Dehumidifying and detoxifying medicine for treating eczema and preparation method thereof

Abstract

The invention discloses a dehumidifying and detoxifying medicine for treating eczema and a preparation method thereof. The medicine is prepared from radix sophorae flavescentis, herba portulacae, cortex phellodendri, fructus cnidii, radix sanguisorbae, cortex dictamni, radix salviae miltiorrhizae, nidus vespae and Sinkiang lithospermum erythrorhizon. The traditional Chinese medicine has the effects of clearing heat, drying dampness, astringing liquid, cooling blood, removing toxicity, diminishing swelling, dispelling wind, arresting itching and astringing sores, can be used for treating symptoms such as skin redness and swelling, scorching hot, papule, blister, erosion and exudation caused by eczema, and is suitable for patients with eczema caused by damp-heat accumulation in skin and exuberance of blood heat and toxin.

Description

Field of the Invention

[0001] The invention relates to a dehumidifying and detoxifying medicine for treating eczema and a preparation method thereof, belonging to the technical field of medicines. Background Art

[0002] Eczema is a common inflammatory skin disease affecting the epidermis and superficial dermis, caused by a variety of internal and external factors. Its primary characteristics are redness, edema, itching, and dryness, which may be accompanied by scabbing, flaking, blistering, cracking, bleeding, or oozing, resulting in intense, spasmodic itching. Lesions are polymorphic, symmetrically distributed, and prone to exudation, with recurring episodes. Eczema is primarily caused by polymicrobial infections, with Staphylococcus aureus and Pityrosporum fumigatus being detected in eczematous lesions. The rash begins in preexisting skin areas, such as the face, neck, upper limbs, and trunk, but can also occur on normal skin. Currently, clinical treatments for eczema primarily rely on glucocorticoids, tar-based preparations, immunomodulators, or topical antibiotics. However, long-term use of corticosteroids and antibiotics can lead to drug resistance, skin atrophy, telangiectasia, and hyperpigmentation, among other local adverse reactions.

[0003] Currently, eczema is mainly treated with Western medicine, and there is a lack of products made from pure Chinese medicine with antibacterial, antipruritic and moisturizing effects. Chinese medicine, as a Chinese treasure, has the advantages of mild properties, no irritation or allergic effects on the skin, no side effects, no drug resistance, easy use and green environmental protection. It is especially suitable for pregnant women, infants and high-risk sensitive groups, and has great social and economic significance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a dehumidifying and detoxifying drug for treating eczema and a preparation method thereof. The present invention has the effects of clearing away heat, drying dampness, astringing fluid, cooling blood, detoxifying and reducing swelling, and dispelling wind, relieving itching and healing sores. It can treat symptoms such as skin redness, swelling, burning sensation, papules, blisters, erosion, and exudation caused by eczema, and is suitable for patients with eczema caused by damp-heat accumulation in the skin and excessive blood heat and toxins.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The invention relates to a dehumidifying and detoxifying medicine for treating eczema. The medicinal active ingredients of the medicine are calculated by weight as follows: 100-300 parts of Dictamni cortex, 100-300 parts of Salvia miltiorrhiza, 100-300 parts of Honeycomb, 100-300 parts of Sophora flavescens, 100-300 parts of Portulaca oleracea, 100-300 parts of Phellodendron chinense, 100-300 parts of Cnidium monnieri, 100-300 parts of Sanguisorba officinalis and 100-300 parts of Lithospermum erythrorhizon.

[0007] The aforementioned dehumidifying and detoxifying medicine for treating eczema, whose medicinal active ingredients are calculated by weight: 150-250 parts of Dictamni cortex, 150-250 parts of Salvia miltiorrhiza, 150-250 parts of honeycomb, 150-250 parts of Sophora flavescens, 150-250 parts of Portulaca oleracea, 150-250 parts of Phellodendron chinense, 150-250 parts of Cnidium monnieri, 150-250 parts of Sanguisorba officinalis and 150-250 parts of Lithospermum officinale.

[0008] Specifically, the aforementioned dehumidifying and detoxifying medicine for treating eczema, its medicinal active ingredients are calculated by weight: 200 parts of Dictamni cortex, 200 parts of Salvia miltiorrhiza, 200 parts of honeycomb, 200 parts of Sophora flavescens, 200 parts of Portulaca oleracea, 200 parts of Phellodendron chinense, 200 parts of Cnidium monnieri, 200 parts of Sanguisorba officinalis and 200 parts of Lithospermum erythrorhizon.

[0009] The preparation method of the aforementioned dehumidifying and detoxifying medicine for treating eczema is to weigh all the medicinal materials in the prescription according to the proportion, and process them according to the conventional preparation method to prepare the corresponding pharmaceutical preparation.

[0010] The aforementioned pharmaceutical preparations include external-use mixtures or creams.

[0011] The external-use mixture is prepared as follows: all the medicinal materials in the prescription are weighed according to the proportion, 5-6 times the amount of water is added, soaked for 0.5 hours, decocted for 0.5-1.5 hours, the decoction is collected, filtered, sterilized, and packaged to obtain the external-use mixture.

[0012] Specifically, the external-use mixture is prepared as follows: all the medicinal materials in the prescription are weighed in proportion, 5.5 times the amount of water is added, soaked for 0.5 hours, and then decocted for 1 hour. The decoction is collected, filtered, sterilized, and packaged to obtain the external-use mixture.

[0013] The aforementioned cream is prepared as follows:

[0014] (1) Weigh each medicinal material according to the prescription ratio, add 5-6 times the amount of water, soak for 0.5 h, and then decoct for 0.5-1.5 h. Collect the decoction, concentrate to 30-40% of the total amount, filter, and obtain the concentrated solution for later use;

[0015] (2) Place 30-40 g of stearic acid and 5-15 g of monostearate in a dry beaker and heat in a constant temperature water bath to 60°C-70°C until all of them dissolve into the oil phase and set aside;

[0016] (3) Add 25-35 g of Tween to 100 mL of distilled water, heat at 70-80°C while stirring, and slowly add 15-25 g of glycerol until all of it dissolves into the aqueous phase and set aside;

[0017] (4) Slowly add the aqueous phase at 70°C-80°C to the oil phase at 60°C-70°C, stir evenly, and cool to room temperature. Then add 30-40 g of the concentrate from step (1) and 0.8-1.2 g of sodium benzoate, stir evenly, and obtain a cream.

[0018] Specifically, the aforementioned cream is prepared as follows:

[0019] (1) Weigh each medicinal material according to the prescription ratio, add 5-6 times the amount of water, soak for 0.5 hours, and then decoct for 1 hour. Collect the decoction, concentrate to 35% of the total amount, filter, and obtain the concentrated solution for later use;

[0020] (2) Place 35 g of stearic acid and 10 g of monostearate in a dry beaker and heat in a constant temperature water bath to 60°C-70°C until all of them dissolve into the oil phase and set aside;

[0021] (3) Add 30 g of Tween to 100 mL of distilled water, heat at 70°C-80°C while stirring, and slowly add 20 g of glycerol until all of it dissolves into the aqueous phase and set aside;

[0022] (4) Slowly add the aqueous phase at 70°C-80°C to the oil phase at 60°C-70°C, stir evenly, and cool to room temperature. Then add 35g of the concentrate from step (1) and 1g of sodium benzoate, stir evenly, and obtain a cream.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a dehumidifying and detoxifying drug for treating eczema, prepared from Sophora flavescens, Portulaca oleracea, Phellodendron chinense, Cnidium monnieri, Sanguisorba officinalis, Dictamni cortex, Salvia miltiorrhiza, Beehives, and Lithospermum officinale. The properties and efficacy of each component are as follows: Sophora flavescens is the dried root of the leguminous plant Sophora flavescens; it is bitter and cold in nature; it enters the heart, liver, stomach, large intestine, and bladder meridians; it has the effects of clearing heat and dampness, killing insects, and promoting diuresis; it is used for heat dysentery, bloody stool, jaundice, urinary retention, leucorrhea, vaginal swelling and itching, eczema, wet sores, itchy skin, scabies, and leprosy; and it is used externally for the treatment of trichomonas vaginitis. Portulaca oleracea L., a plant of the Portulacaceae family, is sour and cold in nature. It enters the liver and large intestine meridians and has the effects of clearing heat and detoxifying, cooling blood and stopping bleeding, and stopping dysentery. It is used to treat dysentery caused by heat toxins, carbuncles, furuncles, eczema, erysipelas, snake and insect bites, blood in stool, hemorrhoids, and metrorrhagia. Phellodendron chinense Schneid., a plant of the Rutaceae family, is bitter and cold in nature. It enters the kidney and bladder meridians and has the effects of clearing heat and dampness, purging fire and eliminating steam, and detoxifying and treating sores. It is used for damp-heat diarrhea, jaundice, dark urine, leucorrhea and itchy genitals, painful stranguria caused by heat, beriberi and paralysis, bone steaming and fatigue fever, night sweats, spermatorrhea, sores, swelling and toxicity, eczema and wet sores. Salted Phellodendron chinense nourishes yin and reduces fire. It is used for yin deficiency and excessive fire, night sweats and bone steaming. Cnidium monnieri (L.) Cuss., a plant of the Umbelliferae family, is a dried, mature fruit. It has a pungent, bitter, warm nature, and is slightly toxic. It enters the kidney meridian and has the effects of drying dampness and dispelling wind, killing insects and relieving itching, and warming the kidneys and strengthening yang. It is used for vaginal itching and leukorrhea, eczema and pruritus, dampness and low back pain, kidney deficiency and impotence, and uterine cold and infertility. Sanguisorba officinalis (Sanguisorba officinalis L.) or Sanguisorba officinalis L. var. longifolia (Bert.) Yüet Li, a plant of the Rosaceae family. It tastes bitter, sour, and astringent, and is slightly cold. It enters the liver and large intestine meridians and has the effects of cooling blood, stopping bleeding, detoxifying, and healing sores. It is used for bloody stools, hemorrhoids, bloody dysentery, metrorrhagia, burns from water or fire, and carbuncles, sores, and ulcers. Dictamnus dasycarpus (Rutaceae) is the dried root bark of Dictamnus dasycarpus (Rutaceae). It is bitter and cold in nature, and enters the spleen, stomach, and bladder meridians. It has the effects of clearing heat and dampness, dispelling wind and detoxifying. It is used to treat damp-heat sores, jaundice, eczema, scabies, scabies, rheumatism, rheumatic arthritis, and jaundice and dark urine. Salvia miltiorrhiza (Danshen) is the dried root and rhizome of Salvia miltiorrhiza (Lamiaceae). It is bitter and slightly cold in nature, and enters the heart and liver meridians. It has the effects of promoting blood circulation, removing blood stasis, promoting menstruation and relieving pain, clearing the heart and relieving restlessness, cooling blood and eliminating carbuncles. It is used to treat chest pain, abdominal pain, flank pain, accumulation of masses, pain caused by heat, restlessness and insomnia, irregular menstruation, dysmenorrhea, amenorrhea, and sores and swelling.Beehives are nests of the fruit wasp Polistes olivaceous (DeGeer), the Japanese long-legged wasp Polistes japonicus Saussure, or the heterogastric wasp Parapolybia varia Fabricius, all members of the Vespidae family. They are sweet in flavor and neutral in nature, entering the Stomach Meridian. They have the effects of attacking toxins, killing insects, dispelling wind, and relieving pain. They are used to treat sores, swellings, mastitis, scrofula, stubborn skin eczema, tinea versicolor, toothache, and rheumatic pain. Xinjiang Lithospermum officinale is the dried root of Arnebia euchroma (Royle) Johnst., a plant of the Boraginaceae family. They are sweet and salty in flavor and cold in nature, entering the Heart and Liver Meridians. They have the effects of clearing heat and cooling blood, promoting blood circulation and detoxifying, and clearing rashes and removing freckles. They are used to treat excessive blood heat and toxicity, purple-black rashes, persistent measles, sores, eczema, and burns caused by water or fire.

[0025] 1. Prescription

[0026] Dictamni clears heat and dampness, dispels wind and detoxifies, and is effective in treating damp-heat sores, making it an essential dermatological herb. Salvia miltiorrhiza cools blood and promotes blood circulation, eliminates carbuncles and resolves stagnation, and improves skin redness, swelling, heat, and pain caused by blood heat and stasis. Beehives attack toxicity and kill parasites, dispel wind and relieve itching, and are effective for infected skin and stubborn itching. These three herbs are collectively known as the monarch herbs, working synergistically to clear heat and dampness, detoxify and relieve itching, and activate blood circulation and reduce swelling. Sophora flavescens clears heat and dampness, kills parasites and relieves itching, making it particularly effective in treating damp-heat-related rashes and eczema. Portulaca oleracea clears heat and detoxifies, cools blood and reduces swelling, and can relieve burning and exudation when applied topically. These two herbs are collectively known as the minister herbs, assisting the monarch herbs in strengthening their heat-clearing, damp-removing, and astringent effects, making them particularly effective for red, swollen, eroded, and exudative lesions. Phellodendron amurense clears heat and dampness, purges fire and detoxifies, and is effective for damp-heat in the lower abdomen and skin infections. Cnidium monnieri dries dampness and dispels wind, kills parasites and relieves itching, and is effective for dermatitis caused by fungi and parasites. The two herbs are adjuvants, assisting in clearing heat and drying dampness, while also taking into account the potential causes of true infection. Sanguisorba officinalis cools blood and stops bleeding, detoxifies and heals sores, and external application can reduce exudation and promote wound healing. Xinjiang Lithospermum officinale cools blood and invigorates blood circulation, detoxifies and clears rashes, and is good at treating rashes caused by heat toxins. The two herbs are guiding herbs, leading the herbs to the surface, enhancing the effects of cooling blood and detoxifying, astringing and promoting tissue regeneration, and relieving skin redness, erosion, and exudation. This prescription has the functions of clearing heat, drying dampness, astringing fluid, cooling blood, detoxifying and reducing swelling, dispelling wind, relieving itching, and healinizing sores, and can treat symptoms of skin redness, swelling, burning, papules, blisters, erosion, and exudation caused by eczema. It is suitable for patients with eczema caused by damp-heat accumulation in the skin and excessive heat and toxins in the blood.

[0027] Features of the whole formula:

[0028] (1) Clearing away heat and dampness: Dictamni, Sophora flavescens, Phellodendron amurense, etc. work together to clear away damp-heat toxins.

[0029] (2) Cooling blood and activating blood circulation: Salvia miltiorrhiza, Lithospermum officinale, and Sanguisorba officinalis improve blood heat and stasis and promote the repair of skin lesions.

[0030] (3) Detoxification and itching relief: Beehive, Cnidium monnieri, and Portulaca oleracea are used to treat infections, itching, and other symptoms.

[0031] (4) Advantages of external wet compresses: The combination of various drugs can act directly on the affected area through local wet compresses, quickly relieving redness, swelling, heat, pain, exudation, and itching.

[0032] 2. The present invention can improve skin damage induced by DNCB, including epidermal mast cell infiltration and proliferation, and downregulate the expression of inflammatory factors TNF-α, IL-6, and IL-2. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Drug-ingredient-disease-target network of Chushi Huadu prescription in the treatment of eczema;

[0034] Figure 2 : PPI network of Chushi Huadu prescription in treating eczema;

[0035] Figure 3 : GO functional analysis bar chart of targets related to Chushi Huadu prescription in treating eczema;

[0036] Figure 4 : Bubble diagram of KEGG pathway enrichment analysis of targets related to Chushi Huadu prescription in the treatment of eczema;

[0037] Figure 5 : Comparison of ear quality differences among mice in each group (X±s, n=10);

[0038] Figure 6 : Comparison of skin pathology and histology of the right ears of mice in each group (n=10);

[0039] Figure 7 : Comparison of serum TNF-α, IL-6, and IL-2 levels in mice of each group (X±s, n=10);

[0040] Figure 8 : Effects of Chushi Huadu recipe on the expression of TNF-α, IL-6, and IL-2 proteins in tissues of mice in each group (×40) (A: representative images of immunohistochemical staining, B: statistical graph of staining results). DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to the examples, but they are not intended to limit the present invention.

[0042] Example 1.

[0043] Prescription: 20g of Dictamni Cortex, 20g of Salvia miltiorrhiza, 20g of Beehive, 20g of Sophora flavescens, 20g of Portulaca oleracea, 20g of Phellodendron amurense, 20g of Cnidium monnieri, 20g of Sanguisorba officinalis and 20g of Lithospermum erythrorhizon.

[0044] Preparation process:

[0045] Weigh all the medicinal materials in the prescription according to the proportion, add 2.5 times the amount of water, soak for 0.5 hours, and then boil for 1 hour. Collect the decoction, filter, sterilize, and package to obtain the external use mixture.

[0046] Dosage and Administration: Apply to the affected area three times a day, morning, noon and evening.

[0047] Example 2.

[0048] Prescription: 10g of Dictamni Cortex, 30g of Salvia miltiorrhiza, 10g of Beehive, 10g of Sophora flavescens, 30g of Portulaca oleracea, 30g of Phellodendron amurense, 10g of Cnidium monnieri, 10g of Sanguisorba officinalis and 30g of Lithospermum erythrorhizon.

[0049] Preparation process:

[0050] Weigh all the medicinal materials in the prescription according to the proportion, add 5 times the amount of water, soak for 0.5 hours, and then decoct for 1.5 hours. Collect the decoction, filter, sterilize, and package to obtain the external use mixture.

[0051] Dosage and Administration: Apply to the affected area three times a day, morning, noon and evening.

[0052] Example 3.

[0053] Prescription: 30g of Dictamni root bark, 10g of Salvia miltiorrhiza, 30g of Beehive, 30g of Sophora flavescens, 10g of Portulaca oleracea, 10g of Phellodendron amurense, 30g of Cnidium monnieri, 30g of Sanguisorba officinalis and 10g of Lithospermum erythrorhizon.

[0054] Preparation process:

[0055] Weigh all the medicinal materials in the prescription according to the proportion, add 6 times the amount of water, soak for 0.5 hours, and then decoct for 0.5 hours. Collect the decoction, filter, sterilize, and package to obtain the external use mixture.

[0056] Dosage and Administration: Apply to the affected area three times a day, morning, noon and evening.

[0057] Example 4.

[0058] Prescription: 20g of Dictamni Cortex, 20g of Salvia miltiorrhiza, 20g of Beehive, 20g of Sophora flavescens, 20g of Portulaca oleracea, 20g of Phellodendron amurense, 20g of Cnidium monnieri, 20g of Sanguisorba officinalis and 20g of Lithospermum erythrorhizon.

[0059] Preparation process:

[0060] (1) Weigh each medicinal material according to the prescription ratio, add 5.5 times the amount of water, soak for 0.5 hours, and then decoct for 1 hour. Collect the decoction, concentrate to 35% of the total amount, filter, and obtain the concentrated solution for later use;

[0061] (2) Place 35 g of stearic acid and 10 g of monostearate in a dry beaker and heat in a constant temperature water bath to 60°C-70°C until all of them dissolve into the oil phase and set aside;

[0062] (3) Add 30 g of Tween to 100 mL of distilled water, heat at 70°C-80°C while stirring, and slowly add 20 g of glycerol until all of it dissolves into the aqueous phase and set aside;

[0063] (4) Slowly add the aqueous phase at 70°C-80°C to the oil phase at 60°C-70°C, stir evenly, and cool to room temperature. Then add 35g of the concentrate from step (1) and 1g of sodium benzoate, stir evenly, and obtain a cream.

[0064] Dosage and Administration: Apply to the affected area three times a day, morning, noon and evening.

[0065] Example 5.

[0066] Prescription: Dictamni Root 30g, Salvia miltiorrhiza 10g, Beehive 20g, Sophora flavescens 15g, Portulaca oleracea 22g, Phellodendron amurense 23g, Cnidium monnieri 15g, Sanguisorba officinalis 25g and Lithospermum officinale 10g.

[0067] Preparation process:

[0068] (1) Weigh each medicinal material according to the prescription ratio, add 6 times the amount of water, soak for 0.5 hours, and then decoct for 1.5 hours. Collect the decoction, concentrate to 40% of the total amount, filter, and obtain the concentrated solution for later use;

[0069] (2) Place 30 g of stearic acid and 15 g of monostearate in a dry beaker and heat in a constant temperature water bath to 60°C-70°C until all of them dissolve into the oil phase and set aside;

[0070] (3) Add 25 g of Tween to 100 mL of distilled water, heat at 70°C-80°C while stirring, and slowly add 25 g of glycerol until all of it dissolves into the aqueous phase and set aside;

[0071] (4) Slowly add the aqueous phase at 70°C-80°C to the oil phase at 60°C-70°C, stir evenly, and cool to room temperature. Then add 30 g of the concentrate from step (1) and 1.2 g of sodium benzoate, stir evenly, and obtain a cream.

[0072] Dosage and Administration: Apply to the affected area three times a day, morning, noon and evening.

[0073] Example 6.

[0074] Prescription: Dictamni Root 22g, Salvia miltiorrhiza 15g, Beehive 10g, Sophora flavescens 18g, Portulaca oleracea 25g, Phellodendron amurense 30g, Cnidium monnieri 12g, Sanguisorba officinalis 14g and Lithospermum erythrorhizon 26g.

[0075] Preparation process:

[0076] (1) Weigh each medicinal material according to the prescription ratio, add 5 times the amount of water, soak for 0.5 h, and then decoct for 0.5 h. Collect the decoction, concentrate to 30% of the total amount, filter, and obtain the concentrated solution for later use;

[0077] (2) Place 40 g of stearic acid and 5 g of monostearate in a dry beaker and heat in a constant temperature water bath to 60°C-70°C until all of them dissolve into the oil phase and set aside;

[0078] (3) Add 35 g of Tween to 100 mL of distilled water, heat at 70°C-80°C while stirring, and slowly add 15 g of glycerol until all of it dissolves into the aqueous phase and set aside;

[0079] (4) Slowly add the aqueous phase at 70°C-80°C to the oil phase at 60°C-70°C, stir evenly, and cool to room temperature. Then add 40 g of the concentrate from step (1) and 0.8 g of sodium benzoate, stir evenly, and obtain a cream.

[0080] Dosage and Administration: Apply to the affected area three times a day, morning, noon and evening.

[0081] The present invention has carried out a lot of experimental research, and the following is the result of the experimental research of the present invention:

[0082] 1 Materials and Methods

[0083] 1.1 Network pharmacology analysis

[0084] Screening of active ingredients of the dehumidification and detoxification formula: The search terms "Sophora flavescens", "Portulaca oleracea", "Phellodendron amurense", "Cnidium monnieri", "Sanguisorba officinalis", "Dictamni cortex", "Salvia miltiorrhiza", "Fengfang", and "Lithospermum officinale" were used, and the oral bioavailability (OB) ≥ 30% and drug similarity (DL) ≥ 0.18 were used as screening criteria. The active ingredients of the dehumidification and detoxification formula were screened through the TCMSP database (https: / / tcmsp-e.com / ), and the screened active ingredients were entered into the DrugBank database (https: / / go.drugbank.com) for target prediction.

[0085] Screening of eczema-related targets: Using "eczema" as the keyword and Gifts>1 as the condition, eczema-related targets were screened through the GeneCards database (http: / / www.genecards.org).

[0086] Target prediction of the Chushi Huadu formula for the treatment of eczema: Based on the Python 3.8 programming environment (https: / / www.python.org), the merge function of the Pandas library was used to analyze the targets related to the active ingredients of the Chushi Huadu formula and the targets related to eczema. The common targets were taken as potential targets of the Chushi Huadu formula for the treatment of eczema.

[0087] Drug-ingredient-disease-target network construction: Compounds related to potential targets, namely the active ingredients of the Chushi Huadu formula for treating eczema, were retained. A drug-ingredient-disease-target network was constructed using Cytoscape 3.6.0 software (https: / / cytoscape.org). Network analysis was performed to obtain the degree values (Degree) of potential targets and active ingredients in the network. Using Degree ≥ 10 as the screening condition, core targets and core active ingredients were obtained.

[0088] Protein-protein interaction (PPI) network construction: PPI analysis was performed on the screened targets using the String database (htps: / / string-db.org / ), with the protein type set to "homo sapiens" and other parameter values kept as default.

[0089] Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis: GO functional analysis and KEGG pathway enrichment analysis were performed using the DAVID database (https: / / davidbioinformatics.nih.gov / ).

[0090] 1.2 Animal Experiment Verification

[0091] 1.2.1 Instruments, test drugs, and animals

[0092] Instruments: Baslc-Q30 laboratory pure water system (Shanghai Hetai Instrument Co., Ltd.); PX224ZH / E electronic balance (Ohaus Instruments (Changzhou) Co., Ltd., accuracy of 0.1 mg); mouse ear punch (Keen Laboratory Products Co., Ltd.); ELX800 microplate reader (BioTek, USA); DHG-9123AS electric constant temperature blast drying oven (Changzhou Langyue Instrument Manufacturing Co., Ltd.); H1650R centrifuge (Changsha High-tech Industrial Development Zone Xiangyi Centrifuge Instrument Co., Ltd.); XH-C vortex mixer (Jintan Baita Xinbao Instrument Factory); Pannoramic MIDI digital slide scanner (3DHISTECH).

[0093] Test drugs: compound dexamethasone acetate cream (China Resources Sanjiu Pharmaceutical Co., Ltd., batch number 2305001S, specifications: 30 g per tube: 22.5 mg); 2,4-dinitrochlorobenzene (DNCB, Shanghai Aladdin Biochemical Technology Co., Ltd., batch number C465514); tumor necrosis factor-α (TNF-α) kit (batch number RRDC2310008), interleukin 6 (IL-6) kit (batch number RRDC2311002), interleukin 2 (IL-2) kit (batch number RRDC2308021), all purchased from Wuhan Sewell Biotechnology Co., Ltd.

[0094] Animals: Fifty 8-week-old female BALB / C mice of clean-grade health were provided by the Animal Experimental Center of Guizhou Medical University under license number SCXK(Beijing)2019-0008. Mice were housed in a well-ventilated environment with a temperature of 23–25°C, a relative humidity of 50%–60%, and a 12-h / 12-h light / dark cycle, maintaining body weight.

[0095] 1.2.2 Methods

[0096] Preparation of the Dehumidification and Detoxification Formula: Take 20g each of Sophora flavescens, Portulaca oleracea, Phellodendron amurense, Cnidium monnieri, Sanguisorba officinalis, Dictamni cortex, Salvia miltiorrhiza, Beehives, and Lithospermum officinale, add 1000mL of water and boil for 1 hour. Collect the decoction to prepare the high-concentration solution (mass concentration: 180mg / mL). Take 200mL of the high-concentration solution and dilute it to 800mL with water to prepare the low-concentration solution (mass concentration: 45mg / mL). Refrigerate the solution until ready for use.

[0097] Grouping, Modeling, and Dosing: Fifty mice were randomly divided into a blank group (Group A), a model group (Group B), a positive drug group (Group C), and low- and high-dose Chushi Huadu Fang groups (Groups D1 and D2), with 10 mice in each group. Mice in Group A were not modeled. Mice in the remaining groups had their abdomens shaved (2 cm × 2 cm) one day before the experiment. On the day of the experiment, 100 μL of a 5% DNCB-acetone solution was applied to the shaved area of the abdomen for sensitization. Sensitization was boosted once the next day. Five days later, 1% DNCB solution was evenly applied to the left auricle of the mice. The mice were stimulated every three days for a total of four times. After successful modeling, 0.1 g of dexamethasone acetate cream was applied to the ears of mice in Group C, and 5 mL of 45 mg / mL and 180 mg / mL Chushi Huadu Fang solutions were applied to the ears of mice in Groups D1 and D2. Mice in Groups A and B received no special treatment. This treatment was repeated twice daily, 4 hours apart, for 12 consecutive days.

[0098] Effect on ear swelling: Mice were killed 24 hours after the last stimulation, and a hole was punched in the middle of the ear with a 0.6 cm metal punch. The mass of each tissue was weighed and the mass difference was calculated. At the same time, the right ear was made into a paraffin block for later use.

[0099] Effect on histopathology: The right ears of mice were fixed in 4% paraformaldehyde solution for 24 hours, and then made into paraffin sections. After the paraffin was dewaxed and hydrated, hematoxylin-eosin (HE) staining was performed and scanned and photographed using a digital slide scanner.

[0100] Effect on serum inflammatory factor levels: 24 hours after the last stimulation, the mice were taken, the eyeballs were removed after fixation, and blood was collected and placed in an EP tube containing sodium heparin. The blood was centrifuged (at a speed of 1500 r / min) for 15 minutes, and the supernatant was collected. The levels of serum TNF-α, IL-6, and IL-2 were detected by radioimmunoassay.

[0101] Immunohistochemical staining of mouse tissue for TNF-α, IL-6, and IL-2: Mouse right ear tissue was obtained and paraffin sections were prepared according to the aforementioned procedures. After routine dewaxing, antigen retrieval, endogenous peroxidase blocking, and addition of primary antibodies, sections were developed with DAB according to the kit instructions. Dehydration was performed with varying concentrations of ethanol, followed by clearing with xylene, and mounting with neutral gum. The intensity of the brownish-yellow stain was used as the observation indicator, depending on the primary antibody used for the target protein being tested, namely, the TNF-α polyclonal antibody, the IL-6 polyclonal antibody, and the IL-2 polyclonal antibody.

[0102] 1.3 Statistical analysis

[0103] SPSS 16.0 statistical software was used for analysis. Measurement data were expressed as x ± s, and comparisons among multiple groups were performed using one-way analysis of variance. P < 0.05 indicated statistically significant differences.

[0104] 2 Results

[0105] 2.1 Network pharmacology screening of active ingredients and targets

[0106] Screening of active ingredients and targets of the dehumidification formula for the treatment of eczema: A total of 227 active ingredients of the dehumidification formula were obtained in the TCMSP database, 144 active ingredients were obtained after deduplication, and 219 active ingredient-related targets were obtained by importing them into the DrugBank database.

[0107] Screening of potential targets for the Chushi Huadu formula in the treatment of eczema: Through the GeneCards database, a total of 731 eczema-related targets were screened out. After further comparison and analysis with the 219 targets of the active ingredients of the Chushi Huadu formula, a total of 41 targets were screened out, which are the potential targets of the Chushi Huadu formula in the treatment of eczema.

[0108] Drug-ingredient-disease-target network construction: 144 active pharmaceutical ingredients and 41 common targets were imported into Cytoscape 3.6.0 software to draw the drug-ingredient-disease-target network. Figure 1A total of 19 core targets with a degree ≥ 10 were screened, including TNF, β2-adrenergic receptor (ADRB2), serine / threonine protein kinase 1 (AKT1), nitric oxide synthase 2 (NOS2), IL-6, and IL-2. Core active ingredients include quercetin, luteolin, kaempferol, and baicalein.

[0109] PPI network analysis of potential targets of the Chushi Huadu formula for the treatment of eczema: 41 common targets were imported into the String database, and independent proteins related to them were retrieved. Target proteins outside the network were removed to construct a PPI network. Figure 2 .

[0110] GO function and KEGG pathway enrichment analysis: The 41 common targets of Dehumidification and Eczema were imported into the DIVID database for GO function analysis and KEGG pathway enrichment analysis. The top 10 GO function analysis bars are shown in the table according to the false discovery rate (FDR). Figure 3 It can be seen that the main processes involved are cell signal transduction (cell proliferation, cell apoptosis, cell migration), inflammatory response, immune response, etc. It is speculated that the dehumidification and detoxification prescription for the treatment of eczema may be the result of a complex multi-pathway synergistic effect. 10 P value Sorting, the top 30 KEGG pathway enrichment analysis bubble chart is shown in Figure 4 It can be seen that the main signaling pathways involved are TNF, IL-2, IL-17, etc., indicating that the dehumidification and detoxification prescription may treat eczema through multiple targets.

[0111] 2.2 Animal experimental verification

[0112] Effect on ear swelling: Compared with group A, the ear mass difference of mice in group B was significantly increased (P<0.05); compared with group B, the ear mass difference of mice in groups C, D1, and D2 was significantly reduced (P<0.05); compared with group C, the ear mass difference of mice in group D1 was significantly increased (P<0.05), and there was no significant difference in ear mass difference of mice in group D2 (P>0.05). Figure 5 (Note: Compared with Group A, # P<0.05; compared with group B, * P<0.05; compared with group C, △ P<0.05. ).

[0113] Effects on right ear skin pathology and histology: Pathological results showed that no significant changes were observed in the right ears of mice in group A; compared with group A, the right ears of mice in group B showed obvious redness, swelling, and scabs; compared with group B, the above symptoms in the right ears of mice in groups C, D1, and D2 were significantly improved, and the lesion area gradually shrank with increasing drug dosage. Figure 6Pathology. Histological results showed that the epidermal structure of the ear pieces of mice in group A was intact and of normal thickness. Compared with group A, the ear tissue of mice in group B was thicker, with obvious edema, increased epidermal cell division, and disordered arrangement. Compared with group B, the inflammatory cell infiltration and edema of mice in groups C, D1, and D2 were reduced, and the pathological changes of the ear skin were improved. The therapeutic effect was better with a high dose. Figure 6 Histological HE staining (×40).

[0114] Effects on serum TNF-α, IL-6, and IL-2 expression levels: Compared with group A, the serum TNF-α, IL-6, and IL-2 levels of mice in group B were significantly increased (P<0.05); compared with group B, the serum TNF-α, IL-6, and IL-2 levels of mice in groups C, D1, and D2 were significantly decreased (P<0.05), and the higher dose was more effective; compared with group C, the serum TNF-α, IL-6, and IL-2 levels of mice in group D1 were significantly increased (P<0.05), and there were no significant differences in the above indicators in the serum of mice in group D2 (P>0.05). Figure 7 (Note: Compared with Group A, # P<0.05; compared with group B, * P<0.05; compared with group C, △ P<0.05. ).

[0115] Effect on the expression level of TNF-α, IL-6, and IL-2 proteins in tissues: The expression level of TNF-α, IL-6, and IL-2 in tissues was judged by the intensity of the brown-yellow color in the cells. It can be seen that the expression of related proteins was low in the blank group, but significantly increased in the model group. The expression of TNF-α, IL-6, and IL-2 in the high-dose group and the positive drug group was significantly lower than that in the model group. Figure 8 .

[0116] Figure 8 In A, blank group: DAB staining was light, reflecting the baseline level under normal conditions; model group: the inflammatory area showed strong brown positive staining; low-dose group: the positive signal was locally reduced and the staining intensity decreased; high-concentration group: the positive signal was significantly reduced and tissue integrity was restored; positive drug group: the positive signal was significantly reduced, exerting the expected therapeutic effect.

[0117] Figure 8 In panel B, quantitative analysis of the staining was performed, with the mean absorbance (OD) of each group displayed as a bar graph. Compared with the model group, the expression levels of inflammatory factors were significantly reduced in both the high-dose and positive drug groups (n = 3, ***p < 0.001, blank group vs. model group; ***p < 0.001, high-dose group vs. model group; ***p < 0.001, positive drug group vs. model group; data are expressed as mean ± SEM).

[0118] 3 Discussions

[0119] This study used a network pharmacology approach to screen 144 active compounds and 41 shared targets. Core active ingredients included quercetin, luteolin, kaempferol, and baicalein. Key targets for the treatment of eczema included TNF, ADRB2, AKT1, NOS2, IL-6, and IL-2. GO functional analysis and KEGG pathway enrichment analysis revealed that the Chushi Huadu formula's treatment of eczema involves processes such as cell signaling, inflammatory responses, and immune responses, primarily involving signaling pathways such as TNF, IL-2, and IL-17. Animal experiments demonstrated that the high-dose Chushi Huadu formula group significantly downregulated the expression of TNF-α, IL-6, and IL-2 in the serum and right ear tissues of mice. The low-dose group showed slight improvement in eczema symptoms. Consistent with the serum and immunohistochemical results, histopathological evaluation also demonstrated that the Chushi Huadu formula group significantly reduced skin damage in the model mice, including epidermal mast cell infiltration and proliferation. TNF-α, a member of the TNF receptor cytokine superfamily, plays an important role in maintaining homeostasis in cells, tissues, and organisms, regulating inflammatory responses. IL-6 and IL-2, members of the interleukin family, are produced by various cell types with pleiotropic effects, and their levels are associated with the onset and progression of inflammatory responses.

[0120] Studies have shown that the mechanism of action for treating eczema can downregulate the expression of inflammatory factors such as TNF-α, thereby achieving antioxidant, anti-inflammatory, and immune-regulating effects. IL-17 is the primary effector cytokine secreted by Th17 cells. It acts on epithelial cells, endothelial cells, and macrophages to stimulate the expression of pro-inflammatory factors TNF-α and IL-6, thereby activating neutrophils, leading to inflammation and tissue damage. It plays a key role in inflammatory and autoimmune diseases.

[0121] In summary, the Chushi Huadu formula can improve DNCB-induced skin damage, including epidermal mast cell infiltration and proliferation, and downregulate the expression of inflammatory cytokines TNF-α, IL-6, and IL-2. Future experimental validation of the predicted core targets and pathways will further elucidate the molecular mechanism of the Chushi Huadu formula in treating eczema.

Claims

1. A dehumidifying and detoxifying drug for treating eczema, characterized by: The medicinal active ingredients are calculated by weight and are prepared from 100-300 parts of Dictamni cortex, 100-300 parts of Salvia miltiorrhiza, 100-300 parts of honeycomb, 100-300 parts of Sophora flavescens, 100-300 parts of Portulaca oleracea, 100-300 parts of Phellodendron amurense, 100-300 parts of Cnidium monnieri, 100-300 parts of Sanguisorba officinalis and 100-300 parts of Lithospermum erythrorhizon.

2. The dehumidifying and detoxifying drug for treating eczema according to claim 1, wherein: The medicinal active ingredients are calculated by weight and are prepared from 150-250 parts of Dictamni cortex, 150-250 parts of Salvia miltiorrhiza, 150-250 parts of honeycomb, 150-250 parts of Sophora flavescens, 150-250 parts of Portulaca oleracea, 150-250 parts of Phellodendron amurense, 150-250 parts of Cnidium monnieri, 150-250 parts of Sanguisorba officinalis and 150-250 parts of Lithospermum erythrorhizon.

3. The dehumidifying and detoxifying drug for treating eczema according to claim 1 or 2, characterized in that: The medicinal active ingredients are calculated by weight and are prepared from 200 parts of Dictamni cortex, 200 parts of Salvia miltiorrhiza, 200 parts of honeycomb, 200 parts of Sophora flavescens, 200 parts of Portulaca oleracea, 200 parts of Phellodendron chinense, 200 parts of Cnidium monnieri, 200 parts of Sanguisorba officinalis and 200 parts of Lithospermum erythrorhizon.

4. A method for preparing the dehumidifying and detoxifying drug for treating eczema according to any one of claims 1 to 3, characterized in that: All medicinal materials in the prescription are weighed in proportion, and processed according to conventional preparation methods to prepare corresponding pharmaceutical preparations.

5. The method for preparing the dehumidifying and detoxifying medicine for treating eczema according to claim 4, wherein: The pharmaceutical preparation includes an external-use mixture or a cream.

6. The method for preparing the dehumidifying and detoxifying pharmaceutical preparation for treating eczema according to claim 5, characterized in that: The external-use mixture is prepared as follows: all medicinal materials in the prescription are weighed according to a proportion, 5-6 times the amount of water is added, soaked for 0.5 hours, decocted for 0.5-1.5 hours, the decoction is collected, filtered, sterilized, and packaged to obtain the external-use mixture.

7. The method for preparing the dehumidifying and detoxifying pharmaceutical preparation for treating eczema according to claim 6, characterized in that: The external-use mixture is prepared as follows: all medicinal materials in the prescription are weighed according to the proportion, 5.5 times the amount of water is added, soaked for 0.5 hours, decocted for 1 hour, the decoction is collected, filtered, sterilized, and packaged to obtain the external-use mixture.

8. The method for preparing the dehumidifying and detoxifying drug for treating eczema according to claim 5, wherein: The cream is prepared as follows: (1) Weigh each medicinal material according to the prescription ratio, add 5-6 times the amount of water, soak for 0.5 h, and then decoct for 0.5-1.5 h. Collect the decoction, concentrate to 30-40% of the total amount, filter, and obtain the concentrated solution for later use; (2) Place 30-40 g of stearic acid and 5-15 g of monostearate in a dry beaker and heat in a constant temperature water bath to 60°C-70°C until all of them dissolve into the oil phase and set aside; (3) Add 25-35 g of Tween to 100 mL of distilled water, heat at 70-80°C while stirring, and slowly add 15-25 g of glycerol until all of it dissolves into the aqueous phase and set aside; (4) Slowly add the aqueous phase at 70°C-80°C to the oil phase at 60°C-70°C, stir evenly, and cool to room temperature. Then add 30-40 g of the concentrate from step (1) and 0.8-1.2 g of sodium benzoate, stir evenly, and obtain a cream.

9. The method for preparing the dehumidifying and detoxifying drug for treating eczema according to claim 5, wherein: The cream is prepared as follows: (1) Weigh each medicinal material according to the prescription ratio, add 5.5 times the amount of water, soak for 0.5 hours, and then decoct for 1 hour. Collect the decoction, concentrate to 35% of the total amount, filter, and obtain the concentrated solution for later use; (2) Place 35 g of stearic acid and 10 g of monostearate in a dry beaker and heat in a constant temperature water bath to 60°C-70°C until all of them dissolve into the oil phase and set aside; (3) Add 30 g of Tween to 100 mL of distilled water, heat at 70°C-80°C while stirring, and slowly add 20 g of glycerol until all of it dissolves into the aqueous phase and set aside; (4) Slowly add the aqueous phase at 70°C-80°C to the oil phase at 60°C-70°C, stir evenly, and cool to room temperature. Then add 35g of the concentrate from step (1) and 1g of sodium benzoate, stir evenly, and obtain a cream.

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