Hangzhou thistle extract for treating atopic dermatitis and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly provides a Hangzhou thistle extract for treating atopic dermatitis (AD), and the Hangzhou thistle extract is prepared by the following steps: weighing Hangzhou thistle, adding an alcoholic solution for extraction, adsorbing and eluting an extracting solution by using resin, and collecting eluent to obtain the Hangzhou thistle extract. When the Hangzhou thistle extract provided by the invention is externally applied to a skin lesion part, AD symptoms can be effectively improved, including improvement of skin tissue form, promotion of skin lesion part recovery, reduction of scratching times, reduction of ear swelling degree and spleen index, reduction of inflammatory factor level, reduction of mastocyte number and inhibition of degranulation of mastocyte; the inflammatory response of atopic dermatitis is improved by regulating and controlling a TLR4 / NF-kappa B pathway. The problem of poor patient compliance caused by tedious operation of traditional Chinese medicine compound oral administration is solved. A novel, effective and safe treatment mode is provided for AD treatment, application and development of Hangzhou thistle are greatly promoted, and a new strategy is provided for local resource development and utilization and economic development.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to an extract of *Cirsium japonicum* for treating atopic dermatitis and its application. Background Technology

[0002] Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by recurrent eczematous lesions and intense itching. Its pathogenesis involves the interaction of genetics and gene susceptibility, skin barrier defects, immune imbalance, abnormal inflammatory signals, microbial dysbiosis, and environmental factors, forming a complex pathological network.

[0003] Current treatment strategies for Alzheimer's disease (AD) include controlling inflammation, repairing the skin barrier, modulating immunity, and regulating the microbiome. Topical corticosteroids (TCS) offer rapid anti-inflammatory and antipruritic effects with significant short-term efficacy, making them a primary treatment for allergic skin diseases. However, long-term use increases the risk of bacterial infection and has side effects such as skin atrophy, telangiectasia, pigmentation, and steroid-dependent dermatitis. Rebound syndromes are severe upon abrupt discontinuation. Topical calcineurin inhibitors (TCIs) are non-hormonal immunomodulators that selectively inhibit key regulatory nodes of the NFAT pathway in T lymphocytes by antagonizing calcium-dependent phosphatase activity, thereby bidirectionally regulating the immune response during the AD inflammatory process. Topical TCIs can be used in combination with or sequentially with TCS as a better option for maintenance therapy. However, some patients experience burning, stinging, and itching after topical application; these are common adverse reactions, and sun protection is necessary.

[0004] With in-depth research into the pathogenesis of allergic skin diseases, targeted drugs are constantly being developed and applied. These drugs target key cytokines in the innate immune pathway, adaptive immunity, pruritus-scratching cycle, and JAK-STAT pathway, among other pathways. Examples include the IL-4Rα inhibitor dupilumab, the IL-13 monoclonal antibody trorolu, and the JAK1 / 2 inhibitor baricitinib. However, biological targeted agents are relatively few in number, have high application costs, and face limitations in clinical use. Furthermore, with increased application, clinical findings suggest that targeted therapy may lead to severe allergic reactions that could endanger patients' lives. Summary of the Invention

[0005] The purpose of this invention is to overcome the limitations and adverse reactions of existing AD treatment strategies.

[0006] Therefore, the present invention provides a *Cirsium japonicum* extract for treating atopic dermatitis, the *Cirsium japonicum* extract being prepared by the following steps: weighing *Cirsium japonicum*, adding an alcohol solution for extraction, adsorbing and eluting the extract with resin, collecting the eluent to obtain the *Cirsium japonicum* extract.

[0007] Specifically, the alcohol solution mentioned above includes ethanol with a concentration of 40-60%; the ratio of the solution to the plant artichoke is 1:10-1:20.

[0008] Specifically, the above extraction conditions are extraction at 70℃-90℃ for 1-2 hours.

[0009] Specifically, the above adsorption and elution are performed using AB-8 macroporous resin.

[0010] Specifically, after the above resin adsorption, it is eluted successively with 10 Bv 30%, 50%, 70%, and 90% ethanol, and the eluted fraction with 30%-50% ethanol is collected.

[0011] The *Cirsium japonicum* extract provided by this invention can be used to prepare drugs for treating atopic dermatitis.

[0012] Specifically, the above-mentioned drugs use thistle extract as the active ingredient and also include pharmaceutically acceptable excipients.

[0013] The present invention also provides a *Cirsium japonicum* gel for treating atopic dermatitis, comprising the above-mentioned *Cirsium japonicum* extract.

[0014] Specifically, by weight percentage, the thistle gel comprises 0.5-1% thistle extract, 0.75-1.25% rheology modifier, 6-12% humectant, 1-3% surfactant, 0.50-1.00% neutralizer, and the balance being water.

[0015] Specifically, the above-mentioned thistle gel also includes 0.1-0.3% preservatives.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0017] This invention provides an external application of *Cirsium japonicum* extract to the affected skin to treat atopic dermatitis (AD). This treatment effectively improves AD symptoms, including improving skin tissue morphology, promoting lesion recovery, reducing scratching frequency, decreasing ear swelling and spleen index, lowering levels of inflammatory factors such as TNF-α, IgE, IL-4, and IL-17, reducing mast cell number and inhibiting degranulation, and inhibiting the protein expression of TLR4 and NF-κB p65. By regulating the TLR4 / NF-κB pathway, it improves the inflammatory response of atopic dermatitis. This overcomes the problem of poor patient compliance caused by the cumbersome operation of traditional oral Chinese medicine formulas. It provides a novel, effective, and safe treatment method for AD and greatly promotes the application and development of *Cirsium japonicum*, offering a new strategy for local resource development and economic growth.

[0018] The present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0019] Figure 1The images show the appearance of the freeze-dried powder of the *Cirsium japonicum* extract before and after purification in Example 1 of this invention; A: morphology before purification; B: morphology after purification.

[0020] Figure 2 This is a flowchart of animal modeling and drug administration in Embodiment 2 of the present invention.

[0021] Figure 3 This describes the effect of the *Cirsium japonicum* extract on the weight changes in mice in Example 2 (n=8).

[0022] Figure 4 This is the effect of *Cirsium japonicum* extract on skin lesions and scratching frequency in mice, as shown in Example 2; A: Photographs of mouse skin lesions; B: Dermatitis scores in mice (n=8); C: Number of scratches in mice (n=6); Compared with the normal group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the model group, # P < 0.05, ## P < 0.01, ### P < 0.001

[0023] Figure 5 This is the effect of *Cirsium japonicum* extract on ear swelling and spleen index in mice (n=8) in Example 2; A: ear swelling; B: spleen index; compared with the normal group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the model group, # P < 0.05 ## P < 0.01, ### P < 0.001.

[0024] Figure 6 This refers to the effect of *Cirsium japonicum* extract on serum IgE, IL-4, IL-17, and TNF-α levels in mice (n=8); A: IgE; B: IL-4; C: TNF-α; D: IL-7; Compared with the normal group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the model group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0025] Figure 7This is the effect of *Cirsium japonicum* extract on HE staining results of mouse skin in Example 2; A: HE staining image; B: Epidermal thickness (n=3); compared with the normal group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the model group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0026] Figure 8 This is the effect of *Cirsium japonicum* extract on mouse skin mast cells in Example 2; A: Toluidine blue staining of skin ( 200); B: Number of mast cells (n=3); Compared with the normal group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the model group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0027] Figure 9 This is the effect of *Cirsium japonicum* extract on TLR4 protein expression in mouse skin, as shown in Example 2; A: TLR4 immunofluorescence map ( 400); B: TLR4 relative fluorescence intensity (n=3); compared with the normal group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the model group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0028] Figure 10 This is the effect of *Cirsium japonicum* extract on the expression of NF-κB p65 protein in mouse skin, as shown in Example 2; A: Immunofluorescence map of NF-κB p65 ( 400); B: Relative fluorescence intensity of NF-κB (n=3); Compared with the normal group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the model group, # P < 0.05, ## P < 0.01, ### P < 0.001.

[0029] Figure 11This is a diagram showing the appearance of the gel in Example 3.

[0030] Figure 12 This is the gel stability evaluation in Example 3. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

[0032] This invention provides a *Cirsium japonicum* extract for treating atopic dermatitis, the *Cirsium japonicum* extract being prepared using the following steps:

[0033] Weigh the *Artemisia annua* powder and add it to an alcohol solution (preferably 40-60% ethanol) at a material-to-liquid ratio of 1:10-1:20. Extract at 70-90℃ for 1-2 h to obtain *Artemisia annua* extract. Concentrate the extract by rotary evaporation and freeze-dry it to prepare a 2.0 mg / mL solution. Use resin (preferably AB-8 macroporous resin) for adsorption and elution. Load 12 Bv of the solution at a flow rate of 1 mL / min. After standing for 2 h, elute successively with 30%, 50%, 70%, and 90% ethanol at a flow rate of 1 mL / min. Collect the 30%-50% ethanol eluent and freeze-dry it to obtain *Artemisia annua* extract.

[0034] The optimal extraction conditions were 60% ethanol concentration, a solid-liquid ratio of 1:10 g / mL, an extraction temperature of 80℃, and an extraction time of 1.5 h.

[0035] The *Cirsium japonicum* extract provided by this invention can be used to prepare drugs for treating atopic dermatitis.

[0036] Specifically, the above-mentioned drugs use thistle extract as the active ingredient and also include pharmaceutically acceptable excipients.

[0037] The present invention also provides a *Cirsium japonicum* gel for treating atopic dermatitis, comprising the above-mentioned *Cirsium japonicum* extract.

[0038] Specifically, by weight percentage, the thistle gel comprises 0.5-1% thistle extract, 0.75-1.25% rheology modifier, 6-12% humectant, 1-3% surfactant, 0.50-1.00% neutralizer, and the balance being water.

[0039] Among them, carbomer is preferred as the rheology modifier, glycerin is preferred as the humectant, Tween 20 is preferred as the surfactant, and triethanolamine is preferred as the neutralizing agent.

[0040] The thistle gel may also include 0.1-0.3% of a preservative, preferably ethylparaben.

[0041] In a detailed embodiment, the above-mentioned *Artemisia annua* gel is prepared using the following steps: Carbomer 940 is dissolved in distilled water and allowed to swell for 24 hours to obtain a 4% gel matrix, which is then set aside. *Artemisia annua* extract is accurately weighed and dissolved in an appropriate amount of 50% anhydrous ethanol to prepare a 20 mg / mL extract solution, which is then set aside. While stirring, glycerol, the *Artemisia annua* extract solution, and Tween 20 are added to the gel matrix. After stirring evenly, an appropriate amount of triethanolamine and ethylparaben dissolved in anhydrous ethanol are added, and distilled water is added to make up the volume. The mixture is stirred until a gel is formed, thus obtaining the *Artemisia annua* gel. It is then placed in a light-proof container and stored in a cool place.

[0042] The following specific embodiments illustrate the effects of the *Ipomoea alopecia areata* gel for treating atopic dermatitis and its application.

[0043] Example 1:

[0044] This embodiment investigated the optimal extraction and adsorption-elution conditions for the extract of Cirsium japonicum, as detailed below.

[0045] Weigh out the *Artemisia annua* powder and divide it into 9 groups. Each group was extracted with ethanol solution of different concentrations at a certain material-to-liquid ratio for 1.5 h to obtain *Artemisia annua* extract. The Pec purity of the *Artemisia annua* extract of each group was determined by HPLC. The results are shown in Table 1.

[0046] Table 1 Experimental conditions and Pec test results for each group

[0047]

[0048] Weigh 100 g of *Artemisia annua* powder and extract it with 60% ethanol at a volume 10 times that of the powder. Extraction temperature: 80℃; extraction time: 1.5 h. Concentrate the extract by rotary evaporation and freeze-dry. Figure 1 As shown in A. Then, a 2.0 mg / mL solution was prepared and adsorbed and eluted using AB-8 macroporous resin. 12 Bv was loaded at a flow rate of 1 mL / min, and after standing for 2 h, it was eluted successively with 10 Bv of 30%, 50%, 70%, and 90% ethanol at a flow rate of 1 mL / min. The 30%, 50%, and 70% ethanol eluates were collected, freeze-dried, and the *Cirsium japonicum* extract was obtained, as shown in Figure A. Figure 1 As shown in B.

[0049] Example 2:

[0050] This embodiment is based on a DNCB-induced AD mouse model to explore the therapeutic effect of Cirsium hangianum extract on atopic dermatitis mice.

[0051] 1. Preparation of Cirsium hangianum extract

[0052] The dried aerial parts of Cirsium hangianum were crushed and passed through a No. 2 sieve to obtain the crude powder of Cirsium hangianum. It was extracted with 10 times the amount of 60% ethanol at 80 °C for 1.5 h. The extract was concentrated by rotary evaporation and freeze-dried to obtain the Cirsium hangianum extract. After pretreatment of AB-8 macroporous resin, the Cirsium hangianum extract powder was dissolved in distilled water to a concentration of 2.0 mg / mL, and loaded onto the column at a flow rate of 1 mL / min for 10 bed volumes, and allowed to adsorb statically for 2 h. First, the polar components were eluted with water, and then eluted with 30% and 50% ethanol for 10 bed volumes. The elution fraction of 30% - 50% ethanol was collected. After rotary evaporation and concentration, it was freeze-dried to obtain the Cirsium hangianum extract powder, which was stored at -20 °C for later use.

[0053] 2. Preparation of experimental drugs

[0054] 1% Carbomer solution: Weigh 5 g of Carbomer 940 and sprinkle it into 500 mL of distilled water, and let it stand for 24 h to absorb water and swell, which is used as a solvent for later use.

[0055] Cirsium hangianum extract solution: Prepared into solutions of 2.5, 5.0, and 10.0 mg / mL with 1% Carbomer, and stored at 4 °C for later use.

[0056] Compound dexamethasone acetate cream solution: Diluted with distilled water to a 0.15 mg / mL solution for later use.

[0057] Preparation of 2% DNCB solution: Weigh 1.00 g of DNCB solid powder and dissolve it in 50 mL of a mixed solvent of acetone: olive oil = 4:1, mix well, and store at room temperature.

[0058] Preparation of 1% DNCB solution: Weigh 0.50 g of DNCB solid powder and dissolve it in 50 mL of a mixed solvent of acetone: olive oil = 4:1, mix well, and store at room temperature.

[0059] 3. Grouping, modeling, and administration of experimental animals

[0060] SPF-grade 6-week-old male KM mice, weighing 35 - 40 g, were purchased from Beijing SpeyFort Bio-Tech Co., Ltd. (production license number: SCXK (Beijing) 2024-0001). They were housed in the Animal Experiment Center of Beijing University of Chinese Medicine, and the housing environment was a standard SPF-grade barrier environment with a 12 h light and dark cycle every day. All animal experiment protocols were approved by the Animal Experiment Ethics Committee of Beijing University of Chinese Medicine, and the ethical review number was BUCM-2024092703-3244.

[0061] Six- to eight-week-old male KM mice were acclimatized for one week and then randomly divided into six groups: a normal group, a model group, a positive control group (1.5 mg / kg / d), a low-dose group of *Cirsium japonicum* extract (25 mg / kg / d), a medium-dose group (50 mg / kg / d), and a high-dose group (100 mg / kg / d). The positive control group received compound dexamethasone acetate cream solution.

[0062] On day 0, mice underwent hair removal and were weighed. The procedure involved shaving the hair short with a razor, applying hair removal cream, allowing it to stand for 5 minutes, and then wiping off the cream. The hair removal area was 2 cm × 3 cm. Starting day 1, except for the normal control group, all other groups were sensitized by applying 100 μL of 2% DNCB to the back skin for 3 consecutive days, and were weighed on day 3. Starting day 6, 50 μL of 1% DNCB was applied to the back and right ear every 2 days for challenge, while the normal control group received an equal volume of DNCB matrix solution. Except for the normal control and model groups, all other groups received the corresponding drug on the hair removal area on the back starting on day 6 of modeling, twice daily at 0.2 mL each time, for 18 consecutive days. The normal control and model groups received an equal volume of distilled water. The experimental procedure is as follows: Figure 2 As shown.

[0063] 4. Effects of thistle extract on body weight changes and skin lesions in mice

[0064] 4.1 Mouse body weight determination

[0065] Mice were weighed regularly during the experiment. They were weighed on day 0 and day 4 before and after sensitization, and every 4 days during the drug administration period. A curve showing the change in mouse body weight was plotted to observe the changes in mouse body weight.

[0066] 4.2 Comparison of scratching frequency in mice

[0067] Two hours after the last administration, the number of scratches was measured. Six mice were randomly selected from each group and placed in cages for 15 minutes to acclimatize. The number of scratches by each group of mice within 10 minutes was then recorded using a camera. Scratching around the ears with the forepaws, scratching the trunk with the hind paws, and biting various areas of the body surface were used as indicators of itching. A scratching behavior was defined as a continuous scratching action followed by a brief pause (≥1 second of cessation of action).

[0068] 4.3 Photographing and scoring of mouse skin lesions

[0069] At 24 hours after each provocation, the skin lesions on the backs of mice were scored and recorded from three perspectives: erythema, edema, and scratch marks, referring to the standards in Table 2. Two independent observers scored the skin lesions each time, and the average score was taken as the result. During the experiment, the skin lesions of mice in each group were photographed at the same time every 4 days to observe the recovery process.

[0070] Table 2 Scoring criteria for skin lesions in mice with atopic dermatitis

[0071]

[0072] 4.4 Mouse sampling and specimen processing

[0073] After the last administration, the weight of each group of mice was measured. After fasting for 16 h but not water, the mice were anesthetized and samples were taken. Whole blood samples were collected by enucleation using pyrogen-free / endotoxin-free centrifuge tubes. After the samples were left to stand at room temperature for 2 h, they were centrifuged at 3000 r / min for 20 min at 4°C. The upper serum layer was collected, aliquoted, and stored at -20°C for later use. After euthanizing the mice by cervical dislocation, the ear tissue of both ears was cut along the auricle baseline. Ear pieces of equal area were cut from both ears using an 8 mm diameter punch. The weight of the ear pieces (mg) was accurately measured. The difference in weight between the right and left ears was used as the degree of ear swelling to assess the degree of local inflammation in AD. The abdominal cavity was opened, the spleen was removed, washed with physiological saline, dried, and weighed. The spleen index (mg / 10g) was calculated according to formula (3). The damaged skin on the back was cut, the hair was removed, and the pieces were cut to an appropriate size and fixed with 4% paraformaldehyde for subsequent pathological experiments.

[0074] Spleen index (mg / 10g) = Spleen mass (mg) / Body mass (g) 10 (3)

[0075] 4.5 ELISA detection of changes in serum IgE, IL-4, IL-17, and TNF-α levels in mice

[0076] Incubate the ELISA kit at room temperature for 20 minutes, then follow the instructions in the manufacturer's manual:

[0077] 4.5.1 Standard Dilution

[0078] Prepare 6 centrifuge tubes and label them. Add 100 μL of standard diluent to each tube. Transfer 100 μL of the original concentration standard to tube 1 and mix well. Then, serially dilute to tube 5. Keep tube 6 as a blank control. The standard concentration is 0.

[0079] 4.5.2 Sample addition

[0080] Set up standard wells and sample wells. Add 50 μL of gradient standards to the standard wells, and add 10 μL of serum and 40 μL of diluent to the sample wells. Keep only the dilution mixture in the blank wells. Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled antibody to each well.

[0081] 4.5.3 Warmth and Incubation

[0082] The reaction wells were coated and incubated at 37°C for 60 min.

[0083] 4.5.4 Washing

[0084] Discard the reaction solution and spin dry, fill with washing solution, let stand for 1 minute, then discard the solution and spin dry. Repeat washing five times.

[0085] 4.5.5 Color Development

[0086] Add 50 μL each of colorimetric reagents A and B to each well and incubate at 37°C in the dark for 15 min.

[0087] 4.5.6 Termination of the reaction

[0088] The reaction was terminated by adding 50 μL of stop solution, and the OD value was measured at 450 nm within 15 min. A standard curve was plotted with the standard concentration on the x-axis and the OD value on the y-axis, and the concentration values ​​of inflammatory factors for each sample were calculated based on the curve equation.

[0089] 4.6 HE staining of mouse dorsal skin tissue

[0090] 4.6.1 Dehydration

[0091] After the skin tissue on the back of the mouse was fixed with 4% paraformaldehyde, it was placed in an automatic dehydrator and dehydrated with gradient ethanol for 12 hours. After the dehydration was completed, the tissue block was removed.

[0092] 4.6.2 Embedding

[0093] The tissue was impregnated with paraffin three times, each time for 1 hour. Then, the cut side was embedded in paraffin with the cut side facing down. After solidification at -20°C, the paraffin block was trimmed and stored at 4°C for later use.

[0094] 4.6.3 Slicing

[0095] After spreading the 4 μm sections in 40℃ warm water, they were attached to glass slides, baked at 60℃ until the wax melted, and stored at room temperature for later use.

[0096] 4.6.4 HE staining

[0097] (1) Dewaxing paraffin sections to water: The sections were treated in sequence with environmentally friendly dewaxing solution I (20 min), dewaxing solution II (20 min), anhydrous ethanol for 5 min, and 75% ethanol (5 min) twice, and rinsed with distilled water for 2 min.

[0098] (2) Staining of hematoxylin semen: After staining with hematoxylin for 5 min, rinse with running water, treat with differentiation solution and then return to blue with blue solution, and rinse with running water.

[0099] (3) Eosin staining: The sections were placed in 95% ethanol for dehydration, and then stained in eosin staining solution for 15 s.

[0100] (4) Dehydration and mounting: The sections were placed in anhydrous ethanol, n-butanol and xylene in sequence to dehydrate and clear them, and then mounted with neutral resin.

[0101] 4.6.5 Image Acquisition and Analysis

[0102] The pathological changes in mouse skin tissue were observed and the epidermal thickness was measured by scanning HE-stained sections using a high-resolution microscopic imaging system.

[0103] 4.7 Toluidine blue staining and mast cell counting observation

[0104] 4.7.1 Toluidine Blue Staining

[0105] Following the procedure in section "4.6", tissue sections were dewaxed to water, followed by toluidine blue staining: immersing the tissue sections in the staining solution for 2-5 minutes, rinsing with running water, then differentiating with 0.1% glacial acetic acid for 10 seconds, rinsing with water to terminate the reaction, monitoring the degree of metachromatic granule development under a microscope, rinsing with running water, drying at 60°C for 10 minutes, clearing with xylene, and then mounting with neutral resin. A high-resolution microscopy system was used to scan the toluidine blue-stained sections to observe mast cells and their degranulation phenomenon.

[0106] 4.7.2 Mast cell count

[0107] Toluidine blue specifically labels mast cell metachromatic granules as blue-purple. Three fields of view were randomly selected from toluidine blue-stained sections under a 200x optical microscope to count the number of mast cells.

[0108] 4.8 Immunofluorescence assay of TLR4 and NF-κB p65 protein expression

[0109] Skin tissues from mice in the normal group, model group, and low, medium, and high dose groups of thistle extract were selected as samples for the immunofluorescence experiment.

[0110] 4.8.1 Dewaxing paraffin sections to water

[0111] The sections were sequentially dehydrated twice with environmentally friendly dewaxing solution I (20 min), dewaxing solution II (20 min), and anhydrous ethanol (5 min), followed by 75% ethanol (5 min) and then rinsed with distilled water for 2 min.

[0112] 4.8.2 Antigen retrieval

[0113] Tissue sections were immersed in EDTA antigen retrieval buffer (pH 8.0) and antigen epitope retrieval was performed using a gradient temperature program. After retrieval, the retrieval chamber was allowed to cool at room temperature for 30 minutes. The slides were then transferred to phosphate-buffered saline (PBS) (pH 7.4) and washed three times with shaking for 5 minutes each time to thoroughly remove any residual retrieval solution.

[0114] 4.8.3 Circle-based serum blocking

[0115] After the sections were moderately dried, an immunohistochemical pen was used to draw a blocking circle around the tissue periphery, followed by the addition of bovine serum albumin (BSA) for 30 min.

[0116] 4.8.4 Primary Antibody Incubation

[0117] The prepared primary antibody was dropped onto the surface of the slide, and the slide was placed horizontally in a humidified chamber and incubated overnight at 4°C.

[0118] 4.8.5 Secondary Antibiotic Incubation

[0119] The slide was placed in phosphate-buffered saline (PBS) and washed by shaking on a shaker three times, each time for 5 minutes. Then, a secondary antibody matching the primary antibody was added, and the slide was incubated at room temperature in the dark for 50 minutes.

[0120] 4.8.6 Nuclear DAPI counterstaining

[0121] After rinsing the slide in PBS by shaking, add DAPI staining solution and incubate at room temperature for 10 min in the dark.

[0122] 4.8.7 Quenching of tissue autofluorescence

[0123] After washing the slide in PBS, add autofluorescence quencher solution B and treat for 5 min, then rinse with running water for 10 min.

[0124] 4.8.8 Sealing

[0125] Antifluorescence quenching mounting medium for mounting.

[0126] 2.8.9 Image Acquisition

[0127] The excitation wavelength range of DAPI is 330 ~ 380 nm, and the emission wavelength is 420 nm; the excitation wavelength range of CY3 is 510 ~ 560 nm, and the emission wavelength is 590 nm.

[0128] 4.9 Statistical Analysis and Graphing

[0129] All experimental data are expressed as mean ± standard deviation. The experimental data were statistically analyzed and plotted using GraphPad Prism 9.0 software. Normality and homogeneity of variance tests were performed on each group of data. One-way ANOVA was used, and when the ANOVA results showed significance, Dunnett's multiple comparison method was used for post-hoc testing to analyze the differences between each group and the model group. A p-value < 0.05 was considered statistically significant.

[0130] 5. Results and Analysis

[0131] 5.1 Effects of Cirsium japonicum extract on changes in mouse body weight

[0132] Changes in mouse body weight were monitored before and after the experiment, and the results were as follows: Figure 3 As shown, the body weight of mice in the normal group showed a continuous increasing trend. In the other groups, after 3 consecutive days of DNCB sensitization, the body weight decreased to some extent, possibly because the DNCB-induced allergic reaction caused skin discomfort in the mice, and the mice became noticeably restless and agitated. During the allergy-inducing and drug administration phase, the body weight of mice in the positive drug group continuously decreased, eventually falling below that of the model group, possibly due to the side effects of long-term dexamethasone corticosteroid use. The body weight of mice in all dose groups of *Cirsium japonicum* extract showed an increasing trend, with the high-dose group showing a trend more similar to that of the normal group. The low and medium-dose groups showed no significant difference in the increasing trend compared to the model group. These results indicate that topical application of *Cirsium japonicum* extract does not significantly affect the body weight changes in mice with atopic dermatitis.

[0133] 5.2 Effects of thistle extract on skin lesions and scratching frequency in mice

[0134] Photos of mouse skin lesions as shown Figure 4As shown in Figure A, during the experiment, the normal group mice had light pink, soft skin with normal hair growth, intact skin morphology, and no scaling. On day 6, all mice except the normal group showed varying degrees of AD-like symptoms, specifically obvious skin erythema, dryness, epidermal erosion and peeling, and crusting, indicating that the atopic dermatitis mouse model was successfully established. With continuous stimulation of allergies with low concentrations of DNCB, the model group mice had a redder skin color, obvious skin surface damage and crusting, and some mice had localized dry skin and severe scaling. However, the AD-like symptoms showed a trend of first worsening and then improving, possibly because as the mice grew older, their autoimmune ability continuously improved, resulting in a certain degree of self-healing. After administration of the positive control drug and the corresponding dose of *Cirsium japonicum* extract, the skin lesions of the mice showed significant improvement. In the positive control drug group, the epidermal color was more pink than before, the skin damage was better, the dryness was reduced, the scaling was reduced, and the rash distribution was reduced; however, some degree of pigmentation was observed, and hair did not grow on the skin in the affected areas. The AD symptoms of mice treated with thistle extract improved. In the low-dose group, the skin color was lighter than before, but there were still a few erythema, a few rashes, and some scaling. In the medium-dose group, the skin was significantly softer, the color was darker, and the erythema was reduced. In the high-dose group, the skin color returned to light pink, the erythema and scaling basically disappeared, and the administration of thistle extract did not significantly affect the hair growth of mice.

[0135] Dermatitis score results as follows Figure 4 As shown in Figure B, after the first allergic reaction, compared with the normal group, the dermatitis scores of all other groups were significantly increased (P < 0.001), and there was no difference between the model group and the treatment group (P > 0.05), indicating that the AD model was successfully established. Ten days after modeling, the dermatitis score in the model group significantly increased and reached its peak. Twenty-three days after modeling, i.e., 18 days after treatment, compared with the model group, the dermatitis scores of mice in each treatment group were significantly decreased (P < 0.001), indicating that both dexamethasone and *Cirsium japonicum* extract can improve AD symptoms in mice.

[0136] Itching is one of the main clinical symptoms in the pathogenesis of atopic dermatitis (AD), and controlling itching is an essential part of AD treatment. The experiment recorded the number of scratches per 10 minutes in each group of mice as follows: Figure 4 As shown in Figure C, compared with the normal group, the number of scratches in the model group mice was significantly increased (P < 0.001). Administration of low, medium and high levels of thistle extract and dexamethasone cream could significantly reduce the number of scratches in mice, and the differences were statistically significant (P < 0.01).

[0137] 5.3 Effects of *Cirsium japonicum* extract on ear swelling and spleen index in mice

[0138] Ear swelling is a common manifestation of localized inflammation in AD mouse models, and the degree of swelling increases with the severity of inflammation. Results Figure 5 As shown in Figure A, compared with the normal group, the ear swelling of the model group mice was significantly increased (P < 0.001), indicating significant local ear swelling in AD mice. Compared with the model group, the ear swelling of mice in the positive control group and each dose group of *Cirsium japonicum* extract was reduced, with significant reductions in the positive control group and the medium and high dose groups of *Cirsium japonicum* extract (P < 0.05), while the reduction in ear swelling in the low dose group of *Cirsium japonicum* extract was not significant (P > 0.05). This indicates that treatment with medium and high doses of *Cirsium japonicum* extract alleviated ear swelling and reduced local ear inflammation.

[0139] As the largest peripheral immune organ in the body, the spleen often exhibits characteristic pathological changes in the progression of immune-related diseases such as Alzheimer's disease (AD). Abnormal infiltration and aggregation of immune cells lead to tissue remodeling, accompanied by vasodilation and congestion, resulting in increased organ volume and weight. The spleens of mice in each group were weighed, and the spleen index results are as follows: Figure 5 As shown in Figure B, compared with the normal group, the spleen index in the model group was significantly increased (P < 0.001), indicating an abnormal immune response in the spleen of AD mice. Compared with the model group, the spleen index of mice in the medium and high dose groups of *Cirsium japonicum* extract was decreased, with statistically significant differences (P < 0.01). The decrease was even more significant in the dexamethasone group (P < 0.001), with the spleen index even lower than that in the normal group. These results indicate that *Cirsium japonicum* extract can reduce the spleen index in AD mice and improve the body's immune status.

[0140] 5.4 Effects of Cirsium japonicum extract on serum IgE, IL-4, IL-17, and TNF-α levels in mice

[0141] The effects of *Cirsium japonicum* extract on the levels of TNF-α, IgE, IL-4, and IL-17 in mouse serum were detected using ELISA. The results are as follows: Figure 6 As shown in the figure, after establishing the AD model by DNCB induction, the levels of the four inflammatory factors were significantly increased compared with the normal group (P < 0.001), and the differences were statistically significant. After treatment with the positive control drug and various doses of *Cirsium japonicum* extract, the serum levels of TNF-α, IgE, IL-4, and IL-17 in mice showed a decreasing trend compared with the model group. The inhibitory effect on inflammation levels became more significant with increasing dose of *Cirsium japonicum* extract, showing a drug dose-dependent effect. The positive control drug group and the high-dose *Cirsium japonicum* extract group showed significant inhibitory effects on the levels of the four inflammatory factors (P < 0.001), with no significant difference from the levels in the normal group.

[0142] 5.5 Effects of Cirsium japonicum extract on mouse skin histopathology

[0143] HE staining results of the back skin are as follows Figure 7As shown in Figure A, the skin structure on the backs of mice in the normal group was normal and intact, with a clear junction between the epidermis and dermis, normal tissue thickness, and normal cell morphology, and no obvious edema or inflammatory response was observed. Compared with the normal group, the model group showed typical AD pathological features: hyperkeratosis of the epidermis with crust formation, and diffuse inflammatory cell infiltration throughout the dermis. Compared with the model group, mice in the positive drug group and each dose group of *Cirsium japonicum* extract showed significant improvement in skin tissue pathology after intervention, specifically a significant reduction in epidermal thickness and a significant inhibition of inflammatory cell infiltration. To further quantify the analysis, the epidermal thickness of the mouse skin tissue was systematically measured. The results are as follows: Figure 7 As shown in Figure B, compared with the normal control group, the epidermal thickness in the model group was significantly increased (P < 0.001), indicating that the DNCB-induced AD model was successfully established. After drug intervention, the positive drug group and all doses of *Cirsium japonicum* extract showed a reduction in epidermal thickness (P < 0.01), with the high-dose *Cirsium japonicum* extract group showing the most significant reduction (P < 0.001). This indicates that *Cirsium japonicum* extract can significantly improve the pathological changes in AD induced by DNCB.

[0144] 5.6 Effects of thistle extract on mouse skin mast cells

[0145] like Figure 8 As shown in Figure A, toluidine blue (TB) staining was used to examine whether *Cirsium japonicum* extract could effectively improve mast cell activation. Mast cells were mainly distributed in the perivascular region, often existing in scattered or aggregated forms, with a typical round or oval morphology. A small, lightly stained oval nucleus was visible in the center of each cell. Under a high-power microscope, the cytoplasm was rich in numerous homogeneous, metastained blue-purple granules. The mast cell count results are shown below. Figure 8 As shown in Figure B, compared with the normal group, the number of mast cells and released granules in the skin tissue of the model group mice was significantly higher than that of normal mice (P < 0.001), indicating that AD mice experienced a significant allergic reaction. Treatment with the positive control drug and *Cirsium japonicum* extract significantly reduced the number of mast cells and granules in the mice (P < 0.001).

[0146] 5.7 Effects of *Cirsium japonicum* extract on the expression of TLR4 and NF-κB p65 proteins in mouse skin

[0147] Immunofluorescence results as follows Figure 9 and Figure 10As shown, red fluorescence labeled the positive expression regions of TLR4 and NF-κB p65 proteins in mouse skin tissue, while blue fluorescence labeled the DAPI-stained cell nuclei for cellular structure localization. The results showed that, compared with the normal group, the positive fluorescence signals of TLR4 and NF-κB p65 in the model group were significantly enhanced (P < 0.001), indicating that the TLR4 / NF-κB signaling pathway was significantly activated in the DNCB-induced AD model. Compared with the model group, the positive drug group and the medium and high dose groups of *Cirsium japonicum* extract showed significantly reduced positive expression of TLR4 and NF-κB p65 (P < 0.001), suggesting that *Cirsium japonicum* extract may exert its anti-inflammatory effect by inhibiting the activation of the TLR4 / NF-κB signaling pathway. Furthermore, compared with the normal group, the fluorescence intensity of the positive drug group and the high dose group of *Cirsium japonicum* extract was close to normal levels, further confirming the therapeutic potential of *Cirsium japonicum* extract.

[0148] Example 3:

[0149] This embodiment provides an atopic dermatitis-treating herb-infused gel.

[0150] 1. Preparation of thistle extract

[0151] The dried aerial parts of *Artemisia annua* were pulverized and passed through a No. 2 sieve to obtain coarse powder. This powder was extracted with 10 times the volume of 60% ethanol at 80℃ for 1.5 h. The extract was concentrated by rotary evaporation and freeze-dried to obtain *Artemisia annua* extract. After pretreatment with AB-8 macroporous resin, the *Artemisia annua* extract powder was dissolved in distilled water to a concentration of 2.0 mg / mL. 10 Bv of the solution was loaded at a flow rate of 1 mL / min and allowed to stand for 2 h for adsorption. Polar components were eluted first with water, followed by elution with 10 Bv of 30% and 50% ethanol. The 30%–50% ethanol eluent was collected. This eluent was concentrated by rotary evaporation and then freeze-dried to obtain *Artemisia annua* extract powder, which was stored at -20℃ for later use.

[0152] 2. Preparation of thistle gel

[0153] Take an appropriate amount of Carbomer 940, add distilled water and allow it to swell completely for 24 hours to obtain a 4% gel matrix, set aside. Accurately weigh the *Cirsium japonicum* extract and dissolve it in an appropriate amount of 50% anhydrous ethanol to prepare an extract solution of 20 mg / mL, set aside. While stirring, add glycerol, the extract solution, and Tween 20 to the gel matrix, stir well, then add an appropriate amount of triethanolamine and ethylparaben dissolved in anhydrous ethanol, add distilled water to a total mass of 20.0 g, and stir until a gel is formed, thus obtaining a gel with a *Cirsium japonicum* extract concentration of 5 mg / g. Store in a light-proof container in a cool place.

[0154] Nine groups of *Ipomoea aquatica* gels were prepared using the above steps, and their properties were evaluated. The scoring criteria are shown in Table 3, and the raw material ratios and scores for each group are shown in Table 4.

[0155] Table 3 Gel Scoring Criteria

[0156]

[0157] Table 4 Gel Formulation and Scoring

[0158]

[0159] Under these process conditions, the gel exhibits a good brownish-yellow translucent gel appearance, is uniform and delicate, has good formability and excellent spreadability, and shows no stratification in centrifugal stability, heat resistance stability and low temperature stability, demonstrating excellent performance.

[0160] 3. Quality evaluation of thistle gel

[0161] Three batches of *Ipomoea aquatica* gel were prepared using the formulation in group 4 of Table 4 for quality evaluation.

[0162] 3.1 Appearance of thistle gel

[0163] The thistle gel was spread onto transparent containers, and its color, texture, and state were observed. Figure 11 All three samples of thistle gel were brownish-yellow, semi-transparent, semi-solid gels with a uniform texture, containing fine air bubbles, and with suitable viscosity, allowing for even application.

[0164] 3.2 pH determination of thistle gel

[0165] The pH values ​​of three batches of *Ipomoea aquatica* gel were measured using an FE28 pH meter. The results are shown in Table 5. The pH range of the gel was 6.65 to 6.84, which is weakly acidic to neutral, meeting the pH requirements of the Chinese Pharmacopoeia and close to the physiological pH value of the skin.

[0166] Table 5. Results of pH value determination of Artemisia annua gel

[0167]

[0168] 3.3 Stability evaluation of thistle gel

[0169] The results of the stability study of three batches of *Ipi* gel are as follows: Figure 12 As shown, the *Artichoke* gel did not exhibit stratification after centrifugation, and the air bubbles inside the gel disappeared, indicating that the *Artichoke* gel has good centrifugal stability. Under the conditions of 55℃ water bath for 6 h and -20℃ freezing for 24 h, the *Artichoke* gel did not exhibit stratification, drying, or liquefaction, indicating that its heat resistance and low temperature stability are both good.

[0170] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.

Claims

1. A *Cirsium japonicum* extract for treating atopic dermatitis, characterized in that, The *Cirsium japonicum* extract is prepared by the following steps: weigh *Cirsium japonicum*, add alcohol solution for extraction, adsorb and elute the extract with resin, collect the eluent, and obtain *Cirsium japonicum* extract.

2. The *Cirsium japonicum* extract for treating atopic dermatitis as described in claim 1, characterized in that: The alcohol solution includes ethanol with a concentration of 40-60%; the ratio of the solution to the styrax is 1:10-1:

20.

3. The *Cirsium japonicum* extract for treating atopic dermatitis as described in claim 1, characterized in that: Extract at 70℃-90℃ for 1-2 hours.

4. The *Cirsium japonicum* extract for treating atopic dermatitis as described in claim 1, characterized in that: AB-8 macroporous resin was used for adsorption and elution.

5. The *Cirsium japonicum* extract for treating atopic dermatitis as described in claim 1, characterized in that: After resin adsorption, the resin was eluted successively with 10 Bv 30%, 50%, 70%, and 90% ethanol, and the eluted fraction with 30%-50% ethanol was collected.

6. The use of the *Cirsium japonicum* extract as described in any one of claims 1-5 in the preparation of a medicament for treating atopic dermatitis.

7. The application as described in claim 6, characterized in that: The drug uses thistle extract as its active ingredient and also includes pharmaceutically acceptable excipients.

8. A *Cirsium japonicum* gel for treating atopic dermatitis, characterized in that: Includes the thistle extract as described in any one of claims 1-5.

9. The thistle gel as described in claim 8, characterized in that: By weight percentage, it includes 0.5-1% of thistle extract, 0.75-1.25% of rheology modifier, 6-12% of humectant, 1-3% of surfactant, 0.50-1.00% of neutralizer, and the balance being water.

10. The thistle gel of Hangzhou as described in any one of claims 9, characterized in that: It also includes 0.1-0.3% preservatives.

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