A traditional Chinese medicine composition for treating rheumatoid arthritis, a medicine, and a preparation method and application thereof

Abstract

The application discloses a traditional Chinese medicine composition for treating rheumatoid arthritis, a medicine and a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine pharmacy. The application aims to relieve joint swelling, inhibit joint bone erosion damage, effectively reduce the concentration of serum proinflammatory factors, and efficiently treat rheumatoid arthritis with damp-heat stagnation syndrome. The traditional Chinese medicine composition is prepared by mixing radix astragali, caulis sinomenii, rhizoma et herba artemisiae scopariae, radix sophorae flavescentis, radix et rhizoma anemarrhenae, radix et rhizoma smilacis glabrae, radix angelicae sinensis, radix paeoniae alba and rhizoma corydalis, and has strict matching of monarch, minister, assistant and messenger, can clear heat and drain dampness, dispel wind and dredge collaterals, tonify qi and nourish blood, activate blood and relieve pain, and has the advantages of treating symptoms and resisting inflammation and detumescence and strengthening the body and protecting bones, and has synergistic effect, is mild in drug property, can be prepared into conventional oral preparations, has simple and stable preparation process, is suitable for industrial production and long-term clinical use, and has good medicinal value and popularization prospect.

Description

Technical Field

[0001] This invention provides a traditional Chinese medicine composition, drug, preparation method and application for treating rheumatoid arthritis, belonging to the field of traditional Chinese medicine technology. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by bone destruction caused by erosion of the synovial membrane of the joints. Clinical manifestations include symmetrical, persistent joint swelling and pain, accompanied by morning stiffness. In advanced stages, it can lead to joint destruction, stiffness, deformity, and even disability. Currently, there is no cure, and effective treatments are still lacking.

[0003] Relevant patent documents retrieved: This document, published in China (CN111317784A) on June 23, 2020, discloses a method for preparing concentrated Danggui Niantong Pills. The method involves using supercritical CO2 fluid extraction technology to obtain extract A from raw medicinal material A, including Notopterygium incisum, Angelica sinensis, Saposhnikovia divaricata, Atractylodes lancea (fried), Atractylodes macrocephala (fried), Artemisia capillaris, and Pueraria lobata. Raw medicinal material B, including Codonopsis pilosula, Scutellaria baicalensis, Cimicifuga foetida, Sophora flavescens, Alisma plantago-aquatica, Anemarrhena asphodeloides, Polyporus umbellatus, and Glycyrrhiza uralensis, is decocted twice and filtered. The filtrate is then concentrated to obtain a thick paste B. Extract A, thick paste B, and excipients are then combined to prepare concentrated Danggui Niantong Pills.

[0004] The publication country is China, publication number CN121466231A, publication date 2026.02.06. This document discloses a traditional Chinese medicine granule for improving joint swelling and pain in rheumatoid arthritis, made from the following raw materials in parts by weight: Principal ingredient: 15g of *Sinomenium acutum* (Qingfengteng). 30 portions, Tripterygium wilfordii (processed) 5 10 portions, white peony root 15g 25 doses; Herbal ingredient: Astragalus membranaceus 20g 30 portions, Angelica sinensis 10g 15 portions, chicken blood vine 15 20 doses; adjuvant: Clematis chinensis 10g 15 portions, Gentiana macrophylla 10 portions 15 portions, 10 portions of Fangji 15 portions, Smilax glabra 20g 30 portions; Used ingredient: licorice root 5g 10 portions, Corydalis rhizome 10 15 copies.

[0005] Relevant non-patent literature retrieved: The journal *Chinese Journal of Gerontology* published an article titled "Network Pharmacology and Molecular Docking Technology of Huangqi Guizhi Wuwu Decoction for the Treatment of Rheumatoid Arthritis" on January 16, 2023. The article discloses that Huangqi Guizhi Wuwu Decoction is composed of Astragalus membranaceus, Cinnamomum cassia, Paeonia lactiflora, Zingiber officinale, and Ziziphus jujuba. The mechanism of action of Huangqi Guizhi Wuwu Decoction in treating rheumatoid arthritis (RA) involves matching effective components with disease targets, constructing a drug-effective component-target network, and performing gene ontology enrichment analysis and pathway enrichment analysis based on the Kyoto Genome and Genomics Encyclopedia. The results show that, after matching, Huangqi Guizhi Wuwu Decoction contains 40 effective components corresponding to 213 RA disease targets during RA treatment.

[0006] The prior art represented by the aforementioned literature has at least the following unresolved technical problems or defects: The effect of inhibiting inflammatory factors is poor, resulting in a poor treatment effect for RA. Relevant evidence includes: after administration of Danggui Niantong Wan (CN111317784A), IL... The concentration of 1β decreased by only 9% compared to the model group, and the concentration of TNF-α decreased by only 11% compared to the model group. Summary of the Invention

[0007] The purpose of this invention is to provide: A traditional Chinese medicine composition, drug, preparation method and application for treating rheumatoid arthritis, and related technologies, to solve technical problems such as reducing joint swelling, reducing bone erosion, reducing the concentration of inflammatory factors, and better treating rheumatoid arthritis and other damp-heat obstruction syndromes, or combinations thereof.

[0008] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0009] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0010] Definitions of standard chemical terms can be found in the reference "Chinese Pharmacopoeia, 2020 Edition".

[0011] Unless otherwise stated, conventional methods within the scope of the art, such as animal experimental modeling, shall be used.

[0012] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0013] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0014] The term "Astragalus" as used in this article refers to the dried root of *Astragalus membranaceus* (Fisch.) Bge.vaar.mongholicus (Bge.) Hsiao or *Astragalus membranaceus* (Fisch.) Bge., belonging to the legume family. It is harvested in spring and autumn, with the fibrous roots and root heads removed, and then dried in the sun.

[0015] The term "Qingfengteng" as used in this article refers to the dried stems of *Sinomenium acutum* (Thunb.) Rehd. et Wils. and *Sinomenium acutum* (Thunb.) Rehd. et Wils. var. *cinereum* Rehd. et Wils., both belonging to the Menispermaceae family. It is harvested in late autumn or early winter, bundled, or cut into long sections, and then sun-dried.

[0016] The term "Atractylodes" as used in this article refers to the dried rhizome of Atractylodes lancea (Thunb.) DC. or Atractylodes chinensis (DC.) Koidz., both belonging to the Asteraceae family. It is harvested in spring and autumn, the soil and sand are removed, it is sun-dried, and the fibrous roots are removed by knocking off.

[0017] The term "kushen" as used in this article refers to the dried root of *Sophora flavescens* Ait., a plant belonging to the legume family. It is harvested in spring and autumn, the root head and small lateral roots are removed, it is washed, and then dried, or sliced ​​while fresh and then dried.

[0018] The term "Anemarrhena asphodeloides" used in this article refers to the dried rhizome of Anemarrhena asphodeloides Bge., a plant in the Liliaceae family. It is harvested in spring and autumn, and after removing the fibrous roots and soil, it is sun-dried and commonly known as "hairy Anemarrhena"; or the outer skin is removed and it is sun-dried.

[0019] The term "Yin Chen" as used in this article refers to the dried aerial parts of *Artemisia scoparia* Waldst. et Kit. or *Artemisia capillaris* Thunb., both belonging to the Asteraceae family. It is harvested in spring when the seedlings are 6-10 cm tall, or in autumn when the flower buds have fully developed and the flowers are just beginning to open. Impurities and old stems are removed, and the harvested parts are then sun-dried. The spring-harvested part is commonly called "Mian Yin Chen," while the autumn-harvested part is called "Hua Yin Chen."

[0020] The term "Notopterygium" as used in this article refers to the dried rhizomes and roots of *Notopterygium incisum* Tingex H.T. Chang or *Notopterygium franchetii* H.de Boiss., both belonging to the Apiaceae family. They are harvested in spring and autumn, with the fibrous roots and soil removed before being sun-dried.

[0021] The term "Angelica sinensis" used in this article refers to the dried root of Angelica sinensis (Oliv.) Diels, a plant in the Apiaceae family. It is harvested in late autumn, and the fibrous roots and soil are removed. After the moisture has evaporated slightly, it is bundled into small bunches, placed on a shed, and slowly smoked with smoke.

[0022] The term "white peony" used in this article refers to the dried root of Paeonia lactiflora Pall., a plant in the Ranunculaceae family. It is harvested in summer and autumn, washed, and the head, tail, and fine roots are removed. The roots are then boiled in boiling water to remove the outer skin, or the outer skin is removed before boiling, and then dried in the sun.

[0023] The term "Yanhusuo" as used in this article refers to the dried tuber of Corydalis yanhusuo W.T.Wang, a plant belonging to the Papaveraceae family. It is harvested in early summer when the stems and leaves wither, the fibrous roots are removed, it is washed, boiled or steamed in boiling water until there is no white core, then removed and dried in the sun.

[0024] In a first aspect, the present invention provides a traditional Chinese medicine composition for treating rheumatoid arthritis, comprising: Astragalus membranaceus, Sinomenium acutum, Atractylodes lancea, Sophora flavescens, Anemarrhena asphodeloides, Artemisia capillaris, Notopterygium incisum, Angelica sinensis, Paeonia lactiflora, and Corydalis yanhusuo.

[0025] The mass ratio of Astragalus membranaceus, Sinomenium acutum and Atractylodes lancea is (15-20): (10-15): (5-10).

[0026] The preferred mass ratio of Astragalus membranaceus, Sinomenium acutum and Atractylodes lancea is (18-19): (11-12): (9-10).

[0027] The mass ratio of Sophora flavescens, Anemarrhena asphodeloides and Paeonia lactiflora is (5-10):(5-10):(10-15).

[0028] The preferred mass ratio of Sophora flavescens, Anemarrhena asphodeloides and Paeonia lactiflora is (9-10):(9-10):(11-12).

[0029] The traditional Chinese medicine composition, by weight, includes: 15-20 parts of Astragalus membranaceus, 10-15 parts of Sinomenium acutum, 5-10 parts of Atractylodes lancea, 5-10 parts of Sophora flavescens, 5-10 parts of Anemarrhena asphodeloides, 5-10 parts of Artemisia capillaris, 10-15 parts of Notopterygium incisum, 5-10 parts of Angelica sinensis, 10-15 parts of Paeonia lactiflora, and 10-15 parts of Corydalis yanhusuo.

[0030] The Astragalus membranaceus, by weight, can be 15, 16, 17, 18, 19, 20 parts or any combination thereof.

[0031] The amount of *Qingfengteng* can be 10, 11, 12, 13, 14, 15 parts by weight, or any combination thereof.

[0032] The Atractylodes lancea, by weight, can be in the range of 5, 6, 7, 8, 9, 10 parts or any combination thereof.

[0033] The amount of Sophora flavescens, by weight, can be 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, or any combination thereof.

[0034] The amount of Anemarrhena asphodeloides, by weight, can be 5, 6, 7, 8, 9, 10 parts or any combination thereof.

[0035] The amount of Artemisia capillaris, by weight, can be 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, or any combination thereof.

[0036] The amount of Qianghuo, by weight, can be 10, 11, 12, 13, 14, 15 parts or any combination thereof.

[0037] The Angelica sinensis, by weight, can be in the range of 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, or any combination thereof.

[0038] The white peony root, by weight, can be 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, or any combination thereof.

[0039] The amount of Corydalis rhizome, by weight, can be 10, 11, 12, 13, 14, 15 parts or any combination thereof.

[0040] Secondly, the present invention provides a method for preparing the above-mentioned traditional Chinese medicine composition, comprising the following steps: (1) Astragalus membranaceus, Sinomenium acutum, Atractylodes lancea, Sophora flavescens, Anemarrhena asphodeloides, Artemisia capillaris, Notopterygium incisum, Angelica sinensis, Paeonia lactiflora and Corydalis yanhusuo were decocted in water, filtered and concentrated to obtain a thick paste; (2) Filter, dry and pulverize the thick paste to obtain the final product.

[0041] The number of times the decoction is performed is 1-3 times.

[0042] The decoction process involves soaking the ingredients in water and then decocting them at 85-100℃ for 1-2 hours.

[0043] The ratio of water added to the liquid is 1g:(8-10)g.

[0044] The relative density of the paste at 60°C is 1.24-1.26.

[0045] The drying process is described as vacuum drying.

[0046] The vacuum drying process involves a temperature of 60-80℃ and a pressure of -0.06MPa to -0.10MPa.

[0047] The moisture content after drying does not exceed 5 wt%.

[0048] Thirdly, the present invention provides the use of the above-mentioned traditional Chinese medicine composition in the preparation of a drug for treating rheumatoid arthritis.

[0049] Fourthly, the present invention provides a medicament for treating rheumatoid arthritis, comprising the above-mentioned traditional Chinese medicine composition.

[0050] The dosage forms of the drug include: tablets, injections, capsules, granules, pills, creams, powders, syrups, oral solutions, oral suspensions, oral emulsions, and mixtures.

[0051] The present invention has at least the following beneficial effects: Compared with existing technologies, the present invention has better technical effects in reducing joint swelling, reducing bone erosion, and reducing the concentration of inflammatory factors.

[0052] According to experimental tests, the present invention reduces the degree of joint swelling and bone erosion compared to existing technologies.

[0053] According to experimental tests, the present invention reduces the concentration of inflammatory factors compared to existing technologies.

[0054] Furthermore, based on the present invention: Based on the comparison of Example 1 and Comparative Examples 1-5, the present invention, by employing specific components and mass ratios of technical means, achieves new technical effects by reducing joint swelling, reducing bone erosion, and reducing the concentration of inflammatory factors. The combined technical effect is superior to the sum of the effects of each individual technical means.

[0055] The relationship between the monarch, minister, assistant, and envoy in this invention: The principal herbs are *Sinomenium acutum* and *Astragalus membranaceus*, one purging and the other tonifying; the assistant herbs are *Atractylodes lancea*, *Sophora flavescens*, *Anemarrhena asphodeloides*, *Notopterygium incisum*, and *Artemisia capillaris*, clearing heat, removing dampness, dispelling wind, and unblocking the meridians; the adjuvant herbs are *Angelica sinensis* and *Paeonia lactiflora*, nourishing blood, promoting blood circulation, soothing the liver, and relieving spasms; the guiding herb is *Corydalis yanhusuo*, guiding the medicine to the meridians and relieving pain. This formula combines purging and tonifying, warming and cooling herbs, relieving exterior symptoms, clearing heat and removing dampness, strengthening the spleen and nourishing blood, promoting blood circulation and removing blood stasis, unblocking the meridians and relieving pain, addressing both the root cause and the symptoms. It is used for patients with rheumatoid arthritis of the qi and blood deficiency type and damp-heat stagnation type. Indications: Damp-heat stagnation type RA, clinical manifestations: red, swollen, hot, and painful joints, burning sensation upon touch, difficulty in flexion and extension, morning stiffness accompanied by fever and thirst. Attached Figure Description

[0056] Figure 1 A scoring chart showing the swelling of the rat joints and the clinical symptoms of the joints. This indicates that p < 0.005. Figure 1 Day 0 in the initial immunization is the first day of immunization.

[0057] Figure 2 Images of rat ankle joints stained with hematoxylin and eosin (H&E), safranin O / fast green (SO / FG), and reconstructed in three dimensions (Radiological Image).

[0058] Figure 3 The image shows the detection results of TNF-α, IL-1β, and IFN-γ. This means p < 0.05. This means p < 0.01. This means p < 0.005. This means p < 0.001.

[0059] Figure 4 Images show hematoxylin-eosin staining of rat liver, spleen, and kidney (scale bar 50 μm) and the results of liver, spleen, and kidney indices in each group of rats. This means p < 0.05. This means p < 0.001. Detailed Implementation

[0060] 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 by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0061] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0062] Example 1 Formula: 18 parts raw Astragalus membranaceus, 12 parts Sinomenium acutum, 9 parts Atractylodes lancea, 9 parts Sophora flavescens, 9 parts Anemarrhena asphodeloides, 9 parts Artemisia capillaris, 12 parts Notopterygium incisum, 9 parts Angelica sinensis, 12 parts raw Paeonia lactiflora, and 12 parts Corydalis yanhusuo.

[0063] Example 2 Formula: 15 parts raw Astragalus membranaceus, 15 parts Sinomenium acutum, 5 parts Atractylodes lancea, 10 parts Sophora flavescens, 5 parts Anemarrhena asphodeloides, 10 parts Artemisia capillaris, 10 parts Notopterygium incisum, 10 parts Angelica sinensis, 10 parts raw Paeonia lactiflora, and 15 parts Corydalis yanhusuo.

[0064] Example 3 Formula: 20 parts raw Astragalus membranaceus, 10 parts Sinomenium acutum, 10 parts Atractylodes lancea, 5 parts Sophora flavescens, 10 parts Anemarrhena asphodeloides, 5 parts Artemisia capillaris, 15 parts Notopterygium incisum, 5 parts Angelica sinensis, 15 parts raw Paeonia lactiflora, and 10 parts Corydalis yanhusuo.

[0065] Comparative Example 1 The difference from Example 1 is that Atractylodes lancea is replaced with an equal amount of Smilax glabra.

[0066] Comparative Example 2 Formula: 18 parts raw Astragalus membranaceus, 12 parts Sinomenium acutum, 12 parts raw Paeonia lactiflora, and 12 parts Corydalis yanhusuo.

[0067] Comparative Example 3 Formula: Atractylodes lancea 9 parts, Sophora flavescens 9 parts, Anemarrhena asphodeloides 9 parts, Artemisia capillaris 9 parts, Notopterygium incisum 12 parts, Angelica sinensis 9 parts.

[0068] Comparative Example 4 The difference from Example 1 is that the proportions of Sophora flavescens, Anemarrhena asphodeloides and raw Paeonia lactiflora are changed.

[0069] Formula: 18 parts raw Astragalus membranaceus, 12 parts Sinomenium acutum, 9 parts Atractylodes lancea, 17 parts Sophora flavescens, 4 parts Anemarrhena asphodeloides, 9 parts Artemisia capillaris, 12 parts Notopterygium incisum, 9 parts Angelica sinensis, 9 parts raw Paeonia lactiflora, and 12 parts Corydalis yanhusuo.

[0070] Comparative Example 5 The difference from Example 1 is that the proportions of raw Astragalus membranaceus, Sinomenium acutum, and Atractylodes lancea were changed.

[0071] Formula: 22 parts raw Astragalus membranaceus, 6 parts Sinomenium acutum, 11 parts Atractylodes lancea, 9 parts Sophora flavescens, 9 parts Anemarrhena asphodeloides, 9 parts Artemisia capillaris, 12 parts Notopterygium incisum, 9 parts Angelica sinensis, 12 parts raw Paeonia lactiflora, and 12 parts Corydalis yanhusuo.

[0072] Preparation methods of Examples 1-3 and Comparative Examples 1-5: Take the above-mentioned medicinal ingredients and decoct them twice with water. For the first decoction, add 10 times the amount of water, soak for 30 minutes, and boil for 1.5 hours. Filter. For the second decoction, add 8 times the amount of water, boil for 1 hour, and filter. Combine the filtrates and concentrate under reduced pressure at below 75°C to a thick paste with a relative density of 1.24 (at 60°C). Filter and set aside. Place the thick paste in a vacuum drying oven, controlling the thickness of the paste to not exceed 0.8 cm, and vacuum dry (drying temperature: 60°C, pressure: -0.10 MPa) until the moisture content is not more than 5 wt%. Crush the paste into a dry extract powder, pass it through an 80-mesh sieve, and seal it in aluminum foil bags, 300g per bag.

[0073] Animal experiments for testing 1. Experimental animals: Animal species: SPF grade SD rats, female, 6-8 weeks old, body weight 180-220g.

[0074] 2. Modeling Method: CIA Model Preparation (1) Establishment of an animal model of type II collagen-induced arthritis (CIA) using incomplete freunds adjuvant (IFA). Rats were anesthetized by inhalation of 15% isoflurane (500 ml / min), and their tails were shaved. (2) Immunograde bovine type II collagen (2 mg / mL) (20022; Chondrex) dissolved in 0.05 M acetic acid was emulsified with an equal volume of incomplete Freund's adjuvant (IFA; 7002); (3) For the initial immunization (day 0), type II collagen + IFA (equal volumes) were mixed evenly on ice and fully emulsified. When the emulsion was dropped into water, it agglomerated and did not disperse for 30 seconds, indicating that the mixing was complete and a CⅡ-IFA emulsion was obtained. 200 μl of the CⅡ-CFA emulsion was injected intradermally into the rat at a distance of 0.5 cm from the tail to form a wheal with a diameter of 1 cm, which can maintain immune stimulation for a longer period of time. (4) Boost immunization (day 7): Inject CⅡ-IFA emulsion intradermally at a dose of 100 μl / animal, following the same procedure as step (3).

[0075] 3. Experimental groups: Example 1 group, Comparative examples 1-5 groups, blank group, model group, positive drug (methotrexate MTX) group, 8 animals in each group.

[0076] 4. Experimental methods: (1) Method of administration: Drug administration began on day 8 after model establishment.

[0077] Control group: Normal animals, without modeling, were given an equal volume of physiological saline by gavage for 28 days; Model group: After modeling, the model was given an equal volume of physiological saline by gavage for 28 days; Methotrexate group (MTX group): After modeling, MTX was administered by gavage at a dose of 10 mg / kg / week (converted according to the clinical equivalent dose) for 28 days; Example 1 group: After modeling, the above-mentioned drugs from Example 1 group were administered at a dose of 35g / kg / d by gavage for 28 days.

[0078] Comparative Examples 1-5: After modeling, the drugs of the above-mentioned Comparative Examples 1-5 were administered to each group at the same dosage as in Example 1, by gavage, for 28 days.

[0079] (2) Drug intervention program Administration route: Gavage, once daily (MTX once a week, with an equal volume of normal saline given at other times).

[0080] Intervention period: 28 days, during which animals' diet, activity and adverse reactions are closely monitored.

[0081] 5. Experimental detection methods: (1) Measurement of CIA rat weight The rats were weighed every 3 days, placed in a stainless steel container, and their weight was recorded.

[0082] (2) Evaluation of paw edema in CIA rats The thickness of the paw pads in CIA rats reflects the degree of paw swelling. The thickest part of the paw pads at the distal interphalangeal joints of both paws was measured every 3 days, and the average value of the two paws was recorded.

[0083] (3) Clinical symptom score of ankle joint in CIA rats Personnel unrelated to and unaware of the contents of this experiment are requested to score the clinical symptoms of the rats' ankle joints. Evaluation should begin at the time of booster immunization, and scores should be recorded weekly. The scoring criteria are shown in Table 1.

[0084] Table 1

[0085] (4) CIA Large Mouse Notebook Collection and Processing Samples were collected after the 28-day intervention experiment. Rats were fasted and given water for 12 hours prior to sample collection. Under inhalation anesthesia with 15% isoflurane at 1500 ml / min, the abdominal cavity was dissected layer by layer, starting from the middle, until the abdominal aorta was located. A 5 ml syringe was connected to a lancet, and blood was slowly drawn by perpendicular puncture. The collected blood was allowed to stand at room temperature for 20 minutes before being centrifuged for 20 minutes (3000 rpm). The supernatant was collected and stored at -20°C. After the rats died, the liver and kidneys were isolated and dissected, ensuring their morphology remained intact. The fur on the rat's hind limbs was removed using ophthalmic scissors. The epidermis was dissected layer by layer, and muscle tissue was removed. The knee and ankle joints were gradually cleared; the ankle joint was taken from the left side, and the entire hind limb from the right side. All specimens were rinsed twice with physiological saline and then fixed in 4% paraformaldehyde for 48 hours. After fixation of the knee and ankle joints, they were transferred to a decalcification solution for 6 weeks in preparation for paraffin embedding.

[0086] (5) HE staining of CIA mouse-sized mice HE staining is a common tissue section staining technique, typically used for observing tissue morphology under a microscope. Hematoxylin and eosin staining allows for the differentiation of different cell and tissue structures. HE staining refers to the staining of tissue sections with hematoxylin and eosin, hence it is also called hematoxylin-eosin staining. Hematoxylin staining primarily affects the cell nucleus, making it appear blue or purple. Eosin staining primarily affects the cytoplasm and extracellular matrix, making it appear pink or orange. The specific steps for joint staining are as follows: 1) Tissue fixation: The tissues to be tested (knee joint, synovial membrane, liver, kidney) were fixed in 4% paraformaldehyde for 48 hours to maintain their morphology and structure. 2) Decalcification: The fixed bone tissue was cleaned with PBS and placed in 14% EDTA decalcification solution for decalcification treatment for 6 weeks, with the decalcification solution being changed regularly (every 3 days). 3) Tissue dehydration: 70v / v% ethanol for 1 hour, 80v / v% ethanol for 1 hour, 90v / v% ethanol for 1 hour, 95v / v% ethanol for 1 hour, 100% ethanol for 1 hour, xylene for 1 hour, xylene for 1 hour, xylene for 1 hour, paraffin for 1 hour; 4) Embedding: Heat and melt the paraffin block, immerse it in the transparent specimen, place it in a paraffin melting box to keep it warm, and after the tissue is completely immersed in paraffin, embed it by placing it in a mold, pouring in the melted paraffin, quickly placing the paraffin-impregnated tissue block in, and waiting for it to cool and solidify. 5) Tissue sectioning: After trimming the fixed paraffin block, cut it into thin sections (5μm) using a microtome, flatten it in warm water (37℃), then attach it to a glass slide and dry it in a slide dryer (55℃); 6) Dewaxing: Before dewaxing, bake the slides in an oven for 30-60 minutes to make them less likely to detach and facilitate dewaxing. Heat the slides with a hairdryer until the wax on the slides melts, then dewax with 100% xylene 5 times, twice. 7) Hydration: from high ethanol to low ethanol, anhydrous ethanol (10 min) → anhydrous ethanol (10 min) → 95% ethanol (5 min) → 80% ethanol (5 min) → 70% ethanol (5 min). 8) Wash with water: Rinse with double-distilled water (DW) for 5 minutes; 9) Hematoxylin staining: Add 0.5% Harris hematoxylin solution and saturate for 5 min; repeat step (8); 10) Differentiation with 1% hydrochloric acid ethanol: Place the slide in a solution containing 1 v / v% hydrochloric acid ethanol for 20 seconds to make the hematoxylin-stained cell nuclei appear blue or purple; repeat step (8); 11) Soak in dilute ammonia water (0.1wt% ammonia water) for 30 seconds; repeat step (8); 12) Eosin staining: Immerse the slide in a 0.5%-1% eosin solution for 1-5 minutes; 13) Dehydration: Place the slide in ethanol of different concentrations: 80% ethanol (30s) → 90% ethanol (30s) → 95% ethanol (30s) → 95% ethanol (1min) → anhydrous ethanol (1min) → anhydrous ethanol (2min) to remove excess staining agent. 14) Transparency: Immerse the glass slide in 100% xylene for 2 minutes for transparency treatment; 15) Mounting: Cover the slide with a layer of neutral resin and seal it with a coverslip. Place it in a fume hood for more than 12 hours to complete the HE staining process.

[0087] 16) Review criteria: The inflammatory cell infiltration scoring criteria are adopted to evaluate the number and distribution of inflammatory cells in the tissue. Scoring criteria: 0 points: no inflammatory cell infiltration; 1 point: mild inflammatory cell infiltration; 2 points: moderate inflammatory cell infiltration; 3 points: significant inflammatory cell infiltration; 4 points: severe inflammatory cell infiltration.

[0088] Bone injury criteria: 0 points: normal bone quality; 1 point: 1-2 cartilage or bone erosions; 2 points: 2-5 cartilage or bone erosions, accompanied by sparse trabeculae; 3 points: narrowing of the joint space, cartilage or bone erosion on multiple articular surfaces, involving the cortex; 4 points: significantly narrowing of the joint space, significantly sparse trabeculae, significantly reduced bone mass, and articular surface damage exceeding 1 / 4 of the entire articular surface.

[0089] (6) Safranin-Fixed Green Staining After routine dewaxing to water, the sections were placed in a humidified chamber and stained with prepared Weigert stain for 5 minutes, followed by 3 washes with water. Then, they were differentiated in acidic differentiation solution (1 wt% hydrochloric acid-ethanol solution) for 15 seconds, washed twice with dd water (double-distilled pure water), 5 minutes each time. Fast Green staining was added for 5 minutes, followed by washing with a weak acid solution from the kit for 15 seconds, the solution was discarded, and the sections were air-dried at room temperature. Safranin staining was added for 5 minutes, followed by 3 washes with water. The sections were then successively immersed in 95% ethanol for 3 seconds, absolute ethanol for 3 seconds, and absolute ethanol for 1 minute, followed by xylene for 45 seconds for clearing. After drying in a fume hood, the sections were mounted with neutral resin and observed and photographed under a microscope.

[0090] (7) Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of TNF-α, IFN-γ, IL-1β, IL-6 and IL-18 in the serum of CIA rats.

[0091] The ELISA kit used in this experiment employs the "sandwich" method, minimizing non-specific background signals by using two specific antibodies and a solid-phase carrier, thereby improving detection accuracy. Before testing, blood was thawed, diluted with ultrapure water, and the procedure was strictly followed according to the kit instructions and reagent dosages. The specific steps are as follows: Sample collection: After the collected blood was left to stand at room temperature for 20 minutes, it was placed in a centrifuge and centrifuged for about 20 minutes (3000 rpm). It was then stored in a -20°C refrigerator. Reagent preparation: 1) After removing the kit, warm it to room temperature and preheat the microplate reader for 20 minutes; 2) Dilute the concentrated washing buffer in the kit with a ratio of concentrated washing buffer to pure water of 1:20 (by mass). 3) Prepare 8 1.5ml centrifuge tubes, numbered 1-8 respectively. Tube 8 is the blank control group for the standard curve. Repeatedly dilute the standard curve from tube 1 to tube 7. The concentrations of the standard curve are: 1000, 500, 250, 125, 62.5, 31.2, 15.6, 0 pg / ml. 4) Both the biotinylated antibody and the SABC complex should be diluted from 100× to 1× before use and prepared immediately. 5) Mix solutions A and B of the TMB colorimetric solution thoroughly at a 1:1 ratio and set aside; Testing procedure: 1) Add serum and standard, react at 37°C for 40 min, and wash 5 times; 2) Add biotinylated detection antibody, react at 37℃ for 30 min, and wash 5 times; 3) Add the SABC complex, react at 37°C for 20 min, and wash 5 times; 4) Add TMB colorimetric solution and react at 37°C for 10–20 min; 5) Add the stop solution and take the reading; Result judgment and calculation: First subtract the OD value of the blank wells that is less than 0.1, calculate the specific content based on the OD value, and then multiply it by the dilution factor to get the final result; All tests were performed using dual-hole methods and had standard curves; all tests were conducted at room temperature; thorough washing reduced accuracy errors.

[0092] (8) Micro-computed tomography (Micro-CT) CIA rat ankle joint After fixing the right hind limb of the rat in 4% paraformaldehyde for one week, a Micro-CT scan was performed. The equipment parameters were set as follows: tube voltage 90kV, tube current 0.07mA, frame rate 10 frames / second, and pixel size 0.012×0.012×0.012mm. Bone microstructure parameters were analyzed using Avatar 3 analysis software, including bone surface area / tissue volume (BS / TV), bone volume / tissue volume (BV / TV), bone surface area / bone volume (BS / BV), trabeculae number (Tb.N), trabeculae thickness (Tb.Th), trabecular separation (Tb.Sp), bone mineral density (BMD), and bone mineral content (BMC), resulting in a three-dimensional reconstructed radiological image.

[0093] 10. Experimental Results (1) The joint swelling and clinical symptom scores of rats are as follows: Figure 1 .

[0094] like Figure 1As shown in the rat joint diagrams, the control group rats were normal, with no redness, swelling, or inflammation in their joints; the model group rats had severe joint swelling and toe deformities, proving the successful establishment of the model; the positive control group rats had mild joint swelling, demonstrating that the inflammation was relieved after administration of the positive control drug, but slight swelling remained; the control group 1 rats had moderate joint swelling, between the model group and the positive control group, indicating a moderate therapeutic effect; the rats in Example 1 group had joints that were close to normal, with significant reduction in swelling and good restoration of joint morphology. The effects of Examples 2-3 were equivalent to those of Example 1.

[0095] like Figure 1 As shown in the joint symptom scores, the joint symptom scores of the Example 1 group decreased after drug administration and showed a significant difference from the model group. The Example 1 group... Figure 1 After 28 days, there was a significant difference between the control group and the positive drug group (p<0.01).

[0096] (2) Images of rat ankle joint hematoxylin-eosin (H&E), safranin O / fast green (SO / FG) staining and three-dimensional reconstruction (Radiological Image) are shown below. Figure 2 .

[0097] H&E staining: In the blank group, the joint structure was intact, the synovium was thin and flat, there was no inflammatory cell infiltration, the cartilage layer was continuous, and there was no bone destruction; In the model group, the synovial tissue showed significant proliferation and thickening, forming pannus, with a large number of inflammatory cells infiltrating, the cartilage layer was destroyed and discontinuous, and the bone tissue showed erosive damage, indicating successful model construction; In the positive drug group, the pathological damage was significantly improved, the synovial proliferation was reduced, the inflammatory cell infiltration was reduced, and the cartilage and bone destruction was alleviated; The improvement effect of the comparative example 1 group was limited: the synovium still showed significant proliferation, the inflammatory infiltration was not completely controlled, and bone erosion still existed, with the efficacy weaker than that of the positive drug; Example 1 group: The pathological improvement was better than that of the positive drug group: the synovial proliferation was significantly inhibited, the inflammatory cell infiltration was greatly reduced, the cartilage structure was intact, and the bone erosion was significantly treated.

[0098] SO / FG staining: Safranin: specifically binds to glycosaminoglycans (GAG) in the cartilage matrix; the deeper the red, the more intact and active the cartilage. Fast Green: stains bone and connective tissue.

[0099] Control group: The cartilage layer was uniformly dark red, with an intact structure and no damage; Model group: The red color of the cartilage layer almost disappeared, and the cartilage was severely damaged and lost, replaced by hyperplastic synovium / pannus; Positive drug group: The red color of the cartilage was partially restored, and the cartilage damage was inhibited; Comparative example 1 group: The red color of the cartilage was partially restored, and there was still significant cartilage loss; Example 1 group: The red color of the cartilage was significantly restored, close to that of the control group, proving that Example 1 can effectively protect cartilage and inhibit cartilage degradation.

[0100] Three-dimensional reconstruction (Radiological Image): Blank group: smooth joint bone surface, intact trabeculae, no bone erosion or osteophytes, and normal joint space; Model group: severely rough bone surface, moth-eaten bone erosion, disordered and broken trabeculae, a large number of osteophytes, and destruction of joint structure; Positive drug group: significantly reduced bone erosion, relatively smooth bone surface, reduced osteophytes, and protected bone structure; Comparative example 1 group: no significant improvement in bone erosion, with a large amount of bone destruction and osteophytes still present; Example 1 group: significantly reduced bone erosion and osteophytes, and good trabecular integrity.

[0101] The effects of Examples 2-3 are equivalent to those of Example 1.

[0102] (3) Detection results of TNF-α, IL-1β, and IFN-γ are as follows Figure 3 .

[0103] ①TNF-α (RA core pro-inflammatory factor) Compared to the control group, the model group showed a significant increase in TNF-α levels, indicating successful model establishment. In the positive control group, TNF-α levels decreased compared to the model group, indicating that the positive control group could inhibit TNF-α-mediated inflammatory responses. In the comparative example group 1, the decrease in TNF-α was similar to that in the positive control group. In the example group 1, TNF-α levels decreased significantly, with no significant difference from the control group, indicating that the intervention could inhibit TNF-α expression and had a better anti-inflammatory effect than the positive control group and the comparative example group 1 (with significant differences).

[0104] ②IFN-γ (regulates immunity and amplifies inflammation) Compared to the control group, the model group showed a significant increase in IFN-γ; the positive control group showed a decrease in IFN-γ, which could regulate abnormal immune responses; the comparative example group 1 showed a decrease in IFN-γ but no significant difference from the positive control group; the example group 1 showed a significant decrease in IFN-γ, which was significantly lower than that of the positive control group and the comparative example group 1.

[0105] ③IL-1β (a key factor in mediating cartilage / bone destruction) Compared to the control group, the model group showed a sharp increase in IL-1β, indicating successful model establishment. In the positive control group, IL-1β decreased, effectively blocking the bone destruction pathway. In the comparative example group 1, IL-1β decreased, but there was no significant difference compared to the positive control group. In the example group 1, IL-1β decreased significantly, far lower than that in the positive control group and the comparative example group 1, indicating that example 1 can effectively block IL-1β-driven cartilage / bone erosion.

[0106] (4) The hematoxylin-eosin staining patterns of rat liver, spleen, and kidney, and the results of liver, spleen, and kidney indices in each group of rats are shown in the figure. Figure 4 This proves that the embodiments of the present invention are non-toxic.

[0107] 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 traditional Chinese medicine composition for treating rheumatoid arthritis, characterized in that, include: Astragalus, Sinomenium acutum, Atractylodes lancea, Sophora flavescens, Anemarrhena asphodeloides, Artemisia capillaris, Notopterygium incisum, Angelica sinensis, Paeonia lactiflora, and Corydalis yanhusuo.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The mass ratio of Astragalus membranaceus, Sinomenium acutum and Atractylodes lancea is (15-20): (10-15): (5-10).

3. The traditional Chinese medicine composition according to claim 1, characterized in that, The mass ratio of Sophora flavescens, Anemarrhena asphodeloides and Paeonia lactiflora is (5-10):(5-10):(10-15).

4. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition, by weight, comprises: 15-20 parts of Astragalus membranaceus, 10-15 parts of Sinomenium acutum, 5-10 parts of Atractylodes lancea, 5-10 parts of Sophora flavescens, 5-10 parts of Anemarrhena asphodeloides, 5-10 parts of Artemisia capillaris, 10-15 parts of Notopterygium incisum, 5-10 parts of Angelica sinensis, 10-15 parts of Paeonia lactiflora, and 10-15 parts of Corydalis yanhusuo.

5. The method for preparing the traditional Chinese medicine composition according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Astragalus membranaceus, Sinomenium acutum, Atractylodes lancea, Sophora flavescens, Anemarrhena asphodeloides, Artemisia capillaris, Notopterygium incisum, Angelica sinensis, Paeonia lactiflora and Corydalis yanhusuo were decocted in water, filtered and concentrated to obtain a thick paste; (2) Filter, dry and pulverize the thick paste to obtain the final product.

6. The preparation method according to claim 5, characterized in that, The number of times the decoction is prepared is 1-3 times.

7. The preparation method according to claim 5, characterized in that, The decoction process involves simmering at 85-100℃ for 1-2 hours.

8. The preparation method according to claim 5, characterized in that, The relative density of the paste at 60°C is 1.24-1.

26.

9. Use of the traditional Chinese medicine composition according to any one of claims 1-4 in the preparation of a medicament for treating rheumatoid arthritis.

10. A medicament for treating rheumatoid arthritis, comprising the traditional Chinese medicine composition according to any one of claims 1-4.

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