External medicine for preventing and / or treating eczema

Abstract

The invention discloses an external medicine for preventing and / or treating eczema. The external medicine is mainly prepared from purslane, golden cypress, mint, folium isatidis, angelica sinensis, raw liquorice, fructus kochiae, radix sophorae flavescentis, gypsum rubrum and coptis chinensis. A mouse eczema model induced by 2, 4-dinitrochlorobenzene (DNCB) is adopted, a positive drug compound dexamethasone acetate emulsifiable paste and different doses of the externally-applied drug are externally applied to the skin, and the treatment effect of the externally-applied drug on eczema mice is observed. Experimental results show that after administration intervention, compared with a model group, the skin lesion condition of mice in each administration group is obviously improved, the ear swelling degree is reduced, the organ index is reduced, the skin inflammatory infiltration is relieved, and the difference has statistical significance. An enzyme-linked immunosorbent assay (ELISA) result shows that compared with a model group, each dose group of the external medicine can reduce the levels of interleukin 4 (IL-4), gamma-interferon (IFN-gamma), immune globulin E (IgE) and IL-4 / IFN-gamma in mouse serum to different degrees, and the difference has statistical significance.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to an external medicine for preventing and / or treating eczema. Background Art

[0002] Eczema is a common inflammatory skin disease with clinical manifestations of severe itching, erythema, redness, swelling, dry skin, and lichenification. With changes in lifestyle and environment, the incidence of eczema has increased year by year. Due to the protracted course of eczema and its stubbornness and difficulty in healing, it seriously affects and reduces the quality of life of patients. Eczema is caused by multiple factors, including abnormal immune function, impaired skin barrier function, genetics, and environmental factors. Modern medicine's treatment for eczema is mostly based on antihistamines, glucocorticoids, and immunosuppressants. The continuous search for safer and more effective drugs is of great practical significance.

[0003] Traditional Chinese medicine has a long history of treating eczema, with advantages such as significant efficacy and few side effects. According to the view of traditional Chinese medicine, the causes of eczema can be attributed to congenital fetal toxicity and residual heat, acquired visceral disorders, and exogenous rheumatism and heat evil. The core pathogenesis is caused by the retention of dampness evil and the loss of qi, blood and body fluids. Therefore, "dampness" is the key pathological factor in the occurrence and development of eczema, and the treatment of dampness should be carried out throughout the treatment. "Suwen Zhizhen Yaoda Lun Pian" states: "For those who are left inside from the outside, treat the outside first and then adjust the inside", which clarifies that the treatment principle of skin diseases should be mainly external treatment. Eczema occurs on the body surface, and external medicines can directly reach the diseased area and relieve symptoms the fastest. Traditional Chinese medicine has unique advantages in treating eczema, such as significant efficacy and few side effects. Summary of the invention

[0004] The purpose of the present invention is to provide a medicine for external use.

[0005] The external-use medicine provided by the present invention is prepared from purslane, phellodendron, mint, isatis indigotica, angelica, raw liquorice, kochia scoparia, sophora flavescens, syringa schrenckii and coptis chinensis as main raw materials, and the weight proportions of the raw materials are as follows: 25-35 parts of purslane, 5-15 parts of phellodendron, 5-15 parts of mint, 10-20 parts of isatis indigotica, 25-35 parts of angelica, 5-15 parts of raw liquorice, 10-20 parts of kochia scoparia, 5-20 parts of sophora flavescens, 10-20 parts of syringa schrenckii and 5-15 parts of coptis chinensis.

[0006] In one embodiment of the present invention, the mass proportions of the raw materials of the external medicine are: 30 parts of Portulaca Oleracea, 10 parts of Phellodendron Amurense, 10 parts of Mentha Piperita, 15 parts of Folium Isatidis, 30 parts of Angelica Sinensis, 10 parts of Raw Licorice, 15 parts of Kochia Fruit, 12 parts of Sophora Flavescentis, 15 parts of Salvia Nitrariae, and 10 parts of Coptis Chinensis.

[0007] The above-mentioned external medicine is prepared by a method comprising the following steps: taking the raw materials: purslane, phellodendron, mint, isatis indigotica, angelica, raw licorice, kochia scoparia, sophora flavescens, calamus, and coptis chinensis, adding 10 times the amount of water and simmering for 30 minutes, boiling with high heat, then boiling with low heat for 1.5 hours, filtering and concentrating under reduced pressure to a concentration equivalent to 1.03g / mL in terms of the amount of raw medicine, filling the treated liquid into a 250mL medical glass bottle, sealing it with a rubber stopper, and sterilizing it by high-pressure steam sterilization (121°C, 30min).

[0008] The amount of water added for moistening is 10 times the total amount of raw materials (i.e. 157g of medicinal materials plus 1570mL of water).

[0009] The soaking time may be 30 minutes.

[0010] The slight boiling time may be 1.5 hours.

[0011] The use of the above-mentioned external medicine in the preparation of medicines for preventing and / or treating eczema also falls within the protection scope of the present invention.

[0012] The application, the eczema includes acute eczema, prickly heat, dermatitis with heavy exudation, and herpes diseases.

[0013] Eczema is a common allergic and inflammatory disease in clinical practice. According to the clinical course of disease, acute, subacute and chronic eczema are divided. Among them, acute eczema is mostly erythema, papule, papulovesicle, exudate and erosion with itching. Its occurrence is mainly attributed to the interweaving of dampness and heat, and is always caused by "the poison of dampness and heat is contained in the skin". Therefore, treatment should be based on clearing away heat and drying dampness, dispelling wind and relieving itching, cooling blood and detoxifying. Externally used medicine of the present invention is composed of purslane, phellodendron, peppermint, isatis indigotica, angelica, raw liquorice, Kochia scoparia, sophora flavescens, cold water stone and coptis root. In the prescription, purslane and phellodendron are both the main drugs. Purslane has the effects of clearing away heat and detoxifying, cooling blood and relieving itching. "Compendium of Materia Medica" records that purslane can "disperse blood, reduce swelling, and detoxify". Phellodendron is bitter and cold in nature and has the effects of clearing away heat and dampness, purging fire and detoxifying. It has the effects of clearing away heat and detoxifying, cooling blood and stopping bleeding; mint, isatis indigotica, and kochia scoparia are the assistant drugs. Mint is pungent and cool in nature and enters the lung and liver meridians, and has the effects of evacuating wind-heat, clearing heat and detoxifying; isatis indigotica is bitter and cold in nature and enters the heart and stomach meridians, and has the effects of clearing heat and detoxifying, cooling blood and eliminating spots; kochia scoparia is bitter and cold in nature and enters the heart and liver meridians, and "Bencao Yuanshi" records that it can: "remove accumulated heat in the skin and eliminate dampness and itching on the skin", and has the effects of clearing heat, promoting diuresis, and relieving itching The combination of the three medicines can not only enhance the power of clearing heat and detoxifying, but also cool blood and eliminate spots, and has a synergistic effect on the treatment of acute eczema; Sophora flavescens clears heat and dries dampness, kills insects and relieves itching, while Angelica sinensis replenishes blood, nourishes blood, and activates blood circulation, and nourishes the spleen. The two medicines are used together, one cold and one hot, one attacking and one replenishing, one drying and one moistening, which can not only play the role of Angelica sinensis in activating blood circulation and moisturizing the skin, but also restrain the bitter and cold nature of Sophora flavescens; Coptis chinensis is bitter and cold, can relieve heart and stomach fire, dry dampness and detoxify, while Hanshuishi is salty and cold, can descend and drain, clear heat and purge fire, and is good at removing Yangming hidden evil. The four medicines are used together to descend and drain blazing fire, clear heat and detoxify, and are collectively adjuvants; Licorice as a guiding medicine can not only reconcile the properties of the medicine and alleviate the side effects of the medicine, but also reduce the cold nature of other medicinal materials and protect the spleen and stomach. This prescription follows the principle of "Suwen Zhizhen Yaoda Lun" that "heat is rampant inside, treat it with salty and cold, supplemented with sweet and bitter, sour to restrain it, bitter to release it. Dampness is rampant inside, treat it with bitter and hot, supplemented with sour and light, bitter to dry it, light to drain it", and at the same time, it also provides wind-clearing and heat-clearing products, giving the evil a way out and promoting the elimination of pathogenic factors. The whole prescription is mainly for clearing away heat and detoxification, supplemented by drying dampness and relieving itching, cooling blood and removing spots, and is suitable for the treatment of acute eczema.

[0014] The present invention adopts the eczema mouse model induced by 2,4-dinitrochlorobenzene (DNCB), which is widely used in drug efficacy research, and the skin is given positive drug dexamethasone and different doses of the topical drug of the present invention for external application to observe its therapeutic effect on eczema mice. The experimental results show that compared with the blank group, the epidermis of the model group mice showed redness, erythema, keratinization, scab and exudation, the skin lesion score increased, the ear swelling degree and the organ index increased significantly, indicating that the modeling was successful. After drug intervention, compared with the model group, the skin lesions of the mice in each dose group were significantly improved, the ear swelling was reduced, the organ index decreased, and the skin inflammatory infiltration was alleviated, and the difference was statistically significant. The results of enzyme-linked immunosorbent assay (ELISA) showed that compared with the model group, each dose group of the topical drug of the present invention could reduce the levels of interleukin 4 (IL-4), gamma-interferon (IFN-γ), immunoglobulin E (IgE) and IL-4 / IFN-γ in mouse serum to varying degrees, and the difference was statistically significant.

[0015] In summary, the present invention investigated the therapeutic effect of the topical medication of the present invention on the DNCB-induced mouse eczema model by observing animal skin, pathological morphological changes, and the expression of related immune cytokines and inflammatory factors, providing necessary experimental evidence for the clinical safety application of the drug and the development of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The results of the methodological specificity test in Example 1 of the present invention are shown in Figure 1. Note: A: matrine reference substance; B: test substance; C: negative control substance lacking matrine; 1: matrine.

[0017] Figure 2 This is the result of the linear relationship experiment conducted by the methodology in Example 1 of the present invention.

[0018] Figure 3 The results of the single factor experiment in Example 1 of the present invention are shown in Figure 1. Note: A: the effect of the amount of water added on the matrine content; B: the effect of the extraction time on the matrine content; C: the effect of the number of extractions on the matrine content; D: the effect of the moistening time on the matrine content.

[0019] Figure 4 This is the HE staining picture of the ear of the mouse with eczema in Example 2 of the invention (HE×200, n=8). Note: A: blank group, B: model group, C: positive drug group, D: high-dose group of the external medicine of the present invention, E: medium-dose group of the external medicine of the present invention, F: low-dose group of the external medicine of the present invention. Compared with the model group, * P<0.05, ** P<0.01.

[0020] Figure 5This is the HE staining image of the back of the mouse with eczema in Example 2 of the present invention (HE×200, n=8). Note: A: blank group, B: model group, C: positive drug group, D: high-dose group of the topical drug of the present invention, E: medium-dose group of the topical drug of the present invention, F: low-dose group of the topical drug of the present invention. Compared with the model group, * P<0.05, ** P<0.01.

[0021] Figure 6 This is the TB staining image of the auricle of eczema mice in Example 2 of the present invention (TB×200, n=8). Note: A: blank group, B: model group, C: positive drug group, D: high-dose group of the topical drug of the present invention, E: medium-dose group of the topical drug of the present invention, F: low-dose group of the topical drug of the present invention. Compared with the model group, * P<0.05, ** P<0.01.

[0022] Figure 7 This is the TB staining image of the back of the eczema mouse in Example 2 of the present invention (TB×200, n=8) Note: A: blank group, B: model group, C: positive drug group, D: high-dose group of the topical drug of the present invention, E: medium-dose group of the topical drug of the present invention, F: low-dose group of the topical drug of the present invention. Compared with the model group, * P<0.05, ** P<0.01.

[0023] Figure 8 These are the immunohistochemical results of IL-1β, IL-6, IL-33, and TNF-α in the skin lesion tissues of eczema mice in Example 2 of the present invention (×200, n=8).

[0024] Fig. 9 The levels of inflammatory factors in the serum of eczema mice in Example 2 of the present invention (n=8). Note: A: IL-4, B: IFN-γ, C: IgE, D: IFN-γ / IL-4. Compared with the model group, * P<0.05, ** P<0.01. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0026] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0027] Example 1 Study on the preparation process of lotion

[0028] 1. Formula

[0029] Purslane 30g Phellodendron 10g Mint 10g

[0030] Isatis indigo leaf 15g Angelica sinensis 30g Raw licorice 10g

[0031] Kochia scoparia 15g Sophora flavescens 12g Cold water stone 15g Coptis chinensis 10g

[0032] 2. Preparation process

[0033] Take the above medicinal materials and add 10 times the amount of water to soak for 30 minutes, boil with high heat, and then simmer for 1.5 hours with low heat, stir and filter out the medicinal liquid while it is hot, concentrate the filtrate, and concentrate it under reduced pressure to the equivalent of 1.03g / mL based on the amount of crude drug. Fill the treated medicinal liquid into a 250mL medical glass bottle, seal it with a rubber stopper, and sterilize it by high-pressure steam sterilization (121℃, 30min).

[0034] 3. Dosage Form Selection

[0035] This prescription is a clinical experience prescription. It has unique advantages for external use. It has the effects of clearing away heat and detoxifying, drying dampness and relieving itching. It is clinically used to treat acute eczema and has achieved significant results. The lotion can promote local blood circulation, reduce the permeability of capillaries in the inflamed area, and accelerate the recovery of eczema through moisturizing and wet compressing. This treatment method can be directly absorbed into the body through the skin barrier, which can not only effectively relieve redness, swelling and itching, but also improve the treatment effect. Therefore, the prescription selects the lotion as the preparation dosage form.

[0036] 4. Extraction process design

[0037] 1. Sample preparation and chromatographic conditions

[0038] 1.1 Sample preparation

[0039] Reference substance: Take an appropriate amount of matrine reference substance, weigh accurately, and add methanol to make a 450 μg / mL solution.

[0040] Test sample: Accurately measure 10 mL of this product, place it in a separatory funnel, add 0.5 mL of concentrated ammonia test solution, shake well, extract with chloroform for 3 times, 20 mL each time, combine the chloroform extracts, evaporate to dryness, add appropriate amount of acetonitrile-anhydrous ethanol (80:20) to dissolve the residue, transfer to a 50 mL volumetric flask, add acetonitrile-anhydrous ethanol (80:20) to the scale, filter, and take the filtrate to obtain.

[0041] Negative control: A negative sample lacking Sophora flavescens, prepared according to the method for the test sample.

[0042] 1.2 Chromatographic conditions

[0043] ChromaiVoyager high performance liquid chromatograph; Elite SupersilNH2 chromatographic column (4.6mm×250mm, 5μm); mobile phase: acetonitrile: anhydrous ethanol: 3% phosphoric acid solution (80:10:10); flow rate: 1.0mL / min; injection volume: 10μL; detection wavelength: 220nm; column temperature: 30℃.

[0044] 2 Methodological investigation

[0045] 2.1 Exclusivity Investigation

[0046] The reference solution, test solution and negative control solution were accurately aspirated and the determination was carried out under the chromatographic conditions of 1.2. The results showed that the peak times of the test sample and the matrine reference were consistent, and no corresponding peak appeared in the negative sample at the same time. The results showed that other ingredients in the prescription had no interference with the determination of matrine content in the lotion. This method has good specificity and meets the experimental requirements. Figure 1 .

[0047] 2.2 Investigation of linear relationship

[0048] The standard curve was drawn with the mass concentration of matrine (μg / mL) as the horizontal axis (X) and the chromatographic peak area as the vertical axis (Y). The regression line equation was calculated as: Y = 7.5092X + 19.13, r = 0.9994. The results showed that the linear relationship of matrine in the range of 14.07 to 450 μg / mL was good. Figure 2 .

[0049] 2.3 Precision investigation

[0050] Take the matrine reference solution, and inject it 6 times continuously according to the chromatographic conditions in 1.2, record the peak area, and calculate the RSD. The results are shown in Table 1, RSD = 0.37%, indicating that the precision of the instrument is good.

[0051] Table 1 Precision investigation results

[0052]

[0053] 2.4 Stability investigation

[0054] Take the test solution, inject it at 0, 2, 4, 8, 12, and 24 hours, record the peak area, and calculate the RSD. The results are shown in Table 2, RSD = 1%, indicating that the test solution is stable within 24 hours.

[0055] Table 2 Stability test results

[0056]

[0057] 2.5 Repeatability study

[0058] Take 6 portions of the same batch of medicinal materials, prepare the test solution according to the method under 2.1, measure under the chromatographic conditions under 1.2, record the peak area, and calculate RSD. The results are shown in Table 3, RSD = 1.72%, indicating that the method has good repeatability.

[0059] Table 3 Repeatability test results

[0060]

[0061] 3 Single factor experimental investigation

[0062] In order to investigate the best extraction process of the selected lotion, a single factor investigation was conducted based on the relevant literature on Chinese medicine extraction, and the content of matrine in the extract (unit: μg / g) was used as the experimental optimization index.

[0063] 3.1 Add multiple times of water

[0064] According to the prescription ratio, 5 portions of Chinese herbal medicine slices were weighed, each portion was 157g, and water was added 4, 6, 8, 10, and 12 times respectively. Extraction was performed twice, each time for 1 hour. The filtrate was combined and concentrated to 1.03g / mL based on the raw drug amount. The obtained drug solution was determined for content. Each group of parallel experiments was performed 3 times. The results are shown in Figure 3 -A.

[0065] 3.2 Extraction time

[0066] According to the prescription ratio, 5 portions of Chinese herbal medicine slices were weighed, each portion was 157g, 8 times the amount of water was added, and extracted twice, with the decoction time being 0.5h, 1h, 1.5h, 2h, and 2.5h respectively. The filtrate was combined and concentrated to 1.03g / mL based on the amount of crude drug. The obtained liquid was tested for content, and each group of parallel experiments was performed 3 times. The results are shown in Figure 3 -B

[0067] 3.3 Number of extractions

[0068] Weigh 3 portions of Chinese herbal medicine slices according to the prescription ratio, 157g each, add 8 times the amount of water, decoct for 1h, extract 1, 2, and 3 times respectively, combine the filtrate and concentrate it to the equivalent of 1.03g / mL in terms of crude drug amount, and determine the content of the obtained liquid medicine. Each group of parallel experiments was performed 3 times. The results are shown in the table. Figure 3 -C.

[0069] 3.4 Stuffing time

[0070] Weigh 5 portions of Chinese herbal medicine slices according to the prescription ratio, each portion is 157g, add 8 times the amount of water, and soak them before decocting. The soaking time is 10, 20, 30, 40, and 50min respectively. Extract twice, each time for 1h. After combining the filtrate, concentrate it to the equivalent of 1.03g / mL in terms of crude drug amount. Determine the content of the obtained drug solution. Each group of parallel experiments is done 3 times. The results are shown in the figure. Figure 3 -D.

[0071] 4 Orthogonal test

[0072] Taking the soaking time (A), water addition times (B), extraction time (C), extraction times (D) as the investigation factors, and the content of matrine as the investigation index, L9 (3 4 ) orthogonal table was used for experiment, and the results are shown in Table 4-Table 6.

[0073] Table 4 Factor level table

[0074]

[0075]

[0076] Table 5 Orthogonal test schedule and test results

[0077]

[0078] Table 6 Variance analysis table

[0079]

[0080]

[0081] From the R value in Table 5, it can be seen that the influence of each factor on the extraction content of matrine is C>A>B>D, that is, the number of extractions>water addition>extraction time>saturation time. Analyzing the results of the orthogonal test, the optimal level of each factor can be obtained from the K value. The larger the K value, the higher the extraction content of this level. By comparing the matrine content, the optimal process combination parameters are determined to be A3B3C1D3, that is, 10 times the amount of water, 1.5h extraction time, 1 extraction, and 30min of saturation time.

[0082] 5 Extraction process verification experiment

[0083] In order to detect the stability of the predicted value of the orthogonal test design, three repeated verification tests were carried out under the optimized conditions. According to the above preferred scheme A3B3C1D3, three parallel extractions were carried out, and the relevant indicators were calculated to verify the reliability of the above preferred extraction scheme. The results are shown in Table 7. The differences in various content indicators are small and the reproducibility is good, indicating that the process is reasonable, stable and feasible.

[0084] Table 7 Extraction process validation experimental results

[0085]

[0086] Example 2 Pharmacodynamics Study

[0087] 1 Materials and Instruments

[0088] 1.1 Experimental animals

[0089] SPF male KM mice, 6-8 weeks old, weighing 30-35 g, were purchased from Sbefor (Beijing) Biotechnology Co., Ltd., license number: SCXK (Beijing) 2019-0010. Room temperature (23±2)℃, humidity (55±2)%, light-dark alternation, free access to water and food. The experiment was approved by the Ethics Committee of Shanxi University of Traditional Chinese Medicine and was conducted in accordance with regulations (ethics approval number: AWE202304296).

[0090] 1.2 Drugs and reagents

[0091] DNCB (Chengdu Aikoda Chemical Reagent Co., Ltd., batch number: 202206281); compound dexamethasone acetate cream (China Resources Sanjiu Pharmaceutical Co., Ltd., batch number: 2305008J); interleukin 4 (IL-4), gamma interferon (IFN-γ), immunoglobulin E (IgE) enzyme-linked immunosorbent assay (ELISA) kits (Shanghai Yingxin Biotechnology Co., Ltd., batch numbers: F2165-A, F2182-A, F2056-A); xylene (Sinopharm Chemical Reagent Co., Ltd., batch number: 10023418); hematoxylin-eosin (HE) staining solution set (Wuhan Sewell Technology Co., Ltd., batch number: G1003); Toluidine blue (TB) stain (Beijing Solebow Technology Co., Ltd., batch number: G3670); primary antibodies: interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), interleukin 33 (IL-33) (Beijing Bioson Biotechnology Co., Ltd., batch number: bs-6319R, bs-0782R, bs-10802R, bs-0732R); HRP-labeled goat anti-rabbit secondary antibody (Wuhan Sewell Technology Co., Ltd., batch number: GB23303); Dolichos biflorus agglutinin (DAB) colorimetric agent (Beijing Solebow Technology Co., Ltd., batch number: DA1010).

[0092] 1.3 Instruments

[0093] Multiskan FC automatic microplate reader (Berteng, USA); Donatello dehydrator, Giotto stainer (Typro Bioscience China Co., Ltd.); JB-P5 embedding machine (Wuhan Junjie Electronics Co., Ltd.); RM2016 pathology slicer (Shanghai Leica Instrument Co., Ltd.); GFL-230 oven (Tianjin Lepore Instrument Equipment Co., Ltd.); Axioscope5 microscope (Carl Zeiss Shanghai Management Co., Ltd.); Archimed X4 fluorescence quantitative PCR instrument (Kunpeng Gene Science Instrument Co., Ltd.).

[0094] 2 Experimental methods

[0095] 2.1 Grouping, modeling and drug administration

[0096] After one week of adaptive feeding, 48 KM mice were randomly divided into 6 groups, each with 8 mice, namely, the blank group (Normal), the model group (Model), the positive drug group (Dex, 37.5 mg / kg), the external drug of the present invention (prepared in Example 1) high dose group (Highdose, 206 g / kg), medium dose group (Mediumdose, 103 g / kg), and low dose group (Lowdose, 51.5 g / kg). The 2 cm × 2 cm area on the back was depilated one day before the experiment. On the day of the experiment, except for the blank group, the rest of the mice were smeared with 5% DNCB solution (solvent is acetone: olive oil = 4:1 (volume ratio)) 100 μL and right ear 25 μL for sensitization on the depilated area on the back. On the 6th, 9th, 12th, and 15th days of the experiment, 0.7% DNCB 100 μL and right ear 25 μL were applied to the depilated area for stimulation, and the blank group was coated with an equal amount of acetone-olive oil solvent. The modeling method in the reference literature was used, and the modeling was successful when the mice showed skin redness, dryness, itching, thickening, etc. Each group began to apply the drug daily on the day of stimulation for 14 consecutive days. Samples were taken 24 hours after the last drug administration to test various indicators.

[0097] 2.2 Detection indicators and methods

[0098] 2.2.1 Back skin lesion scoring

[0099] On the 7th, 10th, 13th and 16th days of the experiment, the skin lesions of mice in each group were observed and photographed before administration, and the scores were calculated according to Table 8. The observation indicators included redness, swelling, keratinization and crusting, and exudation.

[0100] Table 8 Scoring criteria for back skin lesions in mouse eczema model

[0101]

[0102] 2.2.2 Organ index determination

[0103] The thymus and spleen of the mice were weighed and the organ index was calculated: Organ index (%) = [organ mass (g) / body mass (g)] × 100.

[0104] 2.2.3 Determination of ear swelling

[0105] After the treatment cycle, holes were punched in the corresponding parts of the left and right ears of the mice with a 6 mm diameter puncher, the ear pieces were removed and weighed with an analytical balance, and the ear swelling was calculated based on the weight difference between the left and right ears.

[0106] 2.2.4 HE staining to observe pathological morphological changes

[0107] Skin lesions from the right ears and backs of mice in each group were collected, fixed with 4% paraformaldehyde, dehydrated with ethanol, embedded in paraffin, sliced, stained with hematoxylin-eosin (HE), dehydrated, transparent, and sealed. Pathological morphological changes were observed under a 20x objective, images were taken, and epidermal thickness was calculated using Image J software.

[0108] 2.2.5 TB staining to observe mast cell infiltration

[0109] After paraffin sections were dewaxed, stained with toluidine blue (TB), dehydrated, transparent, and sealed. Mast cell infiltration was observed under a 20x objective and images were taken. The number of mast cells was counted using Image J software.

[0110] 2.2.6 Immunohistochemistry to determine the expression of IL-1β, IL-6, IL-33, and TNF-α

[0111] After dewaxing and dehydration, the pathological sections were washed and antigen repaired, blocked and incubated with primary antibody (dilution factor 1:100) at 4°C overnight, washed and incubated with secondary antibody at 37°C for 30 min, covered with DAB, observed under a microscope and analyzed for optical density using Image J software.

[0112] 2.2.7 Serum IL-4, IFN-γ, and IgE levels

[0113] After blood collection, let it stand at room temperature until the plasma is separated and then centrifuge it. Centrifuge it in a low-temperature centrifuge for 15 minutes (4°C, 3000r / min), draw the upper serum, and store it in a -80°C refrigerator for later use. Strictly follow the instructions of the ELISA kit to test the levels of IL-4, IFN-γ, and IgE in the serum.

[0114] 2.2.8 RT-PCR detection of inflammatory factors in skin lesions

[0115] RNA was extracted with Trizol, purity was determined, and reverse transcription was performed to obtain cDNA. 2 μL of each of the upstream and downstream primers for PCR (Table 9) was taken and configured into a 2 μL PCR system according to the instructions of the kit. The reaction was performed according to the following procedure: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 5 s, annealing at 60°C for 35 s, and detection was performed after 45 cycles. -ΔΔCt The expression differences of target genes among the groups were analyzed.

[0116] Table 9 Primer information of inflammatory factors in mouse eczema model

[0117]

[0118] 2.3 Statistical methods

[0119] GraphPad Priam 8.0.2 software was used for data analysis. One-way analysis of variance was used for comparison among multiple groups, and P < 0.05 was considered statistically significant.

[0120] 3 Results and analysis

[0121] 3.1 Effect of skin lesion scores on mice

[0122] After the modeling, the epidermis of the mice in the model group showed redness, swelling, erythema, keratinization, scabs and exudation, and the skin lesion score was significantly increased (P < 0.01); after drug intervention, compared with the model group, different doses of the topical medication of the present invention showed obvious therapeutic effects, and the skin lesion score was significantly reduced (P < 0.05, P < 0.01), indicating that the topical medication of the present invention has a certain therapeutic effect on eczema mice, as shown in Table 10.

[0123] Table 10 Results of skin lesion scoring in mouse eczema model

[0124]

[0125] Note: Compared with the model group, * P<0.05, ** P<0.01.

[0126] 3.2 Effect on mouse ear swelling

[0127] Compared with the blank group, the ear swelling of the mice in the model group was significantly increased (P < 0.01); compared with the model group, the ear swelling of the mice in each dosage group was significantly reduced (P < 0.01), which indicates that the topical medication of the present invention can alleviate the ear swelling of mice with eczema, as shown in Table 11.

[0128] Table 11 Analysis of ear swelling in mouse eczema model

[0129]

[0130]

[0131] Note: Compared with the model group, * P<0.05, ** P<0.01.

[0132] 3.3 Effects on mouse organ indexes

[0133] Compared with the blank group, the spleen and thymus indexes of the mice in the model group were significantly increased (P < 0.01); after drug treatment, compared with the model group, the spleen index of the medium and high dose groups of the topical medication of the present invention decreased (P < 0.05), and the thymus index of the high dose group of the topical medication of the present invention decreased significantly (P < 0.05), as shown in Table 12.

[0134] Table 12 Analysis of organ indexes in mouse eczema model

[0135]

[0136] Note: Compared with the model group, * P<0.05, ** P<0.01.

[0137] 3.4 Effects on histopathology of mouse skin lesions

[0138] The auricle skin cell structure of the mice in the blank group was clear, the epidermis thickness was normal, and no inflammatory cell infiltration was observed; the auricle thickness of the mice in the model group was significantly increased (P<0.01), with mild keratinization and accompanied by inflammatory cell infiltration; after drug intervention, the epidermis thickness of the ears of the mice in each dose group of the topical drug of the present invention was significantly reduced (P<0.01), the keratinization phenomenon was improved, and the inflammatory cell infiltration was reduced. Figure 4 .

[0139] The back skin tissues of mice in the blank group were intact, the epidermis was relatively smooth, and there was no inflammatory cell infiltration; the epidermis of mice in the model group was significantly thickened (P<0.01), keratinization was severe, and a large number of inflammatory cells infiltrated; compared with the model group, the excessive keratinization, edema, and inflammatory cell infiltration in each dose group of the topical drug of the present invention were reduced (P<0.01), and the inhibition degree of the high dose group was the most obvious, as shown in Table 1. Figure 5 .

[0140] 3.5 Effects on mast cells in mouse skin lesions

[0141] A small number of mast cells were observed in the dermis of the blank group. Compared with the blank group, the number of mast cells in the dermis of the model group increased significantly (P < 0.01). After administration of different doses of the drug of the present invention, the accumulation of mast cells in the dermis of each group was significantly reduced (P < 0.01), which indicates that the increase of mast cells may be an important cause of eczema. Figure 6 , Figure 7 .

[0142] 3.6 Effects on inflammatory factors in mouse skin lesions

[0143] The positive expression of IL-1β, IL-6, IL-33, and TNF-α is brown-yellow area, located in the cytoplasm and cell membrane, and mainly expressed in the epidermal spinous layer, dermis, and sebaceous glands. The expression of inflammatory factors in the blank group was less, and the expression in the model group was significantly more; compared with the model group, each dose group of the topical drug of the present invention can significantly inhibit the expression of IL-1β, IL-6, IL-33, and TNF-α (P < 0.01), and the optical density value is dose-dependent, as shown in Figure 2. Figure 8 .

[0144] 3.7 Effects on the levels of inflammatory factors in mouse serum

[0145] Compared with the blank group, the levels of IL-4, IgE, IFN-γ and IFN-γ / IL-4 in the serum of the mice in the model group increased significantly (P<0.05, P<0.01); compared with the model group, the topical drug of the present invention can reduce the levels of each factor to varying degrees and the difference is statistically significant. The results show that the topical drug of the present invention can significantly reduce the levels of IL-4, IgE, IFN-γ and IFN-γ / IL-4, indicating that the drug of the present invention can inhibit inflammatory response and has a significant therapeutic effect. Fig. 9 .

[0146] 3.8 Effects on mRNA expression of related inflammatory factors in mouse skin lesions

[0147] Compared with the blank group, the mRNA levels of IL-1β, IL-6, IL-33, and TNF-α in the skin tissue of mice in the model group were significantly increased (P < 0.05, P < 0.01); compared with the model group, the mRNA levels of inflammatory factors in the high-dose group of the topical medication of the present invention after intervention were decreased (P < 0.05, P < 0.01), as shown in Table 13.

[0148] Table 13 Effect of inflammatory factors IL-1β, IL-6, IL-33, and TNF-α mRNA expression on skin lesions in mice with eczema

[0149]

[0150]

[0151] Note: Compared with the model group, * P<0.05, ** P<0.01.

[0152] The pathogenesis of eczema is complex and may be related to multiple factors such as immunity, genetics, skin barrier, environment, and mental factors. This experiment used the DNCB-induced eczema mouse model, which is widely used in drug efficacy research, and applied the positive drug dexamethasone and different doses of the topical drug of the present invention to the skin to observe its therapeutic effect on eczema mice.

[0153] The external-use medicine of the present invention is composed of purslane, phellodendron, mint, isatis leaf, angelica, raw liquorice, kochia fruit, sophora flavescens, calamus and coptis root. In the prescription, purslane and phellodendron are both monarch drugs, purslane has the effects of clearing away heat and detoxicating, cooling blood and relieving itching, and "Compendium of Materia Medica" records that purslane can "disperse blood and reduce swelling, and detoxify", and phellodendron is bitter and cold in nature and flavor, has the effects of clearing heat and drying dampness, purging fire and detoxifying, and has the effects of clearing heat and detoxifying, cooling blood and stopping bleeding; mint, isatis leaf and kochia fruit are ministerial drugs, mint is pungent and cool in nature and flavor, enters the lung and liver meridians, has the effects of evacuating wind-heat, clearing heat and detoxifying; isatis leaf is bitter and cold in nature and flavor, enters the heart and stomach meridians, has the effects of clearing heat and detoxifying, cooling blood and eliminating spots; kochia fruit is bitter and cold in nature and flavor, enters the heart and liver meridians, and has the effects of "removing accumulated heat in the skin and removing dampness and itching outside the skin", and has the effects of clearing heat and promoting diuresis and relieving itching. The combination of the three medicines can not only enhance the power of clearing heat and detoxifying, but also cool blood and eliminate spots, and has a synergistic effect on the treatment of acute eczema; Sophora flavescens clears heat and dries dampness, kills insects and relieves itching, while Angelica sinensis replenishes blood, nourishes blood, activates blood circulation, and nourishes the spleen. The combination of the two medicines, one cold and one hot, one attacking and one replenishing, one drying and one moistening, can not only play the role of Angelica sinensis in activating blood circulation, warm the skin, but also restrain the bitter and cold nature of Sophora flavescens; Coptis chinensis is bitter and cold, relieves heart and stomach fire, dries dampness and detoxifies, and Hanshuishi is salty and cold, clears heat and purges fire, and is good at removing Yangming hidden evil. The four medicines are used together to reduce and drain blazing fire, clear heat and detoxify, and are adjuvants; Licorice as a guiding medicine can not only reconcile the medicinal properties and relieve the side effects of the drugs, but also reduce the cold nature of other medicinal materials and protect the spleen and stomach. After all the prescriptions are matched, the efficacy can be further exerted to ensure that the clinical symptoms can be effectively improved.

[0154] The experimental results showed that compared with the blank group, the epidermis of the mice in the model group showed redness, erythema, keratinization, scabs and exudation, the skin lesion score increased, and the ear swelling and organ index increased significantly, indicating that the model was successful. After drug intervention, compared with the model group, the skin lesions of the mice in each dose group were significantly improved, the ear swelling was reduced, the organ index decreased, and the skin inflammatory infiltration was alleviated, and the difference was statistically significant.

[0155] It is generally believed that the pathogenesis of eczema is closely related to inflammatory response. Helper T cells (Th) play an important role in inflammatory response, including Th1 cells and Th2 cells. Th1 / Th2 cells are in a state of mutual inhibition. Th1 cytokine IFN-γ antagonizes IL-10 and IL-4, thereby inhibiting the activity of Th2 cells. Conversely, Th2 cytokines IL-10 and IL-4 inhibit Th1 cell activity by antagonizing IL-12 and IFN-γ. The dynamic balance between Th1 and Th2 is very important for maintaining the normal function of the body. IFN-γ and IL-4 are usually used as characteristic factors of Th1 and Th2 cells, and their ratio can reflect whether Th1 / Th2 cells are balanced or not. At the same time, Th2 cells assist B cells in synthesizing IgE. IgE plays a key role in the pathogenesis of eczema. It can bind to its receptor to enhance the release of histamine and inflammatory mediators, thereby aggravating the inflammatory response of eczema lesions. The ELISA results showed that compared with the model group, each dosage group of the topical drug of the present invention could reduce the levels of IL-4, IFN-γ, IgE and IL-4 / IFN-γ in the mouse serum to varying degrees, and the difference was statistically significant.

[0156] In summary, this study investigated the therapeutic effect of the topical medication of the present invention on the DNCB-induced eczema mouse model through observation of animal skin, pathological morphological changes, and analysis of the expression levels of related immune cytokines and inflammatory factors, providing experimental evidence for the clinical application of the topical medication of the present invention and new drug development.

[0157] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. An external medicine, made from purslane, phellodendron, mint, isatis indigotica, angelica, raw liquorice, kochia fruit, sophora flavescens, caltrop and coptis root as main raw materials, wherein: The mass proportions of each raw material drug are as follows: 25-35 parts of Portulaca Oleracea, 5-15 parts of Phellodendron Amurense, 5-15 parts of Mentha Piperita, 10-20 parts of Isatis Indigotica Leaf, 25-35 parts of Angelica Sinensis, 5-15 parts of Raw Licorice, 10-20 parts of Kochia Fruit, 5-20 parts of Sophora Flavescentis, 10-20 parts of Calculus Fructus, and 5-15 parts of Coptis Chinensis.

2. The external medicine according to claim 1, characterized in that The mass proportions of the raw materials of the external medicine are as follows: 30 parts of purslane, 10 parts of phellodendron, 10 parts of mint, 15 parts of isatis indigotica, 30 parts of angelica, 10 parts of raw licorice, 15 parts of kochia seeds, 12 parts of sophora flavescens, 15 parts of calamus, and 10 parts of coptis.

3. The external medicine according to claim 1, characterized in that The external medicine is a lotion.

4. A method for preparing the external medicine according to claim 1 or 2, comprising the following steps: taking the raw materials: purslane, phellodendron, mint, isatis indigotica, angelica, raw licorice, Kochia scoparia, sophora flavescens, calamus, and coptis chinensis, adding 10 times the amount of water to soak for 30 minutes, boiling over high heat, then boiling over low heat for 1.5 hours, filtering and concentrating under reduced pressure to a concentration equivalent to 1.03 g / mL based on the amount of the raw medicine, filling the treated liquid into a 250 mL medical glass bottle, sealing it with a rubber stopper, and sterilizing it by high-pressure steam sterilization (121°C, 30 minutes).

5. The method according to claim 4, characterized in that: The amount of water added for steaming is 10 times the total amount of raw materials, the steaming time is 30 minutes, and the slight boiling time is 1.5 hours.

6. Use of the external-use medicine according to any one of claims 1 to 3 in the preparation of a medicine for preventing and / or treating eczema.

7. The use according to claim 6, characterized in that: The eczema includes acute eczema, prickly heat, dermatitis with more exudation, and herpes diseases.

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