CN118370779A - Use of Bidens pilosa exosome in treating atopic dermatitis - Google Patents

Abstract

The present application relates to the technical field of application of Bidens pilosa exosomes, and specifically discloses a use of Bidens pilosa exosomes for treating specific dermatitis. The Bidens pilosa exosomes are secreted by Bidens pilosa tissue, and the Bidens pilosa tissue is selected from any one or more of Bidens pilosa leaves, stems, and roots. The Bidens pilosa exosomes of the present application have a significantly excellent effect in treating specific dermatitis.

Description

Use of Bidens pilosa exosomes in the treatment of specific dermatitis

Technical Field

The present application relates to the technical field of application of Bidens pilosa exosomes, and more specifically, it relates to a use of Bidens pilosa exosomes in the treatment of specific dermatitis.

Background technique

Atopic dermatitis (AD) is a skin disease with a genetic tendency that is common in children and is characterized by severe itching and a high recurrence rate. The causes of atopic dermatitis are relatively complex and are largely related to environmental factors, autoimmune dysfunction, and genetics. The main clinical manifestations of the disease are acute and chronic eczematoid skin lesions, dry skin, and obvious itching, which have a serious impact on the patient's normal life. At present, topical medications are often used for clinical treatment, among which glucocorticoid drugs are more widely used, but there is a deficiency of easy recurrence after drug withdrawal; in addition, there are drugs such as topical corticosteroids and immunosuppressants, which can relieve the symptoms of atopic dermatitis, but long-term use will bring side effects such as rash, tingling, and atrophy on the local skin. Therefore, it is necessary to develop a treatment for atopic dermatitis that has no side effects and is easy to relapse after drug withdrawal.

In view of its side effects, plant extracts are expected to achieve the treatment of atopic dermatitis with no or low side effects due to their bio-friendly characteristics. Among them, Bidens pilosa L. is routinely used in food and medicine, and it has no obvious side effects. Many parts of Bidens pilosa, such as flowers and stems, can be used to treat various diseases and have multiple medicinal functions, such as antipyretics and analgesics, as well as the treatment of gastrointestinal bleeding and eczema. Pharmacological studies have shown that Bidens pilosa extracts have a wide range of biological activities, including antioxidant, anti-cancer, anti-diabetic, anti-inflammatory, antimicrobial and immunomodulatory activities.

Therefore, it is necessary to develop the therapeutic use of Bidens pilosa exosomes for the treatment of atopic dermatitis.

Summary of the invention

In response to the above problems, the present application provides a method for treating specific dermatitis using Bidens pilosa exosomes.

The use of Bidens pilosa exosomes provided in this application for treating atopic dermatitis adopts the following technical solution:

The use of Bidens pilosa exosomes for treating atopic dermatitis, wherein the Bidens pilosa exosomes are secreted by Bidens pilosa tissue, and the Bidens pilosa tissue is selected from any one or more of Bidens pilosa leaves, stems, and roots.

This application shows through cell experiments that Bidens pilosa exosomes can reduce the expression of chemotactic cytokines (TNFα, CCL22, IL-6 and RANTES) in cells, and the results prove the effect of Bidens pilosa exosomes in alleviating atopic dermatitis. In addition, in animal experiments, the skin lesion scores and the thickness of ear swelling were measured. The results showed that after the Bidens pilosa exosomes were applied to mice with atopic dermatitis, the back lesion area of the mice with atopic dermatitis was reduced, the wound healing was better, the skin lesion score was significantly lower than that of mice not administered with drugs, and the thickness of ear swelling was significantly reduced; this result proves that Bidens pilosa exosomes can significantly alleviate the symptoms of atopic dermatitis. In addition, the results of H&E staining found that the back lesion tissue of mice with atopic dermatitis showed incomplete keratinization or hyperkeratinization, the epidermis was significantly thickened, and a large number of inflammatory cells were infiltrated, and the lesion tissue was significantly edematous; and the above symptoms of mice administered with Bidens pilosa exosomes were significantly improved, proving the improvement effect of Bidens pilosa exosomes on mouse atopic dermatitis.

Based on the above experimental results, it is fully demonstrated that Bidens pilosa L. exosomes can be used to treat atopic dermatitis.

Optionally, the method for preparing Bidens pilosa exosomes comprises the following steps:

The Bidens pilosa tissue was taken, and the buffer solution was added and then crushed, and the supernatant was collected after solid-liquid separation, and the supernatant was microfiltered to obtain the Bidens pilosa pretreatment sample;

The Bidens pilosa pretreated sample is subjected to ultracentrifugation to obtain Bidens pilosa exosomes.

Optionally, the ultracentrifugation specifically comprises the following steps:

The Bidens pilosa pretreated sample was centrifuged at 1500-2500g for 25-35min; the supernatant was centrifuged at 8000-12000g for 40-50min and then microfiltered; the filtrate was centrifuged at 80000-120000g for 65-75min; the precipitate was resuspended in buffer and centrifuged at 80000-120000g for 65-75min to obtain Bidens pilosa exosomes.

Optionally, the average particle size of Bidens pilosa exosomes is 79.0 nm, and the concentration is (1.2-1.4)×10 ⁹ /ml.

Optionally, the solid-liquid separation after the Bidens pilosa tissue is crushed specifically comprises the following steps:

The Bidens pilosa tissue is crushed and filtered, and the supernatant is centrifuged at 8000-12000 rpm for 15-25 minutes.

Optionally, the Bidens pilosa exosomes are used to prepare a composition for treating atopic dermatitis, and the components of the composition further include any one or more of excipients, dispersants, food additives, pharmaceutical additives and probiotics.

Optionally, the Bidens pilosa exosomes are used to prepare a drug for treating atopic dermatitis or an auxiliary treatment drug.

Optionally, the dosage forms of the drug and auxiliary therapeutic drug are selected from any one or more of microneedles, external liquid preparations, external solid preparations, external semisolid preparations, plasters, patches and external gas preparations.

In summary, this application has the following beneficial effects:

The present application provides a new use of Bidens pilosa L. exosomes in the treatment of atopic dermatitis, which can significantly improve the skin symptoms of atopic dermatitis model mice and has an excellent effect in treating atopic dermatitis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the process of cell experiment;

Fig. 2 is a schematic diagram of the process of animal experiment;

FIG3 is an electron micrograph of Bidens pilosa exosomes of Example 2;

FIG4 is a particle size and concentration analysis diagram of Bidens pilosa exosomes of Example 2;

FIG5 is a graph showing the cytokine expression results of different treatment groups;

FIG6 is a photograph of the back and ears of mice in different treatment groups;

FIG7 shows the skin lesion scores and ear swelling thickness of mice in different treatment groups;

FIG8 is a diagram showing the results of H&E staining of the ears and backs of mice in different treatment groups.

Detailed ways

The present application is further described in detail below in conjunction with the accompanying drawings and examples. It is particularly noted that if no specific conditions are specified in the following examples, the experiments are carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can all be sourced from common commercial sources.

Detection method

1. Transmission electron microscopy observation of Bidens pilosa exosomes

The specific experimental steps are as follows: take out 10 μL of the obtained Bidens pilosa exosomes and drop them on a copper grid to precipitate for 1 min, and use filter paper to absorb the floating liquid; add 10 μL of uranyl acetate on a copper grid to precipitate for 1 min, and use filter paper to absorb the floating liquid; then air-dry at room temperature for 5 min, and then place the processed samples on a transmission electron microscope, and perform electron microscopy imaging at 100 kV.

2. Particle size analysis of Bidens pilosa exosomes

The specific method is as follows: take out 5 μL of the obtained Bidens pilosa exosomes and dilute them to 30 μL; first use the standard product to test the instrument performance, and after passing the test, load the Bidens pilosa exosome sample for testing, and the instrument detects the particle size and concentration information of the exosomes.

3. Determination of protein concentration of Bidens pilosa exosomes

The specific method is as follows: quickly melt Bidens pilosa exosomes at 37°C, quickly add 5× RIPA lysis buffer, mix well, lyse on ice for 30 minutes, and mix well to obtain the sample to be tested. Prepare the standard sample for protein concentration measurement by BCA method, take 5μL sample and add it to the BCA mixed solution, mix well; then incubate each sample at 37°C for 30 minutes, and detect the absorbance of the standard and the sample to be tested on the microplate reader at OD _562nm and record it to obtain the standard curve. According to the absorbance of the sample to be tested and the standard curve, calculate the protein concentration of the sample to be tested.

4. Cell experiments

BPNP refers to Bidens pilosa exosomes, TNFα refers to tumor necrosis factor α, IFNγ refers to interferon-γ, CCL22 is also known as macrophage-derived chemokine, and RANTES is also known as chemokine CCL5.

The specific experimental process is shown in Figure 1. HACAT cells (purchased from CyBiotech Co., Ltd.) were seeded in a twelve-well plate at a density of 1×10 ⁵ cells/mL and divided into three groups, namely the control group, the M group and the BPNP group, with three wells of cells in each group, and the volume of the culture medium (high-glucose DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, high-glucose DMEM culture medium and penicillin-streptomycin purchased from Shanghai Peiyuan Biotechnology Co., Ltd., and fetal bovine serum purchased from Yikesai Biotechnology Co., Ltd.) was 1 mL. After overnight culture, 10 μg BPNP was given to the cells in the BPNP group, and 100 μL PBS buffer was given to the other two groups. After 1 hour of culture, 1 μL of TNFα and IFNγ were given to the M group and the BPNP group, each with a concentration of 10 μg/mL. After continuing to culture for 23 hours, RNA was extracted, reverse transcribed into cDNA, and the expression changes of chemokines (TNFα, CCL22, IL-6 and RANTES) in each group were determined by RT-qPCR.

5. Animal experiments

DNFB refers to dinitrofluorobenzene, DXM refers to dexamethasone, and BPNP refers to Bidens pilosa exosomes.

Referring to Fig. 2, 8-week-old BALB/c mice (purchased from the Animal Center of Xiamen University) were divided into four groups, namely, control group, DNFB group, DNFB+DXM group and DNFB+BPNP group, with 5 mice in each group. Except for the control group, the other three groups were sensitized by evenly applying 1.0% DNFB (w/v, the solvent was acetone/olive oil with a volume ratio of 3:1) 100 μL on the depilatory area of the back of the mice on the first day of the experiment, and the sensitization was strengthened on the third day. From the second week to the fourth week, 0.5% DNFB solution (w/v, the solvent was acetone/olive oil with a volume ratio of 3:1) was evenly applied on the back of the mice and the front and back of the right ear on the 7th, 10th, 14th, 17th, 21st, 24th, and 28th days of the experiment; wherein, 50 μL was applied on the back and 20 μL was applied on the right ear to induce dermatitis. The control group applied an equal volume of acetone olive oil matrix (acetone: olive oil = 3:1, v/v) on the back hair removal area, and the treatment time node and dosage were the same as the other three groups. Starting from the 7th day, the control group and the DNFB group were gavaged with blank solvent (blank solvent composition: ethanol: saline = 1:99, v/v) at 0.1mL/10g body weight, 3 times a week; the DNFB+DXM group was injected with 0.1mg/kg dexamethasone (solvent is blank solvent, specific composition is ethanol: saline = 1:99, v/v) at 0.1mL/10g body weight, 3 times a week; the DNFB+BPNP group was gavaged with 200μL BPNP, and the concentration of Bidens pilosa exosomes in BPNP was 0.5μg/μL (solvent is blank solvent, specific composition is ethanol: saline = 1:99, v/v), 3 times a week.

Semi-quantitative scoring of the skin lesions and ear swelling thickness of mice are important indicators of atopic dermatitis. Therefore, the following analysis was performed on the mice.

5.1 Skin lesion analysis

Semi-quantitative scoring was performed on the skin lesions on the back of mice on days 1, 3, 7, 10, 14, 17, 21, 24, and 29 of modeling.

The evaluation was based on the clinical manifestation criteria, as follows: 0 point: no damage; 1 point: mild damage; 2 points: moderate damage; 3 points and above: severe damage. The damage was evaluated based on the following four clinical symptoms: (1) erythema; (2) edema/papule; (3) epidermal peeling/scratching; and (4) dryness/crusting.

5.2. Determination of ear swelling thickness

On the 3rd, 10th, 17th, 24th and 29th days of the modeling experiment, the thickness of the right and left ears of the mice was precisely measured using an electronic micrometer, and the thickness difference (μm) was used to reflect ear swelling.

5.3 H&E staining

The specific experimental process is as follows:

On the 29th day of the experiment, the mice were killed and their skin and ear tissue specimens were obtained.

1. Dehydration and wax dipping

Before embedding, the tissue was washed with running water overnight to remove residual fixative, and then dehydrated, transparentized and wax-impregnated according to the procedures shown in Table 1.

Table 1 Dehydration and wax dipping procedures

2. Dyeing

(1) Preparation of paraffin sections: set the section thickness to 5 μm and slice the sections. After cutting, the sections were rinsed in 30 Vol.% ethanol solution, then spread in 48°C warm water, and baked in a 60°C constant temperature oven for 2 h.

(2) Dewax and rehydrate the sections according to Table 2.

Table 2 Dewaxing and rehydration procedures

(3) Stain with hematoxylin solution for 5 min and wash quickly with tap water;

(4) 0.5 wt% hydrochloric acid ethanol separation for 1-2 seconds, followed by quick washing with distilled water;

(5) 1% ammonia solution to turn blue: Immerse the slices in 1% ammonia solution for a few seconds or until the tissue turns blue, then wash with tap water for 45 seconds and examine the degree of cell nuclear color separation under a microscope;

(6) 1% eosin for 15 seconds, quick wash with distilled water;

(7) Rapid washing with 80%, 90%, and 95% ethanol solutions, 15 seconds at each level. Monitor the color contrast between the cell nucleus and cytoplasm under a microscope;

(8) Wash twice with 100% ethanol, 2 min each time;

(9) Wash twice with xylene, 2 min each time, and seal with neutral gum;

(10) Take photos for archiving, observe the staining results, and calculate the skin epidermal thickness.

7. Statistical analysis

SPSS software was used for statistical analysis. The measurement data were expressed as mean ± standard deviation (x ± s). The t test was used, and one-way analysis of variance was used for comparison among the groups. P < 0.05 indicated a significant difference.

Example

After experimental verification, this application confirms that Bidens pilosa exosomes have the effect of treating atopic dermatitis. As a plant, Bidens pilosa generally secretes exosomes (also called extracellular vesicles) from various tissues; Bidens pilosa tissues that can secrete exosomes can be Bidens pilosa leaf parts, Bidens pilosa stems, including rhizomes, and Bidens pilosa roots. Therefore, in this application, Bidens pilosa exosomes can be extracted from any one or more of the leaves, stems and roots of Bidens pilosa. For example, Bidens pilosa rhizomes are used as raw materials to obtain Bidens pilosa exosomes; for example, Bidens pilosa stems and leaves are used as raw materials to obtain Bidens pilosa exosomes.

The preparation method of Bidens pilosa exosomes comprises the following steps:

Taking Bidens pilosa tissue, adding buffer solution and then crushing, collecting liquid after solid-liquid separation, and obtaining Bidens pilosa pretreated sample;

The Bidens pilosa pretreated sample is subjected to ultracentrifugation to obtain Bidens pilosa exosomes.

In the above method, the buffer can be PBS buffer, and the buffer is added so that the exosomes are distributed into the buffer after crushing, and the exosomes and tissue residues are separated by solid-liquid separation in the later stage. The method for achieving solid-liquid separation can be any one or more combinations of centrifugation, filtration, suction filtration, and microfiltration, which can separate Bidens pilosa exosomes and Bidens pilosa tissue residues. For example, the purpose of solid-liquid separation is achieved by the combination of "filtration + centrifugation + microfiltration". Further, filter with gauze to remove large particles of tissue; further centrifuge, specifically centrifuge at 8000-12000rpm for 15-25min; finally, remove small particles of solid impurities by microfiltration.

On the basis of solid-liquid separation, the exosomes of Herba Lycopodii were further separated from the liquid by ultracentrifugation.

Wherein, ultracentrifugation specifically comprises the following steps:

The Bidens pilosa pretreated sample was centrifuged at 1500-2500g for 25-35min; the supernatant was centrifuged at 8000-12000g for 40-50min and then microfiltered; the filtrate was centrifuged at 80000-120000g for 65-75min; the precipitate was resuspended in buffer and centrifuged at 80000-120000g for 65-75min to obtain Bidens pilosa exosomes.

Based on its therapeutic use for atopic dermatitis, Bidens pilosa exosomes can be used to prepare a composition for treating atopic dermatitis, and Bidens pilosa exosomes serve as the main drug component to achieve the therapeutic effect.

The components of the composition further include any one or more of excipients, dispersants, food additives, pharmaceutical additives and probiotics.

Among them, excipients can also be called auxiliary materials. Excipients can be the matrix part in semi-solid preparations (such as ointments, creams, etc.); excipients can be preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, solvents, solubilizers, osmotic pressure regulators and colorants in liquid preparations. The general requirements for excipients are stable properties, no incompatibility with the main drug, no side effects, no effect on the efficacy, not easy to deform, crack, mildew, or be eaten by insects at room temperature, harmless to the human body, no physiological effects, no chemical or physical effects with the main drug, and no effect on the content determination of the main drug.

Based on its therapeutic use for atopic dermatitis, Bidens pilosa exosomes can be used to prepare drugs or auxiliary therapeutic drugs for the treatment of atopic dermatitis.

The dosage forms of the drug and the auxiliary therapeutic drug are selected from any one or more of microinjections, external liquid preparations, external solid preparations, external semisolid preparations, plasters, patches and external gas preparations.

Example 1

The Bidens pilosa exosomes were prepared as follows:

Take the tender stems and leaves of Bidens pilosa, chop them (1mm×1mm), soak 10g of them in PBS buffer, then filter them with gauze, and centrifuge the liquid under the following conditions: centrifuge at 8000rpm for 25min, and microfilter the liquid (0.45μm) to obtain the Bidens pilosa pretreated sample.

The Bidens pilosa pretreated sample was subjected to ultracentrifugation to obtain Bidens pilosa exosomes. The steps of ultracentrifugation are as follows: the sample was centrifuged at 1500×g and 4°C for 35min. Carefully transfer the supernatant to a new centrifuge tube and centrifuge again at 8000×g and 4°C for 50min to remove larger vesicles. Take the supernatant and filter it through a 0.45μm filter membrane to collect the filtrate. Transfer the filtrate to a new centrifuge tube, select an ultraspeed rotor, and centrifuge at 4°C and 80000×g for 75min. Then remove the supernatant, resuspend with 10mL of pre-cooled 1×PBS, select an ultraspeed rotor, and ultracentrifuge again at 4°C and 80000×g for 75min. Then remove the supernatant and resuspend with 150μL of pre-cooled 1×PBS to obtain.

Example 2

The Bidens pilosa exosomes were prepared as follows:

Take the tender stems and roots of Bidens pilosa, clean them, chop them into pieces (1mm×1mm), soak 10g of them in PBS buffer, filter them with gauze, and centrifuge the liquid under the following conditions: centrifuge at 10000rpm for 20min, and microfilter the liquid (0.45μm) to obtain the Bidens pilosa pretreated sample.

The Bidens pilosa pretreated sample was subjected to ultracentrifugation to obtain Bidens pilosa exosomes. The steps of ultracentrifugation are as follows: the sample was centrifuged at 2000×g and 4°C for 30min. Carefully transfer the supernatant to a new centrifuge tube and centrifuge again at 10000×g and 4°C for 45min to remove larger vesicles. Take the supernatant and filter it through a 0.45μm filter membrane to collect the filtrate. Transfer the filtrate to a new centrifuge tube, select an ultraspeed rotor, and centrifuge at 4°C and 100000×g for 70min. Then remove the supernatant, resuspend with 10mL pre-cooled 1×PBS, select an ultraspeed rotor, and ultracentrifuge again at 4°C and 100000×g for 70min. Then remove the supernatant and resuspend with 150μL pre-cooled 1×PBS to obtain.

Example 3

The Bidens pilosa exosomes were prepared as follows:

Take the tender stems and roots of Bidens pilosa, clean them, chop them into pieces (1mm×1mm), soak 10g of them in PBS buffer, filter them with gauze, and centrifuge the liquid under the following conditions: centrifuge at 12000rpm for 15min, and microfilter the liquid (0.45μm) to obtain the Bidens pilosa pretreated sample.

The Bidens pilosa pretreated sample was subjected to ultracentrifugation to obtain Bidens pilosa exosomes. The steps of ultracentrifugation are as follows: the sample was centrifuged at 2500×g and 4°C for 25min. Carefully transfer the supernatant to a new centrifuge tube and centrifuge again at 12000×g and 4°C for 40min to remove larger vesicles. Take the supernatant and filter it through a 0.45μm filter membrane to collect the filtrate. Transfer the filtrate to a new centrifuge tube, select an ultraspeed rotor, and centrifuge at 4°C and 120000×g for 65min. Then remove the supernatant, resuspend with 10mL of pre-cooled 1×PBS, select an ultraspeed rotor, and ultracentrifuge again at 4°C and 120000×g for 65min. Then remove the supernatant and resuspend with 150μL of pre-cooled 1×PBS to obtain.

Test results

1. Transmission electron microscopy observation of Bidens pilosa exosomes: The Bidens pilosa exosomes of Example 2 were used as samples for the experiment, and the results are shown in Figure 3. Figure 3 shows that Bidens pilosa exosomes were obtained by this method, and the electron microscopic morphology of the Bidens pilosa exosomes was a "cup-and-disc" shape, and the particle size was about 79.0 nm.

2. Particle size analysis of Bidens pilosa exosomes: The Bidens pilosa exosomes of Example 2 were used as samples for the experiment, and the results are shown in Figure 4. Figure 4 shows that the average particle size of the exosomes is 79.0 nm, and the concentration of the exosomes is 1.31×10 ⁹ /ml; the particle size and concentration of the exosomes belong to the category of exosomes, indicating that the exosomes obtained are Bidens pilosa exosomes.

3. The Bidens pilosa exosomes of Example 2 were used as samples to measure the protein concentration of Bidens pilosa exosomes. The protein concentration was 16.57 μg/μL. Subsequent cell experiments were performed based on the concentration results.

4. Cell experiment:

The BPNP group used the Bidens pilosa exosomes of Example 2 as samples for experiments, and measured the expression of TNFα, CCL22, IL-6 and RANTES cytokines. The specific results are shown in Figure 5: The expression of TNFα, CCL22, IL-6 and RANTES in the M group was upregulated, while the BPNP group showed improvement compared with the M group.

Atopic dermatitis is related to the expression of chemokines, and the expression of chemokines in the atopic dermatitis cell model is higher than that in normal cells. Therefore, the above results show that Bidens pilosa exosomes have the effect of alleviating atopic dermatitis.

5. Animal experiments

In the following experiments, the BPNP group was tested using the Bidens pilosa exosomes of Example 2 as samples.

5.1. Photos of the back and ears

Photos of the back and ears of mice were taken on the 29th day, as shown in Figure 6: The back and ear injuries of the DNFB group were severe, while the back and ear injuries of the DNFB+DXM group and the DNFB+BPNP group were improved compared with the DNFB group. This result shows that Bidens pilosa exosomes have a significant effect in improving the symptoms of atopic dermatitis.

5.2. Skin lesion score and ear swelling thickness

As shown in Figure 7, on the 29th day, the skin lesion score and ear swelling thickness of the DNFB group were significantly higher than those of the Control group, with significant differences; the DNFB+DXM group and the DNFB+BPNP group showed significant differences from the DNFB group, indicating that Bidens pilosa exosomes can alleviate the symptoms of atopic dermatitis in mice.

5.3 H&E staining results

As shown in Figure 8, by comparing the H&E staining results of the skin and ear tissue sections of mice in each group, it was found that the structures of the skin layers on the back of the mice in the Control group were clear, the structure and morphology of the epidermis and dermis were normal, and there was no obvious inflammatory cell infiltration between cells and within cells; in the lesion tissue on the back of the mice in the DNFB group, incomplete keratinization or excessive keratinization was observed, the epidermis was significantly thickened, accompanied by a large number of inflammatory cell infiltrations, and the lesion tissue was obviously edematous; the DNFB+DXM group and the DNFB+BPNP group were improved compared with the DNFB group, which proved the improvement effect of BPNP on mouse atopic dermatitis.

This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims (8)

1. The use of Bidens pilosa exosomes for treating atopic dermatitis, characterized in that the Bidens pilosa exosomes are secreted by Bidens pilosa tissue, and the Bidens pilosa tissue is selected from any one or more of Bidens pilosa leaves, stems, and roots. 2. The use of Bidens pilosa exosomes for treating atopic dermatitis according to claim 1, characterized in that the preparation method of the Bidens pilosa exosomes comprises the following steps: The Bidens pilosa tissue was taken, and the buffer solution was added and then crushed, and the supernatant was collected after solid-liquid separation, and the supernatant was microfiltered to obtain the Bidens pilosa pretreatment sample; The Bidens pilosa pretreated sample is subjected to ultracentrifugation to obtain Bidens pilosa exosomes. 3. The use of Bidens pilosa exosomes for treating atopic dermatitis according to claim 2, characterized in that the ultracentrifugation specifically comprises the following steps: The Bidens pilosa pretreated sample was centrifuged at 1500-2500g for 25-35min; the supernatant was centrifuged at 8000-12000g for 40-50min and then microfiltered; the filtrate was centrifuged at 80000-120000g for 65-75min; the precipitate was resuspended in buffer and centrifuged at 80000-120000g for 65-75min to obtain Bidens pilosa exosomes. 4. The use of Bidens pilosa exosomes for treating atopic dermatitis according to claim 3, characterized in that the average particle size of Bidens pilosa exosomes is 79.0 nm and the concentration is (1.2-1.4)×10 ⁹ /ml. 5. The use of Bidens pilosa exosomes for treating atopic dermatitis according to claim 2, characterized in that the solid-liquid separation after the Bidens pilosa tissue is crushed specifically comprises the following steps: The Bidens pilosa tissue is crushed and filtered, and the supernatant is centrifuged at 8000-12000 rpm for 15-25 minutes. 6. The use of Bidens pilosa exosomes for treating atopic dermatitis according to claim 1, characterized in that the Bidens pilosa exosomes are used to prepare a composition for treating atopic dermatitis, and the components of the composition further include any one or more of excipients, dispersants, food additives, pharmaceutical additives and probiotics. 7. The use of Bidens pilosa exosomes for treating atopic dermatitis according to claim 1, characterized in that the Bidens pilosa exosomes are used to prepare a drug for treating atopic dermatitis or an auxiliary treatment drug. 8. The use of Bidens pilosa exosomes for treating atopic dermatitis according to claim 6, characterized in that the dosage forms of the drug and the auxiliary therapeutic drug are selected from any one or more of microinjections, external liquid preparations, external solid preparations, external semisolid preparations, plasters, patches and external gas preparations.