A traditional Chinese medicine extract with antibacterial, anti-inflammatory and antipyretic effects, and an active ingredient self-assembled nanomaterial and application thereof

Abstract

The application discloses a traditional Chinese medicine extract with antibacterial, anti-inflammatory and antipyretic effects and an active component self-assembled nanomaterial of the traditional Chinese medicine extract and application, wherein the polygonum capitatum extract and the active component self-assembled nanomaterial have the effects of inhibiting bacteria and relieving inflammation and can be used for treating atopic dermatitis. The in-vitro bacteriostatic experiment and animal experiment results verify that the polygonum capitatum extract and the active component self-assembled nanomaterial have the effects of resisting bacteria and inflammation and can effectively treat atopic dermatitis.

Description

Technical Field

[0001] This invention relates to a traditional Chinese medicine extract and its application, specifically to a traditional Chinese medicine extract with antibacterial, anti-inflammatory, and antipyretic effects, its active ingredient self-assembled nanomaterials, and their applications. The traditional Chinese medicine extract and its active ingredient self-assembled nanomaterials can be used to treat atopic dermatitis. Background Technology

[0002] Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disease. It is considered a systemic disease due to its common comorbidities with other atopic disorders such as allergic rhinitis and asthma. The exact etiology and pathogenesis of atopic dermatitis are not fully understood. Current research suggests that the pathogenesis of atopic dermatitis involves multiple abnormalities, including epigenetic dysregulation, immune dysfunction, skin barrier impairment, and microbiome dysbiosis, all influenced by the interaction of genetic susceptibility and environmental triggers. Epigenetic modifications primarily affect skin barrier integrity and immune homeostasis; skin barrier dysfunction is the most prominent clinical manifestation of atopic dermatitis pathology. Notably, individuals carrying loss-of-function mutations in keratinocytes (FLG), a core structural protein of the epidermal barrier, have a significantly increased risk of developing atopic dermatitis.

[0003] Studies have found that the pathogenesis of atopic dermatitis (AD) is driven by an imbalanced Th1 / Th2 cell balance. Th1 cells secrete interferon-γ (IFN-γ), activating M1 macrophages to fight intracellular pathogens and mediating cellular immunity. Conversely, Th2 cells produce interleukin-4 (IL-4), stimulating eosinophils, mast cells, and M2 macrophages to coordinate humoral immunity. IFN-γ promotes the differentiation of T cells and B cells, particularly polarizing Th0 precursor cells toward a Th1 phenotype, while inhibiting Th2 cell proliferation. Furthermore, IFN-γ inhibits IgE synthesis, suggesting that in the acute Th2-dominant phase of atopic dermatitis, enhanced Th1-related cytokines may alleviate IL-4-driven cytokine and gene expression.

[0004] This Th2 bias (manifested as elevated IL-4 and decreased IFN-γ levels) contributes to epidermal barrier dysfunction in patients with atopic dermatitis. Both affected and non-affected skin exhibit microbiome dysbiosis, characterized by increased Staphylococcus aureus colonization and decreased microbial diversity, along with associated metabolic disorders, all of which exacerbate skin inflammation. Mechanical trauma from persistent scratching further intensifies the disease by inducing the release of pro-inflammatory mediators and autoantigens from keratinocytes and triggers the production of specific IgE. Non-immune factors, including neuroendocrine interactions, also play a role in regulating inflammatory progression. Currently, there are many types of drugs used clinically to treat atopic dermatitis (AD): Th2-targeted therapies have shown promise, with promising drugs including monoclonal IL-4, IL-13, IL-22, and IL-31 receptor antibodies, phosphodiesterase-4 inhibitors, and JAK inhibitors (topical and systemic), most of which have been tested in phase 2-3 trials. However, all of these drugs have certain drawbacks. In a comparative study, compared with placebo, both the IL-4 antibody dupilumab and the JAK inhibitor abrocitinib reduced signs and symptoms of atopic dermatitis; abrocitinib was superior to dupilumab in reducing itching at 2 weeks, but the results of the two drugs were similar in other aspects. Adverse reactions such as skin infections or asthma attacks require careful evaluation of the future use of these new therapies in sensitive populations (children) and patients with typical complications of atopic dermatitis. For example, many patients have long-term, functionally impaired conjunctivitis after receiving dupilumab treatment, especially when it is combined with seasonal allergic conjunctivitis. JAK inhibitors carry risks of thromboembolism and cancer, and may be associated with respiratory infections, shingles, headaches, diarrhea, and decreased white blood cell counts. Therefore, finding a safe, stable, and effective drug treatment for atopic dermatitis is of great importance. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to address the shortcomings of existing atopic dermatitis treatments, such as strong toxic side effects, by providing a multi-component, multi-target treatment scheme based on the natural drug Polygonum capitatum. This scheme aims to treat atopic dermatitis more safely and effectively, improve patients' quality of life, and exert antibacterial, anti-inflammatory, and antipyretic effects.

[0006] Technical Solution: Based on the above objectives, the present invention provides an extract of the traditional Chinese medicine Polygonum capitatum, wherein the extract is obtained by extracting Polygonum capitatum using pure water or 50%~95% ethanol as a solvent. Preferably, the extract is obtained by extracting Polygonum capitatum using pure water, 50% ethanol, 60% ethanol, 70% ethanol, or 90%~95% ethanol as a solvent. More preferably, the extract is obtained by extracting Polygonum capitatum using pure water, 50% ethanol, 60% ethanol, 70% ethanol, or 95% ethanol as a solvent.

[0007] The preparation of the extract of the traditional Chinese medicine Polygonum capitatum is made by mixing the extract of the traditional Chinese medicine Polygonum capitatum with pharmaceutical excipients to form any one of the following: ointment, liniment, patch, lotion, solution, powder, spray, syrup, suppository, tablet, pill, granule, capsule, injection, and gastrointestinal coagulated sustained-release preparation.

[0008] The preparation method of the extract of the traditional Chinese medicine Polygonum capitatum includes the following steps: pulverizing Polygonum capitatum, adding pure water or 50%~95% ethanol respectively, heating and refluxing for extraction, and concentrating the filtrate. Preferably, the preparation method of the extract of the traditional Chinese medicine Polygonum capitatum includes the following steps: pulverizing Polygonum capitatum, adding pure water, 50% ethanol, 60% ethanol, 70% ethanol or 90%~95% ethanol respectively, heating and refluxing for extraction, and concentrating the filtrate. More preferably, the preparation method of the extract of the traditional Chinese medicine Polygonum capitatum includes the following steps: pulverizing Polygonum capitatum, adding pure water, 50% ethanol, 60% ethanol, 70% ethanol or 95% ethanol respectively, heating and refluxing for extraction, and concentrating the filtrate.

[0009] The method for preparing the extract of the traditional Chinese medicine Polygonum capitatum involves concentrating the filtrate to 1 / 8 to 1 / 15 of its total volume.

[0010] A method for extracting gallic acid and protocatechuic acid from the extract of the traditional Chinese medicine Polygonum capitatum, wherein the extract of Polygonum capitatum is separated by preparative liquid chromatography to obtain gallic acid and protocatechuic acid.

[0011] The self-assembled nanomaterials of active ingredients from the traditional Chinese medicine Polygonum capitatum are formed by the self-assembly of berberine hydrochloride, gallic acid, and protocatechuic acid through non-covalent interactions, with a molar ratio of 1:1:0.2 to 1:1:1. Preferably, the particle size of the self-assembled nanomaterials of active ingredients from Polygonum capitatum is 150-350 nm.

[0012] The self-assembled nanomaterials containing the active ingredients of the traditional Chinese medicine Polygonum capitatum include non-covalent interactions such as hydrogen bonds and π-π stacking; the gallic acid and protocatechuic acid are derived from the extract of the traditional Chinese medicine Polygonum capitatum or are commercially available.

[0013] A pharmaceutical preparation is prepared from self-assembled nanomaterials containing the active ingredients of the traditional Chinese medicine Polygonum capitatum, with or without pharmaceutically acceptable excipients. Preferably, the dosage form of the pharmaceutical preparation includes ointments, liniments, patches, lotions, solutions, powders, sprays, syrups, suppositories, tablets, pills, granules, capsules, injections, and enteric-coated sustained-release formulations.

[0014] The method for preparing the self-assembled nanomaterial includes the following steps: dissolving berberine hydrochloride, gallic acid, and protocatechuic acid separately in an organic solvent to prepare solutions; mixing them in a molar ratio, heating to 40-90°C and stirring; transferring to a 2000-5000 Da dialysis bag for dialyzing, and collecting the solution in the bag to obtain the nanomaterial. Preferably, the organic solvent includes methanol. More preferably, after dissolving berberine hydrochloride, gallic acid, and protocatechuic acid separately in an organic solvent to prepare solutions, the solutions can be filtered through a microporous membrane. More preferably, the pore size of the microporous membrane is 0.1-0.5 μm, preferably 0.22 μm.

[0015] The application of the extract of Polygonum capitatum, the self-assembled nanomaterials of the active ingredients of Polygonum capitatum, or the pharmaceutical preparations thereof in the preparation of drugs for treating atopic dermatitis.

[0016] The application of the aforementioned Polygonum capitatum extract, the aforementioned self-assembled nanomaterials, or the aforementioned pharmaceutical preparations in the preparation of antibacterial, anti-inflammatory, or antipyretic drugs.

[0017] A traditional Chinese medicine extract that can treat atopic dermatitis and also has antibacterial, anti-inflammatory, and heat-clearing properties, and its applications.

[0018] A traditional Chinese medicine extract that can treat atopic dermatitis and also has antibacterial, anti-inflammatory, and heat-clearing properties is obtained by extracting Polygonum capitatum with pure water, 50% ethanol, 60% ethanol, 70% ethanol, and 95% ethanol, respectively.

[0019] Preferably, the herbal extract is obtained by extracting Polygonum capitatum from pure water.

[0020] Preferably, the herbal extract is obtained by extracting Polygonum capitatum with 50% ethanol.

[0021] Preferably, the herbal extract is obtained by extracting Polygonum capitatum with 60% ethanol.

[0022] Preferably, the herbal extract is obtained by extracting Polygonum capitatum with 70% ethanol.

[0023] Preferably, the herbal extract is obtained by extracting Polygonum capitatum with 95% ethanol.

[0024] The present invention also provides a method for preparing the above-mentioned extract of Polygonum capitatum, comprising the following steps: appropriately crushing Polygonum capitatum, adding 5 to 15 times its weight of pure water, 50% ethanol, 60% ethanol, 70% ethanol and 95% ethanol respectively, heating and refluxing for extraction, and concentrating the filtrate to 1 / 10 of the total volume to obtain the corresponding Polygonum capitatum extract.

[0025] This invention further provides a self-assembled nanomaterial of active ingredients from the traditional Chinese medicine Polygonum capitatum, wherein the nanomaterial is formed by the self-assembly of berberine hydrochloride, gallic acid, and protocatechuic acid through non-covalent interactions. The gallic acid and protocatechuic acid are derived from the aforementioned Polygonum capitatum extract.

[0026] Preferably, the molar ratio of the three components is berberine hydrochloride: gallic acid: protocatechuic acid = 1:1:0.2.

[0027] Preferably, the molar ratio of the three components is 1:1:0.4.

[0028] Preferably, the molar ratio of the three components is 1:1:0.6.

[0029] Preferably, the molar ratio of the three components is 1:1:0.8. Under this ratio, the particle size distribution of the nanomaterial is uniform and the system is most stable.

[0030] Preferably, the molar ratio of the three components is 1:1:1.

[0031] The non-covalent forces include hydrogen bonds and π-π stacking interactions.

[0032] The preparation method of the above-mentioned self-assembled nanomaterials is characterized by the following steps: berberine hydrochloride, gallic acid, and protocatechuic acid are dissolved in methanol to prepare solutions of 8-15 mg / mL, which are then filtered through a 0.1-0.5 μm microporous membrane; the three are mixed in a selected molar ratio, heated to 40-70°C, and stirred for 1-5 hours; subsequently, the mixed solution is transferred to a dialysis bag with a molecular weight cutoff of 2000-4000 Da, dialyzed with ultrapure water, and the solution obtained in the bag is collected, which is the self-assembled nanomaterial. Preferably, the mixture is heated to 60°C and stirred for 1-5 hours.

[0033] A schematic diagram illustrating the preparation and verification of self-assembled nanomaterials from traditional Chinese medicine extracts and their active ingredients in this invention is shown below. Figure 1 .

[0034] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The extract of Polygonum capitatum and the self-assembled nanomaterials of the present invention are both derived from natural Chinese medicine, with clear sources, high safety, and no obvious toxic side effects. (2) As verified by in vitro experiments, the 95% ethanol extract provided by the present invention can significantly inhibit Staphylococcus aureus, and the antibacterial activity of the self-assembled nanomaterials is superior to that of each monomer and its physical mixture, showing synergistic antibacterial ability. (3) In animal models, the extract and nanomaterials of the present invention can significantly improve the symptoms of atopic dermatitis, repair the skin barrier, reduce the skin lesion score, and effectively downregulate the levels of key inflammatory factors such as IFN-γ, IL-4 and LTB4 in the body, showing excellent anti-inflammatory and immunomodulatory activities. (4) The present invention has a definite antipyretic effect on febrile model animals, can significantly reduce body temperature, and inhibit the release of pyrogenic inflammatory factors such as TNF-α, IL-6, and IL-1β, which can effectively improve the systemic inflammation and fever symptoms associated with atopic dermatitis. Attached Figure Description

[0035] Figure 1 This is a schematic diagram illustrating the preparation and verification of self-assembled nanomaterials from traditional Chinese medicine extracts and their / her active ingredients according to the present invention.

[0036] Figure 2The pathological changes of the dorsal skin of mice in each group of atopic dermatitis were shown by HE staining. Con: blank group; Mod: model group; A~F: high / low water extract, high / low 50% ethanol extract, and high / low 95% ethanol extract, respectively; G: dexamethasone group.

[0037] Figure 3 The pathological changes in lung tissue of mice in each group of blood-heat syndrome were shown by HE staining. Con: blank group; Mod: model group; A~D: water extract group, 50% alcohol extract group, 95% alcohol extract group, and aspirin group, respectively.

[0038] Figure 4 HPLC chromatograms of chemical components from different extracts of Polygonum capitatum, 1-5 in order: gallic acid, protocatechuic acid, rutin, quercetin, and quercetin;

[0039] Figure 5 The Tyndall effect diagram for self-assembled nanomaterials (BGP nps);

[0040] Figure 6 TEM image of self-assembled nanomaterials (BGP nps);

[0041] Figure 7 The fluorescence spectrum of the self-assembled nanomaterial (BGP nps);

[0042] Figure 8 The image shows the ultraviolet spectrum of a self-assembled nanomaterial (BGP nps).

[0043] Figure 9 Infrared spectrum of self-assembled nanomaterial (BGP nps). Detailed Implementation

[0044] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0045] Example 1 Preparation of Chinese herbal extracts Pulverize appropriate amounts of Polygonum capitatum, take 100 g of each, and add 10 times their weight of water, 50% ethanol, 60% ethanol, 70% ethanol, or 95% ethanol respectively. Heat under reflux for extraction three times, 1 h each time. Filter, combine the filtrates, and concentrate under reduced pressure to 2.5 g / mL of crude drug for later use.

[0046] Antibacterial activity verification Patients with atopic dermatitis are at risk of bacterial, viral, or fungal skin infections due to skin barrier defects, bacterial colonization (especially Staphylococcus aureus), and altered skin microbiome. Staphylococcus aureus-induced skin infections are the most common in atopic dermatitis patients. Therefore, studying the antibacterial activity of Polygonum capitatum extract is an important means of preventing and treating atopic dermatitis. Therefore, this invention establishes an in vitro antibacterial model and examines the antibacterial effect of the herbal extract by detecting the inhibition zone, with kanamycin and ampicillin as positive control drugs.

[0047] 1 Experimental Methods 1.1 Preparation of Nutrient Agar Take 18 g of beef extract peptone medium and 1 L of pure water. To prepare a solid medium, add 15 g of agar powder. Sterilize the solution using an autoclave (121℃, 20 min). Then pour the solution into petri dishes to prepare a solid medium (NA medium). Perform a sterilization check on the medium.

[0048] 1.2 Preparation of test bacterial culture Take a small amount of bacteria sealed in glycerol and inoculate it into liquid culture medium. Incubate at 37°C for 24 hours before use.

[0049] 1.3 Antibacterial zone experiment Add 100 μL of the cultured bacterial suspension to NA solid medium, spread evenly with a spreader, and divide the solid medium into four regions. Make 5 mm diameter wells in each region. Add 100 μL of different concentrations of the test sample solution to the wells and incubate at 37℃ for 24 h. For *E. coli*, add 50 μg / mL kanamycin as a positive control; for *Staphylococcus aureus*, add 50 μg / mL ampicillin as a positive control. Each dilution is repeated twice. After the experiment, seal the petri dishes with sealing film. Incubate at 37℃ for 24 h, and measure the diameter of the inhibition zone. The experiment is repeated three times.

[0050] The diameter of the inhibition zone (mm) = outer diameter and inner diameter.

[0051] 2. Experimental Results As shown in Table 1, the experimental results indicate that the water extract had no inhibitory effect on Escherichia coli and Staphylococcus aureus. However, the 95% ethanol extract of Polygonum capitatum, at a crude drug concentration of 1 g / mL, exhibited inhibition zone diameters of 11 mm and 10 mm against Escherichia coli and Staphylococcus aureus, respectively, both larger than the inhibition zones of other extracts, demonstrating a significant antibacterial effect. Therefore, the antibacterial active substances of Polygonum capitatum extracts are likely mainly concentrated in the 95% ethanol extract.

[0052]

[0053] The results of this embodiment demonstrate that the traditional Chinese medicine extracts provided by this invention can significantly inhibit Staphylococcus aureus and Escherichia coli. The 95% ethanol extract exhibits relatively strong antibacterial activity against Escherichia coli, while the 60%, 70%, and 95% ethanol extracts show stronger antibacterial effects against Staphylococcus aureus than other extracts. This suggests that high-concentration ethanol extracts may have a strong inhibitory effect on Staphylococcus aureus and Escherichia coli, improving skin lesions induced by bacterial colonization in atopic dermatitis, and possessing antibacterial activity and potential as a treatment for atopic dermatitis.

[0054] Example 2 Effects of different extracts of Polygonum capitatum on LPS-induced inflammation in zebrafish 1 Experimental Methods 1.1 Determination of LPS-induced inflammation concentration in zebrafish The collected zebrafish embryos were transferred into 6-well culture plates and randomly divided into 50 embryos per well. After removing residual culture medium, LPS was added to each well of each group to make a final concentration of 0, 5, 10, 16, and 20 μg / mL, respectively. The experiment was repeated 3 times. The embryos were incubated in an incubator at 28.5℃. Dead embryos were removed at intervals. After 48 h of molting, the suspended membrane shell was removed in time. The hatching rate of zebrafish embryos was calculated and the yolk sac area was measured.

[0055] 1.2 Evaluation of the anti-inflammatory activity of Polygonum capitatum extract 1.2.1 Effect of Polygonum capitatum extract on hatching rate The collected zebrafish embryos were transferred into 6-well culture plates and randomly divided into 50 embryos per well. After removing residual culture medium, extracts of Polygonum capitatum were added to each well of each group to make a final concentration of 0, 10, 100, 1000, and 10000 μg / mL. The experiment was repeated 3 times. The embryos were incubated in an incubator at 28.5℃. Dead embryos were removed at intervals. After 48 h of molting, the suspended membrane shell was removed in time, and the hatching rate of zebrafish embryos was calculated.

[0056] 1.2.2 Evaluation of the anti-inflammatory effects of different Polygonum capitatum extracts Collected zebrafish embryos were transferred into 24-well culture plates and randomly divided into groups of 30 embryos per well. Residual culture medium was removed, and *Polygonum capitatum* extract under different extraction conditions was added to each well of each group to achieve final concentrations of 0, 20, 50, and 100 μg / mL. LPS was then added to each well of the juvenile fish to reach the induction concentration. The embryos were incubated at 28.5℃. Dead embryos were removed periodically. After 48 h of molting, the outer membrane was promptly removed for subsequent experiments. ROS and NO production rates were measured 72 h after fertilization. The experiment was repeated three times.

[0057] 2. Experimental Results At concentrations of 1 mg / mL or higher, all zebrafish juveniles of Polygonum capitatum died, while at concentrations of 100 μg / mL or lower, all hatched.

[0058] The results of this embodiment are shown in Tables 2 and 3: At LPS concentration of 10 μg / mL, the zebrafish yolk sac area was the largest, suggesting that at this concentration, the absorption of the yolk sac in zebrafish was slowed due to inflammation, leading to an increased yolk sac area compared to the control group. Different extracts of Polygonum capitatum could inhibit LPS-induced ROS and NO production in zebrafish, exhibiting certain anti-inflammatory activity. The aqueous extract of Polygonum capitatum showed a significant reduction in ROS and NO, and its anti-inflammatory effect was superior to other extracts, suggesting a potential therapeutic effect for acute inflammation.

[0059]

[0060]

[0061] Example 3 The therapeutic effects of different extracts of Polygonum capitatum on atopic dermatitis 1 Experimental Methods 1.1 Grouping, modeling, and drug administration of mice Mouse grouping: A specific dermatitis model was established in mice according to the literature. Ten mice were divided into a blank group, a model group, a water extract group of Polygonum capitatum (high dose, low dose), a 50% alcohol extract group of Polygonum capitatum (high dose, low dose), a 95% alcohol extract group of Polygonum capitatum (high dose, low dose), and a positive control group.

[0062] Mouse modeling and drug administration: Mice were routinely fed for one week. Then, 90 male BALB / c mice were anesthetized with sodium pentobarbital intraperitoneally (80 mg / kg) before the experiment. Hair was removed from the back of mice (approximately 3 cm × 2 cm). For the first week, except for the control group, mice in the first week underwent sensitization by applying 100 μL of 1% DNFB solution to their backs on days 1, 4, and 7. The mice rested in the second week. Starting in the third week, 100 μL of 0.1% DNFB solution was applied to the back of mice every other day to induce and maintain an inflammatory response in the back skin for two weeks. The control group underwent the same treatment at the same time points and locations with an equal amount of matrix (acetone + olive oil, volume ratio 1:4) for four weeks. Samples were collected on day 29. Dexamethasone (positive control) was calculated at 10 mg / kg, with 0.2 mL / 20 g dissolved in distilled water for later use. Extracts of Polygonum capitatum were converted to adult (70 kg) dosages for oral administration to mice. The high-dose (11.7 g / kg crude drug) and low-dose (3.9 g / kg crude drug) extracts were administered in 0.4 mL via gavage for two weeks, once daily. On day 29, mice were euthanized by cervical dislocation within 24 hours of the experiment's end. Blood was collected, allowed to stand at room temperature for 2 hours, then centrifuged at 1500 rpm for 15 min to obtain the supernatant, which was stored at -80℃. Full-thickness skin from the back was collected; a portion was preserved in liquid nitrogen, and the remainder was fixed in formalin.

[0063] 1.2 Evaluation of the therapeutic activity of Polygonum capitatum in treating atopic dermatitis 1.2.1 Manifestations and severity scores of atopic dermatitis-like skin lesions in mice The mice were photographed every other day using a digital camera to record the dynamic changes in the skin lesions on their backs. The severity of the skin lesions was scored on day 1, day 7, day 14, and day 21 of the experiment, with four aspects: erythema / hemorrhagic, edema / exudation, dryness / desquamation, and epidermal peeling. The scores represent the severity and are divided into four levels: none (0), mild (1), moderate (2), and severe (3). The four symptoms are added together to form the final score, which ranges from 0 to 12.

[0064] 1.2.2 HE staining to observe the histopathological features of skin lesions After the experiment, skin from the back of each group of mice was taken, dehydrated, fixed, and embedded in paraffin to prepare paraffin sections. Hematoxylin-eosin (HE) staining was used to observe the histological characteristics of the mouse skin lesions. Image analysis software ImageJ was used to analyze the epidermal thickening. The vertical distance from the stratum corneum to the basal layer of the epidermis was measured under a 200x optical microscope and compared.

[0065] 1.2.3 ELISA method for detecting the levels of IFN-γ and IL-4 in peripheral blood Follow the ELISA test kit instructions for the following procedures. Store all samples at -80℃. Remove the frozen serum and kit. Add 1 mL of standard diluent to the lyophilized sample, dissolve and mix well, and serially dilute to the following concentrations: IL-4 standard concentrations: 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.2 pg / mL, 15.6 pg / mL, 7.8 pg / mL, 0 pg / mL. IFN-γ standard concentrations: 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.62 pg / mL, 7.81 pg / mL, 0 pg / mL. Follow the kit instructions for the remaining procedures.

[0066] 1.2.4 Detection of LBT4 content in skin lesions (1) Take out the frozen skin tissue that was originally stored in liquid nitrogen, weigh 0.1 g, cut it into small pieces and homogenize it, centrifuge at 2000 rpm / min and take the supernatant.

[0067] (2) Dilute the standard to 2000 pg / mL with standard diluent, then dilute by half. The final standard concentrations are 2000 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, and 0 pg / mL. Follow the kit instructions for the remaining steps.

[0068] 2. Experimental Results The results of this embodiment are shown below. Figure 2 As shown in Table 4, the epidermis and dermis of the control group mice were normal, with normal cell morphology and no edema or inflammatory cell infiltration. Drug administration began at day 14, and after one week of administration, the skin condition of the mice gradually improved, and the atopic dermatitis score (SCORAD) gradually decreased. The epidermis of the control group mice was normal, with no edema; the epidermis of the model group mice was significantly hyperplastic, and the epidermal thickness was significantly higher than that of the control group (…). P <0.0001), and severe edema; compared with the model group, the epidermal thickness of mice induced by DNFB was reduced in each treatment group, and all were significant, and the 95% ethanol extract of Polygonum capitatum showed better effect, second only to the positive drug dexamethasone group ( P <0.0001). Therefore, the 95% alcohol extract of Polygonum capitatum showed the best therapeutic effect on mice with atopic dermatitis.

[0069] Atopic dermatitis is generally caused by an imbalance in the ratio of Th1 to Th2 cells, with Th1 cells secreting IFN-γ and Th2 cells secreting IL-4. The LTB4 content in skin tissue reflects the degree of skin inflammation. The experimental results, as shown in Table 5, indicate that compared to the model group, except for the high-dose group of Polygonum capitatum water extract, all extracts of Polygonum capitatum and dexamethasone reduced serum neutralization in mice with atopic dermatitis. Furthermore, the low-dose group of 50% ethanol extract and the high-dose group of 95% ethanol extract of Polygonum capitatum showed more significant effects in reducing IL-4 levels. P <0.01. The low-dose group of 95% ethanol extract and the dexamethasone group showed more significant effects in reducing IFN-γ levels. P <0.0001). Extracts of Polygonum capitatum and dexamethasone both reduced serum LTB4 levels in mice with atopic dermatitis. The low-dose group of the 95% ethanol extract showed a more significant reduction (…). P <0.0001). Taking all factors into account, the 95% alcohol extract of Polygonum capitatum showed good efficacy in treating atopic dermatitis in mice.

[0070]

[0071]

[0072] Example 4 The therapeutic effects of different extracts of Polygonum capitatum on fever in mice 1 Experimental Methods 1.1 Preparation of the drug Weigh 300 g of Polygonum capitatum herb into 3 portions. Extract three times, using water, 50% ethanol, and 95% ethanol respectively, by heating under reflux for 1 h. Combine the filtrates and concentrate to 500 mg / mL for later use.

[0073] Weigh a measured amount of yeast and slowly add distilled water to prepare a 0.2 g / mL suspension. Incubate at 25°C for 24 h. Dissolve aspirin (Asp) in physiological saline to prepare a 20 mg / mL solution.

[0074] 1.2 Grouping, Modeling, and Drug Administration Mouse grouping: Kunming mice were divided into groups of 5 mice each, namely blank group, model group, Polygonum capitatum water extract group, Polygonum capitatum 50% alcohol extract group, Polygonum capitatum 95% alcohol extract group, and Asp (200 mg / kg) group.

[0075] Three days before modeling, the rectal temperature of mice was measured in the morning and evening (t). R (1) Fasting is required for 12 hours before measuring rectal temperature, but water intake is permitted. The first temperature measurement is taken as the basal body temperature. (2) t RMice with temperature changes less than 0.3℃ were used for subsequent experiments. Mice in the blank control group were subcutaneously injected with saline in the neck and back, while mice in the other groups were subcutaneously injected with active yeast suspension (10 mL / kg) in the neck and back. A temperature increase exceeding 0.8℃ was considered a successful model. Four hours after modeling, mice in the corresponding groups were administered medication by gavage. The dosage of each Polygonum capitatum extract was converted to adult (70 kg) dosage for mice, with a dosage of 11.7 g / kg of crude Polygonum capitatum extract, administered via gavage at 0.2 mL. The blank control and model groups were administered the corresponding amount of 0.5% sodium carboxymethyl cellulose (CMCNa) via gavage.

[0076] 1.3 Indicator Evaluation 1.3.1t R Measurement Mice t were measured every 1 hour from 0 to 13 hours after modeling. R The mouse was fixed in place, a small amount of petroleum jelly was applied to the tip of the electronic thermometer, and it was gently inserted into the anus. The reading was taken after the reading stabilized.

[0077] 1.3.2 Determination of fecal moisture content Fresh feces were collected during the modeling process and weighed at a constant wet mass. The feces were dried at 105℃ for 5 hours and cooled to a constant mass, which was then weighed at a constant dry mass. The water content of the feces was calculated.

[0078] Fecal moisture content = (wet mass / dry mass) / wet mass 1.3.3 Pathological observation of mouse lung tissue After the experiment, the mice were euthanized by cervical dislocation, and the lung tissue was immediately removed, fixed with 4% formaldehyde solution, embedded in paraffin, stained with HE as usual, and observed under an optical microscope.

[0079] 1.3.4 Measurement of serum TNF-α, IL-6, IL-1β, and IL-10 levels in mice Take 1 mL of whole blood and let it stand for 30 min, then centrifuge at 3500 r / min for 10 min. Collect the serum and detect TNF-α, IL-6, IL-1β, and IL-10 in the serum according to the kit instructions. The specific experimental steps are the same as in 1.2.3 of Example 3.

[0080] 2. Experimental Results The results of this embodiment are shown in Table 6. After injection of yeast suspension, the rectal temperature of mice gradually increased. At 4 h, all mice in the treatment group were administered the drug via gavage. The rectal temperature of mice in both the control group and the treatment group was significantly lower than that in the model group. This indicates that Polygonum capitatum extract can reduce fever induced by dry yeast suspension in mice, thus playing a role in clearing heat. After fever, the absorption of water in the intestines of mice increases. Since no drug intervention was performed in the model group, the fecal water content of the mice in the model group was lower than that in the control group. P<0.05), the fecal water content in each treatment group was greater than that in the model group, indicating that Polygonum capitatum extract can improve the increased intestinal water absorption caused by fever in mice.

[0081] Depend on Figure 3 It was found that, compared with the model group, the blank group had normal lung tissue structure, abundant alveoli, and uniform alveolar septa, while the model group had thickened alveolar walls and extensive inflammatory cell infiltration. Compared with the model group, the drug-treated group had relatively normal lung tissue structure, normal bronchial structure, and less inflammatory cell infiltration, indicating that different extracts of Polygonum capitatum can improve lung inflammation caused by fever in mice.

[0082] Table 7 shows that different extracts of *Polygonum capitatum* significantly reduced serum TNF-α levels in mice with blood-heat syndrome. Among the different extracts, the 95% ethanol extract showed the most significant reduction. Regarding IL-10, all different extracts of *Polygonum capitatum* reduced serum IL-10 levels in mice. Compared with the model group, only the 50% ethanol extract of *Polygonum capitatum* showed a significant reduction. P <0.05). Different extracts of *Polygonum capitatum*, including 50% and 95% ethanol extracts, significantly reduced serum IL-6 levels in mice with blood-heat syndrome. Among the different extracts, the 50% ethanol extract showed the most significant reduction. Furthermore, all different extracts of *Polygonum capitatum* reduced serum IL-1β levels in mice. Compared with the model group, the 50% ethanol extract of *Polygonum capitatum* showed the best and most significant reduction. Therefore, these experimental results indicate that different extracts of *Polygonum capitatum* have heat-clearing and anti-inflammatory effects, and effectively improve fever induced by dried yeast suspension in mice.

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[0085] Example 5 Component analysis of different extracts of Polygonum capitatum 1 Experimental Methods 1.1 Characteristic Spectrum Study of Polygonum capitatum Extract 1.1.1 Preparation of test and reference solutions Take 200.00 μL of the prepared Polygonum capitatum extract, add 1.80 mL of 50% methanol to dilute to 50.00 mg / mL, and filter the diluted solution through a 0.22 μm filter to obtain the test solution.

[0086] Gallic acid, protocatechuic acid, rutin, quercetin, and quercetin were weighed to 6.65 mg, 2.81 mg, 3.17 mg, 2.95 mg, and 3.00 mg, respectively, and dissolved in 1 mL of methanol using an ultrasonic cleaner. Then, the above reference solutions were prepared into mixed solutions with concentrations of 200.00 μg / mL, 30.00 μg / mL, 60.00 μg / mL, 60.00 μg / mL, and 25.00 μg / mL, respectively, and filtered through a 0.22 μm filter to obtain the reference solutions.

[0087] 1.1.2 Chromatographic conditions and mobile phase conditions Using a C13 column 18 (4.6×250 mm, 5 μm), volumetric flow rate 0.8 mL / min, detection wavelength 254 nm, column temperature 35℃, injection volume 10 μL.

[0088] Chromatographic mobile phase conditions: 0–10 min: 5% acetonitrile-(0.1% formic acid) water ~ 5% acetonitrile-(0.1% formic acid) water; 10–14 min: 5% acetonitrile-(0.1% formic acid) water ~ 14% acetonitrile-(0.1% formic acid) water; 14–20 min: 14% acetonitrile-(0.1% formic acid) water ~ 16% acetonitrile-(0.1% formic acid) water; 20–24 min: 16% acetonitrile-(0.1% formic acid) water ~ 16% acetonitrile-(0.1% formic acid) water; 24–30 min: 16% acetonitrile-(0.1% formic acid) water ~ 20% acetonitrile-(0.1% formic acid) water; 30–40 min: 20% acetonitrile-(0.1% formic acid) water ~ 30% acetonitrile-(0.1% formic acid) water; 40–45 min: [The last part of the text appears to be a repetition of the previous sentence and can be omitted.] min: 30% acetonitrile-(0.1% formic acid) water ~ 40% acetonitrile-(0.1% formic acid) water; 45~50 min: 40% acetonitrile-(0.1% formic acid) water ~ 40% acetonitrile-(0.1% formic acid) water; 50~52 min: 40% acetonitrile-(0.1% formic acid) water ~ 5% acetonitrile-(0.1% formic acid) water; 52~55 min: 5% acetonitrile-(0.1% formic acid) water ~ 5% acetonitrile-(0.1% formic acid) water.

[0089] 1.2 Determination of chemical components in Polygonum capitatum extract According to section 1.1.1, prepare the test solution by dissolving gallic acid, protocatechuic acid, rutin, quercetin, and reference standards into mixed solutions with final concentrations of 400 μg / mL, 60 μg / mL, 120 μg / mL, 120 μg / mL, and 50 μg / mL, respectively. Filter these solutions through a 0.22 μm filter to obtain the reference solutions. Calculate the content of each chemical component in the medicinal material.

[0090] 2. Experimental Results exist Figure 4 By comparing the peak retention times of Polygonum capitatum extract and mixed control solution, the main chemical components in Polygonum capitatum extract were identified as gallic acid, protocatechuic acid, rutin, quercetin, and quercetin.

[0091] .

[0092] Table 8 shows that the standard curves for each reference standard exhibit good linearity. Using the external standard one-point method, the peak areas of the tested samples were calculated to yield gallic acid contents of 2252.0 μg / g, 1545.4 μg / g, and 726.0 μg / g in the water, 50% ethanol, and 95% ethanol extracts of *Polygonum capitatum*, respectively. The contents of protocatechuic acid in these extracts were 249.6 μg / g, 180.8 μg / g, and 143.0 μg / g, respectively. The contents of rutin in these extracts were 791.2 μg / g, 651.2 μg / g, and 522.6 μg / g, respectively. The contents of quercetin in the water extract, 50% ethanol extract, and 95% ethanol extract of Polygonum capitatum were 397.2 μg / g, 509.2 μg / g, and 572.0 μg / g, respectively. The contents of quercetin in the water extract, 50% ethanol extract, and 95% ethanol extract of Polygonum capitatum were 37.6 μg / g, 39.0 μg / g, and 147.4 μg / g, respectively.

[0093] The above results indicate that the contents of gallic acid, protocatechuic acid, and rutin decreased with increasing ethanol concentration, while the contents of quercetin and quercetin increased with increasing ethanol concentration.

[0094] Example 6 Preparation and characterization of self-assembled materials Based on the above spectrum-effect correlation results, gallic acid and protocatechuic acid, which are abundant in water extracts and have clear anti-inflammatory target binding activities, were selected as potential anti-inflammatory components and core assembly units of self-assembled nanomaterials. At the same time, berberine hydrochloride, which has complementary structure and antibacterial activity, was introduced to construct ternary self-assembled nanomaterials (BGP nps).

[0095] 1 Experimental Methods 1.1 Preparation of BGP nps Gallic acid, berberine, and protocatechuic acid were dissolved in methanol to prepare a 10 mg / mL solution, filtered through a 0.22 μm microporous membrane, and the three were mixed, heated (60℃), stirred for 2 h, and then transferred to a 3000 Da dialysis bag. Dialysis was performed with ultrapure water for 12 h, with the water changed every 3 h, and the solution in the dialysis bag was collected.

[0096] Based on the research on self-assembled materials of berberine and gallic acid, berberine hydrochloride, gallic acid, and protocatechuic acid were self-assembled in molar ratios of 1:1:0.2, 1:1:0.4, 1:1:0.6, 1:1:0.8, and 1:1:1, respectively.

[0097] 1.2 Characterization of BGP nps (1) Dynamic light scattering (DLS) and transmission electron microscopy (TEM) characterization of the morphology and size of BGP nps Prepare 1 mL of BGP nps solution with a concentration of 1 mg / mL and ultrapure water as solvent. After sonication for 30 min, take 20 μL of BGP nps solution and drop it onto a carbon film copper grid to form a full water droplet. Place it in an electronic moisture-proof drying oven to dry overnight. Then, observe the overall morphology and size of BGP nps nanoparticles under a large field of view using a transmission electron microscope.

[0098] (2) Optical performance of BGP nps UV-Vis absorption spectrum of BGP nps: First, prepare a 1 mmol BGP nps solution and transfer it to a quartz cuvette. Then, zero the UV-Vis spectrophotometer with ultrapure water, set a 1 cm optical path, and perform a wavelength scan in the 200 nm to 800 nm band to measure its spectrum.

[0099] Fluorescence spectrum (FL) of BGP nps: A BGP nps solution of a certain concentration was placed in a fully transparent quartz colorimeter. A fluorescence spectrophotometer was used with appropriate laser voltage and scanning speed to obtain the three-dimensional fluorescence spectrum of BGP nps at excitation and emission wavelengths of 300-700 nm.

[0100] (3) BGP nps component structure analysis Fourier Transform Infrared Spectroscopy (FTIR) Test: To detect the surface functional groups of BGP nps, BGP nps powder was mixed with potassium bromide powder (1:100), thoroughly ground, and then pressed into transparent thin sheets using a tablet press. The FTIR instrument was started, the sample was placed in the sample chamber of the instrument, and the absorption peaks of the sample at different wavenumbers were recorded. The obtained FTIR spectra were analyzed, and the corresponding functional groups of each absorption peak were identified.

[0101] 2. Experimental Results 2.1 Characterization of BGP nps morphology by DLS, Zeta potential and TEM From Table 9 and Figure 6It can be seen that when the molar ratio of berberine, gallic acid, and protocatechuic acid is 1:1:0.8, the PDI values ​​are all less than 0.3, and the absolute value of the Zeta site is the largest, indicating that the self-assembled nanoparticle system is relatively stable under this molar ratio condition. Furthermore, the TEM image shows that the particle size of the nanoparticles under this condition is basically consistent with the particle size measured by DLS.

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[0103] 2.2 Identification of chemical composition of BGP nps by ultraviolet and fluorescence spectra Depend on Figure 5 It is known that berberine, gallic acid, and protocatechuic acid do not exhibit the Tyndall effect when existing individually, and the physical mixture of the three (BGPM) also does not produce the Tyndall effect. However, after self-assembly into nanomaterials, the Tyndall effect becomes very pronounced. Figure 7 It is evident that the strongest fluorescence emission at 543 nm in the self-assembled nanomaterial BGP nps is the result of the synergistic effect of the following processes: Through π-π stacking and hydrogen bond networks, BBR molecules form a tightly stacked structure dominated by "H-aggregation," which directly leads to a blue shift in its fluorescence emission (from 551 nm to 543 nm). GA and PCA are immobilized in the assembly and effectively transfer the absorbed excitation energy to BBR through the FRET mechanism, resulting in self-fluorescence quenching and enhancing the emission signal of BBR in BGP nps. Therefore, the strong emission peak at 543 nm is not simply a superposition of BBR fluorescence, but rather marks the successful construction of a novel nanoscale fluorescent structure, and its blue shift directly confirms the highly ordered molecular arrangement within the self-assembled structure.

[0104] Depend on Figure 8 As can be seen, the absorption peaks of BGP nps, relative to the other three substances, exhibit a blue shift, with the characteristic absorption peak of BBR shifting from 421.15 nm to 419.90 nm. The absorption of BGP nps relative to GA / PCA in the short-wavelength region also shows a blue shift. This is mainly attributed to the strong π-π packing interactions between molecules, forming H-aggregates. This tight packing increases the excited-state energy of the molecules, leading to a shift of the absorption peak towards shorter wavelengths. After protocatechuic acid (PCA) participates in self-assembly, its phenolic hydroxyl and carboxyl groups form a broad intermolecular hydrogen bond network with BBR or gallic acid (GA). This hydrogen bond network fixes the molecular conformation of PCA, altering the electron distribution on its benzene ring, thus significantly weakening its original characteristic absorption peak or submerging it in other broader and stronger absorption bands.

[0105] In summary, the shift in the BGP nps spectrum provides direct evidence of its self-assembled structure, with π-π stacking dominating the blue shift and hydrogen bonding and electrostatic interactions playing key roles in the co-assembly process.

[0106] 2.3 BGP nps Fourier transform infrared spectral composition and structure analysis Depend on Figure 9 It is known that the infrared peaks of the physical mixture BGPM are a simple superposition of the characteristic peaks of the three monomers, without significant peak shifts, broadening, or disappearance; the only change in peak intensity is due to the concentration superposition. In contrast, the characteristic peaks of BGP nps show significant shifts, broadening, intensity reduction, and even splitting. In the 3600–3200 cm⁻¹ range, the peak shape of BGP nps is completely merged into a broad, blunt peak, and the overall peak shifts to lower wavenumbers by 10–50 cm⁻¹, with a significant decrease in peak intensity. In the 1720–1690 cm⁻¹ range, the characteristic peak of the carboxyl group (C=O) shifts to lower wavenumbers by 5–20 cm⁻¹, with a significant decrease in peak intensity; in some spectra, this peak is almost merged into the aromatic ring C=C peak. Therefore, it can be concluded that BGP nps is essentially the formation of non-covalent intermolecular interactions, replacing some of the weak intramolecular or intermolecular interactions of the monomers, enabling the three monomers to form a stable self-assembled nanostructure, with hydrogen bonding as the core driving force and π-π conjugation as the synergistic force.

[0107] This embodiment successfully constructed a carrier-free self-assembled nanomaterial BGP nps composed of berberine hydrochloride, gallic acid, and protocatechuic acid.

[0108] Example 7 Evaluation of the antibacterial efficacy of self-assembled materials against Staphylococcus aureus 1 Experimental Methods 1.1 Drug Preparation Berberine (BBR), gallic acid (GA), and protocatechuic acid (PCA) were weighed separately, dissolved, and used to prepare stock solutions. These stock solutions were then diluted with water to prepare BBR solutions of 26.9 mM, 13.4 mM, 6.7 mM, and 3.4 mM; GA solutions of 58.8 mM, 29.4 mM, 14.7 mM, and 7.4 mM; and PCA solutions of 64.9 mM, 32.4 mM, 16.2 mM, and 8.1 mM. These solutions were then physically mixed at a molar ratio of BBR:GA:PCA = 1:1:0.8 to prepare 15 mM, 7.5 mM, 3.8 mM, and 1.9 mM solutions. BGP nps were prepared to prepare 7.5 mM, 3.8 mM, 1.9 mM, and 0.9 mM solutions.

[0109] 1.2 Preparation of test bacterial culture Take a small amount of Staphylococcus aureus sealed with glycerol, inoculate it into liquid culture medium, and incubate at 37°C for 24 hours before use.

[0110] 1.3 Staphylococcus aureus Antibacterial Experiment Dilute the cultured bacterial solution to 1×10⁻⁶. 6For each CFU / mL sample, 100 μL was added to NA solid medium and spread evenly using a spreader. The solid medium was divided into four sections, and 5 mm diameter wells were made in each section. 50 μL of different concentrations of the test sample solution was injected into the wells, and the mixture was incubated at 37°C for 12 h. Staphylococcus aureus was treated with 50 μg / mL ampicillin as a positive control. Each dilution was repeated twice. After the experiment, the culture dishes were sealed with sealing film and incubated at 37°C for 24 h. The diameter of the inhibition zone was measured after 24 h.

[0111] Inhibition zone diameter (mm) = outer diameter / inner diameter 2. Experimental Results

[0112] Table 10 shows that at a concentration of 7.5 mM, the diameter of the inhibition zone of BGP is larger than that of other monomers at the same molar concentration, and also larger than that of the inhibition zone of physically mixed BGPM. GA and PCA, at this concentration, have no inhibitory effect on Staphylococcus aureus. This indicates that the antibacterial properties of berberine, gallic acid, and protocatechuic acid are enhanced after self-assembly into nanomaterials.

[0113] Example 8 Evaluation of the antibacterial efficacy of self-assembled materials against Staphylococcus aureus 1 Experimental Methods 1.1 Drug Preparation Berberine (BBR), gallic acid (GA), protocatechuic acid (PCA), and BGP nps were weighed separately, dissolved in a trace amount of methanol to prepare a stock solution, and then diluted with water to prepare 1 μM, 10 μM, 100 μM, 1 mM, and 10 mM solutions sequentially. The three solutions were then physically mixed at a molar ratio of BBR:GA:PCA = 1:1:0.8 to prepare the same concentration gradient as described above.

[0114] 1.2 Zebrafish feeding and embryo collection Adult Tg(mpx:eGFP) transgenic zebrafish were raised at room temperature (28℃) under natural light cycle and fed twice a day. Zebrafish embryos were obtained by natural mating of healthy male and female fish (1:1).

[0115] 1.3 Determination of the concentration of sodium dodecyl sulfate (SDS) induced by zebrafish dermatitis The collected 3-day zebrafish embryos were transferred into 6-well culture plates and randomly divided into 30 embryos per well. After removing residual culture medium, SDS was added to each well of each group to make final concentrations of 0, 0.3 μg / mL, 0.6 μg / mL, 1.2 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 10.0 μg / mL, and 20.0 μg / mL, respectively. The experiment was repeated 3 times. The embryos were incubated in an incubator at 28.5 ℃. Dead embryos were removed at intervals, and the survival rate of zebrafish embryos was calculated. Ten zebrafish juveniles were randomly selected from each group, and neutrophils were counted on the back and yolk sac of the zebrafish.

[0116] 1.4 Evaluation of the anti-inflammatory activity of BGP nps 1.4.1 Investigation of the toxicity concentrations of BGP nps and individual monomers The collected 3-day-old zebrafish larvae were transferred into 6-well culture plates and randomly divided into groups of 30 larvae per well. After removing residual culture medium, BGP nps and various monomers were added to each well of each group to make final concentrations of 0, 1 μM, 10 μM, 100 μM, and 1 mM, respectively. The experiment was repeated 3 times. The embryos were incubated in an incubator at 28.5℃. Dead embryos were removed at intervals. The survival rate of zebrafish was calculated after 24 h.

[0117] 1.4.2 Effects of BGP nps and individual monomers on neutrophil recruitment and reactive oxygen species production in zebrafish with atopic dermatitis The collected 3-day-old zebrafish larvae were transferred into 6-well culture plates and randomly divided into 30 embryos per well. After removing residual culture medium, BGP nps and other monomers were added to each well of each group to achieve final concentrations of 50 μM, 100 μM, and 200 μM, respectively. SDS was then added to each well of the larvae to reach the induction concentration. The plates were cultured in an incubator at 28.5℃ for 12 h. Ten zebrafish larvae were randomly selected from each group, and neutrophils were counted on the back and yolk sac of the zebrafish. The experiment was repeated 3 times.

[0118] The generation of reactive oxygen species (ROS) in zebrafish larvae was detected using the fluorescent probe dye 2,7-dichlorodihydrofluorescein diester (DCF-DA). Ten zebrafish larvae from each group were transferred to 6-well culture plates. After removing residual culture medium, DCF-DA was diluted to 20 μM with embryo culture medium and added to the culture plates. The plates were incubated in a dark incubator at 28.5℃ for 1 h. The plates were gently shaken at intervals to ensure full contact between the probe and the larvae. After incubation, the plates were rinsed 3-5 times with embryo culture medium. After anesthesia with 0.02% tricaine, the generation of ROS was observed under a fluorescence inverted microscope, and quantitative fluorescence analysis was performed using ImageJ software.

[0119] 1.4.3 Effects of BGP nps and individual monomers on IFN-γ, TNF-α, IL-6, and IL-1β levels in zebrafish with atopic dermatitis Three-day-old zebrafish juveniles were transferred into 12-well culture plates and randomly divided into groups of 50 juveniles per well. After removing residual culture medium, BGP nps and other monomers were added to each well of each group to achieve final concentrations of 50 μM, 100 μM, and 200 μM, respectively. SDS was then added to each well of the juveniles to reach the induction concentration. Three wells were set up for each concentration. The plates were incubated at 28.5℃ for 12 h. After induction, the zebrafish juvenile tissue was homogenized, and the supernatant was extracted. Three samples were selected from each group, and inflammatory factors were measured using ELISA. The experiment was repeated three times.

[0120] 2. Experimental Results 2.1 Results of SDS-induced zebrafish dermatitis concentration screening As shown in Table 11, zebrafish survival rates were low at SDS concentrations of 20 μg / mL and 10 μg / mL. Concentrations of 5 μg / mL and below showed a large accumulation of neutrophils in the dorsal and yolk sac regions of the zebrafish compared to the control group. This indicates that SDS stimulation caused inflammatory damage to the zebrafish skin, leading to a large accumulation of neutrophils from immune cells at the inflammatory site. Based on these results, an SDS concentration of 2.5 μg / mL was selected as the optimal induction concentration.

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[0122] 2.2 Effects of BGP nps and individual monomers on survival rate and neutrophil and reactive oxygen species production in zebrafish with atopic dermatitis

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[0124] When the concentration of BBR and BGP nps was 1 mM, all zebrafish died, while all zebrafish survived at other concentrations. When GA, PCA and physically mixed BGPM were in the concentration range of 0~1 mM, all zebrafish survived, with a survival rate of 100%.

[0125] As shown in Table 12, BGP nps, each monomer, and their physical mixtures all reduced the number of neutrophils in zebrafish with atopic dermatitis. BGP nps showed the most significant effect at a concentration of 100 μM. Compared with the monomer groups and the physical mixture groups, the BGP nps group exhibited significantly reduced neutrophil aggregation on the back and abdomen of the zebrafish. These results indicate that BGP nps can alleviate skin inflammatory damage in zebrafish.

[0126] BGP nps, its monomers, and physical mixtures all significantly reduced reactive oxygen species (ROS) levels in zebrafish with SDS-induced atopic dermatitis. Among these, BGP nps showed the most significant effect at a concentration of 200 μM. Compared to the monomer-treated groups and the physical mixture groups, the BGP nps group exhibited a more significant reduction in ROS generation in zebrafish, indicating that this nanomaterial possesses stronger antioxidant capacity and can more effectively alleviate oxidative stress in the atopic dermatitis model.

[0127] 2.3 Effects of BGP nps and individual monomers on TNF-α, IL-1β, and IL-10 in zebrafish with atopic dermatitis

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[0129] Table 13 shows that BGP nps, each monomer, and their physical mixtures significantly reduced IL-10 levels in zebrafish with SDS-induced atopic dermatitis. Protocatechuic acid showed the best effect at a concentration of 50 μM, while BGP nps also exhibited the strongest inhibitory effects at concentrations of 50 μM and 200 μM. Furthermore, BGP nps at a concentration of 200 μM significantly downregulated the levels of TNF-α and IL-1β in zebrafish. Compared to the monomers and physical mixtures, BGP nps exhibited more significant anti-inflammatory activity; therefore, BGP nps can synergistically alleviate the pathological state of atopic dermatitis in zebrafish at multiple inflammatory factor levels.

[0130] Based on the above experimental results, the *Polygonum capitatum* extract and its self-assembled nanomaterials, provided by this invention, can effectively intervene in the pathological process of atopic dermatitis through multiple pathways. At the whole animal level, the *Polygonum capitatum* extract significantly repaired the skin barrier, improved skin lesions, and downregulated the levels of key inflammatory mediators such as serum IFN-γ, IL-4, and LTB4 in skin tissue, suggesting that it may exert anti-inflammatory and immunomodulatory effects by regulating the balance of Th1 / Th2 cytokines. In terms of antibacterial activity, the 95% ethanol extract showed a clear inhibitory effect on *Staphylococcus aureus*, helping to control microbial colonization and secondary infections at AD lesions. Furthermore, the nanomaterials formed by the non-covalent self-assembly of the active components *Polygonum capitatum*, gallic acid and protocatechuic acid, and introduced berberine hydrochloride exhibited stronger antibacterial activity and synergistic anti-inflammatory effects compared to the individual monomers and physical mixtures. They significantly reduced neutrophil infiltration at inflammatory sites, lowered reactive oxygen species levels, and inhibited the release of inflammatory factors such as TNF-α, IL-1β, and IL-10. Zebrafish model and mouse antipyretic experiments further confirmed that the traditional Chinese medicine extract and self-assembled nanomaterials provided by this invention have both heat-clearing and anti-inflammatory effects, and can effectively relieve fever symptoms caused by systemic inflammation.

[0131] In summary, using the extract of Polygonum capitatum or its active ingredients as self-assembled nanomaterials as core raw materials, and with the synergistic effect of multiple components and multiple targets and low toxicity and side effects, it is expected to be developed into a drug, special medical food or functional health product for the treatment of atopic dermatitis and its accompanying fever symptoms.

Claims

1. An extract of the traditional Chinese medicine Polygonum capitatum, characterized in that, The extract of the Chinese herb Polygonum capitatum is obtained by extracting Polygonum capitatum with pure water or 50%~95% ethanol as solvent.

2. A formulation of the extract of Polygonum capitatum as described in claim 1, characterized in that, The extract of Polygonum capitatum, a traditional Chinese medicine, is mixed with pharmaceutical excipients to form any one of the following formulations: ointment, liniment, patch, lotion, solution, powder, spray, syrup, suppository, tablet, pill, granule, capsule, injection, and gastrointestinal sustained-release formulation.

3. A method for preparing the extract of Polygonum capitatum, a traditional Chinese medicine, as described in claim 1, characterized in that, The process includes the following steps: crush Polygonum capitatum, add pure water or 50%~95% ethanol, heat and reflux to extract, and concentrate the filtrate.

4. The method for preparing the extract of Polygonum capitatum according to claim 3, characterized in that, Concentrate the filtrate to 1 / 8 to 1 / 15 of its total volume.

5. A method for extracting gallic acid and protocatechuic acid from the extract of Polygonum capitatum, a traditional Chinese medicine, as described in claim 1, characterized in that, The extract of the traditional Chinese medicine Polygonum capitatum was separated into gallic acid and protocatechuic acid by preparative liquid chromatography.

6. A self-assembled nanomaterial of active ingredients from the traditional Chinese medicine Polygonum capitatum, characterized in that, It is formed by the self-assembly of berberine hydrochloride, gallic acid and protocatechuic acid through non-covalent interactions, with a molar ratio of 1:1:0.2 to 1:1:

1.

7. A pharmaceutical preparation, characterized in that, It is prepared from the self-assembled nanomaterial of active ingredients of Polygonum capitatum as described in claim 6, with or without the addition of pharmaceutically acceptable excipients.

8. A method for preparing the self-assembled nanomaterial according to claim 6, characterized in that, The process includes the following steps: dissolving berberine hydrochloride, gallic acid, and protocatechuic acid separately in organic solvents to prepare solutions; mixing them in molar ratios and heating to 40-90°C with stirring; transferring the solutions to a 2000-5000 Da dialysis bag for dialyzing, and collecting the solutions in the bag to obtain the final product.

9. The use of the extract of Polygonum capitatum according to claim 1, the self-assembled nanomaterial of the active ingredient of Polygonum capitatum according to claim 6, or the pharmaceutical preparation according to claim 7 in the preparation of a drug for treating atopic dermatitis.

10. The use of the extract of Polygonum capitatum according to claim 1, the self-assembled nanomaterial according to claim 6, or the pharmaceutical preparation according to claim 7 in the preparation of antibacterial, anti-inflammatory, or antipyretic drugs.

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