groups were added to an equal volume of culture medium. After 20 min, the reaction was terminated by incubating on ice for 10 min. Cells were collected, and proteins were extracted using RIPA lysis buffer. Protein concentration was determined using the BCA method, and then the β-aminohexosidase content was quantified according to the instructions provided with the ELISA kit. The calculation formula is shown in Equation IV. β-Hex release rate (%) = (OD 给药组 -OD 阴性对照组 ) / (OD 模型组 - OD 正常组 ) ×100%, Formula IV.

[0056] 2. Experimental Results The inhibition rate of β-aminohexosidase release by exosome-like nanovesicles from *Prunus cerasifera* was detected as follows: Figure 7 And as shown in Table 4. According to Figure 7 The results in Table 4 show that, within the concentration range of 6.25–100 μg / mL, *Pyrrosia lingua* exosome-like nanovesicles significantly inhibited the release of β-aminohexosidase (P<0.001), and their regulatory ability was comparable to that of the positive control drug ketotifen fumarate (Keto). Therefore, *Pyrrosia lingua* exosome-like nanovesicles can inhibit the degranulation of RBL-2H3 cells and have good anti-allergic activity.

[0057] Table 4. Inhibition rate of β-aminohexosidase release (%)

[0058] Note: Data are expressed as mean ± standard deviation.

[0059] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of a type of *Prunus cerasifera* exosome-like nanovesicle in the preparation of products with skin care functions.

2. The application according to claim 1, characterized in that, The skin care function includes any one of the following: ① to ③ ① Anti-skin oxidation; ② Inhibit skin inflammation; ③ Improves allergic dermatitis; ④ Promotes skin cell proliferation and repair.

3. The application according to claim 2, characterized in that, The anti-skin antioxidants include scavenging free radicals (ABTS). + And / or free radical DPPH.

4. The application according to claim 2, characterized in that, The inhibition of skin inflammation includes inhibiting the production of the inflammatory mediator NO.

5. The application according to claim 2, characterized in that, The improvement of allergic dermatitis includes inhibiting the release of β-aminohexosidase and / or inhibiting the degranulation process of at least one of mast cells and basophils.

6. The application according to claim 2, characterized in that, The promotion of skin cell proliferation and repair includes promoting the proliferation of human keratinocytes.

7. The application according to any one of claims 1 to 6, characterized in that, The products include cosmetics and / or pharmaceuticals.

8. The application according to any one of claims 1 to 6, characterized in that, The preparation method of the *Prunus cerasifera* exosome-like nanovesicles includes the following steps: ① Crush fresh rock peaches, mix them with buffer solution, and homogenize to obtain a slurry; ② Filter the slurry, perform gradient centrifugation on the obtained filtrate, collect the supernatant and perform membrane filtration to obtain a clear filtrate; ③ Mix the clarified filtrate and polyethylene glycol solution, let stand, and then centrifuge at ultra-high speed to collect the precipitate; ④ The precipitate was resuspended and dialyzed. The dialysate was then purified by ultrafiltration to obtain *Prunus cerasifera* exosome-like nanovesicles.

9. The application according to claim 8, characterized in that, The buffer solution mentioned in step ① is a phosphate buffer solution; The gradient centrifugation in step ② includes centrifugation at 200~400g, 1000~3000g and 10000~15000g in sequence; the membrane filtration uses a 0.22~0.45 μm filter membrane.

10. The application according to claim 8, characterized in that, The polyethylene glycol solution in step ③ has a mass-volume concentration of 10%, and the standing operation is to stand at 4°C overnight for 12 hours. The dialysis described in step ④ is performed at 4°C using phosphate buffer.

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