Coptis total alkaloid self-assembly composition, skin gel thereof, preparation method and application

Abstract

The invention relates to a coptis total alkaloid self-assembly composition and skin gel, a preparation method and application thereof, and belongs to the technical field of self-assembly pharmacy. The preparation method of the coptis total alkaloid self-assembled composition comprises the following steps: preparing a coptis hydrochloric acid extracting solution and preparing the coptis total alkaloid self-assembled composition. The skin gel of the coptis total alkaloid self-assembled composition is prepared from the coptis total alkaloid self-assembled composition. The self-assembled composition or the self-assembled composition skin gel is applied to preparation of a medicine for treating eczema. The preparation method has the following advantages: the particle size distribution is relatively uniform, and the system is relatively stable; the average particle size is 100 to 800 nm, and the PDI is 0.02 to 0.40; sEM (scanning electron microscope) shows that the shape is sphere-like; the skin gel has good biocompatibility, and the coptis total alkaloid self-assembled composition in the gel can relieve inflammatory response and improve eczema symptoms of mice by regulating and controlling an NLRP3 / GSDMD / IL-33 signal channel.

Description

Technical Field

[0001] The invention belongs to the technical field of self-assembly pharmaceutical manufacturing, and particularly relates to a coptis chinensis total alkaloid self-assembly composition and a skin gel thereof, as well as a preparation method and application thereof. Background Art

[0002] Eczema is a common, chronic, recurring inflammatory skin disease that leads to skin barrier damage, chronic itching, and inflammatory reactions, severely impacting patients' daily lives. Clinical treatment options include topical glucocorticoids, calcineurin inhibitors, oral antihistamines, immunosuppressants, and glucocorticoids, but these medications are associated with significant side effects and the development of drug resistance. Therefore, the development of new treatment strategies is urgently needed.

[0003] In recent years, the clinical value of traditional Chinese medicine in the treatment of eczema has gradually gained recognition, particularly in its unique advantages in alleviating itching, reducing inflammation, and promoting skin repair. Coptis chinensis, a traditional Chinese medicine, has attracted considerable attention due to its diverse pharmacological activities. Topical application of Coptis chinensis has significant astringent and antipruritic properties, can rapidly dry wounds, reduce inflammation and swelling, and promote granulation tissue growth and wound repair.

[0004] The main active ingredients in Coptis chinensis are alkaloids, whose hydrophilicity and low skin permeability limit their clinical application. Extensive evidence suggests that self-assemblies formed during the decoction of traditional Chinese medicine can effectively improve the drug delivery of active ingredients. The formation of self-assemblies not only promotes the absorption and target tissue distribution of active ingredients, but also enhances their pharmacological activity and reduces adverse reactions. However, self-assemblies formed by traditional decoction are susceptible to various factors, resulting in low yield and poor stability. Summary of the Invention

[0005] The inventors of this application have for the first time constructed a key technology for the new self-assembly of traditional Chinese medicine based on the pH-driven method. The self-assembly composition of total alkaloids from Coptis chinensis obtained can improve the transdermal penetration ability of the active ingredients, allowing the drugs to reach deeper layers of the skin. Preliminary experiments have shown that the composition has good biological activity both in vivo and in vitro. Therefore, this technology is of great significance to the development and application of new traditional Chinese medicines.

[0006] The first object of the present invention is to disclose a self-assembly composition of total alkaloids of Coptis chinensis.

[0007] The second object of the present invention is to disclose a method for preparing the above-mentioned Coptidis total alkaloid self-assembly composition.

[0008] The third object of the present invention is to disclose a skin gel of the self-assembly composition of total alkaloids of Coptis chinensis.

[0009] The fourth object of the present invention is to disclose a method for preparing the skin gel of the self-assembled composition of total alkaloids of Coptis chinensis.

[0010] The fifth object of the present invention is to disclose the application of the above-mentioned coptis chinensis total alkaloid self-assembly composition.

[0011] The sixth object of the present invention is to disclose the application of the above-mentioned Rhizoma Coptidis total alkaloids self-assembly composition as a gel for skin use.

[0012] The purpose of the present invention is achieved through the following technical solutions:

[0013] A self-assembly composition of total alkaloids from Coptis chinensis, wherein the self-assembly composition of total alkaloids from Coptis chinensis is prepared by the following method:

[0014] (1) Weigh the raw material Coptis chinensis;

[0015] (2) Take Coptis chinensis, add 5.81-29.07 mM hydrochloric acid solution, soak for 0.5 h, extract for 0.5-2 h, filter, repeat the extraction for 0.5-2 h, filter, combine the filtrates, and concentrate to 0.1-0.3 g crude drug / mL for later use;

[0016] (3) Prepare 0.5-2.0 mol / L NaOH solution and set aside;

[0017] (4) adjusting the pH value of the product of step (2) to neutral with the NaOH solution prepared in step (3), stirring at a speed of 100 to 300 r / min for 15 minutes to obtain a self-assembled composition of total alkaloids of Coptis chinensis.

[0018] The self-assembly composition of total alkaloids from Coptis chinensis described in the above technical solution is prepared by the following method:

[0019] (1) Weigh the raw material Coptis chinensis;

[0020] (2) Take Coptis chinensis, add 17.44 mM hydrochloric acid solution and soak for 0.5 h, extract for 1 h, filter, repeat the extraction for 1 h, filter, combine the filtrates, and concentrate to 0.2 g crude drug / mL for later use;

[0021] (3) Prepare 0.5-2.0 mol / L NaOH solution and set aside;

[0022] (4) adjusting the pH value of the product of step (2) to neutral with the NaOH solution prepared in step (3), stirring at a speed of 100 to 300 r / min for 15 minutes to obtain a self-assembled composition of total alkaloids of Coptis chinensis.

[0023] The self-assembly composition of total alkaloids from Coptis chinensis described in the above technical solution, wherein: the average particle size of the self-assembly composition of total alkaloids from Coptis chinensis is 100-800 nm, the PDI is 0.02-0.40; and SEM shows that its morphology is spherical.

[0024] The preparation method of the self-assembly composition of total alkaloids of Coptis chinensis described in the above technical solution comprises the following steps:

[0025] (1) Weigh the raw material Coptis chinensis;

[0026] (2) Take Coptis chinensis, add 5.81-29.07 mM hydrochloric acid solution, soak for 0.5 h, extract for 0.5-2 h, filter, repeat the extraction for 0.5-2 h, filter, combine the filtrates, and concentrate to 0.1-0.3 g crude drug / mL for later use;

[0027] (3) Prepare 0.5-2.0 mol / L NaOH solution and set aside;

[0028] (4) adjusting the pH value of the product of step (2) to neutral with the NaOH solution prepared in step (3), stirring at a speed of 100 to 300 r / min for 15 minutes to obtain a self-assembled composition of total alkaloids of Coptis chinensis.

[0029] The preparation method described in the above technical solution, wherein the preparation method comprises the following steps:

[0030] (1) Weigh the raw material Coptis chinensis;

[0031] (2) Take Coptis chinensis, add 17.44 mM hydrochloric acid solution and soak for 0.5 h, extract for 1 h, filter, repeat the extraction for 1 h, filter, combine the filtrates, and concentrate to 0.2 g crude drug / mL for later use;

[0032] (3) Prepare 0.5-2.0 mol / L NaOH solution and set aside;

[0033] (4) adjusting the pH value of the product of step (2) to neutral with the NaOH solution prepared in step (3), stirring at a speed of 100 to 300 r / min for 15 minutes to obtain a self-assembled composition of total alkaloids of Coptis chinensis.

[0034] A skin gel of a self-assembled composition of total alkaloids from coptis chinensis, comprising the following components in percentage by weight: 40-60% of the self-assembled composition of total alkaloids from coptis chinensis described in the above technical solution, 10-30% of distilled water, 5-15% of propylene glycol as a gel moisturizer, 5-15% of glycerol as a gel moisturizer, 3-5% of SEPINEO P600 (acrylamide / sodium acryloyldimethyltaurate copolymer, isohexadecane, polysorbate 80, and sorbitan oleate) as a thickener, 3-5% of butylene glycol, 2-5% of zinc oxide, 0.5-1% of ethylparaben, and 1-1.5% of 1,2-hexanediol.

[0035] The skin gel of the self-assembled composition of total alkaloids of Coptis chinensis described in the above technical solution, wherein the weight percentages of the components are: 50% of the self-assembled composition of total alkaloids of Coptis chinensis described in any one of claims 1 to 3, 17.5% of distilled water, 10% of propylene glycol as a gel moisturizer, 10% of glycerol as a gel moisturizer, 4% of SEPINEO P600 (acrylamide / sodium acryloyldimethyl taurate copolymer & isohexadecane & polysorbate 80 & sorbitan oleate) thickener, 4% of butylene glycol, 2.5% of zinc oxide, 0.7% of ethylparaben and 1.3% of 1,2-hexanediol.

[0036] The preparation method of the skin gel of the self-assembly composition of total alkaloids of Coptis chinensis described in the above technical solution comprises the following steps:

[0037] (1) Weighing the raw materials according to the weight percentage of claim 6 or 7;

[0038] (2) Distilled water, the self-assembly composition of total alkaloids from Rhizoma Coptidis, SEPINEO P600, zinc oxide and butylene glycol were placed in a beaker, and homogenized and stirred for 20 minutes at 80°C, at a speed of 100-300 r / min and a homogenization condition of 1000-1500 rpm. Glycerin and propylene glycol were then added, and the homogenization and stirring were continued for 20 minutes. The temperature was lowered to 65°C, and ethylparaben and 1,2-hexanediol were added at a speed of 100-300 r / min, and the mixture was homogenized and stirred for 10 minutes to mix evenly. The mixture was then divided into portions to obtain a skin gel of the self-assembly composition of total alkaloids from Rhizoma Coptidis.

[0039] The use of the self-assembly composition of total alkaloids from Coptis chinensis described in the above technical solution or the skin gel of the self-assembly composition of total alkaloids from Coptis chinensis described in the above technical solution in the preparation of drugs for treating skin diseases.

[0040] The application described in the above technical solution, wherein: the skin disease is eczema.

[0041] The application described in the above technical solution, wherein: the application is to reduce inflammatory response and improve eczema symptoms in mice by regulating the NLRP3 / GSDMD / IL-33 signaling pathway.

[0042] The self-assembly formed by traditional water decoction is easily affected by various factors, has low yield and poor stability; the present invention changes the traditional method and adopts a self-assembly technology based on the pH-driven method, adding an alkaline solution to the hydrochloric acid extract of Coptis chinensis and controlling the conditions to form a self-assembly; the specific technology is to use the principle that the drug in the hydrochloric acid extract of Coptis chinensis has a high solubility in acidic solution and a low solubility in neutral solution, and by controlling the conditions such as the process, the concentration of the hydrochloric acid solution, and the concentration of the crude drug in the hydrochloric acid extract of Coptis chinensis, many independent small particles are quickly precipitated under the driving force of supersaturation to obtain a polydisperse self-assembly, which is the self-assembly composition of total alkaloids of Coptis chinensis.

[0043] The present invention has the following beneficial effects:

[0044] 1. The particle size distribution of the total alkaloid self-assembly composition of Coptis chinensis prepared by the present invention is relatively uniform, and the system is relatively stable; its average particle size is 100-800 nm, and PDI is 0.02-0.40; SEM shows that its morphology is spherical.

[0045] 2. The skin gel of the self-assembly composition of total alkaloids from Coptis chinensis of the present invention has good biocompatibility. The self-assembly composition of total alkaloids from Coptis chinensis in the gel can reduce inflammatory response and improve eczema symptoms in mice by regulating the NLRP3 / GSDMD / IL-33 signaling pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Characterization diagram of the self-assembled composition of total alkaloids from Coptis chinensis in Experimental Example 1, wherein A is a scanning electron microscope image; B is a particle size distribution result diagram;

[0047] Figure 2 The results of the CCK8 proliferation experiment in Experimental Example 2 are shown in Figure A, where A shows the cytotoxicity of the coptis chinensis decoction detected by CCK8; and B shows the cytotoxicity of the coptis chinensis total alkaloid self-assembly composition detected by CCK8;

[0048] Figure 3 This is the result of Experimental Example 2 on the effects of the coptis chinensis decoction and the coptis chinensis total alkaloid self-assembly composition on inflammatory factors in RAW 264.7 cells induced by LPS, wherein A is NLRP3; B is TNF-α; C is IL-1β;

[0049] Figure 4 This is a diagram of the dosing method for the drug administration group in Experimental Example 3;

[0050] Figure 5 The figure shows the results of the animal experiment in Experimental Example 3, where A is the skin lesions of the eczema model mice in each group; B is the weight of the mice; and C is the skin lesion index.

[0051] Figure 6The ELISA results in Experiment 3 are shown in Figure 3. A is the serum IgE level; B is the serum IL-1β level; C is the serum TNF-α level; D is the serum IL-6 level;

[0052] Figure 7 The following are the results of mouse skin section staining in Experiment 3. A is the HE staining result; B is the TB staining result; C is the epidermal thickness statistics; D is the mast cell count statistics;

[0053] Figure 8 Figure 3 shows the RNA-seq sequencing results in Experimental Example 3. A is the KEGG analysis diagram; B is the mRNA expression level of the NLRP family in skin tissue.

[0054] Figure 9 This is the RT-PCR test result in Experiment 3. A is NLRP3; B is ACS; C is Caspase-1; D is GSDMD; E is IL-1β; F is IL-33;

[0055] Figure 10 The Western Blot results in Experimental Example 3 are shown in Figure 3. A is a Western blot result; B is NLRP3; C is ACS; D is Caspase-1; E is GSDMD; and F is IL-33. DETAILED DESCRIPTION

[0056] To facilitate understanding of the technical solution of the present invention, the following further describes a self-assembly composition of total alkaloids of Coptis chinensis and its skin gel, preparation method and application of the present invention in conjunction with specific examples.

[0057] Example 1: Preparation of self-assembly composition of total alkaloids from Coptis chinensis:

[0058] The preparation of the self-assembly composition of total alkaloids from Coptis chinensis adopts the pH-driven method, that is, adding NaOH solution to a hydrochloric acid extract of a traditional Chinese medicine at an appropriate concentration, and controlling the concentration of the hydrochloric acid solution and the crude drug concentration of the extract to form the self-assembly composition of total alkaloids from Coptis chinensis. The specific steps are as follows:

[0059] (1) Weigh 362.5 g of raw material Coptis chinensis;

[0060] (2) Add 17.44 mM hydrochloric acid solution and soak for 0.5 h, extract for 1 h, filter, repeat the extraction for 1 h, filter, combine the filtrates, and concentrate to 0.2 g crude drug / mL for later use;

[0061] (3) Prepare 1 mol / L NaOH solution and set aside;

[0062] (4) adjusting the pH of the Coptidis hydrochloric acid extract prepared in step (2) to neutral with the NaOH solution prepared in step (3), stirring at a speed of 200 r / min for 15 minutes to obtain a Coptidis total alkaloid self-assembly composition.

[0063] Example 2: Preparation of self-assembly composition of total alkaloids from Coptis chinensis:

[0064] The preparation of the self-assembly composition of total alkaloids from Coptis chinensis adopts the pH-driven method, that is, adding NaOH solution to a hydrochloric acid extract of a traditional Chinese medicine at an appropriate concentration, and controlling the concentration of the hydrochloric acid solution and the crude drug concentration of the extract to form the self-assembly composition of total alkaloids from Coptis chinensis. The specific steps are as follows:

[0065] (1) Weigh 489.2 g of raw material Coptis chinensis;

[0066] (2) Add 23.26 mM hydrochloric acid solution and soak for 0.5 h, extract for 1 h, filter, repeat the extraction for 1 h, filter, combine the filtrates, and concentrate to 0.1 g crude drug / mL for later use;

[0067] (3) Prepare 2.0 mol / L NaOH solution and set aside;

[0068] (4) adjusting the pH of the Coptidis hydrochloric acid extract prepared in step (2) to neutral with the NaOH solution prepared in step (3), stirring at a speed of 300 r / min for 15 minutes to obtain a Coptidis total alkaloid self-assembly composition.

[0069] Example 3: Preparation of self-assembly composition of total alkaloids from Coptis chinensis:

[0070] The preparation of the self-assembly composition of total alkaloids from Coptis chinensis adopts the pH-driven method, that is, adding NaOH solution to a hydrochloric acid extract of a traditional Chinese medicine at an appropriate concentration, and controlling the concentration of the hydrochloric acid solution and the crude drug concentration of the extract to form the self-assembly composition of total alkaloids from Coptis chinensis. The specific steps are as follows:

[0071] (1) Weigh 380.2 g of raw material Coptis chinensis;

[0072] (2) Add 11.63 mM hydrochloric acid solution and soak for 0.5 h, extract for 1 h, filter, repeat the extraction for 1 h, filter, combine the filtrates, and concentrate to 0.3 g crude drug / mL for later use;

[0073] (3) Prepare 0.5 mol / L NaOH solution and set aside;

[0074] (4) adjusting the pH of the Coptidis hydrochloric acid extract prepared in step (2) to neutral with the NaOH solution prepared in step (3), stirring at a speed of 200 r / min for 15 minutes to obtain a Coptidis total alkaloid self-assembly composition.

[0075] Example 4: Hydrochloric acid solution concentration screening:

[0076] For step (2) in Example 1, extract Coptis chinensis with 5.81, 11.63, 17.44, 23.26, and 29.07 mM hydrochloric acid solutions, respectively, soak for 0.5 h, extract for 1 h, filter, repeat the extraction for 1 h, filter, combine the filtrates, and concentrate to 0.1 g crude drug / mL; adjust the pH of the Coptis chinensis hydrochloric acid extract to neutral with NaOH solution, and stir at a speed of 100-300 r / min for 15 min to obtain a Coptis chinensis total alkaloid self-assembly composition.

[0077] Table 1 Hydrochloric acid water concentration screening results

[0078]

[0079] The results are shown in Table 1. It can be seen from Table 1 that the self-assembled composition of total alkaloids from Coptis chinensis can be obtained when the concentration of the hydrochloric acid solution is 5.81-29.07; when the concentration of the hydrochloric acid solution is 17.44 mM, the particle size of the self-assembled composition of total alkaloids from Coptis chinensis is the smallest.

[0080] Example 5: Extract crude drug concentration screening:

[0081] According to the preparation steps of Example 1, Coptis chinensis was extracted with a 17.44 mM hydrochloric acid solution, soaked for 0.5 h, extracted for 1 h, filtered, and the extraction was repeated for 1 h, filtered, and the filtrates were combined and concentrated to extracts of 0.1, 0.2, and 0.3 g crude drug / mL, respectively. The particle size and appearance properties of the Coptis chinensis total alkaloid self-assembly compositions were compared, and the crude drug concentration of the extracts was screened.

[0082] Table 2 Crude drug concentration screening results

[0083]

[0084] The results are shown in Table 2. According to the results in Table 2, the particle size and PDI of the self-assembled composition of total alkaloids of Coptis chinensis at a concentration of 0.2 g crude drug / mL are relatively small, and the solution is stable without precipitation. Therefore, the gel is prepared at this crude drug concentration.

[0085] Example 6: Preparation of skin gel of self-assembly composition of total alkaloids of Coptis chinensis:

[0086] (1) preparing a self-assembly composition of total alkaloids from Coptis chinensis according to the method of Example 1;

[0087] (2) Weigh the following raw materials: 500 g of the self-assembled composition of total alkaloids of Coptis chinensis, 175 g of distilled water, 25 g of zinc oxide, 40 g of SEPINEO P600, 40 g of butylene glycol, 100 g of glycerol, 100 g of propylene glycol, 7 g of ethylparaben, and 13 g of 1,2-hexanediol;

[0088] (3) Distilled water, the self-assembly composition of total alkaloids from Rhizoma Coptidis, SEPINEO P600, zinc oxide and butylene glycol were placed in a beaker and homogenized and stirred for 20 minutes at 80°C with a stirring speed of 200 r / min (stirring paddle) and a homogenizing speed of 1000 rpm (homogenizer). Glycerin and propylene glycol were then added and homogenized and stirred for 20 minutes. The temperature was lowered to 65°C, and ethylparaben and 1,2-hexanediol were added at a speed of 100-300 r / min and homogenized and stirred for 10 minutes to mix evenly. The mixture was then divided into portions to obtain a skin gel of the self-assembly composition of total alkaloids from Rhizoma Coptidis.

[0089] The thickener SEPINEO P600 (acrylamide / sodium acryloyldimethyl taurate copolymer & isohexadecane & polysorbate 80 & sorbitan oleate) medicinal dressing registration number is F20210000411.

[0090] Example 7: Preparation of skin gel of self-assembly composition of total alkaloids of Coptis chinensis:

[0091] (1) preparing a self-assembly composition of total alkaloids from Coptis chinensis according to the method of Example 1;

[0092] (2) Weigh the following raw materials: 550 g of the self-assembled composition of total alkaloids from Coptis chinensis, 125 g of distilled water, 30 g of zinc oxide, 50 g of SEPINEO P600, 30 g of butylene glycol, 150 g of glycerol, 50 g of propylene glycol, 5 g of ethylparaben, and 10 g of 1,2-hexanediol;

[0093] (3) Distilled water, the self-assembly composition of total alkaloids from Rhizoma Coptidis, SEPINEO P600, zinc oxide and butylene glycol were placed in a beaker and homogenized and stirred for 20 minutes at 80°C, a stirring speed of 300 r / min and a homogenization speed of 1400 rpm. Glycerin and propylene glycol were then added and homogenized and stirred for 20 minutes. The temperature was lowered to 65°C, and ethylparaben and 1,2-hexanediol were added at a speed of 300 r / min and homogenized and stirred for 10 minutes to mix evenly. The mixture was then divided into portions to obtain a skin gel of the self-assembly composition of total alkaloids from Rhizoma Coptidis.

[0094] Example 8: Preparation of skin gel of self-assembly composition of total alkaloids of Coptis chinensis:

[0095] (1) preparing a self-assembly composition of total alkaloids from Coptis chinensis according to the method of Example 1;

[0096] (2) Weigh the following raw materials: 600 g of the self-assembled composition of total alkaloids of Coptis chinensis, 360 g of distilled water, 75 g of zinc oxide, 60 g of SEPINEO P600, 75 g of butylene glycol, 200 g of glycerol, 100 g of propylene glycol, 15 g of ethylparaben, and 15 g of 1,2-hexanediol;

[0097] (3) Distilled water, the self-assembly composition of total alkaloids from Rhizoma Coptidis, SEPINEO P600, zinc oxide and butylene glycol were placed in a beaker and homogenized and stirred for 20 minutes at 80°C, a stirring speed of 200 r / min and a homogenization speed of 1200 rpm. Glycerin and propylene glycol were then added and homogenized and stirred for 20 minutes. The temperature was lowered to 65°C, and ethylparaben and 1,2-hexanediol were added at a speed of 200 r / min and homogenized and stirred for 10 minutes to mix evenly. The mixture was then divided into portions to obtain a skin gel of the self-assembly composition of total alkaloids from Rhizoma Coptidis.

[0098] Example 9: Determination of the content of the main active ingredients in the skin gel of the self-assembly composition of total alkaloids of Coptis chinensis:

[0099] (1) Determination of the contents of epiberberine, coptisine, palmatine and berberine by HPLC:

[0100] Instruments and reagents:

[0101] LCMS-2010EV high performance liquid chromatograph, Shimadzu Corporation, Japan; methanol was of chromatographic grade; other reagents were of analytical grade.

[0102] Chromatographic conditions:

[0103] ODS-3 chromatographic column (4.6×250mm, 5μm); pre-column: GL-CARTRIDGE C18 column; mobile phase: acetonitrile-0.05mol / L potassium dihydrogen phosphate solution (50:50) (add 0.4g of sodium dodecyl sulfate to every 100mL, and then adjust the pH to 4.0 with phosphoric acid); flow rate: 1.0mL / min; detection wavelength: 345nm; column temperature: 35℃; injection volume: 10μL.

[0104] Preparation of reference solution: Take appropriate amount of epiberberine, coptisine, palmatine and berberine reference substances, accurately weigh them, and add methanol to make a solution containing 0.2 mg per 1 mL to obtain the reference solution.

[0105] Preparation of the test solution: Take an appropriate amount of the gel sample prepared in Example 6, place it in a 25 mL volumetric flask, add an appropriate amount of methanol solution, dissolve it by ultrasonication, add methanol solution to the scale, shake well, filter, and take the filtrate to obtain the test solution.

[0106] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into high performance liquid chromatography instrument, and determine.

[0107] (3) It was finally determined that the amounts of the main active ingredients in each gel (20 g) were: epiberberine not less than 10 mg, coptisine not less than 20 mg, palmatine not less than 20 mg, and berberine not less than 80 mg.

[0108] The beneficial effects of the present invention are described below through specific test examples:

[0109] Test Example 1: Characterization of the self-assembled composite of total alkaloids from Coptis chinensis:

[0110] 1. Take the total alkaloid self-assembly composition of Coptis chinensis prepared in Example 1, dilute it 20 times with distilled water, and measure the particle size and PDI using a nanoparticle size analyzer. Repeat the measurement 3 times and take the average value.

[0111] 2. The morphology of the self-assembled composite of total alkaloids from Coptis chinensis was observed by SEM. The sample was diluted 20 times with distilled water and dropped on tin foil. It was then sputter-coated with a gold-palladium conductive layer at a current of 20 mA for 1 min and observed at an accelerating excitation voltage of 10 kV.

[0112] Particle size distribution and morphological analysis of the self-assembled composite of total alkaloids from Coptis chinensis Figure 1 As shown in Figure 2, SEM showed that the morphology of the self-assembled composite of total alkaloids from Coptis chinensis was spherical, and the particle size distribution of the self-assembled composite was relatively uniform, indicating a relatively stable system. The average particle size was 310.23±36.38nm, and the PDI was 0.133±0.059.

[0113] Test Example 2: Cellular pharmacodynamics study of self-assembled composites of total alkaloids from Coptis chinensis:

[0114] 1. Cell line: RAW264.7 macrophages

[0115] 2. Reagents and equipment: DMEM culture medium, highly inactivated fetal bovine serum, penicillin-streptomycin mixture, CCK-8 test kit.

[0116] 3. Experimental methods:

[0117] (1) Sample preparation

[0118] Take 60g of Coptis chinensis, add 10 times the amount of ultrapure water, soak for 30min, reflux and decoct for 1h, filter with non-woven gauze, repeat the extraction of the residue once, filter, combine the filtrate, and concentrate to 0.2g crude drug / mL to obtain Coptis chinensis water decoction.

[0119] The total alkaloid self-assembly composition of Coptis chinensis prepared in Example 1 and the Coptis chinensis decoction were gradiently diluted with DMEM culture medium solution to 300, 200, 100, 50, 20, 10, and 5 μg crude drug / mL and set aside.

[0120] (2) CCK8 proliferation assay:

[0121] RAW264.7 cells in good growth state and in the logarithmic growth phase were seeded in 96-well plates, with the cell density in each well maintained at 1×10 4 96-well plates were placed in a 37°C, 5% CO2 incubator for adherent culture. RAW264.7 cells were treated with different concentration gradients of Coptis chinensis decoction (CR: 300, 200, 100, 50, 20, 10, 5 μg crude drug / mL) and Coptis chinensis total alkaloid self-assembly complex (CR SAN: 300, 200, 100, 50, 20, 10, 5 μg crude drug / mL) for 24 hours.

[0122] (3) RT-PCR detection of inflammatory factor expression levels in RAW264.7 cells:

[0123] RAW264.7 cells in the logarithmic growth phase were taken and seeded in 6-well plates. After the cells were completely attached and stimulated with LPS, different drugs (CR: 100, 200 μg crude drug / mL, CR SAN: 100, 200 μg crude drug / mL) were added and treated for 24 hours. The cells were collected and Trizol reagent was added to extract total RNA. Reverse transcription was performed to prepare cDNA, the culture medium was removed, and the cells were washed once with PBS. The PBS was removed, and 1 mL of Trizol was added to each group and placed on ice. The cells were gently shaken to allow the Trizol reagent to fully contact for 5 minutes. RT-PCR was used to detect the expression of NLRP3, TNF-a, and IL-1β mRNA in the cells. 2 -△△CT The relative mRNA expression was calculated by the method.

[0124] 4. Experimental results: CCK8 results showed that different concentrations of Coptis chinensis decoction and Coptis chinensis total alkaloids self-assembled composites had good cell biocompatibility. Figure 2 The crude drug concentrations of 100 μg / mL and 200 μg / mL of Coptis chinensis decoction and the self-assembly composition of total alkaloids from Coptis chinensis could significantly reduce the levels of NLRP3, TNF-a, and IL-1β in LPS-induced macrophages (P<0.001). However, at the same concentration, the self-assembly composition of total alkaloids from Coptis chinensis had better anti-eczema activity. Figure 3 .

[0125] Test Example 3: Pharmacodynamic study on the anti-eczema effect of the self-assembly composition of total alkaloids from Coptis chinensis for skin application (hereinafter referred to as the self-assembly composition of total alkaloids from Coptis chinensis for skin application):

[0126] 1. Animals: 60 male BALB / c mice, SPF grade, 8 weeks old, weighing 20±1 g.

[0127] 2. Reagents and equipment: 2,4-dinitrochlorobenzene (DNCB); ELISA kits for IgE, TNF-a, and IL-1β; olive oil, acetone.

[0128] 3. Experimental methods:

[0129] (1) Model establishment and drug administration:

[0130] SPF-grade BALB / c mice that passed quarantine were randomly divided into 10 groups in each group, namely, a blank group, a DCNB model group, and a skin gel of the self-assembled composition of total alkaloids from Coptis chinensis (0.5 g / kg, 1 g / kg, and 2 g / kg, calculated as crude drug amount), of which 0.5 g / kg was CR SAN-L, 1 g / kg was CR SAN-M, and 2 g / kg was CR SAN-H; and a dexamethasone cream positive control group (1.0 mg / kg).

[0131] After two days of adaptive feeding, the mice were depilated on their backs (approximately 2 cm × 2 cm area) using a depilatory cream the day before the experiment. The dorsal skin of the mice was smeared with 200 μL of 1% DNCB on days 1 and 3, and 200 μL of 0.6% DNCB (dissolved in a 3:1 mixture of acetone and olive oil) on days 5, 7, 9, 11, and 13. The skin gel group and the dexamethasone group were administered with the self-assembled composition of the total alkaloids of Coptis chinensis starting on day 4, while the blank group and the model group were treated with PBS for 10 consecutive days. Dosing was performed at a fixed time each day, and body weight and skin lesion scoring were performed simultaneously.

[0132] (2) ELISA detection of mouse serum IgE, TNF-a and IL-1β levels:

[0133] The blood was collected from the eyeballs of the mice and placed at room temperature for 30 min. The blood was then heated at 4°C and 5000 r / min. -1 The mixture was centrifuged under the conditions of 40 ° C for 10 min, the upper serum was aspirated, the lower plasma and blood cells were discarded, and the mixture was stored at -20 ° C. The levels of IgE, TNF-a, IL-6 and IL-1β in mouse serum were determined by ELISA.

[0134] (3) Hematoxylin and eosin (H&E) staining:

[0135] The skin from the mouse back was removed and fixed with 4% paraformaldehyde. The fixed skin was dehydrated and embedded in paraffin. The paraffin blocks were cut into 4 μm sections and stained with hematoxylin and eosin (H&E). Microscopic images of the skin sections were obtained, avoiding the location of hair follicles. Five selected sites were selected, and epidermal thickness was measured in each skin section using Image J software.

[0136] (4) Mast cell detection:

[0137] The paraffin sections of mouse skin were stained with toluidine blue, and the changes in the number of mast cells in each group of mice were observed and counted under an optical microscope.

[0138] (5) High-throughput RNA sequencing:

[0139] RNA was extracted from mouse skin lesions and sequenced using the Illumina platform. Significantly altered genes were identified through differential gene analysis. Functional annotation and pathway analysis were performed using the GO and KEGG databases to reveal key biological pathways.

[0140] The RNA-seq sequencing method involves selecting appropriate detection methods based on the sample type and experimental requirements to assess the integrity and purity of the RNA sample. Subsequently, mRNA is enriched from the total RNA using oligo(dT) magnetic beads, and its secondary structure is disrupted by thermophilic denaturation. The resulting mRNA sample is treated with a specific shearing reagent to randomly fragment under appropriate conditions, preparing for subsequent library construction. Next, single-stranded cDNA is synthesized using reverse transcriptase, and double-stranded cDNA is constructed using a second-strand synthesis reaction system. The double-stranded cDNA is then end-repaired, with an A tail added to the 3' end, and ligated with specific adapters to facilitate subsequent amplification and sequencing. Following PCR amplification, the library is quality-checked using appropriate methods to ensure that its quality meets sequencing requirements. Furthermore, the PCR product is denatured into single strands and circularized to form single-stranded circular DNA. Uncircularized linear DNA molecules are simultaneously digested to improve sequencing efficiency. In the subsequent sequencing process, single-stranded circular DNA forms DNA nanoballs (DNBs) containing multiple copies through rolling circle replication, and is fixed in the chip mesh using high-density DNA nanochip technology. It is then sequenced using combined probe anchor polymerization technology (cPAS). The raw data generated by sequencing must be filtered by SOAPnuke (v1.5.6) to remove reads contaminated with adapters, reads containing more than 5% unknown base N, and low-quality reads (the proportion of bases less than 15 is greater than 20%), and finally obtain high-quality clean data. Subsequent data analysis is performed in the Dr.Tom multi-omics data mining system (https: / / biosys.bgi.com), including variant detection and differential gene analysis. First, the clean data is aligned to the reference genome using HISTA2 (v2.1.0) software, and gene fusion detection is performed using Ericscript (v0.5.5). At the same time, rMATS (V3.2.5) is used to detect alternative splicing and differential alternative splicing events. For differentially expressed genes (DEGs), clean data were aligned to the reference gene set using Bowtie2 (v2.3.4.3), and gene expression was quantified using RSEM (v1.3.1). Subsequently, pheatmap (v1.0.8) was used to plot clustered heat maps of gene expression across samples. Differentially expressed genes were detected using DESeq2 (v1.4.5), DEGseq, or PoissonDis. Significantly differentially expressed genes with a Q value ≤ 0.05 or an FDR ≤ 0.001 were screened to provide data support for subsequent biological research.

[0141] KEGG and GO enrichment analysis: To further explore the gene functions related to phenotypic changes, Phyper was used to perform GO and KEGG enrichment analysis on differentially expressed genes based on the hypergeometric test, with a Qvalue ≤ 0.05 as the threshold. Genes that met this condition were defined as significantly enriched in the candidate genes.

[0142] (6) RT-PCR assay:

[0143] Trizol method for isolating and purifying tissue RNA: First, place a 1.5mL EP tube in an ice box, add 200μL Trizol reagent, and weigh 50mg of the tissue sample to be tested (mouse skin lesion tissue) into the EP tube. Subsequently, use a high-speed tissue grinder to grind at 60Hz for 60s, repeat three times, until the tissue is completely ground. After grinding, add 800μl Trizol reagent, place it on ice for 10 minutes, and pipette 30 times to ensure sufficient lysis. Then, add 200μl chloroform, shake vigorously for 15s, and then let it stand on ice for 10 minutes. Centrifuge the sample at 12000rpm for 10 minutes at 4°C, use a pre-cooled centrifuge to separate the aqueous phase and carefully transfer the supernatant to a new EP tube, and record the volume of the supernatant. Then, add an equal volume of isopropanol to the supernatant, gently turn it upside down 10 times, and place it on ice for 10 minutes to promote RNA precipitation. Subsequently, centrifuge at 12000rpm for 10min at 4°C, discard the supernatant, and retain the white precipitate at the bottom of the tube. Add 1mL of pre-cooled 75% DEPC ethanol solution to each tube and gently blow the precipitate to suspend it. Then, centrifuge at 7500rpm for 5 minutes at 4°C, repeat twice to ensure thorough washing of the precipitate. After each centrifugation, discard the supernatant, turn the EP tube upside down on the filter paper, and use the filter paper to absorb the ethanol remaining on the wall of the tube. Finally, add 20μLDEPC water to dissolve the RNA precipitate, and use the NanoDrop 1000 spectrometer to measure the RNA concentration, record the concentration of each sample, and store the remaining samples at -80°C for future use. The reverse transcription reaction was carried out according to the instructions of the Takara reverse transcription kit. The mixed solution was prepared according to the recommended reaction system and transferred to the eight-tube PCR instrument for reaction. The expression of NLRP3, ACS, Caspase-1, GSDMD, IL-1β and IL-33 was detected by real-time quantitative polymerase chain reaction. The GAPDH value was used as a control. 2 -△△CT The relative mRNA expression was calculated by the method.

[0144] (7) Western Blot assay:

[0145] According to the kit instructions, first prepare 10% separation gel and 5% concentration gel, and prepare an appropriate amount of electrophoresis fluid. Next, remove the comb on the gel, add 5 μL of protein marker to the leftmost well of the electrophoresis tank, and then add the protein sample to be tested (protein extracted from mouse skin lesion tissue) to each well in turn. When performing electrophoresis, first treat it with 80V voltage for 30 minutes, then increase it to 120V for 60 to 80 minutes. After completing the electrophoresis, prepare the transfer fluid, add 1 pack of transfer powder to 800mL ultrapure water and 200mL of methanol, and mix well. Use an appropriate amount of transfer fluid to wet the transfer splint, making sure the black side is facing down and the white side is facing up. Remove the gel from the electrophoresis rack, rinse the surface foam with tap water, and cut off the part that does not contain the sample to be tested according to the position of bromophenol blue and protein marker. Ensure that the film is always moist during the whole process. Then, cut a PVDF membrane of appropriate size, soak it with methanol and activate it for 5 minutes. Place the activated PVDF membrane and gel in the order of a black transfer chuck, sponge pad, cut gel, PVDF membrane, sponge pad, and white transfer chuck, and secure the transfer chuck. Next, add pre-chilled transfer buffer to the transfer chuck and place the transfer chamber in a basin filled with ice. Transfer proteins at a rate of 1 kDa per minute at 110 V. After transfer, prepare a blocking buffer containing 5% skim milk: 100 mL of TBST and 5 g of skim milk powder. Mix thoroughly, then place the PVDF membrane in a clean antibody incubation chamber, add 50 mL of blocking buffer, and incubate on a shaker at room temperature for 2 hours. After blocking, discard the blocking buffer and apply 4 mL of the primary antibody dilution to the PVDF membrane. Incubate in a refrigerator at 4°C for 12 to 16 hours. After incubation, wash the PVDF membrane with 1× TBST for 10 minutes, repeating three times, to remove unbound antibody. Next, referring to the instructions for the secondary antibody kit, dilute the secondary antibody in 1× TBST at an appropriate ratio, apply it dropwise to the PVDF membrane, and incubate on a shaker at room temperature for 2 hours. After incubation, wash the membrane again with 1× TBST for 10 minutes, repeating three times. Finally, according to the instructions for the ECL luminescence kit, mix equal volumes of solution A and solution B, store in the dark, and apply it dropwise to the PVDF membrane, ensuring that the membrane surface is completely covered with the luminescence solution. Set the appropriate exposure time according to the experimental needs for development and save the image. After development, use ImageJ software to analyze the grayscale value of the image to assess the relative expression levels of NLRP3, ACS, Caspase-1, GSDMD, and IL-33 in the test samples.

[0146] 4. Experimental results:

[0147] (1) The results of the animal experiment in Test Example 3 are shown in the figure Figure 5 As shown, A is the skin lesion condition of eczema model mice in each group; B is the weight of mice; C is the skin lesion index; Figure 5A shows that compared with the normal group mice, the epidermis of the model group mice showed obvious erythema, hyperkeratosis accompanied by scale formation, and edema appeared in the local tissue of the dermis, indicating that the model was successfully established. Figure 5 B shows that compared with the model group, the skin gel group of the self-assembled composition of total alkaloids from Coptis chinensis can effectively alleviate the weight loss caused by modeling. Figure 5 C shows that compared with the model group, the skin lesions of the Rhizoma Coptidis total alkaloids self-assembly composition skin gel group and the dexamethasone group were improved to varying degrees.

[0148] (2) The ELISA results in Test Example 3 are shown in FIG. Figure 6 shown. Figure 6 AD showed that the skin gel of the self-assembled composition of total alkaloids from Coptis chinensis could significantly reduce the expression of IgE, TNF-a, IL-6 and IL-1β in the serum of eczema mice (P<0.05).

[0149] (3) The results of HE staining and TB staining of mouse skin in Experiment 3 are shown in the figure. Figure 7 shown. Figure 7 AD further showed that the skin gel of the self-assembled composition of total alkaloids from Coptis chinensis could significantly alleviate epidermal thickening and mast cell infiltration in mice with eczema (P<0.001).

[0150] (4) The RNA-seq sequencing results in Experiment 3 are shown in Figure 2. Figure 8 shown. Figure 8 The results showed that the NOD-like receptor signaling pathway was significantly enriched in the skin gel group of the self-assembled composition of total alkaloids from Coptis chinensis, suggesting its important role in the pathological process of eczema. Further screening of NLRP family members revealed that NLRP3 was specifically highly expressed.

[0151] (5) The RT-PCR test results in Experiment 3 are shown in the figure below. Figure 9 As shown, A is NLRP3; B is ACS; C is Caspase-1; D is GSDMD; E is IL-1β; and F is IL-33. Figure 9 AF showed that the skin gel of the self-assembled composition of total alkaloids from Coptis chinensis could significantly reduce the mRNA expression levels of NLRP3, ACS, Caspase-1, GSDMD, IL-1β and IL-33 in lesional skin (P<0.05).

[0152] (6) The Western Blot test results in Experiment 3 are shown in the figure below. Figure 10As shown in the figure, A is the Western blot result; B is NLRP3; C is ACS; D is Caspase-1; E is GSDMD; F is IL-33. 10A-F show that the skin gel of the self-assembly composition of total alkaloids of Coptis chinensis can significantly reduce the protein expression levels of NLRP3, ACS, Caspase-1, GSDMD and IL-33 in lesional skin (P<0.05).

[0153] In summary, the present invention prepares a self-assembled composition of total alkaloids from Coptis chinensis by a pH-driven method. The composition exhibits a spherical structure and a relatively uniform particle size distribution. In vitro studies have shown that the self-assembled composition of total alkaloids from Coptis chinensis has good cell biocompatibility and better anti-eczema activity than Coptis chinensis decoction. In vivo studies have shown that the self-assembled composition of total alkaloids from Coptis chinensis can effectively alleviate inflammatory responses in eczema mice and improve eczema symptoms in mice by regulating the NLRP3 / GSDMD / IL-33 signaling pathway.

[0154] The above description is only a preferred embodiment of the present invention and does not impose any formal or substantial limitation on the present invention. Any technician familiar with the present profession can make use of the technical content disclosed above without departing from the scope of the technical solution of the present invention, and any equivalent changes, modifications and evolutions made by them are equivalent embodiments of the present invention. At the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A self-assembly composition of total alkaloids from Coptis chinensis, characterized in that: The coptis chinensis total alkaloid self-assembly composition is prepared by the following method: (1) Weigh the raw material Coptis chinensis; (2) Take Coptis chinensis, add 5.81-29.07 mM hydrochloric acid solution, soak for 0.5 h, extract for 0.5-2 h, filter, repeat the extraction for 0.5-2 h, filter, combine the filtrates, and concentrate to 0.1-0.3 g crude drug / mL for later use; (3) Prepare 0.5-2.0 mol / L NaOH solution and set aside; (4) adjusting the pH value of the product of step (2) to neutral with the NaOH solution prepared in step (3), stirring at a speed of 100 to 300 r / min for 15 minutes to obtain a self-assembled composition of total alkaloids of Coptis chinensis.

2. The Coptidis total alkaloid self-assembly composition according to claim 1, characterized in that The coptis chinensis total alkaloid self-assembly composition is prepared by the following method: (1) Weigh the raw material Coptis chinensis; (2) Take Coptis chinensis, add 17.44 mM hydrochloric acid solution and soak for 0.5 h, extract for 1 h, filter, repeat the extraction for 1 h, filter, combine the filtrates, and concentrate to 0.2 g crude drug / mL for later use; (3) Prepare 0.5-2.0 mol / L NaOH solution and set aside; (4) adjusting the pH value of the product of step (2) to neutral with the NaOH solution prepared in step (3), stirring at a speed of 100 to 300 r / min for 15 minutes to obtain a self-assembled composition of total alkaloids of Coptis chinensis.

3. The Coptidis total alkaloid self-assembly composition according to claim 1, wherein: The average particle size of the coptis chinensis total alkaloid self-assembly composition is 100-800 nm, and the PDI is 0.02-0.40; SEM shows that the morphology is spherical.

4. The method for preparing the self-assembly composition of total alkaloids from Coptis chinensis according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) Weigh the raw material Coptis chinensis; (2) Take Coptis chinensis, add 5.81-29.07 mM hydrochloric acid solution, soak for 0.5 h, extract for 0.5-2 h, filter, repeat the extraction for 0.5-2 h, filter, combine the filtrates, and concentrate to 0.1-0.3 g crude drug / mL for later use; (3) Prepare 0.5-2.0 mol / L NaOH solution and set aside; (4) adjusting the pH value of the product of step (2) to neutral with the NaOH solution prepared in step (3), stirring at a speed of 100 to 300 r / min for 15 minutes to obtain a self-assembled composition of total alkaloids of Coptis chinensis.

5. The preparation method according to claim 4, characterized in that The preparation method comprises the following steps: (1) Weigh the raw material Coptis chinensis; (2) Take Coptis chinensis, add 17.44 mM hydrochloric acid solution and soak for 0.5 h, extract for 1 h, filter, repeat the extraction for 1 h, filter, combine the filtrates, and concentrate to 0.2 g crude drug / mL for later use; (3) Prepare 0.5-2.0 mol / L NaOH solution and set aside; (4) adjusting the pH value of the product of step (2) to neutral with the NaOH solution prepared in step (3), stirring at a speed of 100 to 300 r / min for 15 minutes to obtain a self-assembled composition of total alkaloids of Coptis chinensis.

6. A skin gel of a self-assembled composition of total alkaloids from Coptis chinensis, characterized in that: The skin gel of the self-assembled composition of total alkaloids of Coptidis rhizome is composed of the following components in weight percentage: 40-60% of the self-assembled composition of total alkaloids of Coptidis rhizome according to any one of claims 1 to 3, 10-30% of distilled water, 5-15% of propylene glycol as a gel moisturizer, 5-15% of glycerol as a gel moisturizer, 3-5% of SEPINEO P600 thickener, 3-5% of butylene glycol, 2-5% of zinc oxide, 0.5-1% of ethylparaben and 1-1.5% of 1,2-hexanediol.

7. The skin gel of the self-assembled composition of total alkaloids from Coptis chinensis according to claim 6, characterized in that: The weight percentages of the components are as follows: 50% of the self-assembled composition of total alkaloids of Coptis chinensis according to any one of claims 1 to 3, 17.5% of distilled water, 10% of propylene glycol as a gel moisturizer, 10% of glycerol as a gel moisturizer, 4% of thickener SEPINEO P600, 4% of butylene glycol, 2.5% of zinc oxide, 0.7% of ethylparaben and 1.3% of 1,2-hexanediol.

8. The method for preparing the skin gel of the self-assembled composition of total alkaloids of Coptidis rhizome according to claim 6 or 7, characterized in that: The preparation method comprises the following steps: (1) Weighing the raw materials according to the weight percentage of claim 6 or 7; (2) Distilled water, the self-assembly composition of total alkaloids from Rhizoma Coptidis, SEPINEO P600, zinc oxide and butylene glycol were placed in a beaker, and homogenized and stirred for 20 minutes at 80°C, at a speed of 100-300 r / min and a homogenization condition of 1000-1500 rpm. Glycerin and propylene glycol were then added, and the homogenization and stirring were continued for 20 minutes. The temperature was lowered to 65°C, and ethylparaben and 1,2-hexanediol were added at a speed of 100-300 r / min, and the mixture was homogenized and stirred for 10 minutes to mix evenly. The mixture was then divided into portions to obtain a skin gel of the self-assembly composition of total alkaloids from Rhizoma Coptidis.

9. Use of the self-assembly composition of total alkaloids from Coptis chinensis according to any one of claims 1 to 3 or the skin gel of the self-assembly composition of total alkaloids from Coptis chinensis according to claim 6 or 7 in the preparation of a drug for treating skin diseases.

10. The use according to claim 9, characterized in that: The skin disease is eczema.

11. The use according to claim 9 or 10, characterized in that: The application is to reduce inflammatory response and improve eczema symptoms in mice by regulating the NLRP3 / GSDMD / IL-33 signaling pathway.

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