An extract for treating psoriasis, its preparation method and application

Abstract

This invention discloses an extract for treating psoriasis, its preparation method, and its application, relating to the field of traditional Chinese medicine extraction technology. The invention discloses a composition for treating psoriasis, the preparation method of which includes the following steps: mixing and extracting ephedra, cinnamon twig, red peony root, polygonum cuspidatum, artemisia annua, and oldenlandia diffusa to obtain an extract; or extracting ephedra, cinnamon twig, red peony root, polygonum cuspidatum, artemisia annua, and oldenlandia diffusa individually, and then mixing the extracts. Compared with the Kai Xuan Jie Du formula, the extract disclosed in this invention omits coptis chinensis, indigo naturalis, and gentian, making the prescription more concise and more advantageous in improving psoriasis-like skin lesions in model mice. Compared with methotrexate, a commonly used drug for treating psoriasis, the extract disclosed in this invention has a better therapeutic effect on psoriasis and can alleviate imiquimod-induced kidney function damage in mice.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine extraction technology, and relates to an extract for treating psoriasis, its preparation method and application. Background Technology

[0002] Psoriasis is a chronic, relapsing, inflammatory, systemic disease mediated by the immune system and induced by both genetic and environmental factors. Clinically, it manifests as scaly erythematous plaques or patches, localized or widespread. Psoriasis can be complicated by systemic diseases, severely impacting patients' quality of life. Although the exact cause of psoriasis is not fully understood, genetic, immune, and environmental factors play important roles in its development. Genetics is a major risk factor for psoriasis, and the immune pathways associated with interleukin-23 (IL-23) and Th17 helper cells are the core mechanisms of psoriasis pathogenesis.

[0003] Methotrexate has anti-inflammatory, anti-proliferative, and immunomodulatory effects, and is one of the most effective traditional drugs for treating psoriasis. Methotrexate is rapidly absorbed in the gastrointestinal tract by binding to a reduced folate carrier protein, and is converted intracellularly into polyglutamate methotrexate to exert its effect. Methotrexate shows good efficacy against all types of psoriasis, including moderate to severe plaque psoriasis, peripheral erythrodermic psoriasis (PsA), erythrodermic psoriasis, globus pharyngeal psoriasis (GPP), and psoriasis that severely affects function (such as palmoplantar psoriasis). Methotrexate can also treat severe nail psoriasis and peripheral PsA, and is particularly suitable when other systemic therapies are ineffective, contraindicated, intolerable, or unaffordable. However, high doses of methotrexate have adverse reactions, mainly bone marrow suppression and hepatotoxicity.

[0004] Jia Qin, Ye Ping, Dai Dan, Xiao Xue, Song Ping. Kai Xuan Jie Du Fang improves imiquimod-induced psoriasis-like mouse skin lesions by regulating S1P levels [J]. Journal of Jiangsu University (Medical Edition), 2023, 33(2): 118-125. This paper discloses that Kai Xuan Jie Du Fang can improve the symptoms and signs of psoriasis in mice in multiple ways, such as improving splenomegaly, thymic atrophy, capillary hyperplasia, reducing the degree of epidermal hyperplasia, repairing the skin barrier, and reducing the expression of local inflammatory factors. On the other hand, this drug can not only regulate S1P expression, but also regulate the expression level of S1PR at the mRNA level. The S1P / S1PR pathway is very likely a potential target of Kai Xuan Jie Du Fang, and S1P / S1PR plays an important role in inflammatory signaling pathways, skin cell proliferation, and skin barrier. However, this paper only discloses the composition of Kai Xuan Jie Du Fang, without disclosing whether the Chinese medicine has been extracted, and without optimizing the ratio of the extract.

[0005] In view of this, and to address the shortcomings of existing technologies, this invention provides an extract for treating psoriasis, its preparation method, and its applications. The extract disclosed in this invention can be used to prepare drugs for treating psoriasis and has excellent research and development prospects. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of previous research and provide an extract for treating psoriasis, its preparation method and application, which can give full play to the efficacy of traditional Chinese medicine, effectively solve the problem of medication for the treatment of psoriasis, and has promotion and development value.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] On one hand, the present invention provides a composition for treating psoriasis, the preparation method of the composition comprising the following steps: mixing ephedra, cinnamon twig, red peony root, polygonum cuspidatum, artemisia annua, and oldenlandia diffusa and then extracting them to obtain an extract; or extracting ephedra, cinnamon twig, red peony root, polygonum cuspidatum, artemisia annua, and oldenlandia diffusa separately to obtain extracts, and then mixing the extracts.

[0009] Preferably, the preparation method of the composition includes the following steps: mixing ephedra, cinnamon twig, red peony root, Japanese knotweed, artemisia annua, and oldenlandia diffusa, then extracting with water to obtain an extract.

[0010] More preferably, the mass ratio of ephedra, cinnamon twig, red peony root, Japanese knotweed, artemisia annua, and oldenlandia diffusa is 1-12:3-18:3-18:5-20:5-40:5-40.

[0011] More preferably, the mass ratio of ephedra, cinnamon twig, red peony root, Japanese knotweed, artemisia annua, and oldenlandia diffusa is 6:9:9:10:20:20.

[0012] More preferably, the preparation method specifically includes the following steps: adding water and heating under reflux for extraction 2-4 times, adding 2-10 times the weight of the medicinal material in water each time, and extracting for 20-120 minutes each time to obtain the extract.

[0013] More preferably, the preparation method specifically includes the following steps: adding water and heating under reflux for extraction twice. In the first extraction, add 5 times the weight of the medicinal material in water and extract for 60 minutes. In the second extraction, add 3 times the weight of the medicinal material in water and extract for 30 minutes to obtain the extract.

[0014] More preferably, the preparation method further includes the following steps: concentrating the extract under reduced pressure into a thick paste, drying it under reduced pressure, and pulverizing it into powder to obtain the extract.

[0015] More preferably, the preparation method further includes the following steps: concentrating the extract under reduced pressure to a thick paste with a relative density of 1.2-1.3 (measured at 50-60℃), drying under reduced pressure at 60-70℃, and pulverizing into powder to obtain the extract.

[0016] In another aspect, the present invention provides an extract composed of ephedra extract, cinnamon twig extract, red peony root extract, Japanese knotweed extract, artemisia annua extract, and oldenlandia diffusa extract.

[0017] In another aspect, the present invention provides a medicine whose active ingredient includes the above-described composition or the above-described extract.

[0018] The drug can be prepared into dosage forms such as pills, capsules, granules, oral liquids, powders, tablets, lozenges, and sugar lozenges. For different dosage forms, a suitable drug carrier in this field can be selected.

[0019] The drug carrier used can be solid, liquid, or gaseous. Examples of solid carriers include lactose, kaolin, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, and stearic acid. Examples of liquid carriers include syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0020] In preparing compositions for oral dosage forms, any convenient pharmaceutical medium can be used. For example, water, ethanol, oils, alcohols, flavoring agents, preservatives, coloring agents, etc., can be used to form oral liquid dosage forms, such as suspensions and solutions; while carriers, such as starch, sugars, microcrystalline cellulose, diluents, granulators, emulsifiers, lubricants, binders, and disintegrants, can be used to form oral solid dosage forms, such as powders, capsules, and tablets. Tablets and capsules are preferred oral dosage units using solid pharmaceutical carriers due to their ease of administration. Tablets can be coated using standard aqueous or non-aqueous techniques.

[0021] Tablets containing the extract of this invention can be prepared by compression or molding, optionally using one or more excipients or adjuvants. The active ingredient can be compressed in a free-flowing form (e.g., powder or granules) in a suitable machine, optionally mixed with binders, lubricants, inert diluents, surfactants, or dispersants. Molded tablets can be molded in a suitable machine, i.e., a mixture of powdered compounds moistened with an inert liquid diluent. Each tablet preferably contains about 0.05 mg to about 5 g of active ingredient, and each sachet or capsule preferably contains about 0.05 mg to about 5 g of active ingredient. For example, formulations intended for oral administration to humans may contain about 0.5 mg to about 5 g of active pharmaceutical ingredient, mixed with a suitable and convenient carrier material, which may comprise about 5% to 95% of the total composition. Unit dosage forms typically contain about 1 mg to about 2 g of active ingredient, typically in doses of 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

[0022] The pharmaceutical compositions of this invention suitable for parenteral administration can be prepared as aqueous solutions or suspensions of the active compound. Suitable surfactants, such as hydroxypropyl cellulose, may be included. Dispersions can also be prepared in mixtures of glycerol, liquid polyethylene glycol, and their oils. Furthermore, preservatives may be added to prevent harmful microbial growth.

[0023] The medicaments of the present invention can be in forms suitable for topical use, such as aerosols, creams, ointments, lotions, powders, or the like. Furthermore, the compositions can be in suitable forms for transdermal drug delivery devices. For example, a cream or ointment with a desired consistency can be prepared by mixing a hydrophilic material and water, and about 5 wt% to about 10 wt% of a compound.

[0024] The medicament of the present invention can be in a form suitable for rectal administration, wherein the carrier is solid. It is preferable to formulate the mixture into a single-dose suppository. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be made by first forming a mixture containing a softened or melted carrier, followed by cooling and shaping in a mold.

[0025] In addition to the carrier components described above, the pharmaceutical formulations may include (if applicable) one or more additional carrier components, such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc. Furthermore, other excipients may be added, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., colorants, and flavoring agents, etc., to make the formulation isotonic with the blood of the intended recipient. Components containing the extracts of this invention can also be prepared in powder or concentrated form.

[0026] Preferably, the drug is an oral pharmaceutical preparation.

[0027] Finally, the present invention provides the use of the above-described composition, the above-described extract, or the above-described drug in the preparation of a drug for treating psoriasis.

[0028] Preferably, the psoriasis is selected from any one of psoriasis vulgaris, psoriatic arthritis, pustular psoriasis, and erythrodermic psoriasis.

[0029] More preferably, the psoriasis is psoriasis vulgaris.

[0030] Preferably, the symptoms of psoriasis include:

[0031] (1) Red patches appear on the skin, covered with silvery-white scales;

[0032] (2) Itching, burning, or pain in the skin;

[0033] (3) Dry, cracked, or bleeding skin;

[0034] (4) Scalp dandruff appears;

[0035] (5) Fingernails and / or toenails are “thimble-like” or dotted pits, onycholysis, or hyperkeratosis of the nail bed;

[0036] (6) Joint swelling.

[0037] More preferably, the symptoms of psoriasis include:

[0038] (1) Red patches appear on the skin, covered with silvery-white scales;

[0039] (2) Itching, burning, or pain in the skin;

[0040] (3) Dry, cracked or bleeding skin.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] (1) Compared with the Kaixuan Detoxification Formula (composed of Artemisia annua, Polygonum cuspidatum, Hedyotis diffusa, Coptis chinensis, Ephedra sinica, Cinnamomum cassia, Indigo naturalis, and Gentiana scabra), the extract disclosed in this invention omits Coptis chinensis, Indigo naturalis, and Gentiana scabra, making the prescription more concise and more advantageous in improving psoriasis-like skin lesions in model mice.

[0043] (2) Compared with methotrexate, a commonly used drug for treating psoriasis, the extract disclosed in this invention can better alleviate the kidney function damage caused by imiquimod in mice and has a better therapeutic effect on psoriasis. Attached Figure Description

[0044] Figure 1 The effects of Examples 1-3 and Comparative Examples 1-3 on the skin lesions of model mice are shown, where a represents the results of the first animal experiment and b represents the results of the second animal experiment.

[0045] Figure 2 PASI scores of dorsal skin lesions in mice of each group were calculated on day 6. Among them, there was no statistically significant difference in ns. ** P<0.01; *** P<0.001.

[0046] Figure 3 Examples 1-3 and Comparative Examples 1-3 show the effects of these examples on the pathological manifestations of skin lesions in psoriasis model mice. In this example, a represents the results of the first animal experiment, and b represents the results of the second animal experiment.

[0047] Figure 4 The effect of the extract from Example 1 on the Baker score in the pathological observation of skin lesions in a psoriasis model mouse was observed. * P<0.05; ** P<0.01; *** P<0.001.

[0048] Figure 5 The effect of the extract of Example 1 on the expression of Ki67 in the epidermis of a mouse model of psoriasis.

[0049] Figure 6 The extract of Example 1 was used to treat Ki67 on the epidermal skin of a mouse model of psoriasis. + The effect of cell number, among which, ** P<0.01; *** P<0.001.

[0050] Figure 7 The effect of the extract of Example 1 on the expression of CK10 in the epidermis of a mouse model of psoriasis is shown in the figure. The area above the black dashed line represents the skin epidermis.

[0051] Figure 8 The effect of the extract of Example 1 on the mean optical density value of CK10 in the epidermis of a psoriasis model mouse, wherein, * P<0.05; *** P<0.001.

[0052] Figure 9 The effect of the extract of Example 1 on the expression of Claudin-1 in the epidermis of a mouse model of psoriasis is shown in the figure. The area above the black dashed line represents the skin epidermis.

[0053] Figure 10 The effect of the extract from Example 1 on the mean optical density of Claudin-1 epidermis in a mouse psoriasis model, wherein, * P<0.05; ** P<0.01; *** P<0.001.

[0054] Figure 11 The effect of the extract of Example 1 on the subcutaneous blood vessel distribution density in a mouse model of psoriasis.

[0055] Figure 12 The effect of the extract of Example 1 on the spleen morphology of a mouse model of psoriasis.

[0056] Figure 13 The effect of the extract from Example 1 on the spleen index of a psoriasis model mouse, wherein, *** P<0.001.

[0057] Figure 14 The effect of the extract from Example 1 on the expression of serum pro-inflammatory cytokines in psoriasis model mice, wherein, ** P<0.01; *** P<0.001.

[0058] Figure 15 The effects of the extract from Example 1 on the morphology of the heart, liver, and kidneys in a mouse model of psoriasis. Detailed Implementation

[0059] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed in this application. Those skilled in the art can make various changes and modifications to the invention based on the disclosed content, and such changes should also fall within the scope of protection claimed in this application.

[0060] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all pharmaceuticals used in the embodiments of the present invention are obtained through conventional commercial channels.

[0061] Ephedra is the herbaceous stem of plants such as Ephedra sinica, Ephedra equisetifolia, or Ephedra intermedium, belonging to the Ephedraceae family.

[0062] Cinnamon twigs are the tender branches of the cinnamon tree, a plant in the Lauraceae family.

[0063] Red peony root is the dried root of the plant *Paeonia lactiflora* or *Paeonia veitchii*, belonging to the Ranunculaceae family.

[0064] Japanese knotweed is the dried rhizome and root of Polygonum cuspidatum Sieb. et Zucc., a plant belonging to the Polygonaceae family.

[0065] Artemisia annua L., a plant in the Asteraceae family, is the dried aerial part of the plant.

[0066] Oldenlandia diffusa, a plant belonging to the Rubiaceae family and the Oldenlandia genus, is a species of Oldenlandia diffusa.

[0067] The whole herb of Hedyotis diffusa Willd. [Oldenlandia diffusa (Willd.) Roxb.].

[0068] Example 1

[0069] The formula is as follows: 6 parts ephedra, 9 parts cinnamon twig, 9 parts red peony root, 10 parts Japanese knotweed, 20 parts artemisia annua, and 20 parts oldenlandia diffusa.

[0070] Preparation method: Take the prescribed amount of medicinal materials, add 5 times the amount of pure water for the first time, soak for 2 hours, heat and reflux for 60 minutes, filter the extract, and continue to extract the remaining residue. Add 3 times the amount of pure water for the second time, extract for 30 minutes, filter the extract, combine the filtrates obtained from the two extractions, and concentrate under reduced pressure to a decoction containing 6.24 g / mL of raw medicinal material.

[0071] Example 2

[0072] The formula is as follows: 1 part ephedra, 3 parts cinnamon twig, 18 parts red peony root, 20 parts Japanese knotweed, 40 parts artemisia annua, and 40 parts oldenlandia diffusa.

[0073] The preparation method is the same as in Example 1.

[0074] Example 3

[0075] The formula is as follows: 12 parts ephedra, 18 parts cinnamon twig, 3 parts red peony root, 5 parts Japanese knotweed, 5 parts artemisia annua, and 5 parts oldenlandia diffusa.

[0076] The preparation method is the same as in Example 1.

[0077] Comparative Example 1

[0078] The formula is as follows: Ephedra 1 part, Cinnamon Twig 3 parts, Polygonum Cuspidatum 20 parts, Artemisia annua 40 parts, Hedyotis diffusa 40 parts, Coptis chinensis 6 parts, Indigo Naturalis 3 parts, Gentiana scabra 9 parts. This formula is referenced in the literature (Qin Yeping, Dai Dan, Xiao Xue, et al. Kaixuan Jiedu Formula improves the skin lesions of imiquimod-induced psoriasis-like mice by regulating S1P level [J]. Journal of Jiangsu University: Medical Edition, 2023, 33(2):118-125.DOI:10.13312 / j.issn.1671-7783.y230038.).

[0079] The preparation method is the same as in Example 1.

[0080] Comparative Example 2

[0081] The formula is as follows: 6 parts ephedra and 9 parts cinnamon twig.

[0082] The preparation method is the same as in Example 1.

[0083] Comparative Example 3

[0084] The formula is as follows: 9 parts of Paeonia lactiflora Pall., 10 parts of Polygonum cuspidatum Sieb. et Zucc., 20 parts of Artemisia annua L., and 20 parts of Hedyotis diffusa Willd.

[0085] The preparation method is the same as that in Example 1.

[0086] Result detection

[0087] 1. Experimental materials and methods

[0088] 1.1. Experimental animals

[0089] Specific pathogen-free (SPF)-level C57BL / 6J mice, male, 6 - 8 weeks old, weighing 18 - 22 g, a total of 72 mice, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK (Beijing) 2021 - 0006. The mice were housed under SPF conditions in the animal house of Guang'anmen Hospital, China Academy of Chinese Medical Sciences. They were all given standard ordinary feed, the environmental humidity was 55 ± 5%, the temperature was controlled at 22 - 25 °C, and the lighting time was 12 h / day. They were cage-raised, 6 mice per cage, a total of 12 cages, and had free access to food and water. The experimental protocol was approved by the Experimental Animal Ethics Committee of Guang'anmen Hospital, China Academy of Chinese Medical Sciences (approval number: IACUC - GAMH - 2021 - 021). All experimental animal protocols were operated in accordance with the guidelines approved by the Animal Ethics Committee.

[0090] 1.2. Experimental drugs

[0091] The examples and comparative examples were provided by the Pharmacy Department of Guang'anmen Hospital, China Academy of Chinese Medical Sciences; Imiquimod Cream (company: 3M HealthCare limited, import drug registration number: H20160079, specification: 12.5 mg / 250 mg), Methotrexate Tablets (company: Shanghai Shangyao Xinyi Pharmaceutical Factory Co., Ltd., batch number:国药准字H31020644, specification: 2.5 mg).

[0092] 1.3. Main experimental reagents

[0093] Table 1 Main experimental reagents

[0094]

[0095]

[0096] 1.4. Experimental methods

[0097] 1.4.1. Establishment of psoriasis mouse model

[0098] After one week of acclimatization, all C57BL / 6J mice in the model group were prepared for modeling. The day before modeling, the fur on the backs of the mice was shaved to create an exposed area of ​​approximately 2cm × 3cm. Depilatory cream was then applied and applied to the exposed area. After the cream remained on the mouse's back for 90 seconds, the cream and remaining hair were removed with a tissue dampened with warm water to fully expose the mouse's back skin. From the start of modeling, 62.5mg of imiquimod cream was applied to the exposed skin at 9:00 AM daily for 5 consecutive days. Mice in the control group received 62.5mg of petroleum jelly on their backs at the same time as a control.

[0099] 1.4.2 Grouping and Administration

[0100] Four hours after model establishment, administration was initiated via gavage. Following the body surface area method in *Pharmacological Experimental Methodology*, and based on the calculation method of mouse dose = [human dose (g crude drug / kg / d) × human conversion coefficient] / mouse conversion coefficient, the equivalent dose for mice was calculated to be approximately 12.3 times that for humans. According to the above prescription, the daily dosage was 0.1 mL / 10 g (calculated based on body surface area between humans and rats). The decoction of the traditional Chinese medicine was sterilized by autoclaving and then refrigerated for later use.

[0101] The specific groupings for the first animal experiment are as follows:

[0102] ① Control group: 62.5mg petroleum jelly was applied to the back and administered by gavage with 0.1mL / 10g distilled water;

[0103] ② Model group: 62.5mg imiquimod cream was applied to the back and administered via gavage with 0.1mL / 10g distilled water;

[0104] ③ Methotrexate group: 62.5 mg imiquimod cream was applied to the back and 1 mg / kg methotrexate solution was administered by gavage, with a dosage of 0.1 mL / 10 g;

[0105] ④ Example 1 group: 62.5mg imiquimod cream was applied to the back and the decoction prepared in Example 1 was administered by gavage, with a dosage of 0.1mL / 10g;

[0106] ⑤ Example 2 group: 62.5mg imiquimod cream was applied to the back and the decoction prepared in Example 2 was administered by gavage, with a dosage of 0.1mL / 10g;

[0107] ⑥ Example 3 group: 62.5mg imiquimod cream was applied to the back and the decoction prepared in Example 3 was administered by gavage, with a dosage of 0.1mL / 10g;

[0108] The specific groupings for the second animal experiment are as follows:

[0109] ⑦ Blank group: Apply 62.5mg petroleum jelly to the back and administer via gavage with 0.1mL / 10g distilled water;

[0110] ⑧ Model group: 62.5mg imiquimod cream was applied to the back and administered via gavage with 0.1mL / 10g distilled water;

[0111] ⑨ Example 1 group: 62.5mg imiquimod cream was applied to the back and the decoction prepared in Example 1 was administered by gavage, with a dosage of 0.1mL / 10g;

[0112] ⑩ Comparative Example 1: 62.5 mg imiquimod cream was applied to the back and the decoction prepared in Comparative Example 1 was administered by gavage at a volume of 0.1 mL / 10 g.

[0113] Comparative Example 2: 62.5 mg imiquimod cream was applied to the back and the decoction prepared in Comparative Example 2 was administered by gavage at a volume of 0.1 mL / 10 g.

[0114] Comparative Example 3: 62.5 mg imiquimod cream was applied to the back and the decoction prepared in Comparative Example 3 was administered by gavage at a volume of 0.1 mL / 10 g.

[0115] Six mice were administered the drug once a day by gavage for five consecutive days. The mice were weighed and their skin lesions were scored using the PASI system before applying imiquimod cream each day. The morphology of the mice's back skin was also recorded by taking photos under consistent lighting and photographic equipment.

[0116] 1.4.3 Sample Collection and Preservation

[0117] After establishing the mouse model and intervention on day 5, the feed was removed, and the mice were starved. On day 6, at 8:00 AM, samples were collected. Mice were anesthetized by intraperitoneal injection of a 0.3% sodium pentobarbital solution prepared with physiological saline at a dose of 0.2 mL / 10 g. After anesthesia, the whiskers were trimmed, the eyeballs were removed with forceps, and blood was collected using serum separation gel coagulation tubes. The collected test tubes were then stored at room temperature.

[0118] After blood collection from the mice, the skin was placed on a pre-sterilized foam board. Skin was then excised from the back using surgical scissors and rinsed in PBS solution. The subcutaneous tissue side of the skin was placed face up on white filter paper, and the subcutaneous vascularization was photographed to record the findings. The skin tissue was divided into four portions. One portion was attached to filter paper and immersed in neutral formalin fixative to prevent tissue curling. The other three portions were placed in labeled cryovials, flash-frozen in liquid nitrogen, and then stored at -80°C for later use.

[0119] Mice were fixed to foam boards, and the skin and ribs of the chest were cut open to expose the heart. A 5mL syringe (with a 1mL needle) filled with physiological saline was inserted into the apex of the heart and the saline was slowly injected to perfuse the heart and help remove the remaining blood from the mouse's body, which would facilitate subsequent morphological analysis.

[0120] After perfusion, the spleen was removed and weighed. One-third of the largest lobe of the heart and liver was removed, and the kidney with its outer membrane removed on one side was injected into neutral formalin fixative.

[0121] After the mice were collected, the coagulated blood was processed centrally. The blood collection tubes were placed in a low-temperature high-speed centrifuge and centrifuged at 4°C, 3000 rpm, and 15 min. After centrifugation, the supernatant was transferred to cryovials using a pipette and stored at -80°C for later use.

[0122] 1.4.4 PASI score for skin lesions

[0123] On days 1-6, before modeling, the changes in skin lesions on the back of mice were photographed and recorded using fixed equipment and relatively consistent lighting conditions. At the same time, the PASI scoring criteria were used to score the erythema (0-4 points), scaling (0-4 points), and infiltration degree (0-4 points) of the mouse skin lesions. The sum of the three scores was the PASI score.

[0124] 1.4.5 Calculation of spleen index and assessment of subcutaneous angiogenesis

[0125] The spleen index of mice is calculated by dividing the weight of the mouse spleen (mg) by the weight of the mouse body (g) and multiplying by 100%. The subcutaneous vascular proliferation of mice is recorded by photographs and assessed macroscopically by the naked eye.

[0126] 1.4.6 Preparation and sectioning of histopathological paraffin blocks

[0127] Tissues fixed for 72 hours or more were removed from the fixative, and the tissue portions to be observed were cut with a scalpel and placed in a marked tissue embedding cassette. The tissues were then sequentially immersed in 75% ethanol for 2 hours, 85% ethanol for 30 minutes, 95% ethanol for 2 hours (with a solution change), 95% ethanol for 2 hours, anhydrous ethanol for 2 hours (with a solution change), and anhydrous ethanol for 2 hours (with a solution change). The tissues were then cleared by immersion in xylene for 1.5 hours (with a solution change), xylene for 1.5 hours (with a solution change), and xylene for 45 minutes (with a solution change). The paraffin melting tank of the tissue embedding machine was started in advance and set to 62°C to melt the paraffin. The dehydrated and cleared tissues were then immersed in paraffin for 3 hours.

[0128] After paraffin infiltration is completed, start the cold stage of the embedding machine, prepare the pathological tissue paraffin block embedding mold in advance, open the embedding box, take out the tissue that has been infiltrated with paraffin, drop 1 / 4 of the paraffin into the mold, use tweezers to place the tissue to be observed face up in the mold, wait for the paraffin in the mold to solidify slightly, quickly drop the mold to fill it with paraffin and cover the bottom of the embedding box, transfer it to the cold stage, wait for the paraffin block to solidify, transfer it from the cold stage to the room temperature laboratory operating table for demolding, and the preparation of the tissue pathology paraffin block is completed.

[0129] Start the slide warmer in advance, setting the water temperature to 55℃. Take the prepared paraffin block and fix it in the paraffin microtome. Turn on the humidifier and aim it at the paraffin block. Initially, set it to coarse trimming at 20μm / cut. Once tissue is reached, set it to fine trimming, with 4.5μm for skin tissue and 4μm for heart, liver, and kidney tissues due to their density. After sectioning, use tweezers to spread the tissue slide in the slide warmer until the tissue is fully expanded and wrinkle-free. Use a regular or high-adhesion slide to retrieve the slide and let it air dry overnight.

[0130] 1.4.7 Eosin-Hematoxylin (HE) Staining

[0131] The slide baking machine was set to 60℃ for 1 hour. After the paraffin was fully melted, xylene was added sequentially for 10 minutes, followed by (replacement) xylene for 5 minutes, (replacement) xylene for 5 minutes, anhydrous ethanol for 10 minutes, (replacement) anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, and then washed with tap water for dewaxing. The dewaxed tissue slides were then stained with hematoxylin for 15 minutes, and excess staining solution was washed away with tap water. Differentiation was performed using 1% hydrochloric acid ethanol. After washing away excess differentiation solution with tap water, eosin staining solution was added to stain the cytoplasm for 5 minutes, and excess staining solution was washed away with tap water. After dehydration, the slides were mounted with neutral resin.

[0132] 1.4.8. Pathological observation of psoriasis skin using the Baker score

[0133] The Baker score was used to assess the pathological manifestations of psoriatic lesions. The specific scoring criteria are shown in the table below.

[0134] Table 2. Baker Scoring Criteria for Psoriasis

[0135]

[0136] 1.4.9 Immunohistochemical detection of the localization and expression of target proteins in mouse epidermis

[0137] Bake the slides at 60℃ for 1 hour in a slide oven until the paraffin is fully melted. Then, perform dewaxing according to step 1.4.7. After dewaxing, wash away excess organic solvent with double-distilled water. Pour 200mL of 1X antigen retrieval solution into the immunohistochemistry antigen retrieval box. Set the microwave oven to 100℃ for 5 minutes to preheat the retrieval solution. Transfer the slides to the preheated retrieval solution, making sure the liquid level is high enough to submerge the tissue. Perform antigen retrieval at 100℃ for 5 minutes and then at 50℃ for 15 minutes. During the retrieval process, add liquid as needed to prevent the slides from drying out due to boiling. After retrieval, allow the slides to cool to room temperature.

[0138] After the slides have cooled to room temperature, wash them three times with PBS solution for 5 minutes each time. Wipe the PBS solution around the tissue dry with a paper towel. Draw a water-blocking circle with an immunohistochemistry pen. Add an appropriate amount of endogenous peroxidase inhibitor to the tissue, just enough to cover it. Incubate at room temperature for 10 minutes to block endogenous peroxidase. Rinse with PBS solution three times for 5 minutes each time. Cover the tissue with blocking solution containing 5% goat serum, 1% BSA, and 0.3% Triton, and block for 1 hour. During the blocking process, prepare antibody dilution solutions containing 1% BSA and 0.3% Triton, diluting Ki67 at 1:400, Claudin1 at 1:800, and CK10 at 1:2000. After blocking, discard the blocking solution, place the slides in an immunohistochemistry humidifier, add the prepared primary antibody to the corresponding tissue surface, add water to the bottom of the chamber, and incubate overnight at 4°C.

[0139] Remove the overnight slides, check for any dried areas, and equilibrate at room temperature for 30 minutes. Wash with PBS solution containing 0.1% Triton for 10 minutes each time, 5 times. Wipe away any fluid around the tissue, add reaction enhancement solution, incubate at room temperature for 20 minutes, wash with PBS solution for 5 minutes each time, 3 times, add secondary antibody, incubate at room temperature for 20 minutes, and wash again with PBS solution for 5 minutes each time, 3 times. Discard excess PBS solution, add DAB chromogenic solution under a microscope, wait for positive areas to appear, record the chromogenic time, quickly discard the chromogenic solution, block tap water, and ensure consistent chromogenic time across different groups for the same target. After chromogenic development, stain the nuclei with hematoxylin and dehydrate, then mount with neutral resin.

[0140] 1.4.10 ELISA detection of inflammatory factors in mouse serum

[0141] Remove the ELISA kit from the 4°C freezer and allow it to equilibrate to room temperature for 30 minutes. Add diluent to the lyophilized standards, let stand for 15 minutes, then mix thoroughly to ensure complete dissolution. Prepare standard diluents of different concentrations using a serial dilution method, approximately 120 μL for each concentration. Thaw serum samples stored at -80°C on ice, and dilute an appropriate amount of serum sample to 320 μL using the sample diluent.

[0142] Add 100 μL of different concentrations of standards and serum samples to each well of the pre-coated microplate. Seal the wells with sealing tape and incubate at 37°C in the dark for 1.5 h. After incubation, discard the liquid from the wells. Use a multipipe to add 350 μL of washing buffer to each well and wash for 30 seconds × 5 times. Note that after discarding the washing buffer, pat the remaining liquid in the wells dry on absorbent paper.

[0143] Add 100 μL of biotinylated antibody diluent to the blank wells and 100 μL of biotinylated antibody working solution to the remaining wells. Seal the reaction wells with adhesive tape and incubate at 37°C in the dark for 1 hour. After incubation, discard the liquid in the wells and wash each well with 350 μL of washing buffer for 30 seconds × 5 times using a multi-pipe syringe.

[0144] Add 100 μL of enzyme conjugate diluent to the blank wells and 100 μL of enzyme conjugate working solution to the remaining wells. Seal the reaction wells with adhesive tape and incubate at 37°C in the dark for 30 min. After incubation, discard the liquid in the wells and wash each well with 350 μL of washing buffer for 30 s × 5 times using a multi-pipe syringe.

[0145] Add 100 μL of chromogenic substrate (3,3',5'-trimethylbenzidine), incubate at 37°C in the dark for 15 min, and after incubation, add 100 μL of reaction stop solution. Measure the absorbance using a microplate reader set to a wavelength of 450 nm.

[0146] 1.4.11 Detection of Liver and Kidney Function Indicators in Mice

[0147] In this study, the liver function indicators ALT and AST, and the kidney function indicators BUN and Cr in mice were tested. The procedures were all performed according to the kit instructions in section 1.3. The tests were conducted using a fully automated biochemical analyzer, with absorbance read at a wavelength of 340 nm, and the content of the relevant indicators was calculated.

[0148] 1.4.12 Statistical Methods

[0149] ImageJ 1.54g was used to count positive cells and score staining intensity on immunohistochemical images. SPSS 25.0 was used for statistical analysis. Quantitative data are expressed as mean ± standard deviation (±s), and qualitative data are expressed as percentages (%). Paired-samples t-tests were used for within-group comparisons conforming to a normal distribution, and paired-samples Kruskal-Wallis tests were used for within-group comparisons not conforming to a normal distribution. Independent-samples t-tests were used for between-group comparisons conforming to a normal distribution, and Kruskal-Wallis tests or chi-square tests were used for between-group comparisons not conforming to a normal distribution. One-way ANOVA was used for comparisons among multiple groups conforming to a normal distribution, and the Kruskal-Wallis test was used for comparisons among multiple groups not conforming to a normal distribution. P < 0.05 was considered statistically significant.

[0150] 2. Results

[0151] 2.1 Effects of Examples and Comparative Examples on Skin Lesions and Pathological Manifestations in Psoriasis Model Mice

[0152] 2.1.1 Effects of the Examples and Comparative Examples on Skin Lesions in a Mouse Model of Psoriasis

[0153] Skin photographs of mice in each group after continuous administration are shown below. Figure 1 As shown.

[0154] First animal experiment: by Figure 1 As shown in 'a', the mice in the control group had smooth skin, exhibiting a healthy pinkish-red color, without obvious scaling or thickening. In contrast, the mice in the model group had thickened epidermis, with more erythema and scaling. The mice in Examples 1-3 had slightly reddened skin with a small amount of scaling, but this was significantly improved compared to the model group.

[0155] Second animal experiment: by Figure 1 As shown in b, compared with the group in Example 1, the mice in the Comparative Examples 2-3 groups showed more erythema and scaling on their skin. Compared with the Comparative Example 1 group, the mice in the Example 1 group showed less scaling on their skin.

[0156] First animal experiment: Statistical analysis was performed on the PASI scores of dorsal skin lesions in mice of the blank group, model group, methotrexate group, and Example 1 group on day 6 (see...). Figure 2 The results showed that the extract of Example 1 and methotrexate improved the skin lesions on the back of psoriasis-like mice and reduced their PASI scores (P<0.001).

[0157] 2.1.2 Effects of the Examples and Comparative Examples on the Pathological Manifestations of the Psoriasis Mouse Model

[0158] First animal experiment: Pathological examination showed that, compared with the control group, the model group mice had significantly thickened stratum corneum and epidermis, thickened spinous processes, elongated and more undulating dermal processes, loosely arranged KCs, and extensive inflammatory cell infiltration in the dermis, which are typical pathological manifestations of psoriasis. The compositions in Examples 1-3 and methotrexate significantly improved the pathological manifestations of psoriasis in the model mice. Figure 3 a. The pathological manifestations of psoriasis in mice were quantified using the Baker score, see [reference needed]. Figure 4 Significant differences were found in Baker's scores among the blank group, model group, methotrexate group, and Example 1 group of mice. Both the extract of Example 1 and methotrexate significantly reduced the Baker's score of skin lesions in psoriasis model mice. The Baker's score of the mice in Example 1 group was lower than that of the methotrexate group (P<0.05), suggesting that the extract of Example 1 was more effective than methotrexate in improving the pathology of psoriasis.

[0159] 2.2 Effects of Example 1 on the proliferation, differentiation, and epidermal barrier function of psoriatic KCs

[0160] 2.2.1 Effect of Example 1 on the proliferation of psoriatic KCs

[0161] First animal experiment: Excessive proliferation of KCs is one of the typical pathological features of psoriasis. See Figure 5 Compared to the control group, the model group mice showed a significant increase in Ki67 expression in their epidermis. The extract from Example 1 and methotrexate reduced Ki67 expression in the epidermis of the model mice. This was determined by analyzing Ki67 expression per millimeter of epidermis. + The cell count was counted, such as Figure 6 Ki67 per millimeter of epidermal tissue in each group of mice + There was a significant difference in cell count; compared to the control group, the model group mice had significantly higher Ki67 levels in their epidermal cells. + The cell number increased significantly (P<0.001), indicating that the model group showed signs of KC overproliferation. The extract from Example 1 and methotrexate both reduced Ki67 in the model mice. + Cell count (P<0.001), among which the Ki67 cells in the epidermis of mice in the Kaixuan Detoxification group were significantly higher. + The number of cells was less than that in the methotrexate group (P<0.01), suggesting that the extract of Example 1 has a better ability to inhibit the proliferation of psoriatic KCs than methotrexate.

[0162] 2.2.2 Effect of Example 1 on the differentiation of psoriatic KCs

[0163] First animal experiment: Psoriatic keratinocytes (KCs) showed signs of incomplete differentiation. CK10 is one of the markers of late-stage keratinocyte differentiation. CK10 in mouse epidermal cells was labeled by immunohistochemistry. The late-stage differentiation of KCs in mice in the blank group, model group, methotrexate group, and Example 1 group was evaluated. (See...) Figure 7 In the model group, CK10 staining on the epidermis of mice was lighter, while in the Example 1 group and the methotrexate group, CK10 staining was darker compared to the model group. The expression of CK10 in mouse epidermis was quantified by the average optical density of the positive area, see [link to data]. Figure 8 Significant differences were observed in the expression of CK10 in the epidermis of mice in each group. The expression of CK10 in the epidermis of the model group was significantly lower than that in the blank group (P<0.001). Both the extract of Example 1 and methotrexate could promote the expression of CK10 in the model mice. The expression of CK10 in the epidermis of the Example 1 group was higher than that in the methotrexate group (P<0.001), suggesting that the extract of Example 1 has a stronger regulatory ability on the differentiation of psoriatic KCs than methotrexate.

[0164] 2.2.3 Effect of Example 1 on the epidermal barrier function of psoriasis patients

[0165] First animal experiment: Psoriasis is caused by disordered keratinocyte arrangement and decreased expression of intercellular junction proteins, leading to impaired epidermal barrier function. Claudin-1, a tight junction protein located at the tight junctions between cells, is a major component of the intercellular barrier. Immunohistochemical staining was used to label Claudin-1 in mouse epidermal cells to evaluate the epidermal barrier function of mice in the blank control group, model group, methotrexate group, and Example 1 group. (See attached data) Figure 9 Compared to the control group, the Claudin-1 staining area in the epidermis of the model group mice was lighter and discontinuous. The Claudin-1 staining area in the Example 1 group and the methotrexate group was darker and more continuous than that in the model group. Claudin-1 expression in mouse epidermis was quantified by the average optical density of the positive area. Figure 10 Significant differences were found in the average optical density of Claudin-1 positive areas in the epidermis of mice across different groups. Claudin-1 levels in the epidermis of the model group were significantly lower than those in the control group (P<0.05). Both methotrexate and the extract from Example 1 promoted Claudin-1 expression in the epidermis of model mice (P<0.01), suggesting that treatment with both methotrexate and the extract from Example 1 can repair the epidermal barrier function in psoriasis patients. Claudin-1 expression in the epidermis of mice in the Example 1 group was higher than that in the methotrexate group (P<0.05), indicating that the extract from Example 1 provides better protection of the epidermal barrier function than methotrexate.

[0166] 2.3 Effects of Example 1 on subcutaneous blood vessel density, spleen index, and serum pro-inflammatory cytokine expression in a mouse model of psoriasis

[0167] 2.3.1 Effect of Example 1 on subcutaneous blood vessel distribution density in psoriasis model mice

[0168] First animal experiment: Excessive angiogenesis is part of the pathological process of psoriasis. During inflammation, the density of subcutaneous capillaries increases, thereby supporting the transport of immune cells and inflammatory mediators to the lesion area. Therefore, the distribution density of subcutaneous capillaries also reflects the severity of the epidermal inflammatory response in psoriasis. The distribution of subcutaneous capillaries in mice in the blank group, model group, methotrexate group, and Example 1 group was recorded during the sampling process. See below. Figure 11 The subcutaneous capillary density of the model mice was significantly increased. The extract of Example 1 and methotrexate could reduce the subcutaneous capillary distribution density of the model mice, suggesting that the extract of Example 1 and methotrexate could reduce the degree of inflammatory response at the skin lesions of the psoriasis mouse model.

[0169] 2.3.2 Effect of Example 1 on spleen index in psoriasis model mice

[0170] First animal experiment: The spleen participates in regulating the body's immune response and inflammatory processes; changes in spleen indices can also be used to monitor the progression of the inflammatory response and the anti-inflammatory effects of drugs. See [link to relevant documentation]. Figure 12 The spleens of the model group mice were significantly enlarged compared to the control group mice. Methotrexate and the extract from Example 1 both reduced the splenomegaly in the model mice. The degree of splenomegaly in mice was quantified using the spleen index, see [link to relevant data]. Figure 13 Significant differences were found in the spleen index among the groups of mice. The spleen index of the model group mice was significantly increased compared with the blank group (P<0.001). The extract of Example 1 and methotrexate could reduce the spleen index of the model group mice (P<0.001), suggesting that the extract of Example 1 and methotrexate can alleviate the inflammatory response of psoriasis.

[0171] 2.3.3 Effect of Example 1 on the expression level of pro-inflammatory cytokines in the serum of psoriasis model mice

[0172] First animal experiment: Elevated expression of serum inflammatory mediators, such as IL-17, IL-22, and TNF-α, during the pathogenesis of psoriasis reflects the severity of the inflammatory response. The levels of IL-17, IL-22, and TNF-α in the serum of mice in the blank group, model group, and Example 1 group were detected by ELISA. The expression levels of inflammatory factors in each group are shown in Table 3. Statistical analysis was performed on the differences in the levels of various inflammatory factors between groups. Figure 14 Compared with the blank group mice, the expression of IL-17, IL-22 and TNF-α in the serum of the model group mice was significantly increased (P<0.01). The extract of Example 1 can reduce the levels of IL-17, IL-22 and TNF-α in the serum of the model mice (P<0.01), suggesting that the extract of Example 1 can significantly reduce the degree of inflammatory response in the serum of psoriasis patients.

[0173] Table 3. Expression levels of inflammatory factors

[0174] Blank group 13.87±2.23 151.31±10.07 39.24±14.09 Model group 38.77±6.26 262.14±73.94 144.42±24.18 Example 1 Group 14.20±3.40 104.64±51.10 58.16±13.88

[0175] 2.4 Safety evaluation of Example 1

[0176] 2.4.1 Effects of Example 1 on the morphology of the heart, liver, and kidneys in a psoriasis model mouse

[0177] First animal experiment: To further evaluate the safety of the extract from Example 1, HE staining was used to observe the effects of the extract from Example 1 on the pathological morphology of the mouse heart, liver, and kidney. Figure 15The mouse heart tissue sections from Example 1 showed tightly packed and orderly arranged cardiomyocytes with consistent intercellular spaces, and no signs of myocardial fibrosis, cell necrosis, inflammatory cell infiltration, or other pathological changes were observed. Similarly, in the liver tissue sections from the mice in Example 1, hepatocytes were tightly packed, with centered nuclei and clearly visible nucleoli; sinusoids were clear without obstruction or abnormal congestion, and no pathological changes such as cell necrosis, excessive inflammation, or fibrosis were observed. In the kidney tissue sections, renal tubular cells were neatly arranged with centered nuclei and abundant cytoplasm; the glomerular capillary walls were intact, with no evidence of inflammation or thrombosis, and no problems such as inflammatory cell infiltration or fibrosis were observed. These results demonstrate that the extract from Example 1 did not cause organic damage to the heart, liver, or kidneys of mice, thus proving its high safety profile.

[0178] 2.4.2 Effects of Example 1 on Liver and Kidney Function in Psoriasis Model Mice

[0179] First animal experiment: ELISA was used to measure liver and kidney function-related biochemical indicators in mice in the blank group, model group, methotrexate group, and Example 1 group. The biochemical indicator levels and statistical analysis results of each group are shown in Table 4. There were no significant differences in serum ALT, AST, and Cr levels among the groups. There were differences in serum BUN levels among the groups (P<0.01). Specifically, the BUN levels in the model group and methotrexate group were significantly higher than those in the blank group (P<0.05), suggesting that the modeling drug and methotrexate intervention may cause kidney damage in mice. However, mice treated with the extract of Example 1 did not show an abnormal increase in serum BUN levels compared to the blank control group (P=0.782). This indicates that the extract of Example 1 protected kidney function in model mice during the treatment of psoriasis, thus demonstrating its safety.

[0180] Table 4. Expression levels of biochemical indicators of liver and kidney function

[0181]

[0182]

[0183] Note: Compared with the blank group 1) P<0.05.

[0184] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A composition for treating psoriasis, characterized in that, The preparation method of the composition includes the following steps: extracting ephedra, cinnamon twig, red peony root, Japanese knotweed, artemisia annua, and oldenlandia diffusa to obtain an extract; or extracting ephedra, cinnamon twig, red peony root, Japanese knotweed, artemisia annua, and oldenlandia diffusa separately to obtain extracts, and then mixing the extracts. The mass ratio of ephedra, cinnamon twig, red peony root, Japanese knotweed, artemisia annua, and oldenlandia diffusa is 1-12:3-18:3-18:5-20:5-40:5-40.

2. The composition according to claim 1, characterized in that, The preparation method of the composition includes the following steps: mixing ephedra, cinnamon twig, red peony root, Japanese knotweed, artemisia annua, and oldenlandia diffusa, then extracting with water to obtain the extract.

3. The composition according to claim 1, characterized in that, The mass ratio of ephedra, cinnamon twig, red peony root, Japanese knotweed, artemisia annua, and oldenlandia diffusa is 6:9:9:10:20:

20.

4. The composition according to claim 2, characterized in that, The preparation method of the composition specifically includes the following steps: adding water and heating under reflux for extraction 2-4 times, adding 2-10 times the weight of the medicinal material in water each time, and extracting for 20-120 minutes each time to obtain the extract.

5. The composition according to claim 4, characterized in that, The preparation method of the composition specifically includes the following steps: adding water and heating under reflux for extraction twice. In the first extraction, add 5 times the weight of the medicinal material in water and extract for 60 minutes. In the second extraction, add 3 times the weight of the medicinal material in water and extract for 30 minutes to obtain the extract.

6. The composition according to claim 5, characterized in that, The preparation method of the composition further includes the following steps: concentrating the extract under reduced pressure into a thick paste, drying under reduced pressure, and pulverizing into powder to obtain the extract.

7. A medicine for treating psoriasis, characterized in that, The active ingredient of the drug includes the composition according to any one of claims 1-6.

8. The use of the composition according to any one of claims 1-6 or the medicament according to claim 7 in the preparation of a medicament for treating psoriasis.

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