A pharmaceutical composition for treating atopic dermatitis, and a preparation method and use thereof

Abstract

The present application discloses a kind of medicine composition for resisting atopic dermatitis and its preparation method and purposes;The medicine composition includes effective components made of the following weight proportion of each raw material: Huanglian 3-50 parts by weight, Huangqin 3-30 parts by weight, Huangbo 3-30 parts by weight and Bai Xianpi 6-50 parts by weight.The present application has good therapeutic effect on atopic dermatitis skin damage and itching.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pharmaceutical composition for treating atopic dermatitis, its preparation method, and its uses. Background Technology

[0002] Atopic dermatitis is a common allergic inflammatory skin disease in clinical practice. Clinically, skin lesions are mainly characterized by papules, vesicles, exudation, erosion, and itching, featuring intense itching, polymorphic lesions, and recurrent, persistent symptoms. Atopic dermatitis usually begins in infants and young children, and its prevalence in adults remains high; currently, approximately 15-30% of infants and 2-10% of adults worldwide suffer from atopic dermatitis. Although atopic dermatitis is widespread, its etiology is not fully understood. Based on current research, it is generally believed that atopic dermatitis is related to allergic reactions, and its pathogenesis is closely related to gene mutations, exposure to exogenous irritants, impaired epidermal barrier function, immune system overreaction, and the interactions between these factors. Upon exposure to exogenous irritants, antigen-specific CD4+ T cells differentiate into type 1 helper T cells (Th1) and type 2 helper T cells (Th2). Th2 cell hyperactivity leads to Th1 / Th2 cell dysfunction, resulting in the infiltration of downstream inflammatory factors and mast cells, further increasing allergic reactions in patients with atopic dermatitis. Significantly elevated levels of immunoglobulin IgE are detectable in the serum of all patients with atopic dermatitis, suggesting that immune system dysregulation plays a crucial role in the course of atopic dermatitis among these underlying factors. Therefore, oral corticosteroids (dexamethasone) or topical steroids are commonly used clinically to treat atopic dermatitis. However, their side effects cannot be ignored, such as acne-like rashes, skin atrophy, telangiectasia, pigmentation, and steroid-dependent dermatitis.

[0003] Traditional Chinese herbal medicines are valued by the medical community due to their high safety profile. However, research on the effects of traditional Chinese medicine compound formulas on atopic dermatitis is relatively limited. Finding natural medicines that can regulate immune system function without toxic side effects is crucial, and developing them into safe and effective drugs or foods for treating atopic dermatitis has significant clinical implications.

[0004] It should be noted that the information disclosed in the background section above is only for understanding the background of the present invention, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0005] The main objective of this invention is to overcome the deficiencies of the above-mentioned background technology and provide a pharmaceutical composition for treating atopic dermatitis, its preparation method, and its uses.

[0006] In a first aspect, the present invention provides a pharmaceutical composition for treating atopic dermatitis, comprising active ingredients made from the following raw materials in the indicated weight ratios: 3-50 parts by weight of Coptis chinensis, 3-30 parts by weight of Scutellaria baicalensis, 3-30 parts by weight of Phellodendron chinense, and 6-50 parts by weight of Dictamnus dasycarpus.

[0007] Furthermore, the weight ratio of each raw material is as follows: 9 parts by weight of Coptis chinensis, 6 parts by weight of Scutellaria baicalensis, 6 parts by weight of Phellodendron chinense, and 9 parts by weight of Dictamnus dasycarpus.

[0008] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0009] Furthermore, the active ingredient and the excipient are formulated together into a pharmaceutically acceptable dosage form.

[0010] Furthermore, the dosage form is granules, capsules, concentrated pills, oral liquid, or tablets.

[0011] Furthermore, the active ingredient in the pharmaceutical composition is prepared by the following steps:

[0012] (1) Weigh each raw material according to the weight ratio, add ethanol, soak at room temperature for a predetermined time, then extract and filter;

[0013] (2) The filtrate from step (1) is recovered, and after removing ethanol, a first extract is obtained. The first extract is used as the active ingredient. Preferably, the process further includes step (3) extracting the first extract with ethyl acetate and drying it to obtain a powdered second extract. The second extract is used as the active ingredient.

[0014] In a second aspect, the present invention also provides a method for preparing a pharmaceutical composition for treating atopic dermatitis, comprising the following steps:

[0015] (1) Weigh each raw material according to the weight ratio described in the first aspect, add ethanol, soak at room temperature for a predetermined time, extract, and filter.

[0016] (2) The filtrate from step (1) is recovered, and after removing ethanol, a first extract is obtained. The first extract is used as the active ingredient of the pharmaceutical composition.

[0017] Furthermore, the method includes step (3) extracting the first extract with ethyl acetate and drying it to obtain a powdered second extract, which is used as the active ingredient of the pharmaceutical composition.

[0018] Furthermore, the preparation method further includes the following steps: (4) preparing a pharmaceutically acceptable dosage form by combining the second extract obtained in step (3) with a pharmaceutically acceptable excipient.

[0019] Furthermore, the dosage form is granules, capsules, concentrated pills, oral liquid, or tablets.

[0020] In a third aspect, the present invention also provides the use of the pharmaceutical composition of the first aspect in the preparation of a medicament for treating or preventing atopic dermatitis in a subject.

[0021] Furthermore, the drug is administered orally.

[0022] The beneficial effects of this invention include:

[0023] 1. The pharmaceutical composition of the present invention has a good therapeutic effect on skin lesions and itching caused by DNCB-induced atopic dermatitis, and can be used to prepare drugs and functional foods with anti-atopic dermatitis properties, providing a new option for the treatment of atopic dermatitis.

[0024] 2. Currently, the pathogenesis of atopic dermatitis, especially the cytokine pathogenesis, is receiving increasing attention. This invention demonstrates that the pharmaceutical composition of this invention can significantly improve atopic dermatitis-like skin lesions and scratching behavior in mice, reduce the levels of IgE, histamine, and TNF-α in serum, and increase the levels of ceramides in the skin. It also inhibits epidermal thickening and the infiltration of inflammatory cells and mast cells, elucidating the anti-atopic dermatitis mechanism of the pharmaceutical composition of this invention from the perspective of inflammatory response. In addition, the pharmaceutical composition of this invention can downregulate the gene expression of overreacting Th2 cytokines and upregulate the gene expression of suppressed Th1 cytokines, elucidating the regulatory mechanism of the pharmaceutical composition of this invention on the immune system. Furthermore, the pharmaceutical composition of this invention can also increase the protein expression levels of filagrin (FIG) and loricrin (LOR) in the skin, elucidating the mechanism of action of MHLJDD and MHLJDD-F in treating atopic dermatitis from the perspective of restoring skin barrier function.

[0025] 3. Experiments have shown that the pharmaceutical composition of the present invention has the advantages of rapid symptom improvement and short treatment course. The pharmaceutical raw materials of the present invention are few in number and the preparation method is simple and easy to operate, and it has wide application value. Attached Figure Description

[0026] Figures 1a-1f Specifically, these are the effects of MHLJDD and HLJDD on: (A) the thickness of mouse ear skin in specific embodiments of the present invention. Figure 1a (B) Effect of scratching behavior scores in mice Figure 1b (C) Effects of IgE and histamine levels in mouse serum ( Figure 1c (D) Effect of histamine levels in mouse serum ( Figure 1d (E) Effect of IL-1β content in mouse dorsal skin ( Figure 1e(F) Effect of IL-6 content in mouse dorsal skin ( Figure 1f Data are expressed as mean ± standard error (mean ± SEM), n = 6. Compared with the control group, *p < 0.05, **p < 0.01.

[0027] Figure 2 This invention describes the effects of MHLJDD and MHLJDD-F on mouse body weight in a specific embodiment. All data are expressed as mean ± standard error (mean ± SEM), n = 12. Compared with the NC group, #p < 0.05, ##p < 0.01; compared with the MC group, *p < 0.05, **p < 0.01.

[0028] Figure 3 This invention describes the effects of MHLJDD and MHLJDD-F on the thickness of the skin on the back of mice in a specific embodiment. All data are expressed as mean ± standard error (mean ± SEM), n = 12. Compared with the NC group, #p < 0.05, ##p < 0.01; compared with the MC group, *p < 0.05, **p < 0.01.

[0029] Figure 4 This invention relates to the effects of MHLJDD and MHLJDD-F on DNCB-induced eczema-like symptoms on the back of mice in a specific embodiment of the invention.

[0030] Figure 5 This invention relates to the effects of MHLJDD and MHLJDD-F on the eczema-like symptom scores of DNCB-induced dorsal skin in mice. All data are expressed as mean ± standard error (mean ± SEM), n = 12. Compared with the MC group, *p < 0.05, **p < 0.01.

[0031] Figure 6 This invention relates to the effects of MHLJDD and MHLJDD-F on the scores of DNCB-induced scratching behavior in mice in a specific embodiment of the invention. All data are expressed as mean ± standard error (mean ± SEM), n = 12. Compared with the NC group, #p < 0.05; ##p < 0.01; compared with the MC group, *p < 0.05, **p < 0.01.

[0032] Figures 7a-7d Specifically, these are the effects of MHLJDD and MHLJDD-F on DNCB-modeled mice in the specific embodiments of the present invention: (A) Serum TNF-α levels ( Figure 7a (B) Effects of serum IgE ( Figure 7b (C) Effects of histamine in serum ( Figure 7c (D) Effects of ceramide content in the skin ( Figure 7dData are expressed as mean ± standard error (mean ± SEM), n = 12; compared with the NC group, #p < 0.05; ##p < 0.01; compared with the MC group, *p < 0.05; **p < 0.01.

[0033] Figure 8a and Figure 8b These are the effects of MHLJDD and MHLJDD-F on the skin physiological structure of DNCB-modeled mice, as described in specific embodiments of the present invention. Figure 8a The influence of epidermal thickness () Figure 8b H&E staining was performed on skin sections from the back of mice, and the sections were observed under an optical microscope at 100x magnification. The arrows indicate the epidermal thickness.

[0034] Figure 9a and Figure 9b This invention describes the effects of MHLJDD and MHLJDD-F on the number of mast cells in the skin of DNCB-modeled mice in a specific embodiment of the invention. Toluidine blue staining was performed on skin sections from the back of the mice, staining the mast cells purple-red. The samples were observed under a light microscope at 100x magnification. All data are expressed as mean ± standard error (mean ± SEM), n = 6. Compared with the NC group, #p < 0.05, ##p < 0.01; compared with the MC group, *p < 0.05; **p < 0.01.

[0035] Figures 10a-10f The effects of MHLJDD and MHLJDD-F on the gene expression levels of inflammatory factors TNF-α, IL-1β, IL-4, IL-6, IL-13 and IFN-γ in the skin of DNCB model mice, as described in the specific embodiments of the present invention, are as follows: data are expressed as mean ± standard error (mean ± SEM), n = 6; compared with the NC group, #p < 0.05; ##p < 0.01; compared with the MC group, *p < 0.05; **p < 0.01.

[0036] Figure 11 This invention describes the effects of MHLJDD and MHLJDD-F on the expression levels of FLG and LOR proteins in the skin of DNCB-induced model mice. Figure (A) shows the protein blot bands of FLG and LOR protein expression, and Figure (B) is a quantitative representation of Figure (A). All data are expressed as mean ± standard error (mean ± SEM), n = 3. Compared with the NC group, #p < 0.05; ##p < 0.01; compared with the MC group, *p < 0.05; **p < 0.01. Specific implementation methods

[0037] The embodiments of the present invention are described in detail below. It should be emphasized that the following description is merely exemplary and not intended to limit the scope or application of the invention. Unless otherwise specified, the embodiments and features described in the embodiments can be combined with each other. In this document, unless otherwise specified, % refers to mass percentage, and "room temperature" refers to 23-25°C.

[0038] This invention provides a pharmaceutical composition for treating atopic dermatitis, comprising active ingredients made from the following raw materials in the indicated weight ratios: 3-50 parts by weight of Coptis chinensis, 3-30 parts by weight of Scutellaria baicalensis, 3-30 parts by weight of Phellodendron chinense, and 6-50 parts by weight of Dictamnus dasycarpus.

[0039] In some preferred embodiments, the weight ratio of each raw material in the pharmaceutical composition is as follows: 9 parts by weight of Coptis chinensis, 6 parts by weight of Scutellaria baicalensis, 6 parts by weight of Phellodendron chinense, and 9 parts by weight of Dictamnus dasycarpus.

[0040] In some preferred embodiments, the pharmaceutical composition further includes pharmaceutically acceptable excipients, which allow the active ingredient and excipients to be formulated together into a pharmaceutically acceptable dosage form, such as granules, capsules, concentrated pills, oral liquids, or tablets. "Pharmaceutically acceptable excipients" refers to additives other than the active ingredient that do not interfere with the biological activity of the active ingredient; these are also known as excipients and are commonly used in the pharmaceutical industry, such as binders, fillers, disintegrants, lubricants, solubilizers, flavoring agents, antibacterial agents, antioxidants, colorants, preservatives, emulsifiers, flavoring agents, osmotic pressure regulators, etc.

[0041] In some preferred embodiments, the active ingredient in the pharmaceutical composition is prepared by the following steps: (1) weighing each raw material according to the weight ratio, adding ethanol, soaking at room temperature for a predetermined time, extracting, and filtering; (2) recovering the filtrate of step (1), removing ethanol, and obtaining the first extract, which is used as the active ingredient.

[0042] In a further preferred embodiment, the method further includes step (3) extracting the first extract with ethyl acetate and drying it to obtain a powdered second extract, which is used as the active ingredient.

[0043] For example, in a specific example, the active ingredient in the pharmaceutical composition is prepared by the following steps: (1) after weighing each raw material, add 80% ethanol, soak at room temperature for 24 hours, extract by ultrasonication for 1 hour, filter, and repeat the above extraction steps twice; (2) combine the three filtrates, recover all ethanol under reduced pressure to obtain the first extract; (3) extract the first extract three times with 10 times the amount of ethyl acetate, discard the ethyl acetate, recover under reduced pressure, and freeze dry to obtain the second extract in powder form.

[0044] The pharmaceutical composition of this invention is a traditional Chinese medicine compound preparation, wherein: Coptis chinensis is the dried rhizome of Coptis chinensis Franch., Coptis deltoidea CYCheng et Hsiao, or Coptis yunnanensis Wall., all belonging to the Ranunculaceae family. It is bitter and cold in nature, and enters the heart, spleen, stomach, liver, gallbladder, and large intestine meridians. It has the effects of clearing heat and drying dampness, purging fire and detoxifying, and is commonly used for damp-heat fullness, vomiting and acid regurgitation, diarrhea, jaundice, high fever and delirium, excessive heart fire, restlessness and insomnia, hematemesis due to blood heat, red eyes, toothache, thirst, carbuncles and boils; externally, it is used to treat eczema, damp sores, and ear discharge. The main chemical components of Coptis chinensis are lignans and alkaloids, as well as volatile oils, phenolic acids, flavonoids, etc. Berberine is the most important alkaloid substance in Coptis chinensis, possessing strong anti-inflammatory, antioxidant, antibacterial, and anticancer activities. Phellodendron bark is the dried bark of the Phellodendron chinense Schneid., a plant in the Rutaceae family. It is bitter and cold in nature, and enters the kidney and bladder meridians. It has the effects of clearing heat and drying dampness, purging fire and eliminating steaming heat, and detoxifying and healing sores. It is used for damp-heat diarrhea, jaundice, dark urine, and leukorrhea. Vulvar itching, painful and burning urination, athlete's foot, bone steaming fever, night sweats, seminal emission, sores and swellings Poisoning, eczema, and weeping sores, Phellodendron bark Its main chemical components are flavonoids and alkaloids, which have anti-allergic, antioxidant, and antibacterial effects. Scutellaria baicalensis is the dried root of the plant Scutellaria baicalensis Georgi (Lamiaceae family). It is bitter and cold in nature. It enters the lung, gallbladder, spleen, large intestine, and small intestine meridians, and has the effects of clearing heat and drying dampness, purging fire and detoxifying, stopping bleeding, and calming the fetus. It is used for damp-heat syndrome, summer-heat dampness, chest tightness and nausea, damp-heat fullness, diarrhea, jaundice, lung-heat cough, high fever and thirst, blood-heat vomiting and nosebleeds, carbuncles and boils, and threatened abortion. Modern research has found that Scutellaria baicalensis has strong antibacterial, anti-inflammatory, antiviral, and anti-allergic effects, and can improve the body's immune function. Dictamnus dasycarpus is the dried root bark of the plant Dictamnus dasycarpus Turcz. (Rutaceae family). According to the 2020 Pharmacopoeia, it is bitter and cold in nature, and enters the spleen, stomach, and bladder meridians. It has the effects of clearing heat and drying dampness, dispelling wind and detoxifying. It is used for damp-heat sores, weeping eczema, urticaria, scabies, rheumatic arthralgia, jaundice and dark urine. Dictamnus dasycarpus is also widely used in dermatology. It can be used to treat tinea pedis, flat warts, pruritus, urticaria, eczema and other skin diseases. It has good anti-allergic and antipruritic effects.

[0045] This invention establishes a mouse model of atopic dermatitis and studies the therapeutic effect of a drug composition extracted from Coptis chinensis, Scutellaria baicalensis, Phellodendron chinense, and Dictamnus dasycarpus in a predetermined weight ratio on DNCB-induced atopic dermatitis-like skin lesions and pruritus. The possible mechanism of action is analyzed. By detecting various indicators in mouse serum and skin, it was found that the drug composition of this invention (hereinafter referred to as MHLJDD and MHLJDD-F, especially MHLJDD-F) can significantly improve skin lesions and pruritus in mice. Its mechanism of action may be related to inhibiting inflammatory responses, regulating immune cell function, and thus repairing the epidermal barrier function. Therefore, in vivo experiments have confirmed that the synergistic combination of Coptis chinensis, Scutellaria baicalensis, Phellodendron chinense, and Dictamnus dasycarpus in the drug composition of this invention has a good therapeutic effect on DNCB-induced atopic dermatitis-like skin lesions and pruritus.

[0046] The present invention also provides a method for preparing a pharmaceutical composition for treating atopic dermatitis, comprising the following steps:

[0047] (1) Weigh each raw material according to the weight ratio described in the first aspect, add ethanol, soak at room temperature for a predetermined time, extract, and filter.

[0048] (2) The filtrate from step (1) is recovered, and after removing ethanol, a first extract is obtained. The first extract is used as the active ingredient of the pharmaceutical composition.

[0049] Preferably, the method further includes step (3) extracting the first extract with ethyl acetate and drying it to obtain a powdered second extract, which is used as the active ingredient of the pharmaceutical composition.

[0050] Optionally, the method further includes step (4): combining the second extract obtained in step (3) with a pharmaceutically acceptable excipient to form a pharmaceutically acceptable dosage form (e.g., granules, capsules, concentrated pills, oral liquids or tablets).

[0051] The research of this invention has confirmed that both the first extract (hereinafter also referred to as MHLJDD) and the second extract (hereinafter also referred to as MHLJDD-F) can significantly improve skin lesions and itching in mice, and have a good therapeutic effect on DNCB-induced atopic dermatitis-like skin lesions and itching. Since both the first and second extracts are non-toxic, they can be administered directly as needed. In this case, the drug composition does not contain pharmaceutically acceptable excipients. When the composition contains pharmaceutically acceptable excipients, they can be mixed according to conventional pharmaceutical methods to prepare the desired dosage form of the drug.

[0052] In the preparation of a medicament for the treatment or prevention of atopic dermatitis in a subject, the medicament may be administered orally.

[0053] The present invention will be further illustrated below through some specific examples and comparative examples.

[0054] Example 1 (Preparation of the pharmaceutical composition of the present invention)

[0055] 9g of Coptis chinensis, 6g of Scutellaria baicalensis, 6g of Phellodendron chinense, and 9g of Dictamnus dasycarpus were soaked in 10 times their volume of 80% ethanol for 24 hours, then ultrasonically extracted for 1 hour. The extraction was repeated twice. The filtrates from the three extractions were combined, recovered under reduced pressure, and freeze-dried to obtain the first extract (MHLJDD), with an extraction rate of 16.45%.

[0056] MHLJDD was extracted three times with 10 times the amount of ethyl acetate. After discarding the ethyl acetate, the extract was recovered under reduced pressure and freeze-dried to obtain the second extract (MHLJDD-F). The extraction rate was calculated to be 11.67%.

[0057] Example 2 (Therapeutic effect of the pharmaceutical composition of the present invention on atopic dermatitis)

[0058] I. Experimental Materials

[0059] 1. Laboratory animals and drugs

[0060] SPF-grade Balb / c mice, female, 22-24g, were purchased from the Laboratory Animal Centre of the Chinese University of Hong Kong. The animals were housed separately, with bedding changed every two days, and underwent a 7-day acclimatization period. During this period, they had free access to water and food. The ambient temperature was 22±2℃, relative humidity was 60%, and the environment featured a 12-hour light-dark cycle.

[0061] 2. Preparation of test reagents: Prepared in the laboratory.

[0062] (1) Preparation of Comparative Sample 1: 9g of Coptis chinensis, 6g of Scutellaria baicalensis, 6g of Phellodendron chinense, and 9g of Gardenia jasminoides were soaked in 10 times the amount of 80% ethanol for 24 hours, ultrasonically extracted for 1 hour, filtered, and the above extraction steps were repeated twice. The filtrates from the three extractions were combined, recovered under reduced pressure, and freeze-dried to obtain the extract, namely Comparative Sample 1 (hereinafter also referred to as HLJDD), with an extraction rate of 50.2%. The above extract HLJDD was prepared into a test solution sample with an extract concentration of 0.03g / mL using distilled water.

[0063] (2) Experimental sample 1: The first extract (MHLJDD) obtained in Example 1 was prepared into a test solution sample with an extract concentration of 0.148 g / mL using distilled water.

[0064] (3) Experimental sample 2: The second extract (MHLJDD-F) obtained in Example 1 was prepared into test solution samples with extract concentrations of 0.026 g / mL, 0.052 g / mL and 0.104 g / mL respectively using distilled water.

[0065] (4) Positive control group test solution (hereinafter also referred to as DXM): Accurately weigh 20 mg of dexamethasone (DXM) and suspend it in 40 mL of 0.5% sodium carboxymethyl cellulose solution to prepare a test solution sample with a concentration of 0.5 mg / mL.

[0066] After the above (1) to (4) drugs are prepared, they should be stored in a refrigerator at 4°C for later use.

[0067] 3. Experimental reagents

[0068] (1) Modeling and drug administration reagents: DNCB, acetone and DXM were purchased from Sigma-Aldrich, USA; distilled water and olive oil were purchased from Watsons, Hong Kong; Coomassie brilliant blue protein quantitative reagent was purchased from Bio-Rad; mouse IgE, histamine, tumor necrosis factor-α (TNF-α), IL-4 and IL-6 kits were purchased from Abcam, USA; mouse ceramide kit was purchased from Wuhan Huamei, China.

[0069] (2) Reagents for paraffin section preparation and staining: eosin, hematoxylin, toluidine blue, xylene, ethanol, hydrochloric acid and neutral resin were all purchased from Sigma-Aldrich, USA.

[0070] (3) Reagents for Western blot (WB) assay: RIPA lysis buffer, protease inhibitor cocktail, PMSF (100mM), phosphorylated protease inhibitor, protein loading buffer, SDS-PAGE gel preparation reagent, TRIS buffer, glycine, SDS and Tween-20 were all purchased from Sigma-Aldrich, USA; PVDF membrane (0.22μm) was purchased from Bio-Rad, USA; BSA was purchased from Thermo Scientific, USA; β-actin was purchased from Santa Cruz, USA; filaggrin (FLG) and lobe protein (LOR) antibodies were purchased from Cell Signaling Technology, USA; protein marker, HRP-labeled goat anti-rabbit antibody and HRP-labeled goat anti-mouse antibody were purchased from Thermo Scientific, USA; RIPA (containing 1% protein inhibitor) was purchased from Thermo Scientific, USA.

[0071] (4) PCR assay reagents: TRIzol reagent was purchased from Thermo Scientific; chloroform and isopropanol were purchased from Sigma-Aldrich; cDNA reverse transcription kit was purchased from Takara; TaqMan rapid amplification PCR kit, TaqMan mouse TNF-α, IL-4, IL-6, IL-1β, IL-13 and IFN-γ primers were purchased from Applied Biosystems.

[0072] 4. Experimental apparatus

[0073] 752-P UV spectrophotometer, purchased from Shanghai Xianke Instrument Co., Ltd.; SHIMADZU analytical balance, purchased from Guangzhou Xiangyi Electromechanical Equipment Co., Ltd.; PL-203 electronic balance, purchased from Mettler Toledo Instruments (Shanghai) Co., Ltd.; IKA10 tissue homogenizer, purchased from IKA GmbH, Germany; BH22 optical microscope, purchased from Olympus GmbH, Japan; FLUOstar Optima microplate reader, purchased from BMG Labtec GmbH, Germany; ABI-7500 real-time PCR instrument, purchased from ABI, USA; FBZ2001-up-p standard reagent pure water system, purchased from Qingdao Fullerm Technology Co., Ltd.; benchtop high-speed refrigerated centrifuge, purchased from Heal Force; DYY-6C electrophoresis apparatus, purchased from Beijing Liuyi Instrument Factory.

[0074] II. Animal Experimentation Process

[0075] 1. Animal grouping and administration

[0076] Before the experiment, each mouse was shaved with a 2cm x 3cm area of ​​hair on its back. Except for the normal control group (NC), each mouse was treated with 200μL of 0.5% DNCB solution (DNCB dissolved in acetone: olive oil = 3:1 (volume ratio)) on its back and 20μL on each ear for 3 consecutive days. After one week, each mouse was treated with 200μL of 1% DNCB solution on its back and 20μL on each ear every 3 days for a total of 7 times. Fourteen days after sensitization with DNCB, mice were randomly divided into the following eight groups: normal group, model control (MC), positive control group (DXM, dose of 5 mg / kg (i.e., the dose concentration of 0.5 mg / mL of the positive control test solution prepared above, with a dose volume of 10 mL / kg for mice)), control sample 1 treatment group (HLJDD, dose of 0.3 g / kg), first extract (i.e., test sample 1) treatment group (MHLJDD, dose of 0.3 g / kg), low dose second extract (i.e., test sample 2) treatment group (MHLJDD-FL, dose of 0.26 g / kg), medium dose second extract (i.e., test sample 2) treatment group (MHLJDD-FM, dose of 0.52 g / kg), and high dose second extract (i.e., test sample 2) treatment group (MHLJDD-FH, dose of 1.04 g / kg), with 12 mice in each group. After grouping, mice were labeled, and their body weight was measured. The corresponding administration volume (10 mL / kg) was recorded and calculated. The normal group and the model group were given the same volume of distilled water. The remaining groups of mice were administered the above concentrations of DXM, HLJDD, MHLJDD, MHLJDD-FL, MHLJDD-FM, and MHLJDD-FH by gavage once daily for 15 days.

[0077] 2. Scoring of eczema symptoms and skin thickness test on the back of mice

[0078] On the day of grouping (day 15 after DNCB sensitization), mouse weight was recorded, and the appearance of the skin on the back was photographed. The mice were scored according to the EASI scoring rules, and the skin thickness at the midline of the mouse's back was measured using calipers. Subsequently, weight, back skin appearance, and back skin thickness were recorded weekly (days 15, 22, and 29 after DNCB sensitization). The EASI scoring rules were as follows: based on the severity of the four major eczema symptoms (erythema, edema, peeling, and lichenification), each symptom was scored from 0 to 3 points, where 0 represents no symptoms, 1 represents mild, 2 represents moderate, and 3 represents severe. The final score for each mouse was the average of the total scores for the four eczema symptoms.

[0079] 3. Mouse scratching behavior experiment

[0080] One hour after the last administration, the mice were placed in a transparent observation box. After they became familiar with the environment, a digital camera was used to record their movements for 20 minutes. The group information was then hidden, and another experimenter observed and recorded the time and number of times the mice scratched their backs with their hind legs within 20 minutes. Each scratch lasting less than 1.5 seconds was scored as 2 points, and each scratch lasting more than 1.5 seconds was scored as 4 points.

[0081] 4. Serum and skin samples

[0082] After the experiment, blood was collected from mice via orbital sampling. The blood samples were allowed to stand at room temperature for 2 hours, then centrifuged at 3000 rpm for 15 minutes, and the supernatant was separated to obtain serum samples. Mice were euthanized by cervical dislocation after blood collection, and a 1cm sample was immediately cut off. 2 Four back skin tissue samples were collected. One sample was fixed in 4% paraformaldehyde for 24 hours for pathological section preparation; one sample was placed in the cell lysis buffer provided in the ELISA kit for subsequent ELISA assays; one sample was placed in TRIzol reagent for RNA extraction and subsequent PCR assays; and one sample was placed in RIPA (containing 1% protein inhibitor) for total cellular protein extraction and subsequent Western blotting. After collecting the required tissue samples, the remaining tissue was stored at -80°C for use in subsequent experiments.

[0083] 5. Western blot detection of related protein expression

[0084] 5.1 Total Protein Extraction

[0085] Wash tissue blocks 2-3 times with cold PBS to remove blood contamination, cut into small pieces, and place in an IKA10 tissue homogenizer. Add 10 times the tissue volume of RIPA lysis buffer (add a protease inhibitor cocktail a few minutes before use) and homogenize thoroughly on ice. Transfer the homogenate to a 1.5 mL centrifuge tube and vortex. Incubate on ice for 30 min, repeatedly pipetting during this time to ensure complete cell lysis. Then centrifuge at 12000g for 10 min and collect the supernatant, which is the total protein solution.

[0086] 5.2 Protein concentration determination: Protein concentration was determined using the Coomassie brilliant blue method.

[0087] 5.3 SDS-PAGE electrophoresis

[0088] Prepare the SDS-PAGE gel according to the instructions of the SDS-PAGE gel preparation kit. After adding sufficient electrophoresis buffer, load the sample into the electrophoresis wells for electrophoresis (stacking gel voltage 70V, 1h; separating gel voltage 120V, 30min). Stop electrophoresis when bromophenol blue just runs out, and then proceed with the membrane transfer.

[0089] 5.4 Transfer of film

[0090] Prepare six 7×9cm filter papers and one appropriately sized PVDF membrane. Activate the PVDF membrane with methanol for 15 seconds before use. Place the transfer clamp, two sponge pads, filter paper, and the activated PVDF membrane in a bowl containing transfer buffer. Open the clamp so the black side is horizontal. Place the sponge pads and three layers of filter paper on the mats. Carefully peel off the separating gel and place it on the filter paper. Place the membrane on the gel, removing any air bubbles. Then cover the membrane with three more layers of filter paper, removing any air bubbles. Finally, cover with the remaining sponge pad. Transfer conditions (wet transfer): fast transfer, 250mA constant current transfer for 45 minutes.

[0091] 5.5 Immune Response

[0092] The transferred membrane was blocked for 2 hours at room temperature on a decolorizing shaker with 5% skim milk (prepared with 0.5% TBST). The primary antibody was diluted (5% skim milk dissolved in TBST; phosphorylated proteins were treated with 5% BSA dissolved in TBST), and incubated overnight at 4°C. The membrane was then washed three times with TBST on a decolorizing shaker at room temperature for 5 minutes each time. The secondary antibody was diluted 2000-fold with TBST, incubated for 2 hours at room temperature, and then washed three times with TBST on a decolorizing shaker at room temperature for 5 minutes each time.

[0093] 5.6. Light emission

[0094] Mix equal volumes of ECLA and ECLB reagents in a centrifuge tube. Place the PVDF membrane, protein side up, into the mixture for 1-2 minutes. Remove any remaining liquid, wrap the membrane, and place it in a dark box for exposure. Finally, develop and fix the membrane using developing and fixing reagents, adjusting the exposure conditions according to different light intensities.

[0095] 6. PCR detection of related gene expression

[0096] 6.1 RNA Extraction

[0097] 100 mg of skin tissue was homogenized in 1 ml of TRIzol reagent using an IKA10 tissue homogenizer and incubated on ice for 5 min. Then, 0.2 ml of chloroform was added to lyse the tissue, followed by incubation for another 2–3 min. After vortexing, the tissue was centrifuged at 12,000 g for 15 min. The supernatant was transferred to another centrifuge tube, and 0.5 ml of isopropanol was added. After incubation for 10 min, the tissue was centrifuged at 12,000 g for 10 min. The white precipitate was RNA. The supernatant was discarded, and the tissue was resuspended in 1 ml of 75% ethanol and centrifuged at 7000 g for 5 min. The supernatant was discarded, and 20–50 μL of DEPC water was added. The tissue was heated in a 55°C hot plate for 10 min to inactivate the RNA. Finally, the RNA content and purity were determined using a microplate reader.

[0098] 6.2 cDNA reverse transcription

[0099] Follow the instructions for the Takara cDNA reverse transcription kit. Load 2 μg of RNA per reaction. Store the transcribed cDNA at -80°C for subsequent detection of related genes.

[0100] 6.3 Real-time quantitative PCR assay

[0101] The TaqMan rapid amplification PCR kit from Applied Biosystems was used according to the instructions. The transcribed cDNA was bound to TaqMan mouse primers for IL-4, IL-6, IL-13, IL-31, IFN-γ, TSLP, and FLG, with β-actin as an internal control. The reaction conditions were 50℃ for 2 min, 95℃ for 10 min, followed by 40 cycles of amplification (95℃ for 15 s and 60℃ for 1 min). The fold increase of the target gene was calculated using the ΔΔCT method: fold increase = 2^-ΔΔCT, where ΔΔCT = (CT value of target gene in experimental group - CT value of β-actin) - (CT value of target gene in control group - CT value of β-actin).

[0102] 7. Pathological and histological observation

[0103] 7.1 Preparation of Paraffin Sections

[0104] Skin tissue was fixed in 4% paraformaldehyde for 24 hours and then automatically dehydrated in a tissue dehydrator. It was then embedded in paraffin to form a paraffin block, which was then dehydrated, cleared, and sealed to form paraffin sections.

[0105] 7.2 Pathological histological observation and epidermal layer thickness measurement

[0106] The prepared paraffin sections are dewaxed with xylene, then stained with ethanol of varying concentrations (100%, 95%, 80%, and 70%), and finally placed in distilled water. They are then ready for staining with hematoxylin and eosin. After staining, the sections are dehydrated with ethanol of varying concentrations (70%, 80%, 95%, and 100%), and then cleared with xylene. Resin is then applied to the cleared sections, which are then sealed with coverslips. Inflammatory cell infiltration in the dermis and epidermal thickness can then be observed and measured under an optical microscope.

[0107] 7.3 Toluidine blue staining to determine mast cell infiltration

[0108] The prepared paraffin sections are dewaxed with xylene, then passed through high-concentration to low-concentration ethanol, and finally immersed in distilled water, ready for staining with toluidine blue. After staining, the sections are dehydrated with low-concentration to high-concentration ethanol, and then cleared with xylene. Resin is then applied to the cleared sections, which are then sealed with coverslips. Under an optical microscope, mast cells in the dermis of the skin can be observed to be stained purplish-red, and the number of mast cells can be automatically counted using ImageJ software.

[0109] 8. Determination of inflammatory factor content in the skin

[0110] Perform the procedure according to the ELISA kit instructions. Accurately weigh the skin tissue, add cell lysis buffer, and prepare a tissue homogenate using a high-speed homogenizer. Centrifuge at 12,000g and 4°C for 15 minutes, and separate the supernatant to determine the content of IL-4 and IL-6 in the skin tissue.

[0111] 9. Statistical Analysis

[0112] Data are expressed as mean ± standard error (mean ± SEM). GraphPad Prism 9 software was used for graphing and statistical analysis. One-way ANOVA was used to compare differences between groups, and Dunnett's method was used for pairwise multiple comparisons between groups. A p-value < 0.05 was considered statistically significant. Furthermore, compared with the normal group, #p < 0.05 and ##p < 0.01; compared with the model group, *p < 0.05 and **p < 0.01.

[0113] III. Experimental Results of the Therapeutic Effects of Each Sample on Atopic Dermatitis

[0114] In the following figures, "Control" or "NC" represents the normal group; "DNCB" or "MC" represents the model group; "DNCB+HLJDD" represents the control group (HLJDD, 0.3 g / kg) treatment group; "DNCB+MHLJDD" or "MHLJDD" represents the experimental group (MHLJDD, 0.3 g / kg) treatment group; "DNCB+MHLJDD-FL" or "MHLJDD-FL" represents the low-dose experimental group (MHLJDD, 0.3 g / kg) treatment group. The treatment group was HLJDD-FL (0.26 g / kg); "DNCB+MHLJDD-FM" or "MHLJDD-FM" both indicate the treatment group with a medium dose of experimental sample 2 (MHLJDD-FL, 0.52 g / kg); "DNCB+MHLJDD-FH" or "MHLJDD-FH" both indicate the treatment group with a high dose of experimental sample 2 (MHLJDD-FL, 1.04 g / kg); "DXM" indicates the positive control group (DXM, 5 mg / kg).

[0115] 1. The effects of experimental sample 1 (MHLJDD) and control sample 1 (HLJDD) on DNCB-induced atopic dermatitis-like lesions in mice were compared. Comparison

[0116] like Figures 1a-1f As shown, the study of this invention found that after two weeks of continuous administration, experimental sample 1 (MHLJDD, 0.3 g / kg) significantly inhibited DNCB-induced ear skin thickening and scratching behavior in mice, and its effect was significantly better than that of control sample 1 (HLJDD, 0.3 g / kg). In addition, MHLJDD also significantly inhibited DNCB-induced serum IgE and histamine levels in mice, and its effect was significantly better than that of HLJDD. Both MHLJDD and HLJDD significantly inhibited the content of IL-4 in the skin of DNCB-induced mice, but MHLJDD was significantly more effective than HLJDD in inhibiting the content of IL-6 in the skin. Therefore, it can be concluded that MHLJDD has a better therapeutic effect on atopic dermatitis-like lesions in mice compared to HLJDD.

[0117] 2. Effects of experimental sample 1 (MHLJDD) and experimental sample 2 (MHLJDD-F) on mouse body weight

[0118] This study found that MHLJDD and MHLJDD-F had no significant adverse effects on mouse body weight, but the positive control drug dexamethasone had a significant inhibitory effect on mouse body weight. The changes in body weight in each group were as follows: Figure 2 As shown ( Figure 2 In the example, on day 15, the bars from left to right represent NC, MC, DXM, MHLJDD, MHLJDD-FL, MHLJDD-FM, and MHLJDD-FH, respectively; the same applies to day 22 and day 29.

[0119] 3. Effects of experimental sample 1 (MHLJDD) and experimental sample 2 (MHLJDD-F) on the thickness of the skin on the back of mice.

[0120] like Figure 3 As shown ( Figure 3 In the diagram, taking day 15 as an example, the columns from left to right represent NC, MC, DXM, MHLJDD, MHLJDD-FL, MHLJDD-FM, and MHLJDD-FH, respectively; the same applies to days 22 and 29. The study of this invention found that after two weeks of oral administration of MHLJDD and MHLJDD-F, the thickening of the dorsal skin of mice induced by DNCB was significantly improved. The inhibitory effect of MHLJDD-F was stronger than that of MHLJDD, and it was dose-dependent (i.e., the inhibitory effect increased with the increase of the dose of MHLJDD-F), indicating that MHLJDD and MHLJDD-F can inhibit the thickening of the epidermis induced by DNCB.

[0121] 4. Experimental Sample 1 (MHLJDD) and Experimental Sample 2 (MHLJDD-F) on DNCB-induced eczema-like symptoms on the back of mice Impact

[0122] like Figure 4 and Figure 5 As shown ( Figure 5 In the diagram, taking day 15 as an example, the columns from left to right represent MC, DXM, MHLJDD, MHLJDD-FL, MHLJDD-FM, and MHLJDD-FH, respectively; the same applies to days 22 and 29. The study of this invention found that DNCB-induced eczema-like symptoms on the back of mice, such as skin erythema, edema, peeling, and lichenification, were significantly improved in the MHLJDD and MHLJDD-F treatment groups. Two weeks after administration, the EASI scores of the MHLJDD treatment group and the high-dose MHLJDD-F treatment group were significantly lower than those of the model group (p < 0.01). The high-dose MHLJDD-F showed the best therapeutic effect, comparable to that of the positive control drug dexamethasone, indicating that both MHLJDD and MHLJDD-F can significantly improve DNCB-induced eczema-like symptoms.

[0123] 5. The effect of experimental sample 1 (MHLJDD) and experimental sample 2 (MHLJDD-F) on DNCB-induced dorsal skin itching symptoms in mice. Influence

[0124] like Figure 6 As shown, the study of this invention found that the pruritus symptoms on the back of mice induced by DNCB were significantly improved in the medium- and high-dose MHLJDD-F treatment group, and the scratching behavior of mice was significantly reduced (p<0.01 compared with the model group). Although MHLJDD also had a certain inhibitory effect, the effect was not as good as that of high-dose MHLJDD-F, indicating that MHLJDD-F has a significant antipruritic effect on DNCB-induced pruritus in mice.

[0125] 6. Experimental Sample 1 (MHLJDD) and Experimental Sample 2 (MHLJDD-F) on the effects of IgE, histamine, and TNF-α levels in the serum of DNCB-induced mice. Effects of α and ceramide content in the skin

[0126] like Figures 7a-7d As shown in the study, the levels of IgE, histamine, and TNF-α in the serum of DNCB-induced mice were significantly reduced in the MHLJDD and MHLJDD-F treatment groups. The high-dose MHLJDD-F showed the most significant inhibitory effect, indicating that MHLJDD-F has a more significant inhibitory effect on DNCB-induced IgE, histamine, and TNF-α compared to MHLJDD, and can more effectively suppress the body's inflammation and immune response. Furthermore, after two weeks of treatment with MHLJDD and MHLJDD-F, the ceramide content in the skin of DNCB-induced mice was significantly increased, indicating that MHLJDD and MHLJDD-F have the function of protecting skin function and maintaining skin hydration.

[0127] 7. Effects of experimental sample 1 (MHLJDD) and experimental sample 2 (MHLJDD-F) on the physiological structure of skin in DNCB-induced model mice.

[0128] like Figures 8a-8b as well as Figures 9a-9bAs shown in the present invention, the study found that the physiological structure of the skin in DNCB-induced model mice was significantly altered, such as a significant increase in epidermal thickness, a noticeable flattening of the wavy line between the epidermis and dermis, and significant infiltration of inflammatory cells and mast cells in the dermis. However, after treatment with MHLJDD and MHLJDD-F, the above-mentioned pathological structural changes were significantly improved, and the epidermal thickness was significantly reduced (p<0.01 compared with the model group). The inhibitory effect was most significant in the high-dose MHLJDD-F treatment group, with the epidermal thickness comparable to that of the normal group mice and less infiltration of inflammatory cells in the dermis. In addition, after treatment with MHLJDD and MHLJDD-F, the number of mast cells in the skin of mice induced by DNCB was significantly reduced (p<0.01 compared with the model group), indicating that both MHLJDD and MHLJDD-F can effectively improve the changes in the physiological structure of mouse skin caused by DNCB, slow down the thickening of the epidermis, and inhibit the inflammatory response in the skin.

[0129] 8. Experimental Sample 1 (MHLJDD) and Experimental Sample 2 (MHLJDD-F) on the inflammatory factor TNF-α in the skin of DNCB-induced mice. Effects of α, IL-1β, IL-4, IL-6, IL-13 and IFN-γ gene expression levels

[0130] like Figures 10a-10f As shown, the study of this invention found that the mRNA expression levels of inflammatory factors TNF-α, IL-1β, IL-4, IL-6, and IL-13 secreted by Th2 cells in the skin of DNCB-induced model mice were significantly increased, while the mRNA expression level of IFN-γ, a cytokine regulated by Th1 cells, was correspondingly decreased. After treatment with MHLJDD and MHLJDD-F, the mRNA expression levels of TNF-α, IL-1β, IL-4, IL-6, and IL-13 in the skin of DNCB-induced model mice decreased, while the mRNA expression level of IFN-γ increased accordingly. The regulatory effect of high-dose MHLJDD-F was the most significant, indicating that MHLJDD and MHLJDD-F can regulate the gene expression levels of cytokines secreted by Th1 and Th2 cells, thereby correcting the Th1 / Th2 cell dysfunction induced by DNCB.

[0131] 9. Experimental Sample 1 (MHLJDD) and Experimental Sample 2 (MHLJDD-F) on FLG and LOR proteins in the skin of DNCB-induced mouse models The impact of expression

[0132] like Figure 11As shown in the study, the protein expression levels of two key protective proteins, FLG and LOR, in the skin of DNCB-induced model mice were significantly decreased, indicating that DNCB caused damage to the epidermal barrier function of the mouse skin. After two weeks of treatment with MHLJDD and MHLJDD-F, the protein expression levels of FLG and LOR in the skin of DNCB-induced model mice significantly rebounded (p<0.01 compared with the model group), with the high-dose MHLJDD-F treatment group showing the most significant effect. This indicates that MHLJDD and MHLJDD-F can increase the protein expression levels of FLG and LOR in the skin of DNCB-induced model mice, thereby achieving the effect of repairing the skin barrier.

[0133] IV. Discussion

[0134] Atopic dermatitis not only causes obvious skin damage such as itching, edema, erythema, peeling, and lichenification, but also significantly impacts the body's immune function. If left untreated, it can develop into a systemic immune disease, threatening health and severely affecting quality of life. Due to the serious side effects of current hormone and steroid medications, traditional Chinese medicine and natural products are increasingly favored by dermatologists. According to traditional Chinese medicine theory, atopic dermatitis is caused by inherent intolerance, internal damp-heat, and external wind-evil, resulting in a battle between wind, dampness, and heat that invades the skin. Current pharmacological research on traditional Chinese medicine treatment of atopic dermatitis mainly focuses on its anti-inflammatory, anti-allergic, and immunomodulatory activities, while research on its ability to repair the skin barrier function is limited.

[0135] In a preliminary experiment, this invention compared the anti-atopic dermatitis effects of experimental sample 1 (MHLJDD) and control sample 1 (HLJDD) in a DNCB-induced mouse atopic dermatitis model. The results showed that MHLJDD had a significant therapeutic effect on DNCB-induced atopic dermatitis-like skin lesions and itching in mice, and its effect was superior to HLJDD. Subsequently, MHLJDD was extracted and optimized using biochromatography (i.e., MHLJDD was extracted with ethyl acetate and its components were optimized to remove many highly ester-soluble components), yielding MHLJDD-F. The research objective of this invention is to find an anti-atopic dermatitis drug with better therapeutic efficacy and fewer side effects. Therefore, this invention uses MHLJDD and MHLJDD-F in animal experiments to explore their therapeutic effects on atopic dermatitis.

[0136] This invention employs an in vivo model of atopic dermatitis-like skin lesions induced by DNCB in mice, serving as a validation model to simulate human atopic dermatitis. In the study, mice with DNCB applied to their backs and ears exhibited typical atopic dermatitis-like lesions. Compared to the model group, mice treated with MHLJDD and MHLJDD-F showed significant improvement in erythema, edema, peeling, and lichenification. Furthermore, the high-dose MHLJDD-F treatment group demonstrated superior therapeutic efficacy compared to the MHLJDD treatment group. These results suggest that MHLJDD-F is more effective than MHLJDD in treating DNCB-induced atopic dermatitis-like skin lesions and itching in mice.

[0137] Inflammation and immune dysregulation are two key factors in the pathogenesis of atopic dermatitis. Current pharmacological research on the treatment of atopic dermatitis mainly focuses on its anti-inflammatory, anti-allergic, and immunomodulatory mechanisms. Helper T cells primarily function to secrete cytokines with immunomodulatory and effector functions, playing a crucial role in the immune response. They are mainly divided into two functional subsets: Th1 and Th2. The main function of Th1 / Th2 cells is to regulate the immune response. Th1 cells mainly secrete cytokines such as IFN-γ and IL-2, while Th2 cells mainly secrete IL-6 and IL-4, primarily mediating humoral immunity. The Th1 / Th2 cell immune process mainly achieves a dynamic balance through the mutual inhibition of different cytokines. Under normal conditions, Th1 / Th2 cells are in a state of dynamic equilibrium. If this balance is disrupted, shifting towards one side, a Th1 / Th2 "drift" state occurs, leading to disease. Current research has confirmed that Th1 / Th2 imbalance can cause atopic dermatitis. Under the stimulation of exogenous factors, antigen-specific CD4+ T cells mainly differentiate into Th2 cells, releasing large amounts of cytokines such as IL-4 and IL-13 to stimulate B cells to secrete IgE. The binding of IgE to mast cells releases large amounts of histamine, further exacerbating the allergic reaction and promoting the entry of lymphokines and macrophage cytokines into the skin lesions. Simultaneously, eosinophils are released into the peripheral blood, leading to a systemic allergic reaction. Therefore, measuring the levels of IgE, histamine, and various inflammatory factors in serum can explain and assess the severity of atopic dermatitis. The research of this invention indicates that the anti-atopic dermatitis effects of MHLJDD and MHLJDD-F may be closely related to their anti-inflammatory and anti-allergic properties. Both can reduce serum levels of IgE, histamine, and TNF-α, and inhibit the gene expression levels of Th2 cytokines and their associated inflammatory factors TNF-α, IL-1β, IL-4, IL-6, and IL-13 in skin tissue, with the high-dose MHLJDD-F showing the most significant regulatory effect. The results of this invention clearly demonstrate that MHLJDD and MHLJDD-F can exert their anti-atopic dermatitis effects by inhibiting inflammatory responses and regulating immune function.

[0138] Impaired epidermal barrier function is also a crucial factor in the pathogenesis of atopic dermatitis. The absence of FLG and LOR proteins can lead to epidermal barrier damage, reduced ceramide content, and accelerated skin moisture loss, resulting in symptoms such as dry skin and peeling. In this invention, both MHLJDD and MHLJDD-F can increase the expression levels of FLG and LOR proteins in the skin and increase the content of ceramides, thereby restoring epidermal barrier function, maintaining skin hydration, and reducing dryness. High-dose MHLJDD-F showed the most significant effect in restoring ceramide levels in mouse skin. These results suggest that restoring epidermal barrier function may be the molecular biological mechanism by which MHLJDD and MHLJDD-F combat atopic dermatitis.

[0139] In summary, MHLJDD and MHLJDD-F exhibit good anti-atopic dermatitis activity, and their effects may be related to inhibiting inflammation, regulating immunity, and protecting skin barrier function. In vivo experiments demonstrated that continuous administration of MHLJDD and MHLJDD-F for two weeks significantly improved atopic dermatitis-like skin lesions and scratching behavior in mice. Biochemical assays using reagent kits to measure serum levels of immunoglobulin E (IgE), histamine, tumor necrosis factor-α (TNF-α), and skin ceramide levels showed that MHLJDD and MHLJDD-F reduced serum levels of IgE, histamine, and TNF-α, and increased skin ceramide levels. Histopathological examination revealed changes in epidermal thickness and the presence of inflammatory cells and mast cells. The results showed that MHLJDD and MHLJDD-F inhibited epidermal thickening and the infiltration of inflammatory cells and mast cells. Real-time polymerase chain reaction (PCR) was used to detect the gene expression levels of helper T cells and inflammation-related factors. Furthermore, Western blotting was used to detect the expression levels of two key proteins, filaggrin (FLG) and lobe-like protein (LOR), in skin tissue. These results indicated that MHLJDD and MHLJDD-F downregulated the gene expression of overreacting Th2 cytokines and upregulated the gene expression of suppressed Th1 cytokines, thereby increasing the protein expression levels of FLG and LOR in the skin and repairing the skin's epidermal barrier function. This invention demonstrates that MHLJDD and MHLJDD-F have good therapeutic effects on DNCB-induced atopic dermatitis-like skin lesions and itching, and show promising potential for development into anti-atopic dermatitis drugs or functional foods.

[0140] The pharmaceutical compositions of the present invention can be formulated together with the active ingredients and excipients into various dosage forms, such as granules, capsules, concentrated pills, oral liquids or tablets. Specific embodiments of the present invention will be further described below.

[0141] Examples 3-20 are examples of preparing the pharmaceutical compositions of the present invention into tablets. The components of each example are shown in Table 1 below:

[0142] Table 1

[0143]

[0144]

[0145] The preparation process of Examples 3-20 above is as follows: Take MHLJDD-F, add at least one of lactose, dextrin, and starch (see Table 1 above), mix evenly, use 7% starch slurry as a binder, wet granulate, dry, add magnesium stearate or talc powder and mix evenly, compress into 10,000 tablets, each containing a predetermined amount of the second extract (see Table 1 above), and a predetermined net weight per tablet (see Table 1 above). For oral administration, take 4 tablets twice daily for the treatment of atopic dermatitis. Symptoms include: skin erythema, edema, peeling, or lichenification.

[0146] Examples 21-32 are examples of preparing the pharmaceutical compositions of the present invention into capsules. The components of each example are shown in Table 2 below:

[0147] Table 2

[0148]

[0149] The preparation process of Examples 21-32 above is as follows: Take MHLJDD-F, add at least one of lactose and starch (see Table 2 above), mix evenly, use 7% starch slurry as a binder, wet granulate, dry, add magnesium stearate and mix evenly, fill into No. 1 capsules to make 10,000 capsules, each capsule containing a predetermined amount of the second extract (see Table 2 above), and each capsule having a predetermined net weight (see Table 2 above). For oral administration, take 4 capsules twice daily for the treatment of atopic dermatitis. Symptoms include: skin erythema, edema, peeling, or lichenification.

[0150] Example 33 (Oral Liquid)

[0151] Take 50g of MHLJDD-F, add 0.5mL of solubilizer prosamin and 15% ethanol, and stir thoroughly. Then add appropriate amounts of adjuvants (such as 0.1% sorbic acid as an antibacterial agent, 0.1% citric acid as an antioxidant, and 0.1% mannitol as a flavoring agent), dissolve evenly, filter to clarify, and fill the contents into ampoules or easy-open bottles, then sterilize. The specification is an oral solution containing 166mg of extract per bottle, with a net content of 10mL per bottle. For oral administration, take 2 bottles twice daily for the treatment of atopic dermatitis. Symptoms include: skin erythema, edema, peeling, or lichenification.

[0152] Example 35 (Oral Liquid)

[0153] Take 100g of MHLJDD-F, add 15% ethanol, and stir thoroughly. Then add appropriate amounts of adjuvants (such as 0.1% sorbic acid as an antibacterial agent, 0.1% citric acid as an antioxidant, and 0.1% mannitol as a flavoring agent), dissolve evenly, filter to clarify, and fill the contents into ampoules or easy-open bottles, then sterilize. The specification is an oral solution containing 300mg of extract per bottle, with a net content of 10mL per bottle. For oral administration, take 2 bottles twice daily for the treatment of atopic dermatitis. Symptoms include: skin erythema, edema, peeling, or lichenification.

[0154] Example 36 (Oral Liquid)

[0155] Take 200g of MHLJDD-F, add 15% ethanol, and stir thoroughly. Then add appropriate excipients (such as 0.1% sorbic acid as an antibacterial agent, 0.1% citric acid as an antioxidant, and 0.1% mannitol as a flavoring agent), dissolve evenly, filter to clarify, and fill the contents into ampoules or easy-open bottles, then sterilize. The specification is an oral solution containing 600mg of extract per bottle, with a net content of 10mL per bottle. For oral administration, take 2 bottles twice daily for the treatment of atopic dermatitis. Symptoms include: skin erythema, edema, peeling, or lichenification.

[0156] Example 37 (Oral Liquid)

[0157] Take 300g of MHLJDD-F, add 15% ethanol, and stir thoroughly. Then add appropriate amounts of adjuvants (such as 0.1% sorbic acid as an antibacterial agent, 0.1% citric acid as an antioxidant, and 0.1% mannitol as a flavoring agent), dissolve evenly, filter to clarify, and fill the contents into ampoules or easy-open bottles, then sterilize. The specification is an oral solution containing 900mg of extract per bottle, with a net content of 10mL per bottle. For oral administration, take 2 bottles twice daily for the treatment of atopic dermatitis. Symptoms include: skin erythema, edema, peeling, or lichenification.

[0158] Example 38 (Oral Liquid)

[0159] Take 400g of MHLJDD-F, add 12% ethanol, and stir thoroughly. Then add appropriate amounts of adjuvants (such as 0.1% sorbic acid as an antibacterial agent, 0.1% citric acid as an antioxidant, and 0.1% mannitol as a flavoring agent), dissolve evenly, filter to clarify, and fill the contents into ampoules or easy-open bottles, then sterilize. The specification is an oral solution containing 1333mg of extract per bottle, with a net content of 10mL per bottle. For oral administration, take one bottle twice daily for the treatment of atopic dermatitis. Symptoms include: skin erythema, edema, peeling, or lichenification.

[0160] Example 39 (Oral Liquid)

[0161] Take 500g of MHLJDD-F, add 10% ethanol, and stir thoroughly. Then add appropriate amounts of adjuvants (such as 0.1% sorbic acid as an antibacterial agent, 0.1% citric acid as an antioxidant, and 0.1% mannitol as a flavoring agent), dissolve evenly, filter to clarify, and fill the contents into ampoules or easy-open bottles, then sterilize. The specification is an oral solution containing 1666mg of extract per bottle, with a net content of 10mL per bottle. For oral administration, take one bottle twice daily for the treatment of atopic dermatitis. Symptoms include: skin erythema, edema, peeling, or lichenification.

[0162] In addition, in other embodiments, the dosage form may also be granules or concentrated pills.

[0163] The above description, in conjunction with specific / preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various substitutions or modifications can be made to these described embodiments without departing from the inventive concept, and all such substitutions or modifications should be considered within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for treating atopic dermatitis, characterized in that, The pharmaceutical composition comprises an active ingredient, which is made from the following raw materials in the following weight ratios: 9 parts by weight of Coptis chinensis, 6 parts by weight of Scutellaria baicalensis, 6 parts by weight of Phellodendron chinense, and 9 parts by weight of Dictamnus dasycarpus. The active ingredient is prepared by the following steps: (1) Weigh each raw material according to the weight ratio, add ethanol, soak at room temperature for a predetermined time, then extract and filter; (2) The filtrate from step (1) was recovered, and after removing the ethanol, the first extract was obtained; (3) The first extract is extracted with ethyl acetate and dried to obtain a powdered second extract, which is used as the active ingredient.

2. The pharmaceutical composition for treating atopic dermatitis according to claim 1, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

3. The pharmaceutical composition for treating atopic dermatitis according to claim 2, characterized in that, The active ingredient and the excipient are formulated together into a pharmaceutically acceptable dosage form.

4. The pharmaceutical composition for treating atopic dermatitis according to claim 3, characterized in that, The dosage form is granules, capsules, concentrated pills, oral liquid, or tablets.

5. A method for preparing a pharmaceutical composition for treating atopic dermatitis, characterized in that, Includes the following steps: (1) Weigh each raw material according to the weight ratio described in claim 1, add ethanol, soak at room temperature for a predetermined time, extract, and filter; (2) The filtrate from step (1) was recovered, and after removing the ethanol, the first extract was obtained; (3) The first extract is extracted with ethyl acetate and dried to obtain a powdered second extract, which is used as the active ingredient of the pharmaceutical composition.

6. The preparation method according to claim 5, characterized in that, It also includes the following steps: (4) The second extract obtained in step (3) is combined with a pharmaceutically acceptable excipient to form a pharmaceutically acceptable dosage form.

7. The preparation method according to claim 6, characterized in that, The dosage form is granules, capsules, concentrated pills, oral liquid, or tablets.

8. Use of the pharmaceutical composition according to any one of claims 1-4 in the preparation of a medicament for treating or preventing atopic dermatitis in a subject.

9. The use as described in claim 8, characterized in that, The drug is administered orally.

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