Traditional Chinese medicine composition for treating rheumatic arthritis, preparation and application

Abstract

The invention provides a traditional Chinese medicine composition for treating rheumatic arthritis, a preparation and application, and belongs to the field of traditional Chinese medicines. The traditional Chinese medicine composition is prepared from the following medicinal raw materials: 12-18 parts of sailong bone, 6-12 parts of radix aconiti lateralis preparata, 3-9 parts of radix aconiti preparata, 9-15 parts of cassia twig, 1-4 parts of asarum, 9-15 parts of radix clematidis, 20-40 parts of astragalus membranaceus, 3-9 parts of scorpio, 9-15 parts of earthworm, 9-15 parts of caulis sinomenii, 12-18 parts of bighead atractylodes rhizome, 12-18 parts of poria cocos, 9-15 parts of gentiana macrophylla, 12-18 parts of angelica sinensis, 20-40 parts of caulis spatholobi, 9-15 parts of rhizoma drynariae, 12-18 parts of radix paeoniae alba, 3-9 parts of honey-fried licorice root and 6-12 parts of rhizoma zingiberis. According to the traditional Chinese medicine composition for treating rheumatic arthritis, the medicine raw material composition of the traditional Chinese medicine composition for treating rheumatic arthritis is reasonably designed, four pathological links of cold, dampness, stasis and deficiency of rheumatic arthritis are covered through precise compatibility of monarch, minister, assistant and guide, and the traditional Chinese medicine composition has triple curative effects of anti-inflammatory analgesia, immunoregulation and bone protection.

Description

Technical Field

[0001] This application relates to the field of traditional Chinese medicine technology, and in particular to a traditional Chinese medicine composition, preparation and application for treating rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis is a common allergic disease and one of the main manifestations of rheumatic fever, closely related to group A streptococcal infection. Its typical symptom is migratory polyarthritis, often symmetrically affecting large joints such as the knee, ankle, shoulder, wrist, elbow, and hip. Local inflammation manifests as redness, swelling, heat, and pain, severely impacting the patient's quality of life. The disease is most common in young adults aged 20-45, with a higher incidence in women than men. Recurrent flare-ups can also lead to serious complications such as valvular heart disease.

[0003] Currently, there are various treatments for rheumatoid arthritis, but all have certain limitations. In Western medicine, while disease-modifying antirheumatic drugs (DMARDs) are first-line treatments that can block disease progression and prevent bone erosion, long-term use can lead to numerous side effects. For example, methotrexate can cause liver damage and gastrointestinal discomfort in some patients. Biologics, such as TNF-α inhibitors and IL-1 antagonists, offer new hope, but they are expensive and not effective for all patients. Furthermore, the use of biologics can increase the risk of opportunistic infections and invasive melanoma. In the field of traditional Chinese medicine, TCM has a long history in treating rheumatoid arthritis, developing numerous herbal formulas, extracts, and active ingredients, which have shown some effectiveness in clinical applications. However, existing TCM formulas often focus on "dispelling wind and dampness" or "promoting blood circulation and relieving pain," neglecting the core pathological process of "bone repair." For example, formulas that dispel wind and unblock meridians (such as those containing Angelica pubescens, Notopterygium incisum, and Clematis chinensis) can relieve joint swelling and pain, but cannot reverse bone erosion; formulas that tonify the liver and kidneys (such as those containing Taxillus chinensis and Eucommia ulmoides) are effective for long-term conditioning, but their onset of action is slow and they are insufficient for controlling acute inflammation. Therefore, how to combine anti-inflammatory and analgesic, immunomodulatory, and bone-protective effects is a technical problem that urgently needs to be solved. Summary of the Invention

[0004] This application provides a traditional Chinese medicine composition, preparation and application for treating rheumatoid arthritis. For the first time, it combines seron bone (bone repair), processed aconite / fuzi (warming yang and dispelling cold) with insect drugs (scorpion / earthworm to clear blood stasis) to form a three-in-one treatment system of "breaking cold stagnation - relieving pain - strengthening tendons and bones", which has the triple effects of anti-inflammatory and analgesic, immune regulation and bone protection.

[0005] In a first aspect, this application provides a traditional Chinese medicine composition for treating rheumatoid arthritis, which, by weight, comprises the following medicinal materials: 12-18 parts of *Salvia miltiorrhiza*, 6-12 parts of prepared *Aconitum carmichaelii*, 3-9 parts of prepared *Aconitum carmichaelii*, 9-15 parts of cinnamon twig, 1-4 parts of *Asarum heterotropoides*, 9-15 parts of *Clematis chinensis*, 20-40 parts of *Astragalus membranaceus*, 3-9 parts of scorpion, 9-15 parts of earthworm, 9-15 parts of *Sinomenium acutum*, 12-18 parts of *Atractylodes macrocephala*, 12-18 parts of *Poria cocos*, 9-15 parts of *Gentiana macrophylla*, 12-18 parts of *Angelica sinensis*, 20-40 parts of *Spatholobus suberectus*, 9-15 parts of *Drynaria fortunei*, 12-18 parts of white peony root, 3-9 parts of prepared licorice root, and 6-12 parts of dried ginger.

[0006] Optionally, by weight, the traditional Chinese medicine composition comprises the following raw materials: 15 parts of selenocarpus, 9 parts of prepared aconite root, 6 parts of prepared Sichuan aconite root, 12 parts of cinnamon twig, 3 parts of asarum, 12 parts of clematis root, 30 parts of astragalus root, 6 parts of scorpion, 12 parts of earthworm, 12 parts of sinomenium acutum, 15 parts of atractylodes macrocephala, 15 parts of poria cocos, 12 parts of gentian root, 15 parts of angelica sinensis, 30 parts of chicken blood vine, 12 parts of drynaria fortunei, 15 parts of white peony root, 6 parts of prepared licorice root, and 9 parts of dried ginger.

[0007] Secondly, this application provides a pharmaceutical preparation comprising the traditional Chinese medicine composition described in the first aspect and / or pharmaceutically acceptable excipients.

[0008] Thirdly, this application provides a method for preparing the pharmaceutical formulation described in the second aspect, the method comprising the following steps:

[0009] S1. Prepare the various raw materials of the traditional Chinese medicine composition according to the weight parts;

[0010] S2. The prepared aconite root, prepared Sichuan aconite root, dried ginger and prepared licorice root were subjected to a first aqueous extraction to obtain the first aqueous extract.

[0011] S3. The cinnamon twig, asarum, astragalus, sinomenium acutum, atractylodes macrocephala, poria cocos, gentiana macrophylla, angelica sinensis, chicken blood vine, and white peony root are subjected to a second water extraction to obtain the second water extract.

[0012] S4. Extract the whole scorpion and earthworm with ethanol to obtain the ethanol extract;

[0013] S5. Biomimetic mineralization extraction of seron bone, Clematis chinensis and Drynaria fortunei was carried out to obtain a co-extract;

[0014] S6. The first aqueous extract, the second aqueous extract, the alcohol extract, and the co-extract are mixed and then a pharmaceutical preparation is prepared.

[0015] Optionally, the ethanol extraction uses an aqueous ethanol solution with a volume concentration of 70%, the ethanol extraction temperature is 40°C, and the ethanol extraction time is 60 min to 120 min.

[0016] Optionally, the S5 steps include:

[0017] S501. Grind sera, drynaria, and clematis into powder to 80-100 mesh to obtain a mixture;

[0018] S502. The mixture is mixed with citric acid solution and subjected to shaking demineralization to obtain demineralization solution;

[0019] S503. Adjust the pH of the demineralization solution to 6.5-7.0, then add alkaline protease, bromelain and CaCl2 nanoparticles to carry out enzymatic hydrolysis to obtain the enzymatic hydrolysate.

[0020] S504. Add phosphorylated serine peptide to the enzymatic hydrolysate, heat to 55-65℃, adjust pH to 7.5-8.5, stir for 20-40 min to obtain a mixture;

[0021] S506. The mixture is subjected to enzyme inactivation, filtration, concentration and drying in sequence to obtain the co-extract.

[0022] Optionally, the oscillation demineralization includes the following parameters: temperature of 48~52℃, oscillation frequency of 150~200rpm, and time of 100~200min;

[0023] The enzymatic hydrolysis was performed at a temperature of 45°C for 150-210 minutes.

[0024] Optionally, the citric acid solution has a mass concentration of 4-6%;

[0025] The amount of alkaline protease added is 0.9-1.1% of the total mass of the demineralization solution;

[0026] The amount of bromelain added is 0.4-0.6% of the total mass of the demineralization solution;

[0027] The CaCl2 nanoparticles have a particle size of 60~80nm and an addition concentration of 0.08~0.12mM.

[0028] Optionally, the amount of phosphorylated serine peptide added is 0.04~0.06% of the total mass of the enzymatic hydrolysate.

[0029] Fourthly, this application provides the use of the traditional Chinese medicine composition described in the first aspect in the preparation of a medicament for treating rheumatoid arthritis of the cold-dampness obstruction type, the use comprising: administering the traditional Chinese medicine composition together with leflunomide.

[0030] The technical solutions provided in this application have the following advantages compared with the prior art:

[0031] This application provides a traditional Chinese medicine composition for treating rheumatoid arthritis. Through the rational design of the medicinal ingredients, the composition uses *Cynanchum paniculatum*, prepared aconite root, and prepared *Aconitum carmichaelii* as the principal herbs to warm the yang, dispel cold, and strengthen tendons and bones; *Cinnamomum cassia*, *Asarum heterotropoides*, *Clematis chinensis*, *Astragalus membranaceus*, scorpion, earthworm, and *Sinomenium acutum* as the assistant herbs to dispel dampness, unblock meridians, guide the medicine to the meridians, enhance efficacy, and promote yang and blood circulation; *Atractylodes macrocephala*, *Poria cocos*, *Gentiana macrophylla*, *Angelica sinensis*, *Spatholobus suberectus*, *Drynaria fortunei*, and white peony root as the adjuvant herbs to dispel dampness, strengthen the spleen, invigorate blood, remove blood stasis, and counteract the warming and drying effects; and *Glycyrrhiza uralensis* and dried ginger as the guiding herbs to harmonize, detoxify, warm the middle jiao, and assist yang. This compound, through the precise combination of principal, assistant, adjuvant, and guiding herbs, covers the four major pathological aspects of rheumatoid arthritis: cold, dampness, blood stasis, and deficiency, and possesses triple therapeutic effects of anti-inflammatory analgesia, immune regulation, and bone protection. Attached Figure Description

[0032] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0033] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 A schematic flowchart illustrating a method for preparing a pharmaceutical formulation provided in this application embodiment;

[0035] Figure 2 The average foot thickness of each group of mice at different time points provided in the embodiments of this application;

[0036] Figure 3 The absorbance of peritoneal effusion from each group of mice provided in the embodiments of this application;

[0037] Figure 4 The concentrations of inflammatory factors in the serum of mice in each group provided in the embodiments of this application. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0039] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0040] This application provides a traditional Chinese medicine composition for treating rheumatoid arthritis. By weight, the composition comprises the following medicinal ingredients: 12-18 parts of *Salvia miltiorrhiza*, 6-12 parts of prepared *Aconitum carmichaelii*, 3-9 parts of prepared *Aconitum carmichaelii*, 9-15 parts of cinnamon twig, 1-4 parts of *Asarum heterotropoides*, 9-15 parts of *Clematis chinensis*, 20-40 parts of *Astragalus membranaceus*, 3-9 parts of scorpion, 9-15 parts of earthworm, 9-15 parts of *Sinomenium acutum*, 12-18 parts of *Atractylodes macrocephala*, 12-18 parts of *Poria cocos*, 9-15 parts of *Gentiana macrophylla*, 12-18 parts of *Angelica sinensis*, 20-40 parts of *Spatholobus suberectus*, 9-15 parts of *Drynaria fortunei*, 12-18 parts of white peony root, 3-9 parts of prepared licorice root, and 6-12 parts of dried ginger.

[0041] This application rationally designs the pharmaceutical raw material composition of a traditional Chinese medicine composition for treating rheumatoid arthritis. The principal herbs are: *Salmonella sibiricum*, *Aconitum carmichaelii* (processed), and *Aconitum carmichaelii* (processed) for warming yang, dispelling cold, and strengthening tendons and bones; *Cinnamomum cassia*, *Asarum heterotropoides*, *Clematis chinensis*, *Astragalus membranaceus*, *Buthus martensii*, *Pheretima aspergillum*, and *Sinomenium acutum* (processed) for dispelling dampness, unblocking meridians, guiding the medicine to the meridians, enhancing efficacy, and promoting yang and blood circulation; *Atractylodes macrocephala*, *Poria cocos*, *Gentiana macrophylla*, *Angelica sinensis*, *Spatholobus suberectus*, *Drynaria fortunei*, and *Paeonia lactiflora* (processed) for dispelling dampness, strengthening the spleen, promoting blood circulation, removing blood stasis, and counteracting the warming and drying properties; and *Glycyrrhiza uralensis* (processed) and *Zingiber officinale* (dried) for harmonizing detoxification, warming the middle jiao, and assisting yang. The functions of each component are as follows:

[0042] Seron bone (12-18 parts): Rich in collagen and calcium-phosphorus complex, it directly repairs articular cartilage, alleviates osteoporosis, and also has antirheumatic effects. Simultaneously, it is combined with Clematis chinensis (oleanolic acid) and Drynaria fortunei (flavonoids) to form a "bone metabolism triple," promoting osteoblast differentiation and inhibiting osteoclast activity.

[0043] Prepared aconite root (6-12 parts) + prepared Sichuan aconite root (3-9 parts): After processing, the diester aconitine is hydrolyzed into a monoester, retaining its analgesic activity and specifically targeting cold pain caused by cold-dampness obstruction. At the same time, it forms the "Si Ni Tang" structure with prepared licorice root, and the glycyrrhizic acid complexes with aconitine, reducing cardiotoxicity.

[0044] Cinnamon twig (9-15 parts) + Asarum (1-4 parts): Cinnamon twig contains cinnamaldehyde, which dilates microvessels and accelerates the clearance of inflammatory metabolites. The volatile oil (methyleugenol) of Asarum inhibits the release of substance P and blocks the transmission of pain nerves. The two constitute a "warming and penetrating pathway," with cinnamon twig dispersing cold from the exterior and asarum penetrating the interior to relieve pain, covering both internal and external lesions of joint pain.

[0045] Clematis chinensis (9-15 parts) + Sinomenium acutum (9-15 parts): The oleanolic acid in Clematis chinensis inhibits TNF-α production and reduces joint swelling. The sinomenine in Sinomenium acutum regulates the Th17 / Treg balance and reduces autoantibody titers. The combined "dampness-removing and immunity-boosting" effects of Clematis chinensis and Sinomenium acutum are effective; Clematis chinensis primarily targets inflammatory pathways, while Sinomenium acutum regulates immune imbalance.

[0046] Astragalus (20-40 parts) + Insect-derived medicines (3-9 parts scorpion, 9-15 parts earthworm): Astragalus polysaccharides increase the CD4+ / CD8+ ratio to 1.5-2.0, enhancing immune homeostasis. Scorpion venom polypeptides from scorpion and earthworm inhibit COX-2 activity, while earthworm plasmin dissolves fibrin. Simultaneously, astragalus tonifies qi and supports detoxification, while insect-derived medicines attack pathogens and unblock meridians, embodying the classic principle of "strengthening the body's resistance and eliminating pathogens."

[0047] Atractylodes macrocephala (12-18 parts) + Poria cocos (12-18 parts): Atractylodes macrocephala and atractylone inhibit the expression of aquaporin AQP3, reducing joint effusion; Poria cocos polysaccharides regulate intestinal flora and reduce the level of inflammatory factor IL-6. With a dual target of "strengthening the spleen and promoting diuresis," Atractylodes macrocephala dries dampness and strengthens the spleen, while Poria cocos drains dampness and promotes diuresis, thus eliminating dampness.

[0048] Gentiana macrophylla (9-15 parts) + Paeonia lactiflora (12-18 parts): Gentiana macrophylla's gentiopicroside inhibits the NF-κB pathway, blocking the inflammatory cascade. Paeonia lactiflora's paeoniflorin relaxes smooth muscle and relieves joint contractures. Gentiana macrophylla clears heat and promotes diuresis, while Paeonia lactiflora soothes the liver and relieves spasms, thus mitigating the warming and drying properties of the principal herb.

[0049] Angelica sinensis (12-18 parts) + Spatholobus suberectus (20-40 parts) + Drynaria fortunei (9-15 parts): The ferulic acid in Angelica sinensis inhibits platelet aggregation and improves microcirculation. The flavonoids in Spatholobus suberectus promote angiogenesis. The psoralen in Drynaria fortunei induces osteoblast differentiation. The combination of "blood-activating and foundation-strengthening" effects, with Angelica sinensis and Spatholobus suberectus activating blood circulation and clearing the meridians, and Drynaria fortunei tonifying the kidneys and strengthening bones, prevents bone erosion.

[0050] Prepared licorice root (3-9 parts): Glycyrrhizic acid combines with aconite alkaloids to form a stable complex, reducing cardiotoxicity. The sweet and mild nature of licorice root harmonizes the overall properties of the formula, balancing the cold and hot effects of the herbs and prolonging the half-life of the medicinal effects.

[0051] Dried ginger (6-12 parts): Gingerol activates the TRPV1 channel, enhancing the warming effect of aconite. Dried ginger and prepared aconite form a "ginger-aconite combination," with dried ginger protecting the middle jiao and promoting the rise of yang qi, while prepared licorice root strengthens the spleen and stomach, forming a closed loop of "warming yang without harming yin."

[0052] Thus, this compound, through the precise combination of principal, assistant, adjuvant, and guide herbs, covers the four major pathological aspects of rheumatoid arthritis: "cold, dampness, blood stasis, and deficiency," and has a triple therapeutic effect of anti-inflammatory and analgesic, immune regulation, and bone protection.

[0053] In some embodiments, the traditional Chinese medicine composition, by weight, comprises the following raw materials: 15 parts of selenocarpus, 9 parts of prepared aconite root, 6 parts of prepared Sichuan aconite root, 12 parts of cinnamon twig, 3 parts of asarum, 12 parts of clematis root, 30 parts of astragalus root, 6 parts of scorpion, 12 parts of earthworm, 12 parts of sinomenium acutum, 15 parts of atractylodes macrocephala, 15 parts of poria cocos, 12 parts of gentian root, 15 parts of angelica sinensis, 30 parts of chicken blood vine, 12 parts of drynaria fortunei, 15 parts of white peony root, 6 parts of prepared licorice root, and 9 parts of dried ginger.

[0054] Based on a general inventive concept, this application provides a pharmaceutical preparation comprising the above-described traditional Chinese medicine composition and / or pharmaceutically acceptable excipients.

[0055] The compositions described in this application can be administered via various routes, such as oral tablets, capsules, powders, oral solutions, injections, and transdermal formulations. According to conventional pharmaceutical practice, pharmaceutically acceptable carriers include diluents, fillers, disintegrants, wetting agents, lubricants, colorants, flavoring agents, or other conventional additives. Typical pharmaceutically acceptable carriers include, for example, microcrystalline cellulose, starch, crospovidone, povidone, polyvinylpyrrolidone, maltitol, citric acid, sodium dodecyl sulfate, or magnesium stearate.

[0056] Figure 1 This is a schematic flowchart illustrating a method for preparing a pharmaceutical formulation, as provided in an embodiment of this application.

[0057] like Figure 1 As shown, this application provides a method for preparing the above-described pharmaceutical preparation, the method comprising the following steps:

[0058] S1. Prepare the various raw materials of the traditional Chinese medicine composition according to the weight parts;

[0059] S2. The prepared aconite root, prepared Sichuan aconite root, dried ginger and prepared licorice root were subjected to a first aqueous extraction to obtain the first aqueous extract.

[0060] It should be noted that the parameters for the first water extraction are as follows: the amount of water added is 9 times the weight of the medicinal materials, the number of decoctions is 2 (after the first decoction, the dregs are filtered and then decocted again to fully extract the dregs), the decoction time is 50 minutes each time (for Aconitum carmichaelii and Aconitum kusnezoffii, a longer time is needed to hydrolyze diester alkaloids to reduce toxicity), the temperature is boiling water at normal pressure (100℃), to avoid excessive heating of Chinese medicinal materials with less loss of volatile oils, after filtration, the two decoctions are combined and filtered with gauze or filter paper to remove the dregs.

[0061] Aconite and Sichuan aconite contain aconitine, which needs to be reduced in toxicity through water extraction. The combination of dried ginger (gingerol) and prepared licorice (glycyrrhizic acid) can neutralize the toxicity. This combination can hydrolyze diester aconitine into a less toxic monoester.

[0062] S3. The cinnamon twig, asarum, astragalus, sinomenium acutum, atractylodes macrocephala, poria cocos, gentiana macrophylla, angelica sinensis, chicken blood vine, and white peony root are subjected to a second water extraction to obtain the second water extract.

[0063] It should be noted that the parameters for the second water extraction are as follows: the amount of water added is 11 times the weight of the medicinal materials (due to the variety of medicinal materials, it is necessary to increase the solvent immersion area), the number of decoctions is 2 (the dregs are extracted again after the first decoction to avoid the residue of effective components), the decoction time is 35 minutes for the first decoction (to reduce the evaporation of volatile components), and 25 minutes for the second decoction. The temperature is kept at a gentle boil (95~100℃) to avoid violent boiling that would cause the volatile components to escape. After filtration, the two decoctions are combined, and the supernatant is collected after centrifugation or settling.

[0064] The second water extraction can avoid the loss of volatile components (such as Asarum volatile oil) while ensuring the efficient dissolution of water-soluble components (such as Astragalus polysaccharide and Paeoniflorin).

[0065] S4. Extract the whole scorpion and earthworm with ethanol to obtain the ethanol extract;

[0066] In some embodiments, the ethanol extraction uses an aqueous ethanol solution with a volume concentration of 70%, the ethanol extraction temperature is 40°C, and the ethanol extraction time is 60 min to 120 min.

[0067] Ethanol extraction of insect-derived drugs: A 70% ethanol aqueous solution can effectively extract fat-soluble active ingredients (such as scorpion venom and earthworm kinase) from whole scorpions and earthworms, while inhibiting enzymatic reactions that destroy the efficacy of the drugs.

[0068] S5. Biomimetic mineralization extraction of seron bone, Clematis chinensis and Drynaria fortunei was carried out to obtain a co-extract;

[0069] It should be noted that due to the special structure of sera, direct crushing of sera makes it difficult for the human body to digest and absorb. Therefore, it is necessary to use solvent extraction to dissolve its effective components before preparation.

[0070] In some implementations, step S5 includes:

[0071] S501. Grind sera, drynaria, and clematis into powder to 80-100 mesh to obtain a mixture;

[0072] Pulverizing selenophora, drynaria fortunei, and clematis chinensis to 80-100 mesh significantly disrupts bone tissue and plant cell wall structure, greatly increasing the specific surface area of ​​the material.

[0073] S502. The mixture is mixed with citric acid solution and subjected to shaking demineralization to obtain demineralization solution;

[0074] In some embodiments, the oscillation demineralization includes the following parameters: temperature of 48~52℃, oscillation frequency of 150~200rpm, and time of 100~200min.

[0075] Citric acid, as an organic acid, can react chemically with calcium salts in salmon bones, dissolving the calcium salts and releasing a large number of calcium ions.

[0076] S503. Adjust the pH of the demineralization solution to 6.5-7.0, then add alkaline protease, bromelain and CaCl2 nanoparticles to carry out enzymatic hydrolysis to obtain the enzymatic hydrolysate.

[0077] In some embodiments, the enzymatic hydrolysis is performed at a temperature of 45°C for a time of 150-210 minutes.

[0078] In some embodiments, the citric acid solution has a mass concentration of 4-6%;

[0079] The amount of alkaline protease added is 0.9-1.1% of the total mass of the demineralization solution;

[0080] The amount of bromelain added is 0.4-0.6% of the total mass of the demineralization solution;

[0081] The CaCl2 nanoparticles have a particle size of 60~80nm and an addition concentration of 0.08~0.12mM.

[0082] The alkaline protease targets the non-helical peptide bonds of collagen, specifically cleaving the non-helical peptide bonds of collagen fibers in seldomton bone, breaking down large collagen fibers into small peptide molecules, providing an organic framework for the nano-hydroxyapatite-collagen peptide-saponin complex, and releasing bioactive small peptide molecules at the same time.

[0083] Bromelain targets hydrophobic amino acids. It targets bitter peptides in materials and reduces the bitterness of extracts by hydrolyzing hydrophobic amino acids (such as proline and leucine), thereby improving taste and helping to improve the compliance of subsequent formulations.

[0084] CaCl2 nanoparticles play multiple key roles in this step. On the one hand, they mimic the osteogenic microenvironment in the human body, providing calcium ion concentration conditions and guiding collagen peptides to fold in a specific manner to form an ordered structure, providing a suitable template for the deposition of nano-hydroxyapatite. On the other hand, with a particle size of 60-80 nm, they can penetrate the micropores (approximately 100 nm in diameter) of demineralized seldomite and enter the bone tissue to participate in the reaction more effectively.

[0085] S504. Add phosphorylated serine peptide to the enzymatic hydrolysate, heat to 55-65℃, adjust pH to 7.5-8.5, stir for 20-40 min to obtain a mixture;

[0086] In some embodiments, the amount of phosphorylated serine peptide added is 0.04 to 0.06% of the total mass of the enzymatic hydrolysate.

[0087] Phosphorylated serine peptides (PSPs) possess unique functional properties. Their phosphate groups (-PO4) 3- It can react with calcium ions (Ca) released and added during the enzymatic hydrolysis step. 2+ The specific binding of hydroxyapatite acts as an initiation point for crystal nucleus formation, guiding the crystallization process of nano-hydroxyapatite and enabling its orderly deposition on the surface of collagen peptides. Simultaneously, the arginine (Arg) in the PSP amino acid sequence (such as Ser(P)-Glu-Ser(P)-Arg-Leu) imparts anti-inflammatory activity, adding extra therapeutic efficacy to the complex.

[0088] A suitable environment was created for the mineralization reaction under conditions of 55-65℃ and pH 7.5-8.5, with stirring for 20-40 minutes. The appropriate temperature and pH promoted the reaction of phosphorylated serine peptides with calcium ions, phosphate ions, etc., ensuring the smooth progress of the mineralization process. Stirring ensured thorough mixing of the reactants, allowing nano-hydroxyapatite to be uniformly deposited on the collagen peptides. Simultaneously, it promoted the combination of saponin components from *Drynaria fortunei* and *Clematis chinensis* with the nano-hydroxyapatite-collagen peptides, ultimately forming a nano-hydroxyapatite-collagen peptide-saponin complex with an average particle size of 80-150 nm.

[0089] S506. The mixture is subjected to enzyme inactivation, filtration, concentration and drying in sequence to obtain the co-extract.

[0090] The enzyme inactivation process rapidly terminates enzyme activity through heating and other methods, preventing further enzyme action during subsequent processing and avoiding further decomposition of active ingredients such as collagen peptides, thus ensuring the stability of the complex structure. The filtration step removes unreacted impurities and insoluble particles, improving the purity of the complex and providing a pure solution system for subsequent concentration and drying. The concentration process reduces the solution volume, increases the concentration of active ingredients, reduces drying costs and time, and makes the active ingredients easier to dry into a solid form. The drying step transforms the concentrate into a stable solid co-extract, facilitating storage, transportation, and mixing with other Chinese herbal extracts and excipients to prepare formulations, meeting the needs of subsequent pharmaceutical processes and helping to maintain the nanoparticle size and structural stability of the complex.

[0091] Therefore, *Cynanchum paniculatum*, *Drynaria fortunei*, and *Clematis chinensis* each play important roles in treating rheumatoid arthritis of the cold-dampness obstruction type, but their effects are complementary. *Cynanchum paniculatum* strengthens tendons and bones, dispels wind and dampness; *Drynaria fortunei* invigorates blood circulation, heals injuries, tonifies the kidneys, and strengthens bones; *Clematis chinensis* dispels wind and dampness, and unblocks meridians. When processed simultaneously, the effective components of the three herbs can interact and synergistically exert their effects during the biomimetic mineralization extraction process. For example, the calcium ions released by *Cynanchum paniculatum*, together with components in *Drynaria fortunei* and *Clematis chinensis*, participate in the formation of a nano-hydroxyapatite-collagen peptide-saponin complex. The resulting complex has stronger anti-inflammatory, analgesic, and joint function-improving effects, which is more effective than using the herbs alone or in simple mixtures.

[0092] In some embodiments, the co-extract contains a nano-hydroxyapatite-collagen peptide-saponin complex with an average particle size of 80~150nm; the X-ray diffraction pattern of the complex has characteristic peaks at 2θ=25.9°±0.2° and 31.8°±0.2°.

[0093] The X-ray diffraction pattern of the composite exhibits characteristic peaks at 2θ = 25.9° ± 0.2° and 31.8° ± 0.2°, indicating that the formed nano-hydroxyapatite possesses typical crystal structure characteristics. The characteristic peak at 2θ = 25.9° ± 0.2° corresponds to the diffraction of the (002) crystal plane of the nano-hydroxyapatite, and the peak at 31.8° ± 0.2° corresponds to the diffraction of the (300) crystal plane. These characteristic peaks demonstrate that well-crystallized nano-hydroxyapatite was successfully synthesized during the biomimetic mineralization process, and that the composite structure formed by its combination with collagen peptides and saponins is stable, exhibiting a specific crystal orientation and arrangement.

[0094] The structural characteristics of the nano-hydroxyapatite-collagen peptide-saponin complex can promote the slow release of active ingredients such as saponins and prolong the drug's action time. At the same time, the nano-sized particles increase the specific surface area, which is beneficial for the binding of drugs to targets and improves drug bioavailability and therapeutic effect.

[0095] S6. The first aqueous extract, the second aqueous extract, the alcohol extract, and the co-extract are mixed and then a pharmaceutical preparation is prepared.

[0096] First, combine the aqueous extracts (to avoid interference from alcohol precipitation), then add the alcohol extract and co-extracts to maximize the retention of active ingredients in each phase. Simultaneously, add excipients according to the target dosage form (e.g., capsules, tablets).

[0097] Based on a general inventive concept, this application provides the use of the traditional Chinese medicine composition described in the first aspect in the preparation of a medicament for treating rheumatoid arthritis of the cold-dampness obstruction type, the use comprising: administering the traditional Chinese medicine composition together with leflunomide.

[0098] The traditional Chinese medicine composition, containing herbs such as *Salvia miltiorrhiza*, *Clematis chinensis*, and *Drynaria fortunei*, utilizes a biomimetic mineralization extraction process to form a nano-hydroxyapatite-collagen peptide-saponin complex, characterized by multiple components and targets. The saponin component inhibits the production of inflammatory factors (such as TNF-α and IL-6), reducing synovial inflammation; the nano-hydroxyapatite and collagen peptides promote cartilage repair and improve joint function. Leflunomide, an immunosuppressant, has an active metabolite, A771726, that inhibits the activity of dihydroorotate dehydrogenase, blocking the de novo synthesis of pyrimidines and selectively inhibiting the proliferation of activated lymphocytes, thereby regulating the body's immune response. The combined use of these two components allows the traditional Chinese medicine composition to holistically regulate the body's Qi and blood, as well as the functions of the internal organs, while leflunomide precisely inhibits abnormal activation of immune cells. This synergistic effect in anti-inflammation and immunomodulation more effectively controls the inflammatory progression of rheumatoid arthritis and alleviates symptoms such as joint pain and swelling.

[0099] While leflunomide is effective for rheumatoid arthritis, long-term or high-dose use may cause adverse reactions such as liver damage, gastrointestinal reactions, and hematological abnormalities. The traditional Chinese medicine composition contains herbs like Astragalus membranaceus and Atractylodes macrocephala, which have qi-tonifying and spleen-strengthening effects, enhancing the body's immunity and reducing the damage of leflunomide to the gastrointestinal tract and liver. Herbs like Paeonia lactiflora and Rehmannia glutinosa, which nourish yin and moisten dryness, can counteract the side effects of warming and drying drugs, while also alleviating discomfort symptoms such as dryness and heat that may be caused by leflunomide. When used in combination, the dosage of leflunomide can be appropriately reduced while ensuring therapeutic efficacy, thereby lowering the risk of toxic side effects and improving patient safety and tolerability.

[0100] Furthermore, based on traditional Chinese medicine theory, the herbal composition utilizes the principles of warming yang and dispelling cold, eliminating dampness and unblocking meridians, and promoting blood circulation and relieving pain. It can effectively improve symptoms such as joint pain, limited range of motion, and worsening pain upon exposure to cold in patients with cold-dampness obstruction type rheumatoid arthritis. Leflunomide, on the other hand, focuses on controlling disease progression and slowing joint damage. The combined use of these two herbs can quickly relieve patients' clinical symptoms such as pain and swelling, while also fundamentally regulating immunity, repairing joint damage, slowing disease progression, improving joint mobility and self-care ability, and significantly improving patients' quality of life.

[0101] In summary, the traditional Chinese medicine composition for treating rheumatoid arthritis of the cold-dampness obstruction type provided in this application has the following advantages:

[0102] (1) Scientific and reasonable formulation design: Based on the theory of monarch, minister, assistant and guide in traditional Chinese medicine, multiple medicinal materials such as selenophora, prepared aconite, and prepared aconitum carmichaelii are precisely matched. Selenophora and other herbs are used as the monarch herbs to warm yang and dispel cold, strengthen tendons and bones; cinnamon twig and other herbs are used as minister herbs to dispel dampness and unblock collaterals, guide the medicine to the meridians and enhance efficacy; atractylodes macrocephala and other herbs are used as assistant herbs to dispel dampness and strengthen the spleen, promote blood circulation and remove blood stasis; prepared licorice and other herbs are used as guide herbs to harmonize and detoxify, warm the middle and assist yang, comprehensively covering the four major pathological links of "cold, dampness, blood stasis and deficiency", and having the triple effects of anti-inflammatory and analgesic, immune regulation and bone protection. The herbs are synergistically combined, such as selenophora, clematis and drynaria forming a "bone metabolism triple", and prepared aconite, prepared aconitum carmichaelii and prepared licorice reducing toxicity and giving full play to the efficacy.

[0103] (2) Innovative preparation process: Differentiated extraction methods are adopted for different medicinal materials. For aconitine-containing processed Aconitum carmichaelii and processed Aconitum kusnezoffii, toxicity is reduced by specific water extraction parameters; for Cinnamomum cassia, volatile components are retained by appropriate water extraction conditions; and fat-soluble active ingredients are extracted from scorpion and earthworm using 70% ethanol aqueous solution. In particular, the biomimetic mineralization extraction technology for selenocarpus, Clematis chinensis and Drynaria fortunei, through steps such as crushing, demineralization, enzymatic hydrolysis and biomimetic mineralization, forms a nano-hydroxyapatite-collagen peptide-saponin complex with an average particle size of 80-150nm. This complex has a stable structure, which can promote the slow release of active ingredients and improve bioavailability.

[0104] (3) Advantages of combined administration of traditional Chinese and Western medicine: Combining traditional Chinese medicine composition with leflunomide achieves complementary advantages of both. The traditional Chinese medicine composition regulates the body's qi and blood and the functions of the internal organs as a whole, while leflunomide precisely inhibits the abnormal activation of immune cells, working synergistically in anti-inflammatory and immunomodulatory effects to more effectively control the progression of inflammation. At the same time, the traditional Chinese medicine composition can reduce adverse reactions such as liver damage and gastrointestinal reactions caused by leflunomide, reduce the dosage of Western medicine, and improve medication safety. The combination of the two can not only quickly relieve clinical symptoms such as joint pain and swelling, but also delay the progression of the disease, reduce the disability rate, and significantly improve the quality of life of patients.

[0105] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to industry standards. If there is no corresponding industry standard, then common international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0106] Example 1

[0107] This embodiment provides a traditional Chinese medicine composition for treating rheumatoid arthritis. By weight, the traditional Chinese medicine composition consists of the following raw materials: 15 parts of selenocarpus, 9 parts of prepared aconite root, 6 parts of prepared Sichuan aconite root, 12 parts of cinnamon twig, 3 parts of asarum, 12 parts of clematis root, 30 parts of astragalus root, 6 parts of scorpion, 12 parts of earthworm, 12 parts of sinomenium acutum, 15 parts of atractylodes macrocephala, 15 parts of poria cocos, 12 parts of gentian root, 15 parts of angelica sinensis, 30 parts of chicken blood vine, 12 parts of drynaria fortunei, 15 parts of white peony root, 6 parts of prepared licorice root, and 9 parts of dried ginger.

[0108] Based on the above-mentioned traditional Chinese medicine composition, this embodiment also provides a method for preparing a pharmaceutical preparation comprising the above-mentioned traditional Chinese medicine composition, the method comprising the following steps:

[0109] S11. Prepare each of the medicinal raw materials of the traditional Chinese medicine composition according to the weight parts;

[0110] S21. The prepared aconite root, prepared Sichuan aconite root, dried ginger and prepared licorice root are subjected to a first aqueous extraction to obtain the first aqueous extract.

[0111] S31. The cinnamon twig, asarum, astragalus, sinomenium acutum, atractylodes macrocephala, poria cocos, gentiana macrophylla, angelica sinensis, chicken blood vine, and white peony root are subjected to a second water extraction to obtain the second water extract.

[0112] S41. Extract the whole scorpion and earthworm with ethanol to obtain an alcohol extract;

[0113] The ethanol extraction uses a 70% (v / v) aqueous ethanol solution, the extraction temperature is 40°C, and the extraction time is 80 min.

[0114] S51. Pulverize selenophora, drynaria fortunei, and clematis chinensis to 80-100 mesh to obtain a mixture; mix the mixture with citric acid solution and demineralize by shaking to obtain a demineralizing solution; adjust the pH of the demineralizing solution to 6.7, then add alkaline protease (CAS No. 9001-75-6), bromelain (CAS No. 37189-34-7), and CaCl2 nanoparticles for enzymatic hydrolysis to obtain an enzymatic hydrolysate; add phosphorylated serine peptide (CAS No. 183582-09-4) to the enzymatic hydrolysate, heat to 60℃, adjust the pH to 8.0, and stir for 30 min to obtain a mixture; subject the mixture to enzyme inactivation, filtration, concentration, and drying in sequence to obtain a co-extract.

[0115] The oscillation demineralization includes the following parameters: temperature 50℃, oscillation frequency 175rpm, and time 150min;

[0116] The enzymatic hydrolysis was performed at a temperature of 45°C for 180 minutes.

[0117] Optionally, the citric acid solution has a mass concentration of 5%;

[0118] The amount of alkaline protease added is 1.0% of the total mass of the demineralization solution;

[0119] The amount of bromelain added is 0.5% of the total mass of the demineralization solution;

[0120] The CaCl2 nanoparticles have a particle size of 60-80 nm and an addition concentration of 0.10 mM.

[0121] The amount of phosphorylated serine peptide added is 0.05% of the total mass of the enzymatic hydrolysate.

[0122] S61. The first aqueous extract, the second aqueous extract, the alcohol extract, and the co-extract are mixed and then processed using a vacuum drying process. After drying, the mixture is pulverized and sieved. Subsequently, pharmaceutically acceptable excipients (such as dextrin, starch, etc.) and flavoring agents (such as stevioside, sucrose, etc.) are added, and the mixture is thoroughly mixed, granulated, and dried to obtain granules. In this granule, each 1g of granules contains 0.65g of crude drug.

[0123] Example 2

[0124] This embodiment provides a traditional Chinese medicine composition for treating rheumatoid arthritis. By weight, the traditional Chinese medicine composition consists of the following medicinal materials: 18 parts of selenocarpus, 6 parts of prepared aconite root, 9 parts of prepared Sichuan aconite root, 9 parts of cinnamon twig, 4 parts of asarum, 9 parts of clematis root, 40 parts of astragalus root, 3 parts of scorpion, 15 parts of earthworm, 9 parts of sinomenium acutum, 18 parts of atractylodes macrocephala, 12 parts of poria cocos, 15 parts of gentian root, 12 parts of angelica sinensis, 40 parts of chicken blood vine, 9 parts of drynaria fortunei, 18 parts of white peony root, 3 parts of prepared licorice root, and 12 parts of dried ginger.

[0125] Based on the above-mentioned traditional Chinese medicine composition, this embodiment also provides a method for preparing a pharmaceutical preparation comprising the above-mentioned traditional Chinese medicine composition, the method comprising the following steps:

[0126] S11. Prepare each of the medicinal raw materials of the traditional Chinese medicine composition according to the weight parts;

[0127] S21. The prepared aconite root, prepared Sichuan aconite root, dried ginger and prepared licorice root are subjected to a first aqueous extraction to obtain the first aqueous extract.

[0128] S31. The cinnamon twig, asarum, astragalus, sinomenium acutum, atractylodes macrocephala, poria cocos, gentiana macrophylla, angelica sinensis, chicken blood vine, and white peony root are subjected to a second water extraction to obtain the second water extract.

[0129] S41. Extract the whole scorpion and earthworm with ethanol to obtain an alcohol extract;

[0130] The ethanol extraction uses a 70% (v / v) aqueous ethanol solution, the extraction temperature is 40°C, and the extraction time is 120 min.

[0131] S51. Crush selenophora, drynaria fortunei, and clematis chinensis to 80-100 mesh to obtain a mixture; mix the mixture with citric acid solution and demineralize by shaking to obtain a demineralizing solution; adjust the pH of the demineralizing solution to 7.0, then add alkaline protease (CAS No. 9001-75-6), bromelain (CAS No. 37189-34-7), and CaCl2 nanoparticles for enzymatic hydrolysis to obtain an enzymatic hydrolysate; add phosphorylated serine peptide (CAS No. 183582-09-4) to the enzymatic hydrolysate, heat to 65℃, adjust the pH to 8.5, and stir for 20 min to obtain a mixture; sequentially inactivate enzymes, filter, concentrate, and dry the mixture to obtain a co-extract.

[0132] The oscillation demineralization includes the following parameters: temperature of 52℃, oscillation frequency of 200rpm, and time of 100min;

[0133] The enzymatic hydrolysis was performed at a temperature of 45°C for 210 minutes.

[0134] Optionally, the citric acid solution has a mass concentration of 6%;

[0135] The amount of alkaline protease added is 1.1% of the total mass of the demineralization solution;

[0136] The amount of bromelain added is 0.6% of the total mass of the demineralization solution;

[0137] The CaCl2 nanoparticles have a particle size of 60-80 nm and an addition concentration of 0.12 mM.

[0138] The amount of phosphorylated serine peptide added is 0.06% of the total mass of the enzymatic hydrolysate.

[0139] S61. The first aqueous extract, the second aqueous extract, the alcohol extract, and the co-extract are mixed and then processed using a vacuum drying process. After drying, the mixture is pulverized and sieved. Subsequently, pharmaceutically acceptable excipients (such as dextrin, starch, etc.) and flavoring agents (such as stevioside, sucrose, etc.) are added, and the mixture is thoroughly mixed, granulated, and dried to obtain granules. In this granule, each 1g of granules contains 0.66g of crude drug.

[0140] Example 3

[0141] This embodiment provides a traditional Chinese medicine composition for treating rheumatoid arthritis. By weight, the traditional Chinese medicine composition consists of the following medicinal materials: 12 parts of selenocarpus, 12 parts of prepared aconite root, 3 parts of prepared Sichuan aconite root, 15 parts of cinnamon twig, 1 part of asarum, 15 parts of clematis root, 20 parts of astragalus root, 9 parts of scorpion, 9 parts of earthworm, 15 parts of sinomenium acutum, 12 parts of atractylodes macrocephala, 18 parts of poria cocos, 9 parts of gentian root, 18 parts of angelica sinensis, 20 parts of chicken blood vine, 15 parts of drynaria fortunei, 12 parts of white peony root, 9 parts of prepared licorice root, and 6 parts of dried ginger.

[0142] Based on the above-mentioned traditional Chinese medicine composition, this embodiment also provides a method for preparing a pharmaceutical preparation comprising the above-mentioned traditional Chinese medicine composition, the method comprising the following steps:

[0143] S11. Prepare each of the medicinal raw materials of the traditional Chinese medicine composition according to the weight parts;

[0144] S21. The prepared aconite root, prepared Sichuan aconite root, dried ginger and prepared licorice root are subjected to a first aqueous extraction to obtain the first aqueous extract.

[0145] S31. The cinnamon twig, asarum, astragalus, sinomenium acutum, atractylodes macrocephala, poria cocos, gentiana macrophylla, angelica sinensis, chicken blood vine, and white peony root are subjected to a second water extraction to obtain the second water extract.

[0146] S41. Extract the whole scorpion and earthworm with ethanol to obtain an alcohol extract;

[0147] The ethanol extraction uses a 70% (v / v) aqueous ethanol solution, the extraction temperature is 40°C, and the extraction time is 60 min.

[0148] S51. Crush selenophora, drynaria fortunei, and clematis chinensis to 80-100 mesh to obtain a mixture; mix the mixture with citric acid solution and demineralize by shaking to obtain a demineralizing solution; adjust the pH of the demineralizing solution to 6.5, then add alkaline protease (CAS No. 9001-75-6), bromelain (CAS No. 37189-34-7), and CaCl2 nanoparticles for enzymatic hydrolysis to obtain an enzymatic hydrolysate; add phosphorylated serine peptide (CAS No. 183582-09-4) to the enzymatic hydrolysate, heat to 55℃, adjust the pH to 7.5, and stir for 40 min to obtain a mixture; subject the mixture to enzyme inactivation, filtration, concentration, and drying in sequence to obtain a co-extract.

[0149] The oscillation demineralization includes the following parameters: temperature 48℃, oscillation frequency 150rpm, and time 200min;

[0150] The enzymatic hydrolysis was performed at a temperature of 45°C for 150 minutes.

[0151] Optionally, the citric acid solution has a mass concentration of 4%;

[0152] The amount of alkaline protease added is 0.9% of the total mass of the demineralization solution;

[0153] The amount of bromelain added is 0.4% of the total mass of the demineralization solution;

[0154] The CaCl2 nanoparticles have a particle size of 60-80 nm and an addition concentration of 0.08 mM.

[0155] The amount of phosphorylated serine peptide added is 0.04% of the total mass of the enzymatic hydrolysate.

[0156] S61. The first aqueous extract, the second aqueous extract, the alcohol extract, and the co-extract are mixed and then processed using a vacuum drying process. After drying, the mixture is pulverized and sieved. Subsequently, pharmaceutically acceptable excipients (such as dextrin, starch, etc.) and flavoring agents (such as stevioside, sucrose, etc.) are added, and the mixture is thoroughly mixed, granulated, and dried to obtain granules. In this granule, each 1g of granules contains 0.66g of crude drug.

[0157] Comparative Example 1

[0158] This comparative example is based on the disclosure in Example 1, with the following modifications:

[0159] No CaCl2 nanoparticles are added in step S51.

[0160] Comparative Example 2

[0161] This comparative example is modified from the one disclosed in Example 1 as follows:

[0162] Phosphorylated serine peptides are not added in step S51.

[0163] Comparative Example 3

[0164] This comparative example is based on the disclosure in Example 1, with the following modifications:

[0165] Alkaline protease is not added in step S51.

[0166] The content of active substances in the co-extracts obtained by step S51 of Examples 1-3 and Comparative Examples 1-3 was determined, and the results are shown in Table 1.

[0167] Table 1. Content of effective substances in the co-extracts of Examples 1-3 and Comparative Examples 1-3

[0168] Ingredient type Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Collagen peptides (mg / g) 202.4 190.4 183.5 144.8 161.9 80.1 Flavonoids (mg / g) 52.1 50.3 46.7 42.7 42.6 35.6 Triterpenoid saponins (mg / g) 33.1 32.6 32.9 28.5 29.0 20.1 Free amino acids (mg / g) 32.9 30.3 30.5 21.1 22.5 12.7 Calcium (mg / g) 16.5 14.9 15.7 8.6 10.4 11.7

[0169] As shown in Table 1, Comparative Example 3 lacked alkaline protease, and the hydrolysis efficiency relying solely on bromelain was low, resulting in a significant decrease in peptide dissolution. Comparative Example 1 lacked CaCl2, leading to a reduced enzymatic hydrolysis rate and a decrease in peptide formation. Comparative Example 2, due to the lack of modification by phosphorylated serine peptides, saw some peptides aggregate and precipitate, affecting the dissolution rate. Meanwhile, in Comparative Examples 1 and 2, the lack of CaCl2 or phosphorylated serine peptides resulted in decreased dissolution and stability of the active ingredients, with contents slightly lower than in the examples. Comparative Example 3, due to insufficient enzymatic hydrolysis and low plant cell breakage rate, resulted in a significant decrease in the dissolution of saponins and flavonoids. Furthermore, Comparative Example 1, lacking CaCl2, had reduced demineralization efficiency and decreased calcium dissolution. Comparative Example 2, lacking a complexing agent, resulted in the loss of some calcium as precipitation. Although the enzymatic hydrolysis conditions in Comparative Example 3 did not directly affect calcium dissolution, insufficient cell breakage led to a decrease in bone mineral release.

[0170] Meanwhile, in Comparative Example 1, no CaCl2 nanoparticles were added, Ca 2+ The absence of this template leads to insufficient mineralization, preventing the directional deposition of nano-hydroxyapatite (n-HAP), resulting in disordered folding of collagen peptides and difficulty in embedding saponins into the complex lattice. Comparative Example 2, without the addition of phosphorylated serine peptides (PSPs), shows that the absence of PSPs leads to insufficient Ca2+ mineralization. 2+ -PO4 3- Nucleus formation was hindered, n-HAP crystallinity decreased, and the anti-inflammatory function of PSP itself was lost. Comparative Example 3 did not include alkaline protease; the lack of alkaline protease prevented celecoxib collagen from being broken down into small peptides, resulting in a lack of an organic framework for n-HAP deposition, and saponins could not be anchored in the complex via peptides. Consequently, the content of the nano-hydroxyapatite-collagen peptide-saponin complex in Comparative Examples 1-3 was reduced, specifically manifested in a decrease in collagen peptides, triterpenoid saponins, and calcium in the co-extracts of the comparative examples.

[0171] The granules obtained in Examples 1-3 and Comparative Example 2 were used in a study of rheumatoid arthritis in mice. The specific experimental procedures are as follows:

[0172] I. Experimental Preparation

[0173] Experimental animals: Five-week-old ICR mice weighing 18±2g were selected, with half males and half females. Before the experiment, they were acclimatized in a standard animal room for 3 days, with an ambient temperature of 22±2℃ and humidity of 50-60%, and a 12-hour day-night cycle. They were allowed free access to food and water.

[0174] Experimental drugs: Granules prepared in Examples 1-3 and Comparative Example 2; leflunomide (as a positive control drug); physiological saline (used for drug preparation, gavage, and control group treatment).

[0175] Instruments and reagents: Vernier calipers (for measuring the degree of joint swelling in mice); ELISA reader and ELISA kit (for detecting inflammatory factors); centrifuge, pipette, EP tubes and other routine laboratory equipment; bovine type II collagen, complete Freund's adjuvant, and incomplete Freund's adjuvant (for constructing a mouse model of rheumatoid arthritis).

[0176] II. Construction of a mouse model of rheumatoid arthritis

[0177] Primary immunization: Bovine type II collagen was dissolved in 0.05M acetic acid solution to prepare a 2 mg / mL solution, and incubated overnight at 4°C. Before use, it was thoroughly mixed with an equal volume of complete Freund's adjuvant and prepared as a water-in-oil emulsion using ultrasonic emulsification (it did not diffuse when dropped into water). On day 0 of the experiment, 50 μL of the emulsion was injected subcutaneously into the right hind paw of each mouse to induce an immune response.

[0178] Booster immunization: On day 21 after the initial immunization, bovine type II collagen and incomplete Freund's adjuvant were mixed and emulsified in a 1:1 ratio. 50 μL of the emulsion was injected subcutaneously into the base of the tail of mice to complete the booster immunization.

[0179] Model screening: Starting from the initial immunization, the thickness of the right hind paw of mice was measured daily using calipers, and the data were recorded. Mice with a paw swelling rate (swelling rate = (post-immunization thickness - pre-immunization thickness) / pre-immunization thickness × 100%) exceeding 20% ​​on day 7 post-immunization were selected for subsequent experiments to ensure successful model construction.

[0180] III. Grouping and Administration

[0181] Grouping: Mice meeting the model criteria were randomly divided into 7 groups, with 12 mice in each group:

[0182] Control group: No model was established; normal saline was administered by gavage.

[0183] Model group: After modeling, the model was given physiological saline by gavage;

[0184] Positive control group: After modeling, the contents of leflunomide were administered by gavage (the dosage was calculated at 0.5 g / kg body weight and dissolved in physiological saline).

[0185] Example 1 Granule Group: After modeling, the drug of Example 1 granules was administered by gavage (total dose 0.5g / kg body weight, suspended in physiological saline).

[0186] Example 2 Granule Group: After modeling, the drug of Example 2 granules was administered by gavage (total dose 0.5 g / kg body weight, suspended in physiological saline).

[0187] Example 3 Granule Group: After modeling, the drug of Example 3 granules was administered by gavage (total dose 0.5g / kg body weight, suspended in physiological saline).

[0188] Comparative Example 2 Granule Group: After modeling, the drug of Comparative Example 2 granules was administered by gavage (total dose 0.5 g / kg body weight, suspended in physiological saline).

[0189] Administration procedure: Starting from day 7 after model establishment, mice in each group were administered the drug by gavage at the same time every day. The gavage volume for each mouse was adjusted to 0.2 mL / 10 g according to body weight, and the administration was continued for 14 days.

[0190] IV. Detection Indicators and Methods

[0191] (1) Monitoring of joint swelling: On day 0 (before immunization), day 7 (before drug administration), day 14 (end of drug administration), and day 21 (experimental endpoint), the thickness of the mid-plantar portion of the right hind paw of mice was measured using vernier calipers, accurate to 0.01 mm. The average swelling degree and swelling inhibition rate of each group of mice were calculated using the following formula:

[0192] Swelling degree = (plantar thickness at measurement time point - plantar thickness before immunization)

[0193] Swelling inhibition rate = (1 - average swelling degree of the treatment group / average swelling degree of the model group) × 100%

[0194] (2) Peritoneal capillary permeability test: On day 14 after the end of drug administration, mice in each group were tested. The specific steps are as follows:

[0195] First, inject 0.5% Evans blue solution (200 μL / vial) via tail vein.

[0196] Ten minutes later, inject 0.6% acetic acid solution (100μL / vial) into the peritoneum.

[0197] Twenty minutes later, the mice were euthanized by cervical dislocation, 5 mL of physiological saline was injected into the abdominal cavity, the abdomen was gently massaged for 2 minutes, and the peritoneal exudate was collected.

[0198] Centrifuge the exudate at 3000 rpm for 10 minutes, collect the supernatant, and measure the absorbance (A value) at 620 nm using an ELISA reader. The higher the A value, the stronger the permeability of the peritoneal capillaries.

[0199] (3) Detection of serum inflammatory factors: On day 21, the endpoint of the experiment, blood was collected from mice in each group by ocular blood collection. The blood was placed in centrifuge tubes and centrifuged at 3000 rpm for 15 minutes to separate the serum. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in the serum were detected using an ELISA kit. The operation was strictly carried out according to the kit instructions. The absorbance at a wavelength of 450 nm was measured using an ELISA reader, and the concentration of inflammatory factors was calculated based on the standard curve.

[0200] The results of the average foot thickness of mice at different time points in each group are shown in Table 2. The results of the absorbance of peritoneal effusion of mice in each group (day 14) are shown in Table 3. The serum inflammatory factor concentration (pg / mL) of mice in each group is shown in Table 4.

[0201] Table 2. Average paw thickness of mice in each group at different time points.

[0202] Group Pre-immunization (Day 0) thickness (mm) Thickness (mm) before drug administration (day 7) Thickness (mm) at the end of treatment (day 14) Experimental endpoint (day 21) thickness (mm) Swelling inhibition rate (day 21) Blank group 0.80±0.03 0.82±0.03 0.83±0.03 0.85±0.03 - Model group 0.81±0.03 1.35±0.08 1.55±0.10 1.70±0.12 - Positive control group 0.80±0.03 1.33±0.07 1.10±0.06 0.95±0.05 44.10% Example 1 Group 0.81±0.03 1.32±0.06 0.85±0.04 0.70±0.03 58.80% Example 2 group 0.80±0.03 1.30±0.07 0.95±0.05 0.80±0.04 52.90% Example 3 Group 0.81±0.03 1.31±0.06 1.00±0.05 0.85±0.04 50.00% Comparative Example 2 0.80±0.03 1.32±0.06 1.07±0.06 0.93±0.05 45.30%

[0203] Table 3 Absorbance of peritoneal effusion from mice in each group

[0204] Group Absorbance (A620) Blank group 0.10±0.02 Model group 0.65±0.05 Positive control group 0.35±0.03 Example 1 Group 0.25±0.02 Example 2 group 0.28±0.02 Example 3 Group 0.30±0.02 Comparative Example 2 0.32±0.03

[0205] Table 4. Serum inflammatory factor concentrations in mice of each group

[0206] Group TNF-α (pg / mL) IL-6 (pg / mL) IL-1β (pg / mL) Blank group 10.0±1.5 8.0±1.2 6.0±1.0 Model group 90.0±8.0 75.0±7.0 60.0±6.0 Positive control group 45.0±4.0 35.0±3.5 28.0±3.0 Example 1 Group 30.0±3.0 25.0±2.5 20.0±2.0 Example 2 group 35.0±3.5 30.0±3.0 23.0±2.5 Example 3 Group 38.0±3.8 32.0±3.2 25.0±2.8 Comparative Example 2 42.0±3.0 34.0±3.2 26.0±2.0

[0207] In Tables 2-4, the indicators of the model group were significantly higher than those of the blank group, indicating that the rheumatoid arthritis model was successfully constructed.

[0208] like Figure 2 As shown, the swelling inhibition rate of Group 1 reached 58.8% on day 21, which was 33.3% higher than that of the positive control group (44.1%), and the plantar thickness (0.70 mm) was close to that of the blank group, indicating that it had the best inhibitory effect on joint inflammation and tissue damage. The swelling inhibition rates of Groups 2 and 3 were 52.9% and 50.0% respectively, both higher than that of the positive control group, suggesting that all three groups of granules could effectively relieve joint swelling in RA.

[0209] like Figure 3 As shown, the absorbance of Examples 1 to 3 were 0.25, 0.28 and 0.30, respectively, all lower than the positive control group (0.35). Among them, the absorbance of Example 1 was the lowest, indicating that it can more effectively inhibit the release of inflammatory mediators (such as histamine and bradykinin), stabilize the tight junctions of vascular endothelial cells, and reduce capillary permeability.

[0210] like Figure 4 As shown, the concentration in Group 1 (30.0 pg / mL) was 33.3% lower than that in the positive control group (45.0 pg / mL), suggesting that it may reduce TNF-α secretion by monocytes and macrophages by inhibiting the NF-κB or MAPK signaling pathway. The concentrations of IL-6 (25.0 pg / mL) and IL-1β (20.0 pg / mL) in Group 1 were the lowest among all groups, indicating that it has a stronger inhibitory effect on Th17 cell differentiation and neutrophil activation, and can block the inflammatory cascade.

[0211] This indicates that the granules in Examples 1-3 all exhibited superior anti-inflammatory, anti-swelling, and immunomodulatory effects compared to the positive control drug (leflunomide) in the RA model. The granules in Examples 1-3 may exert their therapeutic effects through multiple pathways, including inhibiting the release of inflammatory factors, reducing vascular permeability, and protecting articular cartilage. Their compound formulation and preparation process are key factors contributing to the differences in efficacy.

[0212] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the referred range.

[0213] Furthermore, in the description of this application, the terms "comprising," "including," etc., mean "including but not limited to." In this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. The above descriptions are merely specific embodiments of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A traditional Chinese medicine composition for treating rheumatoid arthritis, characterized in that, The traditional Chinese medicine composition, by weight, comprises the following medicinal materials: 12-18 parts of *Salvia miltiorrhiza*, 6-12 parts of prepared *Aconitum carmichaelii*, 3-9 parts of prepared *Aconitum carmichaelii*, 9-15 parts of cinnamon twig, 1-4 parts of *Asarum heterotropoides*, 9-15 parts of *Clematis chinensis*, 20-40 parts of *Astragalus membranaceus*, 3-9 parts of scorpion, 9-15 parts of earthworm, 9-15 parts of *Sinomenium acutum*, 12-18 parts of *Atractylodes macrocephala*, 12-18 parts of *Poria cocos*, 9-15 parts of *Gentiana macrophylla*, 12-18 parts of *Angelica sinensis*, 20-40 parts of *Spatholobus suberectus*, 9-15 parts of *Drynaria fortunei*, 12-18 parts of white peony root, 3-9 parts of prepared licorice root, and 6-12 parts of dried ginger.

2. The traditional Chinese medicine composition for treating rheumatoid arthritis according to claim 1, characterized in that, By weight, the traditional Chinese medicine composition comprises the following raw materials: 15 parts of selenocarpus, 9 parts of prepared aconite root, 6 parts of prepared Sichuan aconite root, 12 parts of cinnamon twig, 3 parts of asarum, 12 parts of clematis root, 30 parts of astragalus root, 6 parts of scorpion, 12 parts of earthworm, 12 parts of sinomenium acutum, 15 parts of atractylodes macrocephala, 15 parts of poria cocos, 12 parts of gentian root, 15 parts of angelica sinensis, 30 parts of chicken blood vine, 12 parts of drynaria fortunei, 15 parts of white peony root, 6 parts of prepared licorice root, and 9 parts of dried ginger.

3. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises the traditional Chinese medicine composition as described in claim 1 or 2 and / or pharmaceutically acceptable excipients.

4. A method for preparing the pharmaceutical formulation according to claim 3, characterized in that, The method includes the following steps: S1. Prepare the various raw materials of the traditional Chinese medicine composition according to the weight parts; S2. The prepared aconite root, prepared Sichuan aconite root, dried ginger and prepared licorice root were subjected to a first aqueous extraction to obtain the first aqueous extract. S3. The cinnamon twig, asarum, astragalus, sinomenium acutum, atractylodes macrocephala, poria cocos, gentiana macrophylla, angelica sinensis, chicken blood vine, and white peony root are subjected to a second water extraction to obtain the second water extract. S4. Extract the whole scorpion and earthworm with ethanol to obtain the ethanol extract; S5. Biomimetic mineralization extraction of seron bone, Clematis chinensis and Drynaria fortunei was carried out to obtain a co-extract; S6. The first aqueous extract, the second aqueous extract, the alcohol extract, and the co-extract are mixed and then a pharmaceutical preparation is prepared.

5. The method for preparing the pharmaceutical formulation according to claim 4, characterized in that, The ethanol extraction uses a 70% (v / v) aqueous ethanol solution, the extraction temperature is 40°C, and the extraction time is 60 min to 120 min.

6. The method for preparing the pharmaceutical formulation according to claim 4, characterized in that, The S5 steps include: S501. Grind sera, drynaria, and clematis into powder to 80-100 mesh to obtain a mixture; S502. The mixture is mixed with citric acid solution and subjected to shaking demineralization to obtain demineralization solution; S503. Adjust the pH of the demineralization solution to 6.5-7.0, then add alkaline protease, bromelain and CaCl2 nanoparticles to carry out enzymatic hydrolysis to obtain the enzymatic hydrolysate. S504. Add phosphorylated serine peptide to the enzymatic hydrolysate, heat to 55-65℃, adjust pH to 7.5-8.5, stir for 20-40 min to obtain a mixture; S506. The mixture is subjected to enzyme inactivation, filtration, concentration and drying in sequence to obtain the co-extract.

7. The method for preparing a pharmaceutical formulation according to claim 6, characterized in that, The oscillation demineralization includes the following parameters: temperature 48~52℃, oscillation frequency 150~200rpm, and time 100~200min; The enzymatic hydrolysis was performed at a temperature of 45°C for 150-210 minutes.

8. The method for preparing a pharmaceutical formulation according to claim 6, characterized in that, The citric acid solution has a mass concentration of 4-6%; The amount of alkaline protease added is 0.9-1.1% of the total mass of the demineralization solution; The amount of bromelain added is 0.4-0.6% of the total mass of the demineralization solution; The CaCl2 nanoparticles have a particle size of 60~80nm and an addition concentration of 0.08~0.12mM.

9. The method for preparing a pharmaceutical formulation according to claim 6, characterized in that, The amount of phosphorylated serine peptide added is 0.04~0.06% of the total mass of the enzymatic hydrolysate.

10. The use of the traditional Chinese medicine composition according to claim 1 or 2 in the preparation of a medicament for treating rheumatoid arthritis of the cold-dampness obstruction type, characterized in that, The application includes administering the traditional Chinese medicine composition together with leflunomide.

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