Application of purslane-derived extracellular vesicles in preparation of anti-inflammatory soothing efficacy products

Abstract

The present application relates to a new application of a cell extracellular vesicle derived from Portulaca oleracea L., and in particular to an application of the cell extracellular vesicle derived from Portulaca oleracea L. in preparation of an anti-inflammatory soothing efficacy product. The present application extracts and purifies a cell extracellular vesicle derived from Portulaca oleracea L. creatively, and studies the physiological efficacy thereof, finds that the cell extracellular vesicle can reduce the expression level of inflammatory factors in macrophages, plays an anti-inflammatory role, and can be internalized by HaCaT cells, and animal tests prove that the cell extracellular vesicle can relieve and treat the skin lesion symptoms of a mouse model of atopic dermatitis, reduce the spleen index of the mouse model of atopic dermatitis, and reduce the expression levels of TNF-α and IL-4 in skin tissues. The cell extracellular vesicle has potential for use in preparation of cosmetics or drugs with anti-inflammatory soothing efficacy, and provides a new strategy for soothing skin sensitivity and irritation or preventing and treating inflammatory skin diseases.

Description

Technical Field

[0001] This invention belongs to the field of plant extracellular vesicle technology, and relates to new applications of purslane-derived extracellular vesicles, specifically the application of purslane-derived extracellular vesicles in the preparation of anti-inflammatory and soothing products. Background Technology

[0002] Extracellular vesicles (EVs) are 40-150 nm bio-nanoparticles produced by cells. They have a phospholipid bilayer membrane structure and contain active substances such as mRNA, proteins, and nucleic acids. EVs can transmit information between plants, animals, and microorganisms. Initially, EVs were considered cell debris, thus their value was overlooked. Today, however, EVs are recognized as key carriers for intercellular information transmission and regulation of cellular physiological functions. Plant EVs can serve as effective carriers for communication between plant and pathogen cells, participate in plant defense responses against pathogens, participate in the formation of plant cell wall components, and play an important role in regulating plant immunity.

[0003] Purslane (Portulaca oleracea L.) is an ancient Chinese plant used for both medicinal and edible purposes, possessing various biological activities such as antibacterial, anti-inflammatory, anti-diabetic, antioxidant, and anti-tumor effects. However, extracellular vesicles (EVs) derived from purslane have received little attention. Whether it is feasible to extract extracellular vesicles from purslane, and whether the extracted purslane-derived EVs possess biological activity, remains unknown. Therefore, it is significant to develop a method for extracting high-purity, high-quality extracellular vesicles from purslane and to conduct in-depth research on the functions of the extracted EVs to explore their potential applications in cosmetics or pharmaceuticals.

[0004] With the increasing number of people with sensitive skin, consumers have a growing demand for skin-soothing products (such as cosmetics or pharmaceuticals). Factors influencing skin irritation include internal factors such as race, age, skin health, and genetics, as well as external factors such as climate and inferior cosmetics. Skin irritation can cause dryness, tightness, redness, itching, peeling, burning and stinging sensations, or damage to the skin barrier, abnormal nerve function, and in severe cases, inflammatory reactions.

[0005] Atopic dermatitis (AD) is a chronic, recurrent inflammatory skin disease that can affect people of any age and has a genetic predisposition. It is characterized by intense itching and eczematous lesions. Most patients experience their first onset in infancy, and due to its chronic and recurrent nature, some patients may continue to have the disease into adulthood. Severe cases can significantly impact daily life and even lead to sleep disturbances. The main pathogenesis of AD is currently unclear, but it may involve immune system signaling imbalances, genetics, skin dysfunction, and psychological factors. Current treatments for atopic dermatitis primarily include topical corticosteroids, glucocorticoids, oral antihistamines, biologics, and ultraviolet therapy. However, these treatments all have drawbacks to varying degrees; for example, long-term use of corticosteroids can cause serious adverse reactions, and biologics are expensive. Therefore, the search for new drugs and treatments is particularly necessary. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a new application of purslane-derived extracellular vesicles, specifically the application of purslane-derived extracellular vesicles in the preparation of products with anti-inflammatory and soothing effects.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides the application of purslane-derived extracellular vesicles in the preparation of products with anti-inflammatory and soothing effects.

[0009] This invention creatively extracts and purifies extracellular vesicles derived from purslane and studies their physiological effects. It was found that they can reduce the expression levels of inflammatory factors in macrophages, exerting an anti-inflammatory effect, and can be internalized by HaCaT cells. Animal experiments demonstrated that they can alleviate and treat skin lesions in a mouse model of atopic dermatitis, reduce the spleen index in the mouse model of atopic dermatitis, and reduce the expression levels of TNF-α and IL-4 in skin tissue. They have potential for use in the preparation of cosmetics or pharmaceuticals with anti-inflammatory and soothing effects, providing a new strategy for soothing sensitive skin irritation or preventing and treating inflammatory skin diseases.

[0010] In this invention, the product includes cosmetics or pharmaceuticals.

[0011] Based on the in vivo and in vitro anti-inflammatory research results of this invention, those skilled in the art can prepare purslane-derived extracellular vesicles into different forms of anti-inflammatory and soothing products, especially topical (transdermal) anti-inflammatory and soothing products, such as cosmetics or pharmaceuticals, according to different needs.

[0012] Preferably, the cosmetic is used to soothe sensitive skin, that is, the cosmetic has the effect of soothing sensitive skin.

[0013] Preferably, the medicine is used to prevent or treat inflammatory skin diseases, that is, the medicine is a medicine for preventing or treating inflammatory skin diseases; preferably, the inflammatory skin diseases include atopic dermatitis.

[0014] In this invention, the product is a transdermal dosage form.

[0015] Preferably, the transdermal dosage form includes: liniment, ointment, lotion, paste, gel, coating, film, patch, and microneedle.

[0016] Preferably, the product further contains excipients, which include any one or a combination of at least two of the following: diluents, dispersants, binders, fillers, thickeners, lubricants, pH adjusters, flavor maskers, colorants, antioxidants, or antibacterial agents.

[0017] The purslane-derived extracellular vesicles involved in this invention can be prepared by any method known to those skilled in the art, but the following extraction method is preferred. The purslane-derived extracellular vesicles obtained by this method have higher purity and better anti-inflammatory and soothing effects.

[0018] Preferably, the method for preparing the purslane-derived extracellular vesicles includes ultracentrifugation or polyethylene glycol precipitation.

[0019] Preferably, the ultracentrifugation method specifically includes the following steps:

[0020] (1) Filter the juice of purslane raw material, centrifuge the filtrate and collect the supernatant;

[0021] (2) After filtering the supernatant, centrifuge it at high speed, collect the precipitate and resuspend it to obtain the final product.

[0022] Preferably, step (1) involves centrifugation and collection of the supernatant at least three times, with centrifugation speeds successively of 800-1800g (e.g., 800g, 900g, 1000g, 1200g, 1300g, 1400g, 1500g, 1600g, 1800g, etc.), 2000-5000g (e.g., 2000g, 2500g, 3000g, 3500g, 4000g, 4500g, 5000g, etc.), and 8000-15000g (e.g., 8000g, 9000g, 10000g, 11000g, 1200g, 1200g, etc.). Centrifugation times are 5-35 min (e.g., 5 min, 10 min, 15 min, 20 min, 25 min, 35 min, etc.), 40-90 min (e.g., 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, etc.) for 00g, 13000g, 15000g, etc., respectively; other specific values ​​within the above ranges can be selected, which will not be elaborated here.

[0023] Preferably, the ultracentrifugation speed in step (2) is 100,000-200,000g (e.g., 100,000g, 120,000g, 140,000g, 150,000g, 160,000g, 170,000g, 180,000g, 200,000g, etc.), and the time is 60-120min (e.g., 60min, 70min, 80min, 90min, 100min, 110min, 120min, etc.); other specific point values ​​within the above ranges can be selected, and will not be elaborated here.

[0024] The centrifugation and ultracentrifugation processes described above will affect the purity and particle size of the purslane-derived extracellular vesicles to some extent. When extraction is performed under the specific preparation conditions described above, the purslane-derived extracellular vesicles obtained will have higher purity and smaller particle size.

[0025] Preferably, the polyethylene glycol precipitation method specifically includes the following steps:

[0026] (1) Filter the juice of purslane raw material, centrifuge the filtrate and collect the supernatant;

[0027] (2) After filtering the supernatant, mix it with polyethylene glycol, let it stand, centrifuge to collect the precipitate and resuspend it;

[0028] (3) The resuspension is purified to obtain the final product.

[0029] Preferably, step (1) involves centrifugation and collection of the supernatant at least three times, with centrifugation speeds successively of 3000-7000g (e.g., 3000g, 4000g, 4500g, 5000g, 5500g, 6000g, 7000g, etc.), 7000-10000g (e.g., 7000g, 7500g, 8000g, 8500g, 9000g, 9500g, 10000g, etc.), and 10000-15000g (e.g., 10000g, 11000g, 11500g, 12000g, 12500g, 13...). Centrifugation times are 5-20 min (e.g., 5 min, 10 min, 15 min, 20 min), 15-45 min (e.g., 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min), and 40-90 min (e.g., 40 min, 45 min, 50 min, 55 min, 60 min, 70 min, 80 min, 90 min), depending on the weight range of the above values. Other specific values ​​within the above ranges can be selected, and will not be elaborated here.

[0030] Preferably, the number average molecular weight of the polyethylene glycol is 4000-8000, such as 4000, 5000, 6000, 7000, 8000, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0031] Preferably, the final concentration of polyethylene glycol after mixing in step (2) is 5-15%, such as 5%, 6%, 7%, 8%, 10%, 12%, 13%, 15%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0032] The final concentration of polyethylene glycol and the centrifugation process settings mentioned above will affect the purity and particle size of the purslane-derived extracellular vesicles to a certain extent. When the final concentration is 6-9%, the purity of the purslane-derived extracellular vesicles is significantly improved and the particle size is significantly reduced when the specific centrifugation process is used.

[0033] Preferably, the settling time is 8-16 hours, such as 8 hours, 9 hours, 10 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0034] Preferably, the centrifugation speed in step (2) is 6000-10000g (e.g., 6000g, 7000g, 7500g, 8000g, 9000g, 10000g, etc.), and the time is 10-50min (e.g., 10min, 15min, 20min, 25min, 30min, 40min, 50min); other specific point values ​​within the above ranges can be selected, and will not be elaborated here.

[0035] Preferably, the purification method includes size exclusion chromatography.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] This invention creatively extracts and purifies extracellular vesicles derived from purslane and studies their physiological effects. It was found that they can reduce the expression levels of inflammatory factors in macrophages, exerting an anti-inflammatory effect, and can be internalized by HaCaT cells. Animal experiments have demonstrated that they can alleviate and treat skin lesions in a mouse model of atopic dermatitis, reduce the spleen index in the mouse model of atopic dermatitis, and reduce the expression levels of TNF-α and IL-4 in skin tissue. Based on the in vivo and in vitro anti-inflammatory research results of this invention, those skilled in the art can prepare purslane-derived extracellular vesicles into different forms of anti-inflammatory and soothing products, especially topical (transdermal) anti-inflammatory and soothing products, such as cosmetics or pharmaceuticals, according to different needs. Attached Figure Description

[0038] Figure 1 This is a transmission electron microscope image of the purslane-derived extracellular vesicles prepared in Example 1;

[0039] Figure 2 This is a particle size distribution diagram of the purslane-derived extracellular vesicles prepared in Example 1;

[0040] Figure 3 This is a potential characterization diagram of the purslane-derived extracellular vesicles prepared in Example 1;

[0041] Figure 4 This is a standard curve for BCA protein assay;

[0042] Figure 5 This is an SDS-PAGE image of the purslane-derived extracellular vesicles prepared in Example 1;

[0043] Figure 6 This is a transmission electron microscope image of the purslane-derived extracellular vesicles prepared in Example 2;

[0044] Figure 7 This is a particle size distribution diagram of the purslane-derived extracellular vesicles prepared in Example 2;

[0045] Figure 8 This is a potential characterization diagram of the purslane-derived extracellular vesicles prepared in Example 2;

[0046] Figure 9 This is an SDS-PAGE image of the purslane-derived extracellular vesicles prepared in Example 2;

[0047] Figure 10 This is a statistical graph showing the results of CCK8 assay for detecting the toxic effects of purslane-derived extracellular vesicles on HaCaT cells (n=5).

[0048] Figure 11 This is a map showing the distribution of purslane-derived extracellular vesicles in HaCaT cells (scale bar = 25 μm);

[0049] Figure 12 This is a statistical graph showing the results of CCK8 assay for detecting the toxic effects of purslane-derived extracellular vesicles on macrophages (n=6).

[0050] Figure 13 It is a flowchart of animal experiments;

[0051] Figure 14 These are images of skin lesions on the backs of mice in each group;

[0052] Figure 15 This is a graph showing the statistical results of the skin damage scores on the backs of mice in each group;

[0053] Figure 16 These are anatomical diagrams of the spleens of mice in each group;

[0054] Figure 17 This is a graph showing the statistical results of spleen indices in each group of mice;

[0055] Figure 18 These are HE sections of skin lesions on the backs of mice in each group (scale bar = 100 μm);

[0056] Figure 19 This is a graph showing the statistical results of the effects of each drug administration group on TNF-α in the skin tissue of mice with atopic dermatitis;

[0057] Figure 20 This is a graph showing the statistical results of the effects of each drug administration group on IL-4 in the skin tissue of mice with atopic dermatitis;

[0058] Figure 21 This is a graph showing the statistical results of the expression levels of TBIL, TP, ALT, AST, CR, and UR in the serum of mice in each group;

[0059] Figure 22 The in vivo transdermal fluorescence distribution of extracellular vesicles derived from purslane (scale bar = 1000 μm). Detailed Implementation

[0060] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the examples described are merely illustrative of the invention and should not be construed as limiting the invention.

[0061] The purslane raw material involved in the following preparation examples has been identified as the dried aerial parts of the Portulacaceae plant Portulaca oleracea.

[0062] Preparation Example 1

[0063] Purslane-derived extracellular vesicles (PELNs) were extracted using ultracentrifugation.

[0064] (1) Take purslane raw material, wash it, cut it into sections, weigh it, soak it for 1 hour, and juice it according to the ratio of 150mL of pure water per 100g.

[0065] (2) Filter the juice through double-layer nylon gauze, take 1200g of juice, centrifuge at 20min and 4℃, then take the supernatant and centrifuge at 3000g, 20min and 4℃, and continue to take the supernatant and centrifuge at 10000g, 60min and 4℃.

[0066] (3) Filter the supernatant through a 0.8 μm filter membrane and centrifuge at 150,000 g for 90 min at 4 °C. Remove the supernatant and take the precipitate. Resuspend the precipitate with a small amount of supernatant to obtain purslane-derived extracellular vesicles (abbreviated as PELNs).

[0067] Test Example 1

[0068] Characterization of PELNs prepared in Example 1:

[0069] The morphology, particle size, potential, protein concentration, and protein molecular weight distribution of the PELNs extracted in Preparation Example 1 were characterized.

[0070] (1) Take PELNs solution, dilute it with an appropriate amount of pure water, add it dropwise onto a copper grid, let it dry naturally, stain with phosphotungstic acid, and observe the morphology under a transmission electron microscope. For example... Figure 1 As shown, the extracellular vesicles derived from purslane exhibit a typical teacup shape, with a distinct phospholipid bilayer. They are complete and plump, consistent with the morphology of general plant extracellular vesicles, and vary in size. Their particle size also falls within the range measured by the Malvern particle size analyzer.

[0071] (2) The particle size and potential distribution of PELNs were analyzed using a Malvern particle size analyzer, such as... Figure 2 and Figure 3As shown, the average particle size of PELNs is 112.4±1.1 nm, the PDI is 0.345±0.037, and the average potential is -1.6±0.4 mV. Their particle size falls within the typical range of 30–150 nm for plant extracellular vesicles, and the negative charge of the particles is consistent with the charged properties of plant extracellular vesicles.

[0072] (3) Prepare samples according to the kit instructions and measure their absorbance. Establish a BCA standard curve and calculate the protein content by substituting the values ​​into the BCA standard curve. Plot a linear regression between absorbance value y and mass concentration x, as shown below. Figure 4 As shown, the protein concentration in the range of 0–2.0 μg / μL showed a good linear relationship with the absorbance value. Then, the protein content in PELNs was determined by the BCA kit method to characterize the amount of PELNs used. The protein concentration of PELNs was 1.54 ± 0.18 μg / μL.

[0073] (4) Protein expression levels in PELNs were analyzed by SDS-PAGE electrophoresis, such as... Figure 5 As shown in the figure, the molecular weight of its protein is mainly distributed below 75kDa.

[0074] Preparation Example 2

[0075] Extracellular vesicles (PELNs) derived from purslane were extracted using the polyethylene glycol precipitation method.

[0076] (1) Take purslane raw material, wash it, cut it into sections, weigh it, soak it for 1 hour, and juice it according to the ratio of 150mL of pure water per 100g.

[0077] (2) Let the juice stand for 2 hours. Filter the upper layer solution with a double layer of 400-mesh nylon sieve, and filter the lower layer solution with double layers of 100, 200 and 400-mesh nylon sieves in sequence. Mix the filtered upper and lower layers of solution, and centrifuge the filtrate in three steps: 5000g, 4℃, 10min; 8000g, 4℃, 30min; 10000g, 4℃, 1h. Collect the supernatant.

[0078] (3) Filter through a 0.8 μm filter membrane, transfer the supernatant to a new centrifuge tube, and add 16%, 20%, 24%, and 30% polyethylene glycol 6000 solution to the supernatant in equal proportions to make final concentrations of 8%, 10%, 12%, and 15%, respectively. Mix thoroughly and incubate vertically at 4°C for 12 hours. Then centrifuge at 8000g, 4°C, for 30 minutes, remove the supernatant, and dissolve the precipitate thoroughly with an appropriate amount of PBS buffer to form a suspension of purslane extracellular vesicles.

[0079] The yield, purity, and particle size of four purslane extracellular vesicle samples were evaluated (yield = mass of lyophilized purslane extracellular vesicles / mass of fresh product; purity = protein concentration × reconstitution volume / mass of lyophilized purslane extracellular vesicles). The results are shown in the table below:

[0080]

[0081] Therefore, it can be concluded that the product has the best overall properties when the final concentration of polyethylene glycol is 8%.

[0082] (4) The extracellular vesicle suspension of purslane obtained with 8% PEG concentration was purified by agarose gel CL-2B filtration chromatography, specifically as follows:

[0083] Take 10 mL of gel and wash with an appropriate amount of ultrapure water to remove the 20% ethanol solvent used to preserve the gel. Add 3 times the volume of PBS buffer (pre-filtered through a 0.22 μm membrane) to homogenize the gel. Take the chromatography column, fix it on the iron stand, and wet the inside and bottom of the column with buffer, maintaining a small liquid level, ensuring there are no air bubbles at the bottom. Use a glass rod to guide the homogenate slowly and continuously pour it into the column along the inner wall, being careful not to generate air bubbles, allowing the gel to settle naturally under gravity. After complete sedimentation, slowly add PBS buffer from the top, open the column outlet, and equilibrate the two column volumes. Close the bottom outlet, slowly add the sample in a ring from the top, open the column outlet, and when the sample settles to the level of the liquid surface, slowly add about 10 mL of PBS elution buffer for separation and purification (gel column length 97 mm, column inner diameter 15 mm).

[0084] Test Example 2

[0085] Characterization of PELNs prepared in Example 2:

[0086] The morphology, particle size, potential, protein concentration, and protein molecular weight distribution of the PELNs extracted in Preparation Example 2 were characterized.

[0087] (1) Take PELNs solution, dilute it with an appropriate amount of pure water, add it dropwise onto a copper grid, let it dry naturally, stain with phosphotungstic acid, and observe the morphology under a transmission electron microscope. For example... Figure 6 As shown, the extracellular vesicles derived from purslane exhibit a typical teacup shape, with a distinct phospholipid bilayer. They are complete and plump, consistent with the morphology of general plant extracellular vesicles, and vary in size. Their particle size also falls within the range measured by the Malvern particle size analyzer.

[0088] (2) The particle size and potential distribution of PELNs were analyzed using a Malvern particle size analyzer, such as... Figure 7 and Figure 8As shown, the average particle size of PELNs is 115.6±7.0 nm, the PDI is 0.436±0.036, and the average potential is -1.46±0.11 mV. Their particle size falls within the typical range of 30–150 nm for plant extracellular vesicles, and the negative charge of the particles is consistent with the charged properties of plant extracellular vesicles.

[0089] (3) Samples were prepared according to the kit instructions and their absorbance was measured. A BCA standard curve was established and the protein content was calculated by substituting the samples into the BCA standard curve. The results showed that the protein concentration of PELNs was 2.066±0.024μg / μL.

[0090] (4) Protein expression levels in PELNs were analyzed by SDS-PAGE electrophoresis, such as... Figure 9 As shown in the figure, the molecular weight of its protein is mainly distributed below 75kDa.

[0091] Test Example 3

[0092] Cytotoxic and uptake effects of PELNs on HaCaT cells:

[0093] (1) Detection of the effect of PELNs on HaCaT cytotoxicity by CCK-8 assay: HaCaT cells were cytotoxic at a density of 1×10⁻⁶ cells / year. 4 pcs / cm 2 Cells were seeded in 96-well plates. Different concentrations of PELNs prepared in Preparation Example 1 (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, and 800 μg / mL) were co-cultured with HaCaT cells. After 24 h of culture, the absorbance of each group of cells was measured at 450 nm, and the cell viability (%) was calculated. Figure 10 As shown, the results indicated that cell viability was greater than 100% when the protein concentration of PELNs ranged from 50 to 800 μg / mL, indicating that PELNs did not produce cytotoxicity in HaCaT cells and had good biocompatibility. At concentrations greater than 600 μg / mL, cell viability decreased compared to 400 μg / mL.

[0094] (2) PELNs were labeled with the cell membrane dye Dio, and HaCaT cell nuclei were labeled with DAPI dye. The distribution of intracellular PELNs was observed under a laser confocal microscope. Figure 11 As shown, the green fluorescence carried by PELNs can be taken up by HaCaT cells and exists in the cell membrane and cytoplasm, while no green fluorescence was observed in the cell nucleus. The results indicate that PELNs can be internalized and absorbed by HaCaT cells.

[0095] Test Example 4

[0096] Cytotoxic and anti-inflammatory effects of PELNs on RAW264.7 cells:

[0097] (1) CCK-8 assay to detect the effect of PELNs on the cytotoxicity of RAW264.7 cells: RAW264.7 cells were cytotoxic at a cell density of 1×10⁻⁶. 4 pcs / cm 2 The cells were seeded in 96-well plates. Different concentrations of PELNs prepared in Preparation Example 1 (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, and 800 μg / mL) were co-cultured with RAW264.7 cells. The absorbance of each group of cells was measured at 450 nm after 24 hours of culture. Figure 12 As shown, the results indicate that the selected concentrations of PELNs did not inhibit RAW264.7 cells, and the survival rate of each group was greater than 100%, indicating that PELNs did not produce cytotoxicity to RAW264.7 cells and had good biosafety.

[0098] (2) Evaluation of in vitro anti-inflammatory effect: RAW264.7 cells in logarithmic growth phase with good morphology were selected and administered at a concentration of 2×10⁻⁶ cells / cells. 5 pcs / cm 2 Cells were seeded at high density in 24-well plates and incubated for 24 h. A blank control group, an LPS-induced stimulation group, a positive control group (dexamethasone, dextromethorphan, 500 nM), and sample groups (protein concentrations of 50 μg / mL and 200 μg / mL) were set up and incubated at 37°C in a 5% CO2 incubator for 24 h. Except for the blank control group, LPS was added to each well and incubated for 24 h. Cell supernatants were analyzed according to the instructions of the ELISA kit (Thermo Fisher Scientific (China) Co., Ltd.) and the nitric oxide assay kit (Thermo Fisher Scientific (China) Co., Ltd.). The levels of TNF-α, IL-6, and NO in the collected cell supernatants were measured, and the inhibition rate of inflammatory factors in each group relative to the LPS-induced stimulation group was calculated. The results are shown in the table below.

[0099]

[0100] The data in the table above show that PELNs have good anti-inflammatory effects at the cellular level.

[0101] Test Example 5

[0102] The therapeutic effects of PELNs on mice with atopic dermatitis:

[0103] (1) Modeling method: 2,4-Dinitrochlorobenzene (DNCB) was dissolved in a mixture of acetone and olive oil (4:1 volume ratio) to prepare 0.5% and 1% DNCB solutions. One day before model establishment, a 2cm x 2cm area of ​​hair was removed from the back of mice. On days 1, 2, 3, and 4, 200μL of 0.5% DNCB solution was evenly applied to the back to sensitize the mice. On days 5 and 7, 100μL of 1% DNCB solution was applied to the back to stimulate sensitization. This process was repeated every 2 days for a total of 4 times to establish a mouse atopic dermatitis model. 48 hours after the 4th stimulation, the back skin of the mice was harvested. (See schematic diagram below.) Figure 13 As shown.

[0104] (2) Grouping and Administration: Twenty-four healthy six-week-old BALB / c mice were used, divided into six groups: a control group, a model group, a positive control group (Dex), and a purslane-derived extracellular vesicle group (PELNs). Administration began on day 8. Mice in the control and model groups were treated with 200 μL of ultrapure water on their backs. Mice in the positive control group were treated with a pea-sized amount of dexamethasone acetate ointment on their backs. Mice in the purslane extracellular vesicle group were treated with 200 μL of the PELNs prepared in Example 1. Administration was once daily for 14 consecutive days. (See schematic diagram below.) Figure 13 As shown.

[0105] (3) Score of skin lesions on the back: Starting from day 7 of the experiment, the severity of skin lesions in each mouse was assessed every 3 days. Based on the Area and Severity Index of Dermatitis (EASI), a score standard for the severity of mouse skin lesions was established, and a total of 5 assessments were conducted. Finally, the cumulative total score changes of each group over 5 days were plotted, and the condition of the skin lesions on the back of the mice was photographed and recorded on days 7, 9, 11, 13, 15, 17, 19, and 21 of the experiment.

[0106] The scoring criteria for the severity of skin lesions in mice are as follows:

[0107] (3.1) Erythema / darkening of color

[0108] 0 points: No erythema observed; 1 point: A few erythema spots, light in color; 2 points: Patchy erythema, bright red in color; 3 points: Widespread erythema, bright red or purplish-red in color, with elevated skin temperature.

[0109] (3.2) Exudation / Scabbing

[0110] 0 points: No exudation; 1 point: Less exudation, accompanied by punctate crusts; 2 points: Exudation accompanied by patchy crusts; 3 points: More exudation, with obvious serous fluid and crusts.

[0111] (3.3) Drying / Desquamation

[0112] 0 points: No dryness / scaling observed; 1 point: Mild dryness / scaling of the epidermis; 2 points: Moderate dryness / scaling of the epidermis; 3 points: Severe dryness / scaling of the epidermis.

[0113] (3.4) Exfoliation / Erosion

[0114] 0 points: No skin peeling / erosion observed; 1 point: Spotted peeling / erosion visible on the skin lesion; 2 points: Skin peeling / erosion is relatively obvious, appearing as small scattered patches; 3 points: Skin peeling / erosion is obvious, distributed in large areas.

[0115] (3.5) Skin thickening

[0116] 0 points: No obvious thickening of the skin; 1 point: Partial thickening of the skin; 2 points: Significant thickening of the skin; 3 points: Large-area thickening of the skin.

[0117] The effects of PELNs on the dorsal skin lesions and dorsal dermatitis scores in mice with DNCB-induced atopic dermatitis were observed. Images of the dorsal skin lesions in each group are shown below. Figure 14 As shown in the figure, the skin lesion scoring charts on the backs of mice in each group are as follows. Figure 15 As shown, in the first week of the experiment, DNCB induced dry skin, erythema, epidermal peeling, and thickening in mice, indicating that DNCB successfully induced atopic dermatitis in mice. Two weeks after administration, the skin of mice in the positive drug group was significantly thinner, and no erythema or epidermal peeling was observed. The PELNs group showed varying degrees of improvement in skin lesions compared to the model group. This indicates that topical application of PELNs to mice can effectively improve the symptoms of atopic dermatitis.

[0118] (4) Spleen index:

[0119] Weigh the spleen and body weight of each group of mice. Calculate the spleen index for each group using the formula: Spleen Index = Spleen weight (g) / Mouse body weight (g) × 100.

[0120] Anatomical diagrams of the spleen and statistical results of spleen index in each group of mice are shown below. Figure 16 and Figure 17 As shown in the figure (compared to the Control group, ###p<0.001, ####p<0.0001; compared to the Model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=6), it can be seen that after continuous DNCB stimulation, the spleen index of mice in the model group increased significantly, showing a significant difference compared to the normal group (p<0.0001). After treatment with the positive control drug dexamethasone cream, the spleen index of mice in the model group was significantly decreased compared to the model group (p<0.001). The spleen index of mice in the PELNs group was significantly decreased compared to the model group (p<0.0001).

[0121] (5) Observation of HE staining of back skin pathological sections:

[0122] After drug administration, skin samples were cut from the lesions, fixed with perforated aluminum foil, and then immersed in formalin for fixation. After dehydration, the samples were embedded in paraffin to prepare 3 μm thick paraffin sections, stained with hematoxylin and eosin (HE), and mounted. The histopathological changes of the HE-stained sections in each group were observed under a microscope.

[0123] The results are as follows Figure 18 As shown in the HE results, the epidermis of the normal group mice was smooth and without inflammatory infiltration. Compared with the normal group, the model group showed significant thickening of the epidermis and dermis, with significant inflammatory cell infiltration in the dermis. Compared with the model group, the epidermis and dermis of the positive drug group and the PELNs group mice were significantly thinner, accompanied by mild inflammatory cell infiltration.

[0124] (6) ELISA detection of TNF-α and IL-4 expression levels in skin tissue:

[0125] On day 21 of the experiment, the levels of TNF-α and IL-4 in the skin tissue of mice in each group were detected by ELISA kit.

[0126] The results are as follows Figure 19 , Figure 20 As shown in the figure (compared with the Control group, ###p<0.001, ####p<0.0001; compared with the Model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=6), it can be seen from the figure that compared with the normal group, the levels of TNF-α and IL-4 in the skin tissue of mice in the model group were significantly increased (p<0.0001); while compared with the model group, all treatment groups showed a decrease. Among them, except for the positive drug group, applying PELNs can significantly reduce the content of TNF-α in the skin tissue (p<0.01), and applying PELNs can also significantly reduce IL-4 (p<0.001).

[0127] (7) Serum biochemical markers determination:

[0128] 100 μL of serum was collected from each group of mice, and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), creatinine (CR), urea (UR), and total protein concentration (TP) in the serum were detected using a fully automated biochemical analyzer. These serum biochemical indicators were used to evaluate the liver and kidney function of the mice after drug administration.

[0129] The results are as follows Figure 21 As shown, among serum biochemical indicators, TBIL and TP levels reflect bilirubin metabolism and liver synthesis function. Elevated TBIL and TP levels suggest potential liver damage. ALT and AST, mainly found within hepatocytes, are sensitive indicators for evaluating liver function; elevated levels of both indicate hepatotoxicity. CR and UR are important indicators for assessing kidney function. Except for the positive control group, no significant pathological changes were observed in the PELNs group compared to the normal group. This suggests that hepatotoxicity and nephrotoxicity should be considered when using dexamethasone cream. Furthermore, the study demonstrates the good biosafety of topical application of PELNs.

[0130] (8) Evaluation of in vivo transdermal efficacy:

[0131] Normal mice were treated with Dio-labeled PELNs on purslane extracellular vesicles under light-protected conditions. At 10 min, 30 min, and 1 h, the effective transdermal skin was dissected, fixed in formalin for 2 h, embedded in OCT, and rapidly frozen in liquid nitrogen. The skin was then longitudinally sectioned using a cryostat and quickly observed under a fluorescence microscope. The results are shown below. Figure 22 As shown in the figure, within 10 minutes to 1 hour after applying Dio-labeled PELNs to the surface of mice, the transdermal amount and depth increased over time.

[0132] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product of this invention, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0133] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0134] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. Application of purslane-derived extracellular vesicles in the preparation of drugs for the prevention or treatment of atopic dermatitis; The methods for preparing the purslane-derived extracellular vesicles include ultracentrifugation or polyethylene glycol precipitation. The ultracentrifugation method specifically includes the following steps: (1) Filter the juice of purslane raw material, centrifuge the filtrate and collect the supernatant; (2) After filtering the supernatant, centrifuge it at ultraspeed, collect the precipitate and resuspend it to obtain the product; The polyethylene glycol precipitation method specifically includes the following steps: (1) Filter the juice of purslane raw material, centrifuge the filtrate and collect the supernatant; (2) After filtering the supernatant, mix it with polyethylene glycol, let it stand for 8-16 h, centrifuge to collect the precipitate and resuspend it; (3) The resuspension was purified by size exclusion chromatography to obtain the final product; In the polyethylene glycol precipitation method, the centrifugation and collection of supernatant in step (1) includes at least three centrifugations, with centrifugation speeds of 3000-7000 g, 7000-10000 g, and 10000-15000 g, and centrifugation times of 5-20 min, 15-45 min, and 40-90 min, respectively. In the polyethylene glycol precipitation method, the final concentration of polyethylene glycol after mixing in step (2) is 6-9%; The number-average molecular weight of the polyethylene glycol used in the polyethylene glycol precipitation method is 4000-8000; In the polyethylene glycol precipitation method, the centrifugation speed in step (2) is 6000-10000 g and the time is 10-50 min.

2. The application according to claim 1, characterized in that, The drug is in a transdermal dosage form.

3. The application according to claim 2, characterized in that, The transdermal dosage forms include: liniments, ointments, lotions, pastes, gels, coatings, films, patches, and microneedles.

4. The application according to claim 1, characterized in that, The drug also contains excipients, which include any one or a combination of at least two of the following: diluents, dispersants, binders, fillers, thickeners, lubricants, pH adjusters, flavor masking agents, colorants, antioxidants, or antibacterial agents.

5. The application according to claim 1, characterized in that, In the ultracentrifugation method, step (1) involves centrifuging and collecting the supernatant at least three times, with centrifugation speeds of 800-1800 g, 2000-5000 g, and 8000-15000 g, and centrifugation times of 5-35 min, 5-35 min, and 40-90 min, respectively.

6. The application according to claim 1, characterized in that, In the ultracentrifugation method, step (2) involves ultracentrifugation at a speed of 100,000-200,000 g for a time of 60-120 min.

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