Extract for treating psoriasis as well as preparation method and application thereof

Abstract

The invention discloses an extract for treating psoriasis as well as a preparation method and application thereof, and relates to the technical field of traditional Chinese medicine extraction. The invention discloses a composition for treating psoriasis, and a preparation method of the composition comprises the following steps: mixing herba ephedrae, cassia twig, radix paeoniae rubra, polygonum cuspidatum, artemisia apiacea and oldenlandia diffusa, and extracting to obtain an extract; or separately extracting the ephedra, the cassia twig, the radix paeoniae rubra, the polygonum cuspidatum, the artemisia apiacea and the oldenlandia diffusa to obtain extracts, and then mixing the extracts. Compared with a Kuan detoxification prescription, the extract disclosed by the invention omits coptis chinensis, indigo naturalis and felwort, is simpler in prescription, and has more advantages in the aspect of improving psoriasis-like skin lesion of model mice. Compared with methotrexate which is a common medicine for treating psoriasis, the extract disclosed by the invention has a better treatment effect on psoriasis and can relieve mouse renal function impairment caused by imiquimod.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extraction, and relates to an extract for treating psoriasis, a preparation method and application thereof. Background Art

[0002] Psoriasis is an immune-mediated, chronic, recurrent, inflammatory, systemic disease induced by the combined effects of genetics and the environment. Clinical manifestations include scaly erythema or plaques, which are localized or widespread. Psoriasis can be complicated by systemic diseases, seriously affecting the patient's quality of life. Although the exact cause of psoriasis has not yet been fully elucidated, genetic, immune and environmental factors play an important role in the occurrence of psoriasis. Genetics is the main risk factor for the onset of psoriasis, and the immune pathways related to interleukin 23 (IL-23) and helper T cell 17 (Th17) cells are the core mechanisms of psoriasis.

[0003] Methotrexate has anti-inflammatory, antiproliferative and immunomodulatory effects and is currently one of the most effective traditional drugs for the treatment of psoriasis. Methotrexate is rapidly absorbed in the gastrointestinal tract by binding to reduced folate carrier protein and is converted into polyglutamic acid methotrexate in cells to exert its effects. Methotrexate has shown good efficacy in all types of psoriasis, and is indicated for moderate to severe plaque psoriasis, PsA, erythrodermic psoriasis, GPP, and psoriasis that seriously affects function (such as palmoplantar psoriasis). Methotrexate can also treat severe nail psoriasis and peripheral PsA, and is particularly suitable when other systemic treatments are ineffective, contraindicated, intolerant or unaffordable. However, high doses of methotrexate have adverse reactions, mainly manifested as bone marrow suppression and hepatotoxicity.

[0004] Jiaqin Yeping, Dai Dan, Xiao Xue, Song Ping. Kaixuan Jiedu Fang improves the symptoms of imiquimod-induced psoriasis-like mice by regulating S1P levels [J]. Journal of Jiangsu University (Medical Edition), 2023, 33(2): 118-125. It is disclosed that Kaixuan Jiedu Fang can improve the symptoms and signs of psoriasis mice in many aspects, such as improving splenomegaly, thymus 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, the 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 to be a potential target of Kaixuan Jiedu Fang, and S1P / S1PR plays an important role in inflammatory signaling pathways, skin cell proliferation, and skin barrier. However, the document only discloses the composition of Kaixuan Jiedu Fang, and does not disclose whether the Chinese medicine has been extracted, nor does it optimize the ratio of the extract.

[0005] In view of this, in order to solve the deficiencies of the prior art, the present invention provides an extract for treating psoriasis and a preparation method and application thereof. The extract disclosed in the present invention can be used to prepare a drug for treating psoriasis, and has a good research and development prospect. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies in previous studies, provide an extract for treating psoriasis and a preparation method and application thereof, give full play to the efficacy of traditional Chinese medicine, effectively solve the problem of medication for the treatment of psoriasis, and have promotion and development value.

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

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

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

[0010] Further preferably, the mass ratio of ephedra, cinnamon twig, red peony root, 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, knotweed, artemisia annua and oldenlandia diffusa is 6:9:9:10:20:20.

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

[0013] More preferably, the preparation method specifically comprises the following steps: adding water, heating and refluxing for extraction twice, adding 5 times the weight of the medicinal material of water for the first time, extracting for 60 minutes; adding 3 times the weight of the medicinal material of water for the second time, extracting for 30 minutes, to obtain an extract.

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

[0015] More preferably, the preparation method further comprises the following steps: concentrating the extract under reduced pressure to a thick paste with a relative density of 1.2-1.3 (thermal measurement at 50-60°C), drying under reduced pressure at 60-70°C, and crushing into powder to obtain an extract.

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

[0017] In yet another aspect, the present invention provides a medicine, the active ingredient of which comprises the above composition or the above extract.

[0018] The drug can be prepared into dosage forms such as pills, capsules, granules, oral liquids, powders, tablets, lozenges, and lozenges, and suitable drug carriers in the art can be selected for different dosage forms.

[0019] The pharmaceutical carrier used can be solid, liquid or gas. 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] When preparing the composition for oral dosage form, any convenient pharmaceutical medium can be used. For example, water, ethanol, oil, alcohol, flavoring agent, preservative, coloring agent, etc. can be used to form oral liquid preparations, such as suspensions and solutions; while carriers, such as starch, sugars, microcrystalline cellulose, diluents, granulating agents, emulsifiers, lubricants, binders, disintegrants can be used to form oral solid preparations, such as powders, capsules and tablets. Tablets and capsules are the 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 the present invention can be prepared by tableting or molding, and one or more auxiliary ingredients or adjuvants can be used. The active ingredient can be tableted in a free-flowing form (such as powder or granules) in an appropriate machine, and can be mixed with a binder, lubricant, inert diluent, surfactant or dispersant to prepare a tablet. Molded tablets can be molded in an appropriate machine, that is, a powdered compound mixture 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, a preparation intended for oral administration to humans may contain about 0.5 mg to about 5 g of active drug, mixed with an appropriate and convenient carrier material, which may account for about 5% to 95% of the total composition. The unit dosage form usually contains about 1 mg to about 2 g of active ingredient, usually 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg or 1000 mg.

[0022] The pharmaceutical composition suitable for parenteral administration of the present invention can be prepared as an aqueous solution or suspension of the active compound. Suitable surfactants such as hydroxypropylcellulose can be included. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and oil mixtures thereof. In addition, preservatives can be added to prevent the harmful growth of microorganisms.

[0023] The medicament of the present invention can be in a form suitable for topical use, such as an aerosol, cream, ointment, lotion, powder or the like. In addition, the composition can be in a suitable form for transdermal administration devices. For example, a cream or ointment with a desired consistency is prepared by mixing a hydrophilic material and water, and about 5wt% to about 10wt% of the compound.

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

[0025] In addition to the above-mentioned carrier components, the above-mentioned pharmaceutical preparations may include (if applicable) one or more additional carrier components, such as diluents, buffers, flavoring agents, adhesives, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc. In addition, other excipients, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., colorants and flavoring agents, etc., may be added. The preparation is made isotonic with the blood of the intended recipient. The components containing the extract of the present invention can also be prepared in the form of a powder or a concentrated solution.

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

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

[0028] Preferably, the psoriasis is selected from any one of psoriasis vulgaris, psoriasis arthritis, psoriasis pustularis, and psoriasis erythroderma.

[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 of the skin;

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

[0034] (4) Scaly skin on the scalp;

[0035] (5) The fingernails and / or toenails are pin-shaped or pitted, onycholysis, or subungual hyperkeratosis;

[0036] (6) Joint swelling.

[0037] Further 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 of 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 Jiedu prescription (composed of Artemisia annua, Polygonum cuspidatum, Hedyotis diffusa, Coptis chinensis, Ephedra sinica, Cinnamon twig, Indigo naturalis, and Gentiana scabra), the extract disclosed in the present invention omits Coptis chinensis, Indigo naturalis, and Gentiana scabra, making the prescription more concise and having more advantages 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 the present invention can better alleviate the renal function damage of mice caused by imiquimod and has a better therapeutic effect on psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The effects of Examples 1-3 and Comparative Examples 1-3 on the skin lesions of model mice, wherein a is the result of the first animal experiment, and b is the result of the second animal experiment.

[0045] Figure 2 PASI scores of the back skin lesions of mice in each group on day 6, where ns, no statistical difference; ** , P<0.01; *** , P<0.001.

[0046] Figure 3 The effects of Examples 1-3 and Comparative Examples 1-3 on the pathological manifestations of skin lesions in psoriasis model mice, wherein a is the result of the first animal experiment, and b is the result of the second animal experiment.

[0047] Figure 4 The effect of the extract of Example 1 on the Baker score of skin lesion pathological observation in psoriasis model mice, wherein: * , P<0.05; ** , P<0.01; *** , P<0.001.

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

[0049] Figure 6 The extract of Example 1 has an effect on Ki67 in the epidermis of psoriasis model mice + The effect of cell number, among which, ** , P<0.01; *** , P<0.001.

[0050] Figure 7 The figure shows the effect of the extract of Example 1 on the expression of CK10 in the epidermis of psoriasis model mice, wherein the area above the black dotted line is the skin epidermis.

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

[0052] Fig. 9 The figure shows the effect of the extract of Example 1 on the expression of Claudin-1 in the epidermis of psoriasis model mice, wherein the area above the black dotted line is the skin epidermis.

[0053] Fig.10 The effect of the extract of Example 1 on the average optical density of Claudin-1 in the epidermis of psoriasis model mice, wherein: * , P<0.05; ** , P<0.01; *** , P<0.001.

[0054] Fig.11 The figure shows the effect of the extract of Example 1 on the subcutaneous blood vessel distribution density in psoriasis model mice.

[0055] Fig.12 The figure shows the effect of the extract of Example 1 on the spleen morphology of psoriasis model mice.

[0056] Fig.13 The effect of the extract of Example 1 on the spleen index of psoriasis model mice, wherein: *** , P<0.001.

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

[0058] Fig.15 The following is the effect of the extract of Example 1 on the morphology of heart, liver and kidney in psoriasis model mice. DETAILED DESCRIPTION

[0059] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of protection claimed in this application, and those skilled in the art can make various changes and modifications to the invention of this application based on the disclosed content, which should also fall within the scope of protection claimed in this application.

[0060] The present invention is further described below by way of specific examples. Unless otherwise specified, the various drugs used in the examples of the present invention are obtained through conventional commercial channels.

[0061] Ephedra is the herbaceous stem of Ephedra sinica, Ephedra equisetum or Ephedra intermedia, all of the Ephedraceae family.

[0062] Cinnamon twigs are the young branches of the cinnamon plant of the Lauraceae family.

[0063] Red peony root is the dried root of red peony root or Sichuan red peony root of the Ranunculaceae family.

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

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

[0066] Hedyotis diffusa, a plant of the genus Hedyotis diffusa in the family Rubiaceae

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

[0068] Example 1

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

[0070] Preparation method: Take the formulated amount of medicinal materials, add 5 times the amount of pure water for the first time, soak for 2 hours, heat under reflux for extraction for 60 minutes, filter the extract, continue to extract the remaining residues, 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 water decoction containing 6.24 g / mL of the crude drug.

[0071] Example 2

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

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

[0074] Example 3

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

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

[0077] Comparative Example 1

[0078] The formula is as follows: 1 part of ephedra, 3 parts of cinnamon twig, 20 parts of knotweed, 40 parts of artemisia annua, 40 parts of oldenlandia diffusa, 6 parts of coptis root, 3 parts of indigo, and 9 parts of gentian. This formula is referenced by (Qin Yeping, Dai Dan, Xiao Xue, et al. Kaixuan Jiedu Recipe improves the symptoms of psoriasis-like mice induced by imiquimod by regulating S1P levels [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 Example 1.

[0080] Comparative Example 2

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

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

[0083] Comparative Example 3

[0084] The formula is as follows: 9 parts of red peony root, 10 parts of knotweed, 20 parts of artemisia annua, and 20 parts of oldenlandia diffusa.

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

[0086] Results

[0087] 1. Experimental Materials and Methods

[0088] 1.1 Experimental Animals

[0089] Specific pathogen-free (SPF) grade C57BL / 6J mice, male, 6-8 weeks old, weighing 18-22g, a total of 72 mice, were purchased from Beijing Weitonglihua Animal Technology Co., Ltd., license number: SCXK (Beijing) 2021-0006. Mice were raised under SPF conditions in the animal room of Guang'anmen Hospital of China Academy of Chinese Medical Sciences, and were given standard ordinary feed, environmental humidity of 55±5%, temperature controlled at 22-25℃, and light time of 12h / day. Cage, 6 per cage, a total of 12 cages, free access to food and water. The experimental protocol was approved by the Experimental Animal Ethics Committee of Guang'anmen Hospital of 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 embodiments and comparative examples were provided by the pharmacy of Guang'anmen Hospital, China Academy of Chinese Medical Sciences; imiquimod cream (company: 3M Health Care limited, imported drug registration certificate number: H20160079, specification: 12.5 mg / 250 mg), methotrexate tablets (company: Shanghai Shangyao Xinyi Pharmaceutical Factory Co., Ltd., batch number: National Medicine Standard 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] All C57BL / 6J mice in the model group were adaptively raised for one week before modeling. The hair on the back of the mice was shaved with a razor one day before modeling to form an exposed area of ​​about 2 cm × 3 cm, and then the exposed area was covered with depilatory cream. After the depilatory cream stayed on the back of the mouse for 90 seconds, a paper towel was dipped in warm water to remove the depilatory cream and the remaining hair on the back to fully expose the back skin of the mouse. After the modeling started, 62.5 mg of imiquimod cream was applied to the exposed skin at 9:00 every day, once a day, for 5 consecutive days. At the same time, 62.5 mg of vaseline was applied to the back of the blank group mice as a control.

[0099] 1.4.2 Grouping and Dosing

[0100] 4 hours after modeling, oral administration was started. According to the body surface area administration method in "Pharmacological Experimental Methodology", the mouse dose = [human dose (g crude drug / kg / d) × human conversion coefficient] / mouse conversion coefficient calculation method, the mouse equivalent dose was calculated to be about 12.3 times that of humans. According to the above prescription, the daily dose is 0.1mL / 10g (converted according to the body surface area between humans and rats). The Chinese medicine decoction was sterilized by high pressure and refrigerated for future use.

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

[0102] ① Blank group: 62.5 mg of vaseline + 0.1 mL / 10 g of distilled water were applied to the back and then intragastrically administered;

[0103] ② Model group: 62.5 mg imiquimod cream applied on the back and 0.1 mL / 10 g distilled water was gavaged;

[0104] ③Methotrexate group: 62.5 mg imiquimod cream applied on the back + 1 mg / kg methotrexate solution gavage, the administration volume was 0.1 mL / 10 g;

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

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

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

[0108] The specific groups of the second animal experiment are as follows:

[0109] ⑦ Blank group: 62.5 mg of vaseline + 0.1 mL / 10 g of distilled water were applied to the back and then intragastrically administered;

[0110] ⑧Model group: 62.5 mg imiquimod cream applied on the back and 0.1 mL / 10 g distilled water was gavaged;

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

[0112] ⑩Comparative Example 1 group: 62.5 mg of imiquimod cream was applied to the back and the decoction prepared in Comparative Example 1 was gavaged, with a dosing volume of 0.1 mL / 10 g;

[0113] Comparative Example 2 group: 62.5 mg of imiquimod cream was applied to the back and the decoction prepared in Comparative Example 2 was gavage, with a dosing volume of 0.1 mL / 10 g;

[0114] Comparative Example 3 group: 62.5 mg of imiquimod cream was applied on the back and the decoction prepared in Comparative Example 3 was gavage, with a dosing volume of 0.1 mL / 10 g.

[0115] Each group of 6 mice was gavaged once a day for 5 consecutive days. The mice were weighed and the PASI score of the skin lesions was calculated before applying imiquimod cream every day, and the skin morphology on the back of the mice was recorded by photographing under consistent lighting and shooting tools.

[0116] 1.4.3. Sample collection and storage

[0117] After the mouse modeling and intervention on the 5th day, the feed was taken away and the mice were starved. The samples were collected at 8 am on the 6th day. The mice were anesthetized by intraperitoneal injection of 0.2 mL / 10 g of 0.3% sodium pentobarbital solution prepared with normal saline. After anesthesia, the whiskers of the mice were trimmed, the eyeballs were removed with tweezers, and the blood of the mice was collected with serum separation gel coagulation tubes. After the blood collection was completed, the test tubes were stored at room temperature.

[0118] The mice after blood collection were placed on a pre-sterilized foam board, and the back skin was cut with surgical scissors and transferred to PBS solution for rinsing. The skin subcutaneous tissue was placed facing up and spread on white filter paper, and photos were taken to record the subcutaneous vascular proliferation. The skin tissue was divided into 4 parts, one of which was attached to the filter paper and put into neutral formalin fixative to prevent the skin tissue from curling. The other three parts were put into marked tissue cryopreservation tubes, and after quick freezing with liquid nitrogen, they were transferred to a -80℃ refrigerator for storage.

[0119] The mouse was fixed on a foam board, the chest skin and ribs were cut open to expose the heart, and a 5 mL syringe (1 mL needle) filled with normal saline in advance was used to pierce the apex of the heart and slowly inject normal saline to perform cardiac perfusion to help remove excess blood in the mouse body and facilitate subsequent morphological analysis.

[0120] After the perfusion was completed, the spleen was removed and weighed, and the heart, 1 / 3 of the largest lobe of the liver, and one side of the kidney with the outer membrane peeled off were placed in neutral formalin fixative.

[0121] After the mouse samples were collected, the coagulated blood was processed centrally, and the blood collection tubes were placed in a low-temperature high-speed centrifuge, and the parameters were set to 4°C, 3000rpm, and 15min for centrifugation. After the centrifugation was completed, the supernatant was transferred to a cryopreservation tube 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, fixed equipment and relatively consistent lighting conditions were used to photograph and record the changes in skin lesions on the back of mice. The PASI scoring standard was used to score the erythema (0-4 points), scaling (0-4 points), and degree of infiltration (0-4 points) of the skin lesions of mice. The sum of the three scores was the PASI score.

[0124] 1.4.5. Calculation of spleen index and assessment of subcutaneous vascular proliferation

[0125] The mouse spleen index was calculated by dividing the mouse spleen weight (mg) by the mouse body weight (g) and multiplying the quotient by 100%. The proliferation of subcutaneous blood vessels in mice was evaluated by photographing and macroscopic observation.

[0126] 1.4.6. Preparation and sectioning of histopathological wax blocks

[0127] Remove the tissues fixed for 72 hours or more from the fixative, use a scalpel to cut the tissue part to be observed, put them into the marked tissue embedding box, and dehydrate them in 75% ethanol for 2 hours → 85% ethanol for 30 minutes → 95% ethanol for 2 hours → (change liquid) 95% ethanol for 2 hours → anhydrous ethanol for 2 hours → (change liquid) anhydrous ethanol for 2 hours, and make the tissue transparent by xylene for 1.5 hours → (change liquid) xylene for 1.5 hours → (change liquid) xylene for 45 minutes. Start the wax melting cylinder of the tissue embedding machine in advance, set the parameters to 62℃ to melt the paraffin, and immerse the dehydrated and transparentized tissue in wax for 3 hours.

[0128] After the wax dipping is completed, start the embedding machine cold stage, prepare the pathological tissue wax block embedding mold in advance, open the embedding box, take out the wax-dipping tissue, drip 1 / 4 paraffin wax into the mold, use tweezers to place the tissue to be observed face up in the mold, wait for the paraffin wax in the mold to solidify slightly, quickly drip paraffin wax into the mold and buckle the bottom of the embedding box, transfer it to the cold stage, and after the wax block has solidified, transfer it from the cold stage to the room temperature laboratory operating table for demolding, and the tissue pathology wax block is completed.

[0129] Start the slicer in advance and set the water temperature to 55°C. Take the prepared wax block, fix it on the paraffin slicer, turn on the air humidifier and aim at the wax block, first set it to rough trimming 20μm / time, and set it to fine trimming after cutting the tissue. The skin tissue is 4.5μm, and the heart, liver and kidney are all set to 4μm because of their dense tissue. After the slice is completed, spread the tissue wax slice in the slicer with tweezers, wait until the tissue is fully stretched without wrinkles, use an ordinary slide or a high-adhesion slide to pick up the slice, and dry it overnight.

[0130] 1.4.7. Eosin-Hematoxylin (HE) staining

[0131] The baking machine was set to 60℃ for 1h. After the paraffin was fully melted, the slices were placed in xylene for 10min→(change liquid) xylene for 5min→(change liquid) xylene for 5min→anhydrous ethanol for 10min→(change liquid) anhydrous ethanol for 5min→95% ethanol for 5min→80% ethanol for 5min→70% ethanol for 5min→wash with tap water for dewaxing. The dewaxed tissue slices were placed in hematoxylin for nuclear staining for 15min, the excess stain was washed off with tap water, and 1% hydrochloric acid ethanol was used for differentiation. After the excess differentiation solution was washed off with tap water, the slices were placed in eosin stain for cytoplasm for 5min, the excess stain was washed off with tap water, and the slices were sealed with neutral gum after dehydration.

[0132] 1.4.8 Baker score for psoriasis skin pathology

[0133] The Baker score is used to evaluate the pathological manifestations of psoriasis lesions. The specific scoring criteria are as follows:

[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 1h in a slide baker. After the paraffin is fully melted, dewax according to the steps in 1.4.7. After dewaxing, wash off the excess organic solvent with double distilled water. Pour 200mL of 1X antigen repair solution into the immunohistochemistry antigen repair box. Set the microwave oven to 100℃ for 5min to preheat the repair solution. Transfer the slides to the preheated repair solution, paying attention to the liquid height to immerse the tissue. Set 100℃ for 5min and 50℃ for 15min for antigen repair. Pay attention to rehydration during the repair process to prevent dry slides caused by violent boiling of the liquid. Let cool at room temperature after repair.

[0138] The slides were cooled to room temperature, washed with PBS solution for 5 min × 3 times, and the PBS liquid around the tissue was wiped dry with a paper towel. The immunohistochemical pen was used to draw a water blocking circle, and an appropriate amount of endogenous peroxidase blocker was dripped into the tissue to just cover the tissue. The endogenous peroxidase was incubated at room temperature for 10 min to block the endogenous peroxidase. The PBS solution was rinsed for 5 min × 3 times, and the tissue was covered with a blocking solution containing 5% goat serum, 1% BSA and 0.3% Triton, and the blocking solution was used for 1 h. During the blocking process, an antibody diluent containing 1% BSA and 0.3% Triton was prepared, and Ki67 was diluted at 1:400, Claudin1 was diluted at 1:800, and CK10 was diluted at 1:2000. After the blocking was completed, the blocking solution was shaken off, and the slides were placed in an immunohistochemical humidification box, and the prepared primary antibody was added to the corresponding surface of the tissue. Water was added to the bottom of the box and kept at 4°C overnight.

[0139] Take out the slides that have been left overnight, check for dry slides, and equilibrate at room temperature for 30 minutes. Use PBS solution containing 0.1% Triton to wash for 10 minutes × 5 times. Wipe dry the liquid around the tissue, drip the reaction enhancement solution, incubate at room temperature for 20 minutes, wash with PBS solution for 5 minutes × 3 times, drip the secondary antibody, incubate at room temperature for 20 minutes, and wash again with PBS solution for 5 minutes × 3 times. Shake off the excess PBS liquid, drip DAB color development solution under the microscope, wait for the positive area to appear, record the color development time, quickly pour out the color development solution, block with tap water, and control the color development time of the same target in different groups to be consistent. After the color development is completed, the nucleus is stained with hematoxylin and dehydrated, and the slide is sealed with neutral resin.

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

[0141] Take out the ELISA kit from the 4℃ refrigerator and equilibrate at room temperature for 30 minutes. Add diluent to the lyophilized standard, let it stand for 15 minutes, mix it evenly, and prepare different concentrations of standard diluents by gradient dilution method, each concentration is about 120μL liquid. Transfer the serum sample stored at -80℃ to ice to melt, take an appropriate amount of serum sample and dilute it to 320μL with specimen diluent.

[0142] Add different concentrations of standard products and serum samples to the pre-coated ELISA plate, 100 μL per well, seal the reaction wells with sealing tape, and incubate at 37°C in the dark for 1.5 hours. After incubation, shake off the liquid in the wells, and use a spray gun to add 350 μL of washing solution to each well for 30 seconds × 5 times. Note that after shaking off the washing solution, pat dry the residual liquid in the wells on absorbent paper.

[0143] Add 100 μL of biotinylated antibody diluent to the blank well, add 100 μL of biotinylated antibody working solution to the remaining wells, seal the reaction wells with sealing tape, incubate at 37°C in the dark for 1 h, shake off the liquid in the wells after incubation, and use a spray gun to add 350 μL of washing solution to each well for 30 s × 5 times.

[0144] Add 100 μL of enzyme conjugate diluent to the blank wells, add 100 μL of enzyme conjugate working solution to the remaining wells, seal the reaction wells with sealing tape, incubate at 37°C in the dark for 30 min, shake off the liquid in the wells after incubation, and use a spray gun to add 350 μL of washing solution to each well for 30 s × 5 times.

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

[0146] 1.4.11. Detection of liver and kidney function indexes in mice

[0147] The liver function indicators ALT and AST and kidney function indicators BUN and Cr of mice were tested in this test. The steps were all carried out according to the instructions of the kit in 1.3. The test was carried out using a fully automatic biochemical analyzer, the absorbance was read at a wavelength of 340nm, and the content of related indicators was calculated.

[0148] 1.4.12 Statistical methods

[0149] ImageJ1.54g was used to count positive cells and score staining intensity in immunohistochemical images. SPSS25.0 was used for statistical analysis. Quantitative data were expressed as ±s, and qualitative data were expressed as percentages (%). Paired sample t-test was used for intra-group comparisons that met the normal distribution, and paired sample Kruskal-Wallis test was used for intra-group comparisons that did not meet the normal distribution. Independent sample t-test was used for inter-group comparisons that met the normal distribution, and Kruskal-Wallis test or chi-square test was used for inter-group comparisons that did not meet the normal distribution. One-way analysis of variance was used for comparisons between multiple groups that met the normal distribution, and Kruskal-Wallis test was used for comparisons between multiple groups that did not meet the normal distribution. P<0.05 indicated that the difference was 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 Examples and Comparative Examples on Skin Lesions in Psoriasis Mouse Model

[0153] After continuous drug administration, the skin photos of mice in each group are as follows: Figure 1 shown.

[0154] The first animal experiment: Figure 1 As shown in a, the skin of the mice in the blank group was smooth, showing a healthy flesh-pink color, without obvious accumulation and thickening of scales. In contrast, the epidermis of the mice in the model group was thickened, with more erythema and scales. The skin of the mice in Example 1-3 groups was slightly red and accompanied by a small amount of scales, but there was a significant improvement compared with the model group.

[0155] The second animal experiment: Figure 1 As shown in b, the skin of mice in Comparative Examples 2-3 showed more erythema and scaling than that in Example 1. Compared with Comparative Example 1, the skin of mice in Example 1 showed less scaling.

[0156] The first animal experiment: The PASI scores of the back skin lesions of the mice in the blank group, model group, methotrexate group, and Example 1 group on the 6th day were statistically analyzed (see Figure 2 ), there were significant differences in the PASI scores of mice in each group. The results showed that the extract of Example 1 and methotrexate improved the back skin lesions of psoriasis-like model mice and reduced their PASI scores (P<0.001).

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

[0158] The first animal experiment: Pathological results showed that compared with the blank group mice, the stratum corneum and epidermis of the model group mice were significantly thickened, the spinous processes were thickened, the skin processes were elongated and undulated, the KCs were loosely arranged, and a large number of inflammatory cells infiltrated the dermis, which are typical pathological manifestations of psoriasis. The composition of Examples 1-3 and methotrexate can significantly improve the pathological manifestations of psoriasis in the model mice, as shown in Figure 1. Figure 3 a. Quantification of psoriasis pathology in mouse skin by Baker score, see Figure 4 There were significant differences in the Baker scores of mice in the blank group, model group, methotrexate group, and Example 1 group. Both the extract of Example 1 and methotrexate could significantly reduce the Baker scores of skin lesions in psoriasis model mice, and the Baker score of mice in Example 1 group was lower than that in the methotrexate group (P<0.05), indicating that the extract of Example 1 was better than methotrexate in improving the pathology of psoriasis.

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

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

[0161] First animal experiment: Excessive proliferation of KCs is one of the typical pathological features of psoriasis. Figure 5 Compared with the blank group mice, the expression of Ki67 in the epidermis of the model group mice was significantly increased. The extract of Example 1 and methotrexate can reduce the expression of Ki67 in the epidermis of the model mice. + The number of cells was counted, such as Figure 6 , Ki67 per mm epidermis of each group of mice + There was a significant difference in the number of cells. Compared with the blank group, the epidermal Ki67 + The number of cells increased significantly (P<0.001), indicating that the model group had excessive proliferation of KCs. The extract of Example 1 and methotrexate could reduce the expression of Ki67 in the model mice. + The number of cells in the epidermis of mice in the Kaixuan Jiedu group was + The number of cells was less than that in the methotrexate group (P<0.01), indicating that the extract of Example 1 was better than methotrexate in inhibiting the proliferation of psoriasis KCs.

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

[0163] The first animal experiment: Psoriasis KCs showed incomplete differentiation. CK10 is one of the markers of the terminal differentiation of keratinocytes. CK10 in mouse epidermal cells was marked by immunohistochemistry to evaluate the terminal differentiation of KCs in the blank group, model group, methotrexate group, and Example 1 group. Figure 7 The positive staining of CK10 in the epidermis of mice in the model group was relatively light, and the CK10 staining in the Example 1 group and the methotrexate group was relatively darker than that in the model group. The expression of CK10 in the epidermis of mice was quantified by the average optical density of the positive area. Figure 8 There were significant differences in the expression of epidermal CK10 in each group of mice. The expression of epidermal CK10 in 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, among which the expression of epidermal CK10 in the mice of Example 1 was higher than that in the methotrexate group (P<0.001), indicating that the extract of Example 1 has a stronger regulatory ability on the differentiation of psoriasis KCs than methotrexate.

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

[0165] The first animal experiment: Psoriasis is caused by disordered arrangement of keratinocytes and decreased expression of intercellular junction proteins, which lead to damage of epidermal barrier function. Claudin-1 is a tight junction protein located at the tight junctions between cells and is one of the main components of the intercellular barrier. By using immunohistochemistry, the epidermal barrier function of mice in the blank group, model group, methotrexate group, and Example 1 group was evaluated by labeling Claudin-1 in mouse epidermal cells. Fig. 9 Compared with the blank group mice, the epidermal Claudin-1 staining area of ​​the model group mice was lighter and discontinuous. The Claudin-1 staining area of ​​the Example 1 group and the methotrexate group was darker than that of the model group, and the staining area was relatively continuous. The expression of Claudin-1 in the epidermis of mice was quantified by the average optical density of the positive area. Fig.10 There were significant differences in the average optical density of the Claudin-1 positive area in the epidermis of mice in each group. The Claudin-1 in the epidermis of mice in the model group was significantly lower than that in the blank group (P<0.05). Both methotrexate and the extract of Example 1 could promote the expression of Claudin-1 in the epidermis of model mice (P<0.01), indicating that both methotrexate and the extract of Example 1 could repair the epidermal barrier function of psoriasis. The expression of Claudin-1 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 of Example 1 was better than methotrexate in protecting the epidermal barrier function.

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

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

[0168] First animal experiment: Excessive angiogenesis is part of the pathological process of psoriasis. During the inflammatory process, 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 indirectly reflects the severity of the inflammatory response of the psoriasis epidermis. During the sampling process, the distribution of subcutaneous capillaries in the blank group, model group, methotrexate group, and Example 1 group of mice was recorded. Fig.11 The density of subcutaneous capillaries in the model group mice was significantly increased, and the extract of Example 1 and methotrexate could reduce the distribution density of subcutaneous capillaries in the model mice, indicating that the extract of Example 1 and methotrexate could reduce the degree of inflammatory response in the skin lesions of the psoriasis mouse model.

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

[0170] The first animal experiment: The spleen is involved in regulating the immune response and inflammatory process in the body. Changes in spleen index can also be used to monitor the progression of inflammatory response and the anti-inflammatory effect of drugs. Fig.12 The spleen of the model group mice was significantly enlarged compared with that of the blank group mice. Both methotrexate and the extract of Example 1 could reduce the enlargement of the spleen in the model mice. The enlargement of the spleen in mice was quantified by the spleen index. Fig.13 There were significant differences in the spleen index of mice in each group. The spleen index of mice in the model group was significantly increased compared with that in the blank group (P<0.001). Both the extract of Example 1 and methotrexate could reduce the spleen index of mice in the model group (P<0.001), indicating that both the extract of Example 1 and methotrexate could 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] The first animal experiment: During the onset of psoriasis, the expression of serum inflammatory mediators, such as IL-17, IL-22 and TNF-α, increased, reflecting 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. The differences in the levels of inflammatory factors among the groups were statistically analyzed. Fig.14 Compared with the blank group mice, the expressions of IL-17, IL-22 and TNF-α in the serum of the model group mice were 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), indicating that the extract of Example 1 can significantly reduce the degree of inflammatory response in the serum of psoriasis mice.

[0173] Table 3 Expression levels of inflammatory factors

[0174] Group IL-17 (pg / mL) IL-22 (pg / mL) TNF-α(pg / mL) 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 Effect of Example 1 on the Morphology of Heart, Liver and Kidney in Psoriasis Model Mice

[0177] First animal experiment: In order to further evaluate the safety of the extract of Example 1, HE staining was used to observe the effect of the extract of Example 1 on the pathological morphology of the heart, liver and kidney of mice. Fig.15It was shown that the mouse heart tissue sections of Example 1 group showed that the myocardial cells were arranged tightly and orderly, the intercellular spaces were consistent, and no signs of myocardial fibrosis, cell necrosis, inflammatory cell infiltration or other pathological changes were observed. Similarly, in the liver tissue sections of the mice in Example 1 group, the hepatocytes were tightly arranged, the cell nucleus was centered and the nucleolus was clearly visible, the sinusoids were clear without obstruction or abnormal congestion, and no pathological changes such as cell necrosis, excessive inflammatory response or fibrosis were found. In terms of kidney tissue sections, the renal tubular cells were neatly arranged, the cell nucleus was centered, the cytoplasm was abundant, the glomerular capillary walls were intact, there was no evidence of inflammation or thrombosis, and no problems such as inflammatory cell infiltration or fibrosis were observed. The above results show that the extract of Example 1 did not cause organic damage to the heart, liver or kidneys of mice, thereby proving its high safety.

[0178] 2.4.2 Effect of Example 1 on liver and kidney function in psoriasis model mice

[0179] The first animal experiment: ELISA was used to measure the biochemical indices related to liver and kidney function of the blank group, model group, methotrexate group, and Example 1 group of mice. The levels of biochemical indices of each group of mice and the statistical analysis results are shown in Table 4. There were no significant differences in the levels of serum ALT, AST, and Cr in each group of mice. There were differences in the BUN levels in the serum of each group of mice (P<0.01), among which the BUN levels in the model group and methotrexate group were significantly increased compared with the blank group (P<0.05), indicating that the modeling drug and methotrexate intervention may cause renal function damage in mice, while the BUN levels in the serum of mice treated with the extract of Example 1 did not show abnormal increases compared with the blank control group (P=0.782), which showed that the extract of Example 1 protected the renal function of the model mice during the treatment of psoriasis, thereby proving 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, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate 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 comprises the following steps: extracting after mixing ephedra, cinnamon twig, red peony root, knotweed, artemisia annua and oldenlandia diffusa to obtain an extract; or extracting ephedra, cinnamon twig, red peony root, knotweed, artemisia annua and oldenlandia diffusa separately to obtain an extract, and then mixing the extracts.

2. The composition according to claim 1, characterized in that The preparation method of the composition comprises the following steps: mixing ephedra, cinnamon twig, red peony root, polygonum cuspidatum, artemisia annua and oldenlandia diffusa, and then adding water to extract to obtain an extract.

3. The composition according to claim 2, characterized in that The mass ratio of ephedra, cassia twig, red peony root, polygonum cuspidatum, artemisia annua and oldenlandia diffusa is 1-12:3-18:3-18:5-20:5-40:5-40.

4. The composition according to claim 3, characterized in that The mass ratio of ephedra, cassia twig, red peony root, polygonum cuspidatum, artemisia annua and oldenlandia diffusa is 6:9:9:10:20:

20.

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

6. The composition according to claim 5, characterized in that The preparation method of the composition specifically comprises the following steps: adding water, heating and refluxing for extraction twice, adding water 5 times the weight of the medicinal material for the first time and extracting for 60 minutes; adding water 3 times the weight of the medicinal material for the second time and extracting for 30 minutes to obtain an extract.

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

8. An extract for treating psoriasis, characterized in that: The extract consists of ephedra extract, cassia twig extract, red peony root extract, knotweed extract, artemisia annua extract and oldenlandia diffusa extract.

9. A drug, characterized in that The active ingredient of the drug comprises the composition according to any one of claims 1 to 7 or the extract according to claim 8.

10. Use of the composition according to any one of claims 1 to 7, the extract according to claim 8 or the drug according to claim 9 in the preparation of a drug for treating psoriasis.

Images