PREPARATIONS FOR TREATING RHEUMATOID ARTHRITIS

Abstract

The invention relates to a pharmaceutical for the treatment of rheumatoid arthritis and its use in the preparation of drugs for the treatment of rheumatoid arthritis, anti-inflammatory and analgesic drugs, drugs to inhibit increased vascular permeability in capillaries, and drugs to inhibit splenic lymphocyte proliferation and serum production of collagen type II antibodies, to reduce the levels of inflammatory cytokines IL-1 and TNF-α, and to inhibit the production of rheumatoid factors IgG and IgM.The pharmaceutical preparation is made from the following medicinal herbs, in parts by weight: 120-190 parts of Astragalus root, 120-190 parts of Gentianae Macrophyllae root, 45-80 parts of Stephaniae Tetrandrae root, 45-80 parts of Aconiti Lateralis Preparata root, 110-180 parts of Polygoni Cuspidati rhizome and root, 110-180 parts of Spatholobi stem, 110-180 parts of Clematis rhizome and root, 20-40 parts of Sinapis Albae seeds, and 45-80 parts of Paeonia Alba root. 120-190 parts of Rehmannia root (Radix Rehmanniae Recens), 45-80 parts of Angelica root (Radix Angelicae Sinensis), 40-65 parts of Myrrh, 40-65 parts of Cyperus root (Rhizoma Cyperi), 45-80 parts of Cinnamon bark (Ramulus Cinnamomi), and 45-80 parts of Achyranthes root (Radix Achyranthis Bidentatae).

Description

A pharmaceutical composition for treating rheumatoid arthritis and its preparation method

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application with application number 202110513825.3 filed with the China Patent Office on May 12, 2021, and with the invention name “A pharmaceutical composition for treating rheumatoid arthritis and its preparation method”. The entire contents of these applications are incorporated by reference into this application. Technical Field

[0003] The present invention relates to a traditional Chinese medicine composition for treating rheumatoid arthritis and a preparation method thereof, belonging to the field of application of Chinese herbal medicines. The composition is mainly clinically used for rheumatoid arthritis, which has symptoms such as swelling, pain, tenderness in limb joints, morning stiffness, difficulty in joint flexion and extension, limited movement, numbness and heaviness of limbs, subcutaneous nodules, fear of cold and wind, fatigue and weakness, etc. Background Art

[0004] Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by erosive arthritis. The prevalence of RA is 0.8% (range, 0.3% to 2.1%) of the population, with a higher incidence in women, approximately three times that of men. RA can occur at any age, with a peak incidence between the ages of 30 and 50. According to a survey conducted by the Chinese Society of Rheumatology in Beijing and Guangzhou, the prevalence of RA is 0.34% in northern China and 0.32% in southern China. The primary clinical manifestations of RA are symmetrical, persistent joint swelling and pain, often accompanied by morning stiffness. The proximal interphalangeal joints, metacarpophalangeal joints, wrists, elbows, and toes are most commonly affected; the cervical spine, temporomandibular joints, sternoclavicular joints, and acromioclavicular joints may also be affected. In the middle and late stages of the disease, patients may develop "swan neck" and "boutonniere" deformities of the fingers, as well as joint ankylosis and metacarpophalangeal subluxation, manifesting as ulnar deviation of the metacarpophalangeal joints. In addition to joint symptoms, subcutaneous nodules, known as rheumatoid nodules, may also develop. Some patients may also experience extra-articular manifestations, including those affecting the lungs, eyes, cardiovascular system, and nervous system. The etiology of RA is unknown, but it may be related to autoimmunity induced by infection, genetic factors, and other factors. Its pathogenesis is complex. Studies have shown that RA is a T-cell-mediated autoimmune disease involving macrophages, dendritic cells, B cells, and inflammatory cells. Its pathological manifestations primarily include chronic inflammation of the synovial membrane, pannus formation, and destruction of cartilage and bone in the joints, ultimately leading to joint deformity and loss of function. RA is extremely harmful, affecting a large population and causing significant disability. The disability rate is 60% after a 5-10-year course of disease and 90% after a 30-year course. The five-year mortality rate for patients with extra-articular manifestations can reach 50%, making it a leading cause of labor force loss and disability in my country. Furthermore, most patients lack access to effective and timely treatment, making active research into the prevention and treatment of RA urgent.

[0005] The treatment of RA has long been a challenging and hot topic in rheumatology. Current Western medical treatment aims to alleviate or eliminate joint swelling, pain, morning stiffness, and other extra-articular symptoms caused by arthritis; control disease progression, prevent and reduce joint bone destruction, achieve prolonged clinical remission, and maintain function of affected joints as much as possible; promote repair of damaged joint bone and improve its function; and control systemic involvement. However, currently used medications for RA are considered palliative, aimed at relieving clinical symptoms. RA pharmacological treatments generally fall into five categories: nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (ADAs), glucocorticoids, anti-cytokine agents, and immunomodulators. Ibuprofen, naproxen, indomethacin, and diclofenac are nonspecific symptomatic treatments, but these drugs cannot prevent disease progression or the development of complications. They also carry side effects such as gastrointestinal irritation, azotemia, platelet dysfunction, allergic rhinitis, and exacerbated asthma. Long-term use of these medications can lead to renal interstitial damage. Antirheumatic drugs that improve the condition, such as methotrexate, sulfasalazine, leflunomide, penicillamine, azathioprine, cyclophosphamide, and cyclosporine, often have adverse reactions such as liver and kidney damage, gastrointestinal reactions, and bone marrow suppression. Long-term use of glucocorticoids cannot prevent joint structural damage or improve lesion progression. Long-term use increases tolerance, making withdrawal difficult and prone to relapse, accelerating joint damage. For patients whose condition remains uncontrolled despite standard medical treatment, surgical treatment may be considered to correct the deformity and improve quality of life. Common procedures include synovectomy, artificial joint replacement, arthrodesis, and soft tissue repair. However, surgery cannot cure the disease, and postoperative drug therapy is still required. In addition, autologous stem cell transplantation, T cell vaccines, and mesenchymal stem cell therapy may be effective in alleviating RA, but these are only suitable for a small number of patients and require further clinical research. Therefore, seeking new treatment methods and measures, developing highly effective and low-toxic drugs and effective and reasonable treatment plans are important measures to significantly improve patients' symptoms and reduce the occurrence of RA disability. It is also a cutting-edge topic that is widely valued by the medical community at home and abroad.

[0006] Current Western medications for RA primarily include symptom-modifying antirheumatic drugs and disease-controlling antirheumatic drugs. The latter category is still in the exploratory and experimental stages, while the former primarily includes nonsteroidal anti-inflammatory drugs, disease-modifying antirheumatic drugs, biologics, and glucocorticoids.

[0007] Nonsteroidal anti-inflammatory drugs (NSAIDs) primarily inhibit cyclooxygenase (COX) activity and reduce prostaglandin synthesis, resulting in anti-inflammatory, analgesic, antipyretic, and joint swelling-reducing effects. They are the most commonly used drugs for the treatment of RA in clinical practice. However, this type of drug can cause gastrointestinal symptoms, liver and kidney damage, and potentially increase adverse cardiovascular events. Therefore, their use is restricted in patients with the above-mentioned diseases. Even ordinary patients require the minimum effective dose and a short course of treatment.

[0008] Disease-modifying antirheumatic drugs (DMARDs) lack significant analgesic and anti-inflammatory effects, hence their name: slow-acting antirheumatic drugs. However, they can delay or control disease progression. Common adverse reactions associated with these drugs include gastrointestinal discomfort, hair loss, rash, liver and kidney damage, leukopenia, and bone marrow suppression. Clinically, early use is emphasized. For complex conditions involving multiple joints or extra-articular symptoms, a combination of multiple drugs may be necessary.

[0009] Biological agents: mainly include tumor necrosis factor (TNF)-α antagonists: etanercept, infliximab, adalimumab; interleukin (IL)-1 antagonist: anakinra; anti-CD20 monoclonal antibody: rituximab; and T cell co-stimulatory signal inhibitors, etc. Because they are mostly injected subcutaneously or intravenously, there are adverse reactions such as infection, infusion reaction, hypertension, rash, itching, fever, nausea, joint pain, drug-induced lupus-like syndrome, and demyelinating lesions.

[0010] Glucocorticoids: They can rapidly improve joint pain and systemic symptoms. For patients with severe RA accompanied by cardiac, pulmonary, or nervous system involvement, short-acting steroids can be given. The dosage depends on the severity of the disease. However, steroid use has drawbacks such as difficulty in withdrawal and a high risk of relapse after discontinuation.

[0011] Traditional Chinese medicine (TCM) has extensive research on the syndrome differentiation and treatment of rheumatoid arthritis, accumulating rich experience. Its unique advantages in alleviating clinical symptoms, improving quality of life, and preventing / mitigating the development of complications complement the shortcomings of Western medicine and are increasingly receiving high attention from the medical community both domestically and internationally. Currently, a wide variety of TCM patent medicines are available for the treatment of RA, with varying effects and properties. These include: wind-dispelling, dampness-removing, meridian-warming, and cold-dispersing herbs, such as Hanshibi Granules, Hanbiting Tablets, Fufang Xuelian Capsules, Zhuifeng Tougu Pills, and Xiaohuoluo Pills; liver-tonifying, kidney-tonifying, meridian-draining, and collateral-activating herbs, including Fengshihantong Tablets, Wangbi Granules, Zhuanggujie Pills, and Yishenjuanbi Pills; tendon-relaxing, collateral-activating, blood-activating, and pain-relieving herbs, including Tongren Dahuoluo Pills, Tongluo Kaibi Tablets, Duhuo Jisheng Pills, and Panlongqi Tablets; and heat-clearing and dampness-removing herbs, including Shirebi Granules. Additionally, there are single-ingredient extracts, such as Tripterygium wilfordii Polyglycosides Tablets and White Peony Total Glucosides Capsules.

[0012] Comprehensive analysis shows that most of the currently marketed Chinese patent medicines for RA are positioned to dispel wind and dampness, warm the meridians and dissipate cold, nourish the liver and kidneys, promote blood circulation and activate the collaterals, relax the muscles and tendons, activate the collaterals, promote blood circulation and relieve pain, as well as clear away heat and dampness. However, each type of medicine has its own focus, and there are not many medicines that comprehensively consider the causes and pathogenesis of RA from multiple perspectives such as wind, cold, dampness, heat, phlegm, blood stasis, and deficiency. In addition, there is no understanding of the etiology, pathogenesis and treatment of this disease from the perspective of collateral disease theory.

[0013] Summary of the Invention

[0014] The pharmaceutical composition of the present invention was created by Professor Wu Yiling, an academician of the Chinese Academy of Engineering, vice president of the China Association of Traditional Chinese Medicine, vice president of the Chinese Society of Integrated Traditional Chinese and Western Medicine, and honorary dean of Hebei Yiling Hospital, based on the theory of collateral disease in traditional Chinese medicine. He comprehensively analyzed the pathogenesis and treatment of rheumatoid arthritis in traditional Chinese medicine from the perspective of "wind, cold, dampness, heat, phlegm, blood stasis, and deficiency" leading to obstruction of the collateral vessels, and combined with many years of clinical experience in treating arthritis to create an empirical prescription for treating rheumatoid arthritis. The prescription has been clinically used for nearly 20 years and has definite clinical efficacy.

[0015] The pharmaceutical composition of the present invention is innovative in its cubic principle and composition of prescriptions and herbs. First, the etiology and pathogenesis of the disease are comprehensively considered from the perspective of multiple pathogenic factors such as wind, cold, dampness, heat, phlegm, blood stasis, and deficiency. Second, guided by the theory of collateral disease, it is proposed that deficiency of vital energy and invasion of the collaterals by pathogenic factors are the pathological basis of the disease, and that phlegm and blood stasis and obstruction of the collaterals are the main pathological links of the disease. Finally, it is proposed that dispelling pathogenic factors, relieving numbness, and invigorating qi and unblocking the collaterals are the main treatment principles and the main treatment methods for the disease are dispelling wind, dispersing cold, removing dampness, invigorating qi, removing blood stasis, and unblocking the collaterals. The medicine is composed of astragalus, gentian, stephania tetrandra, heishunpian, polygonum cuspidatum, millet blood vine, clematis root, mustard seed, white peony root, rehmannia root, angelica root, myrrh, cyperus rotundus, cassia twig, and rhizoma cyperi. Astragalus and Gentiana macrophylla are the main herbs, which work together to dispel wind, cold and dampness, invigorate qi, dredge meridians and relieve pain; at the same time, Stephania tetrandra is used to strengthen the dispelling of dampness, dredge meridians and relieve pain, Heishunpian warms the meridians, dispels cold and dredges meridians, Polygonum cuspidatum clears heat, promotes dampness and relieves pain, Millettia reticulata harmonizes blood, removes blood stasis and dredges meridians, and together they serve as assistant herbs to assist the main herbs in dispelling evil; Clematis chinensis is used to dispel rheumatism, dredge meridians, and relieve numbness and pain, Mustard seed is used to dispel phlegm and dredge meridians, promote qi and disperse stagnation, White Peony Root and Rehmannia glutinosa nourish blood and nourish yin, Angelica sinensis replenishes blood, activates blood circulation and dredges meridians, Myrrh with vinegar activates blood circulation, dredges meridians and relieves pain, Cyperus rotundus regulates qi and activates blood circulation; Cinnamon twig and Rhizoma Cyperi are guiding herbs to lead all the herbs to the upper and lower joints, dredge meridians and relieve numbness. This prescription comprehensively treats the internal and external causes of rheumatoid arthritis, which not only helps to improve the patient's symptoms such as local joint swelling and pain, morning stiffness, difficulty in flexion and extension, and limited movement, but also can significantly improve systemic clinical symptoms such as numbness and heaviness of limbs, chills and wind, fatigue and weakness, improve the patient's quality of life, and show the unique advantages of this prescription. Compared with similar products, the pharmaceutical composition of the present invention is innovative in terms of etiology and pathogenesis, cubic principle and prescription composition.

[0016] Rheumatoid arthritis is a disease caused by the obstruction of collaterals by external factors such as wind, cold and dampness, which, on the basis of deficiency of vital energy, cause phlegm and blood stasis to block the collaterals and form formations. Doctors of all dynasties have discussed this in detail. For example, "Suwen·Bi Lun" states: "Wind, cold and dampness, the three qi, mixed together, form bi"; "Jingui Yaolue" records: "Pain in all limbs, thin body, and swollen feet as if they are falling off" should be treated with Guizhi Shaoyao Zhimu Decoction; Zhu Danxi's "Danxi Xinfa" points out: "Pain in the limbs of obese people is mostly caused by rheumatism and phlegm flowing into the meridians and causing pain"; Ye Tianshi discussed in "Clinical Guide Medical Cases": "Wind, cold and dampness, the three qi, combined together, form bi. However, over the years, the external evil remains, and both qi and blood are damaged, which turn into stagnant blood stasis and condensed phlegm, which mix with the meridians. Therefore, it is not difficult to find that the occurrence and development of this disease are related to multiple pathological factors such as wind, cold, dampness, heat, phlegm, blood stasis, and deficiency, and are closely related to the collaterals.

[0017] Looking back at the records of the etiology and pathogenesis of Bi syndrome over the past two thousand years, we can summarize them into the following aspects:

[0018] (1) Insufficient righteousness: The Neijing states that “where evil gathers, the qi must be weak” and “wind, rain, cold and heat cannot make the qi weak, and evil will not only harm people”. Insufficient righteousness plays a decisive role in the onset of arthritis. Insufficient righteousness mainly includes insufficient congenital endowment, kidney deficiency in old age, improper work and rest, and damage to righteousness after childbirth and illness. As the Zhengzhi Zhunsheng states, “arthritis can be caused by wind, dampness, cold, heat…all of which are symptoms, while kidney deficiency is the root cause”. The kidney governs the bones. In old age, kidney deficiency leads to insufficient essence and marrow, which results in poor bone nourishment, and can manifest as pain in the waist, spine, joints, and heels. At the same time, in old age, kidney deficiency leads to insufficient righteousness, weak defense against external diseases, and empty pores, making it easy for wind, cold, dampness and other evils to invade the body and cause the disease. "People who are overworked often suffer from internal and external weakness, weakening their blood and qi, and their pores, making it easy for wind pathogens to invade. Depending on the pathogens, various types of arthritis may arise" (Wang Huaiyin of the Song Dynasty, in his "Taiping Shenghui Fang"). This suggests that excessive fatigue can also lead to insufficient vital energy, hindering the ability to defend the body from the elements. Similarly, women experience a significant loss of vital energy and blood after childbirth, weakening their vital energy. If not properly cared for, external pathogens such as wind, cold, and dampness can easily invade, also causing arthritis. Furthermore, after illness, the weakened qi is unable to expel pathogens, allowing them to linger and become trapped in the joints, tendons, bones, and blood vessels of the limbs, blocking the collaterals and making recovery difficult.

[0019] (2) External pathogens invasion: Wind, cold, dampness and other external pathogens invade the body, and the struggle between the righteous and the evil. When the evil prevails and the righteous retreats, the disease will occur. Therefore, it is believed that wind, cold, dampness and other external pathogens are the direct factors and objective conditions for the occurrence of this disease. Although a weak body is prone to invasion by external pathogens, if one is still in good physical condition but does not pay attention to health preservation, or works in damp places and sleeps in the open air; or works in water and wades in the rain; or lives in damp places and sleeps in the wind; or the climate changes suddenly, with alternating hot and cold weather; or sweats in the wind after work, or takes a cold shower after sweating, etc. At this time, the body's defense against external pathogens is relatively insufficient, and external pathogens take the opportunity to attack the body. Over time, the wind, cold and dampness will block the meridians, leading to the occurrence of this disease. As stated in "Suwen·Generation of the Five Zang Organs": "When lying down and being blown by wind, blood will coagulate in the skin and cause numbness." Zhang Zihe's "Rumen Shiqin" records: "This disease often occurs when it is cloudy and rainy in all seasons... Therefore, when the grass is dry and it is very cold, or when it is near water... People who work hard are exposed to wind and rain, and sleep in a damp place, then numbness will come from the outside."

[0020] (3) Phlegm stagnation and blood stasis: Phlegm turbidity and blood stasis are both pathological products produced by pathogenic factors acting on the body, and can also act on the body as a sole cause of disease. As Yu Chang said in "Medical Laws": "The three evils of wind, cold and dampness often use the phlegm in the chest as a support." As the disease lasts for a long time, it affects the circulation of qi and blood, causing blood stagnation to become blood stasis, and fluid to condense into phlegm. Phlegm and blood stasis intertwine, blocking the collaterals and penetrating deep into the bones and joints. Or if there is external evil that is not resolved, the internal and external evils will be trapped and glued to the collaterals, making the disease lingering and difficult to cure. Common symptoms in the late stage of this disease include obvious joint swelling and pain, difficulty in flexion and extension, limited movement, and subcutaneous nodules. As stated in "Introduction to Medicine": "The joints are extremely painful, and after a long time, the hands and feet will curl up... and even the body will be covered with lumps and scrofula." These are the signs of phlegm coagulation, blood stasis, and obstruction of the collaterals. "Long-term arthritis must be caused by phlegm and dampness, blood stasis, and the stagnation of the meridians" ("Classification of Symptoms and Treatments: Discussion on the Treatment of Arthritis"). It can be seen that with a long period of illness, the circulation of qi and blood is not smooth, and they stagnate and become stasis. In addition, they are affected by external pathogens. The internal and external factors combine to cause obstruction of the meridians, resulting in arthritis. Moreover, due to long-term illness and physical weakness, it is difficult to expel the pathogens, and the arthritis is difficult to heal.

[0021] In summary, doctors of all dynasties have realized that the pathogenesis of Bi disease is insufficient vital energy, invasion of external evil, combination of internal and external evil, obstruction of evil energy, or phlegm and blood stasis flowing into muscles, joints, tendons and bones, poor circulation of Qi and blood, and obstruction of collateral vessels. The disease is characterized by deficiency in the root and excess in the superficial, a mixed syndrome of deficiency and excess. However, various theories mostly understand the disease from one or two aspects such as deficiency, evil, phlegm, stasis, and heat, and rarely discuss it from the perspective of collateral disease. However, the symptoms of Bi disease are complex, the disease is prolonged and difficult to cure, and the analysis of collateral disease comprehensively analyzing the multiple pathogenesis of "wind, cold, dampness, heat, phlegm, stasis, and deficiency" has important clinical guiding value for fully revealing the disease.

[0022] The inventor is in view of above-mentioned understanding, rheumatoid arthritis is with joint swelling and pain as main manifestation, its pathogenesis is that the pathogenic wind, cold and dampness due to deficiency of vital energy attacks from outside, and phlegm and blood stasis glue block the collaterals due to, the occurrence and development of primary disease is relevant with multiple pathological factors such as wind, cold, dampness, heat, phlegm, blood stasis, deficiency, and is closely related to collaterals. Although its card has the difference of cold, heat, deficiency and excess, yet always with the collaterals qi and blood stasis of the pores, fascia, joints is common pathological characteristics, so "unobstructed collaterals" is its total treatment principle, expelling pathogenic wind and promoting numbness, invigorating qi and unblocking the collaterals is the main treatment principle of primary disease, and establishing that dispelling wind, cold and dampness, invigorating qi and removing blood stasis are the main treatment methods of primary disease. Doctors in the Ming and Qing dynasties elaborated on this. For example, Wu Jutong believed in Treatise on Warm Diseases that "the disease is caused by mixed feelings and is not caused by one single cause. The key point is that Qi cannot be promoted". Therefore, he emphasized that in treating Bi syndrome, "promotion and unblocking are the most important". Ye Tianshi pointed out in Clinical Guide to Medical Cases: Bi syndrome: "even if the sweating does not stop, promotion and unblocking are important. Only by unblocking can the remaining evil be removed." In short, if the body's resistance is strengthened and the evil is eliminated, Qi and blood can circulate smoothly, the Ying and Wei systems return to normal, and the collaterals are unobstructed, Bi syndrome will disappear.

[0023] The inventors believe that based on the TCM pathogenesis characteristics of rheumatoid arthritis, which is that "wind, cold, dampness, heat, phlegm, blood stasis, and deficiency" lead to obstruction of the collaterals, the treatment method is to "dispel wind, cold, dampness, invigorate qi, resolve blood stasis and dredge the collaterals", and use complex prescriptions to treat complex symptoms. Therefore, this medicine has more medicinal ingredients and adopts the principle of two monarch ingredients and four minister ingredients.

[0024] The composition for treating rheumatoid arthritis of the present invention comprises the following components in parts by weight: 120-190 parts of astragalus, 120-190 parts of gentiana, 45-80 parts of stephania tetrandra, 45-80 parts of aconite, 110-180 parts of polygonum cuspidatum, 110-180 parts of millettia reticulata, 110-180 parts of clematis, 20-40 parts of mustard, 45-80 parts of white peony root, 120-190 parts of rehmannia root, 45-80 parts of angelica, 40-65 parts of myrrh, 40-65 parts of cyperus, 45-80 parts of cinnamon twig, and 45-80 parts of chyranthes bidentata.

[0025] The composition for treating rheumatoid arthritis of the present invention preferably includes the following components in parts by weight: 120 parts of Astragalus, 190 parts of Gentiana macrophylla, 45 parts of Stephania tetrandra, 80 parts of Aconite, 110 parts of Polygonum cuspidatum, 180 parts of Millettia reticulata, 110 parts of Clematidis, 40 parts of Mustard seeds, 45 parts of White Peony Root, 190 parts of Rehmannia glutinosa, 45 parts of Angelica sinensis, 65 parts of Myrrh, 40 parts of Cyperus rotundus, 80 parts of Cinnamon Twig, and 45 parts of Achyranthes bidentata.

[0026] The composition for treating rheumatoid arthritis of the present invention preferably includes the following components in parts by weight: 150 parts of astragalus, 160 parts of gentiana, 60 parts of stephania tetrandra, 65 parts of aconite, 120 parts of polygonum cuspidatum, 130 parts of millettia reticulata, 135 parts of clematis, 35 parts of mustard, 50 parts of white peony root, 160 parts of rehmannia, 60 parts of angelica, 55 parts of myrrh, 45 parts of cyperus, 60 parts of cinnamon twig, and 60 parts of achyranthes bidentata.

[0027] The composition for treating rheumatoid arthritis of the present invention preferably includes the following components in parts by weight: 146 parts of astragalus, 139 parts of gentiana, 58 parts of stephania tetrandra, 75 parts of aconite, 120 parts of polygonum cuspidatum, 125 parts of millettia reticulata, 135 parts of clematis, 28 parts of mustard, 60 parts of white peony, 150 parts of rehmannia, 70 parts of angelica, 60 parts of myrrh, 60 parts of cyperus, 70 parts of cinnamon twig, and 70 parts of Achyranthes bidentata.

[0028] The composition for treating rheumatoid arthritis of the present invention preferably includes the following components in parts by weight: 156 parts of Astragalus, 156 parts of Gentiana macrophylla, 63 parts of Stephania tetrandra, 63 parts of Aconite, 146 parts of Polygonum cuspidatum, 146 parts of Millettia reticulata, 146 parts of Clemati radix, 31 parts of Mustard seeds, 63 parts of White Peony, 156 parts of Rehmannia glutinosa, 63 parts of Angelica sinensis, 52 parts of Myrrh, 52 parts of Cyperus rotundus, 63 parts of Cinnamon twig, and 63 parts of Achyranthes bidentata.

[0029] Among the components of the composition for treating rheumatoid arthritis of the present invention, the root of the aconite is preferably Heishunpian, the myrrh is preferably vinegar myrrh, and the root of the ox-bone is preferably Sichuan ox-bone.

[0030] The composition of the present invention can be in the form of capsules, tablets, pills, oral liquids, granules or powders.

[0031] The active ingredient of the pharmaceutical composition of the present invention is prepared by the following steps:

[0032] A. Grind 60 portions of Stephania tetrandra into fine powder, sterilize by gamma irradiation, and set aside.

[0033] B. Take cinnamon twig, vinegar myrrh, and fragrant angelica and add 5-8 times the amount of water, and extract the volatile oil by steam distillation for 5-10 hours continuously. Collect the volatile oil, filter the distilled aqueous solution, collect it in another container, and set aside;

[0034] C. Take Gentiana macrophylla, Millettia reticulata, Chinese angelica, mustard seeds, white peony root, and polygonum cuspidatum, add 8-12 times the amount of 50-80% ethanol, reflux extraction 1-3 times, each time for 1 hour, filter the extract, recover ethanol under reduced pressure, and concentrate to a clear paste with a relative density of 1.15±0.05, and set aside;

[0035] D. Take Astragalus, Heishun Pian, Clematidis, Rehmannia glutinosa, and Cyathula, add 8-12 times the amount of water, decoct 2-5 times, each time for 1.5 hours, filter the decoction, and concentrate under reduced pressure to a clear paste with a relative density of 1.10±0.05, then combine with the aqueous solution after distillation of volatile oil, and concentrate under reduced pressure to a clear paste with a relative density of 1.15±0.05, and set aside;

[0036] E. Combine the pastes from steps C and D, mix well, dry, and grind to obtain a mixed paste powder;

[0037] The fine powder obtained in step A, the volatile oil obtained in step B, and the clear paste powder obtained in step E together constitute the active component of the pharmaceutical composition of the present invention.

[0038] The preparation process of the capsule in the pharmaceutical composition preparation of the present invention is as follows:

[0039] A. Grind 60 portions of Stephania tetrandra into fine powder, sterilize by gamma irradiation, and set aside.

[0040] B. Take cinnamon twig, vinegar myrrh, and fragrant root with 8 times the amount of water, and extract the volatile oil by steam distillation for 10 hours. Collect the volatile oil, filter the distilled water solution, collect it in another container, and set aside;

[0041] C. Take Gentiana macrophylla, Millettia reticulata, Chinese Angelica sinensis, Mustard seeds, White Peony Root, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, each time for 1 hour, filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear paste with a relative density of 1.15±0.05, and set aside;

[0042] D. Take Astragalus, Heishun Pian, Clematidis, Rehmannia glutinosa, and Cyathula officinalis, add 8 times the amount of water, decoct 4 times, each time for 1.5 hours, filter the decoction, and concentrate under reduced pressure to a clear paste with a relative density of 1.10±0.05. Then combine it with the aqueous solution after distillation of volatile oil, and concentrate under reduced pressure to a clear paste with a relative density of 1.15±0.05, and set aside;

[0043] E. Combine the clear pastes obtained in step C and step D, mix them evenly, dry them, and grind them to obtain a mixed clear paste powder. Spray the volatile oil obtained in step B into it, add the sterilized powder in step A, spray granulate it, shape the granules, and fill them into capsules.

[0044] The preparation process of the tablets in the pharmaceutical composition preparation of the present invention is as follows:

[0045] A. Grind 60 portions of Stephania tetrandra into fine powder, sterilize by gamma irradiation, and set aside.

[0046] B. Take cinnamon twig, vinegar myrrh, and fragrant spice with 6 times the amount of water, and extract the volatile oil by steam distillation for 8 hours continuously. Collect the volatile oil, filter the distilled water solution, collect it in another container, and set aside;

[0047] C. Take Gentiana macrophylla, Millettia reticulata, Chinese Angelica sinensis, Mustard seeds, White Peony Root, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, each time for 1 hour, filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear paste with a relative density of 1.15±0.05, and set aside;

[0048] D. Take Astragalus, Heishun Pian, Clematidis, Rehmannia glutinosa, and Cyathula officinalis, add 11 times the amount of water, decoct 3 times, each time for 1.5 hours, filter the decoction, and concentrate under reduced pressure to a clear paste with a relative density of 1.10±0.05. Then combine it with the aqueous solution after distillation of volatile oil, and concentrate under reduced pressure to a clear paste with a relative density of 1.15±0.05, and set aside;

[0049] E. Combine the clear pastes obtained in step C and step D, mix well, dry, and grind to obtain a mixed clear paste powder;

[0050] F. Take the volatile oil obtained in step B and carry out inclusion complexation, and the inclusion complex is ready for use;

[0051] The sterilized powder obtained in step A and the clear paste powder obtained in step E are granulated, the granules are shaped, the inclusion compound obtained in step F is added, and tablets are obtained by conventional methods.

[0052] The process for inclusion of volatile oil in the pharmaceutical composition of the present invention is as follows: taking 6-10 times the amount of cyclodextrin as the volatile oil, adding 6-10 times the amount of water as the cyclodextrin, grinding, adding a mixture of volatile oil and 90-95% ethanol in equal volumes, continuing to grind evenly, and drying to obtain the composition.

[0053] Preferably, the present invention also protects the use of the pharmaceutical composition of the present invention in the preparation of anti-inflammatory drugs.

[0054] Preferably, the present invention also protects the use of the pharmaceutical composition of the present invention in the preparation of a drug for inhibiting increased capillary permeability.

[0055] Preferably, the present invention also protects the use of the pharmaceutical composition of the present invention in the preparation of analgesic drugs.

[0056] Preferably, the present invention also protects the use of the pharmaceutical composition of the present invention in the preparation of a drug for inhibiting the proliferation of spleen lymphocytes and the production of serum type II collagen antibodies, reducing the content of inflammatory cytokines IL-1 and TNF-α, and inhibiting the production of rheumatoid factors IgG and IgM.

[0057] Preferably, the rheumatoid arthritis treated by the pharmaceutical composition of the present invention is adjuvant arthritis or type II collagen-induced arthritis.

[0058] In the pharmaceutical composition of the present invention, the specific drug compatibility is as follows: Sovereign drugs: Astragalus and Gentiana macrophylla. Astragalus: sweet in taste and slightly warm in nature, it is specialized in replenishing Qi, and is a holy medicine for replenishing Qi. It can greatly replenish the Qi of the lungs and spleen internally, and can consolidate the exterior and strengthen the Wei externally. Just as "Compendium of Materia Medica" states: "Astragalus, a medicine for replenishing the lungs and strengthening the spleen, strengthening the Wei and restraining sweat, and expelling wind and transporting poison"; "Compendium of Materia Medica" says: "(Astragalus) has a strong flavor and thick texture, and its power is all in the skin, so it can directly reach the skin and muscles of the human body, consolidate and protect the Yang, and enrich the exterior, which is its specialty." The medicine has a thin aroma and a strong flavor, can replenish the three burners and strengthen the Wei, and fill the skin. It is most suitable to use it as the sovereign drug to replenish the Qi of the lungs and spleen internally, replenish Qi and consolidate the exterior to prevent the invasion of exogenous pathogens. Gentiana macrophylla: spicy, sweet and neutral in nature, it can dispel wind and dampness, dispel cold and relax muscles, activate collaterals and relieve pain. "Compendium of Materia Medica" says: "Qinjiao, bitter can purge, pungent can disperse, slightly warm can promote bowel movement, so it is used to treat cold and heat evil spirits, cold-dampness and rheumatism, pain in the limbs, diarrhea, and promote urination. It can dispel wind and dampness." Qinjiao dispels wind and dampness, and is combined with Astragalus to replenish Qi and dispel evil spirits, dispelling evil spirits without harming the body. Astragalus, with the help of Qinjiao, replenishes Qi without retaining evil spirits. The body has something to rely on, and the external evil spirits will not return. The two herbs are used together to replenish Qi, dispel wind and dampness, and relieve pain. It is used as the main medicine.

[0059] Assistant herbs: Stephania tetrandra, Heishunpian, Polygonum cuspidatum, and Millettia reticulata. Stephania tetrandra: bitter in taste and cold in nature, potent in dispelling dampness, unblocking meridians, and alleviating pain. The Materia Medica Treatise states that it "treats facial paralysis caused by dampness and wind, pain in the hands and feet, and dissipates retained phlegm," aided by Gentiana macrophylla to enhance its dampness-removing effects. Heishunpian: pungent, sweet, and hot in nature, its pungent, warming properties unblock meridians, warming the meridians and dispersing cold. Aconite root: warm in nature, mobile but not stationary, warms the meridians, dissipates cold, eliminates dampness, and relieves pain. It possesses the efficacy of dispelling wind and dampness, dispersing cold, and relieving pain. The Compendium of Materia Medica describes it as "a potent herb that restores yang energy, disperses yin and cold, expels cold phlegm, and unblocks joints." Zhang Xichun believes that "Aconite root is pungent in taste...its power can both ascend and descend, reach internally and disperse externally. It can unblock and unclog any accumulated cold that has accumulated in the internal organs and in the meridians and blood vessels," assisting the other herbs in unblocking meridians, relieving numbness, and dispersing cold. Polygonum cuspidatum: Slightly bitter and slightly cold in nature, it dissipates blood stasis and relieves pain, dispels wind and unclogs the meridians, and clears heat and dampness. "Compendium of Materia Medica Supplement" states that it "treats wind in the joints and blood stasis." It is used in prescriptions to clear heat and stasis, and is used to treat arthralgia that has accumulated over time and has transformed into heat. Millettia reticulata: Bitter and sweet in nature, it promotes blood circulation and unclogs the meridians, and relaxes the tendons. "New Reference to Medicinal Pieces" states that it can "remove blood stasis, generate new blood, and circulate the meridians." This is precisely what is meant by "tonifying blood and strengthening the body's health, dispelling blood stasis and unblocking the meridians." These four herbs, when used together with their cold and warm properties, dispel dampness and cold, clear heat and dampness, harmonize blood and unclog the meridians, and synergistically aid the main herb in dispelling pathogenic factors.

[0060] Auxiliary drugs: Clematis chinensis, mustard seeds, white peony root, Rehmannia glutinosa, Chinese angelica, myrrh, and Cyperus rotundus.

[0061] Clematis root: Spicy and salty in flavor, warm in nature, it dispels wind and dampness, unblocks meridians, and relieves pain. Clematis root can dispel wind and rheumatism, unblock meridians, and relieve pain. It is a key herb for treating stiffness and spasms in the tendons and vessels of arthritis syndrome. "Compendium of Materia Medica" states: "Clematis root treats all kinds of wind and is the most suitable wind-clearing herb for promoting mobility." It is combined with Gentiana macrophylla and Stephania tetrandra to enhance its wind-clearing and dampness-removing effects, leveraging its mobility to unblock meridians and relieve pain. Mustard seed: Pungent in flavor and warm in nature, it clears and dissipates phlegm accumulation internally and externally, dispelling phlegm and unblocking the meridians, promoting qi and dispersing stagnation. It can treat phlegm-dampness and swelling, pain, and numbness in the limbs. "Compendium of Materia Medica" states: "Its pungent nature penetrates the lungs, and its warm nature disperses the exterior. Phlegm trapped beneath the ribs and outside the skin and membranes can be cleared with this pungent and warming property, clearing the interior and exterior, eliminating the risk of stagnation." "Compendium of Materia Medica" states: "It promotes qi and dissipates phlegm, dispels cold and warms the middle, dissipates swelling and relieves pain. It can also treat asthma, cough, nausea, numbness, beriberi, and pain in the tendons, bones, and waist joints." It also warms the interior and dispels cold, aided by aconite to enhance its cold-dispersing properties. White peony root: Bitter, sour, and cooling in nature, it nourishes blood, unblocks the meridians, and relieves pain. "Shennong's Classic of Materia Medica" states that it "removes blood stasis," while "Bielu" states that it "unblocks the blood vessels." Rehmannia root: Sweet in flavor and cold in nature, it nourishes blood and yin, unclogs blood vessels, and dispels blood stasis. Its yin-nourishing properties complement warming herbs like Heishun Pian and Millettia reticulata, working together with white peony root to nourish and activate blood circulation, relieving blood stasis and numbness. Angelica root: Sweet and pungent in flavor and warm in nature, it treats wind by first treating blood, and blood circulation dissipates wind. It nourishes blood, activates blood circulation, and unclogs the meridians. "Compendium of Materia Medica" states: "Danggui, with its sweet and pungent flavor, specifically nourishes blood. Its light and pungent qi also circulates blood, tonifying while moving, activating while nourishing. It is truly a qi-boosting herb and a sacred herb for the blood... When used with Millettia reticulata and Rehmannia root, it dispels pain, promotes bowel movements, and strengthens the tendons and bones." Its combination with Millettia reticulata and Rehmannia root further implies that treating wind requires first treating blood, and blood circulation dissipates wind. Vinegar-infused myrrh: Bitter in flavor and neutral in nature, it activates blood circulation, unclogs the meridians, and relieves pain. "Compendium of Materia Medica" states: "Long-term use relaxes fascia and unclogs blood vessels." "Compendium of Materia Medica" states: "Myrrh generally relieves stagnant blood." Cyperus rotundus: Spicy, slightly sweet, and neutral in nature, it promotes qi circulation, relieves depression, and relieves pain. Qi is the leader of blood, and when qi moves, blood moves. Cyperus rotundus promotes qi circulation and activates blood circulation, working together with myrrh to regulate qi, activate blood circulation, and unclog the meridians.

[0062] Guiding herbs: Cinnamon Twig and Rhizoma Cynoglossi. Cinnamon Twig: Spicy, sweet, and warm in nature, it dredges the meridians, enhancing its ability to warm and dredge the collaterals. As stated in the Ben Jing Shu Zheng (Explanation of Symptoms in the Classic of Compendium of Materia Medica), "Cinnamon Twig can benefit the joints, warm the meridians, and dredge the blood vessels; this is its essence." It can also dispel wind and cold and expel external pathogens. The Changsha Yao Jie (Explanation of Medicinal Herbs) states that it "excels in relieving wind pathogens, regulates wood energy, relieves muscle and vein spasms, and relieves joint congestion... It dredges the meridians and relieves numbness and stiffness, and even dispels dampness and cold." It is used as a guiding herb in this formula, also for its ability to guide the herbs upward. Rhizoma Cynoglossi: Sweet, slightly bitter, and neutral in nature, it dispels wind and dampness, dredges the joints, activates blood circulation, and dredges the collaterals, relieving muscle and bone pain. The Ben Jing states that it "treats cold and dampness-induced paralysis, stiffness and cramps in the limbs, and knee pain that cannot be bent." It is used as a guiding herb in this formula, also for its ability to guide the herbs downward. Together, these two herbs guide the other herbs to the upper and lower joints, dredge the collaterals, and relieve pain, thus serving as guiding herbs.

[0063] The combination of these herbs combines cold and warm properties, combining healing and nourishment. They combine wind-dispelling, cold-dispelling, dampness-clearing, heat-clearing, qi-invigorating, blood-activating, phlegm-removing, and collateral-drying properties in one prescription, aiming to "dispel pathogenic factors, relieve numbness, and replenish qi and unclog the collaterals." This allows the pathogenic factors of wind, cold, dampness, phlegm, blood stasis, and heat to dissipate, restoring vital energy, removing phlegm and blood stasis, relieving numbness, and unblocking the collaterals, allowing qi and blood to flow freely, ultimately leading to a natural recovery.

[0064] The pharmaceutical composition of the present invention has the effects of dispelling wind, cold and dampness, invigorating qi, removing blood stasis and dredging collaterals, and is mainly used to treat rheumatoid arthritis, wind-cold-dampness arthritis, qi deficiency and collateral stasis, and phlegm and blood stasis syndrome.

[0065] Traditional decoctions are difficult to prepare, have a poor taste, and are unstable in quality. They are also inconvenient for patients to carry and take, significantly limiting their use. Therefore, they are often formulated into capsules, tablets, granules, oral liquids, or other dosage forms. Tablets, in particular, require fewer excipients than other dosage forms, are easier to form, are smaller in size, offer accurate dosages, and are more convenient to carry, transport, and take.

[0066] In addition, tablets are dry solids with stable and controllable quality. Certain drugs that are easily oxidized, deteriorated, and deliquesced can be protected by coating. Light, air, moisture, etc. have little effect on them, and the unpleasant odor of traditional Chinese medicine can also be covered up.

[0067] Furthermore, tablet production is highly mechanized and automated, making it suitable for industrial promotion. In order to better maximize the social and economic value of the drug, we combine the properties of the Chinese medicine in the prescription and prefer tablets as the dosage form of this prescription.

[0068] During the research process for the preparation process, we approached the process selection from multiple perspectives. We first conducted a separate study of the medicinal properties of Astragalus membranaceus. Astragalus membranaceus primarily contains triterpenoid saponins, flavonoids, and polysaccharides. Modern research indicates that Astragalus membranaceus decoctions possess significant tonic effects, and that saponins, flavonoids, and polysaccharides are all active ingredients that are readily soluble in hot water. Therefore, water decoction is preferred. Gentiana macrophylla: It primarily contains iridoid glycosides such as gentiopicroside and danshinone. These iridoid glycosides are readily soluble in water and ethanol, and the alcohol extract of Gentiana macrophylla exhibits significant anti-inflammatory properties. Based on the results of solvent research, we chose to use 70% ethanol for extraction. Stephania tetrandra: It primarily contains tetrahydroisoquinoline alkaloids such as teridine, fangchinoline, and cyclopentane. Stephania tetrandra alkaloids exhibit analgesic and anti-inflammatory properties. They are not readily soluble in water but readily soluble in organic solvents such as ethanol. Based on the results of solvent research, we chose to use alcohol for extraction. However, considering that the ethanol-extracted Radix Paeoniae Alba and Radix Polygoni Cuspidati contain significant amounts of tannins and anthraquinones, these components can easily precipitate with alkaloids and be lost. Experimental investigations have shown that Stephania Tetraphyllae is easily pulverized, and the fine powder, when used as an excipient, yields better formulation results. Furthermore, since Stephania Tetraphyllae accounts for a relatively small proportion of the formula, pulverization before addition to the formula prevents excessive dosage. Furthermore, pulverizing Stephania Tetraphyllae retains its active ingredients while reducing the amount of excipients required for formulation, making it more convenient for patients. Therefore, pulverizing Stephania Tetraphyllae is appropriate for this prescription. Aconite root: Primarily contains diterpenoid diester alkaloids. Aconite root decoction exhibits inhibitory effects in various animal models of acute and chronic inflammation, so decoction is recommended. Millettia reticulata: Primarily contains isoflavones, flavonoids, triterpenes, and sterols. Its primary alcohol-soluble components are extracted using 70% ethanol. Angelica sinensis: Primarily contains volatile oils, organic acids, and polysaccharides. Modern research indicates that the volatile oil in angelica sinensis has effects such as inhibiting uterine smooth muscle contractions. Currently, angelica sinensis volatile oil is primarily used to treat dysmenorrhea and menstrual irregularities. The angelica sinensis in this recipe primarily activates blood circulation. The ferulic acid contained in it has anti-platelet aggregation and anti-thrombotic properties, making it the primary component that activates blood circulation. Based on solvent evaluation, 70% ethanol was used for extraction. Clematis chinensis: It primarily contains oleanane-type triterpenoid saponins. Saponins are readily soluble in water, so water decoction is preferred. Mustard seeds: It primarily contains various isothiocyanates such as sinapines and their glycosides, fatty acids, nitrogen-containing compounds, and amino acids. Modern research indicates that sinapines thiocyanates have anti-inflammatory properties. Ethanol extraction yields are high. Based on solvent evaluation, 70% ethanol was used for extraction. Rehmannia glutinosa: It primarily contains iridoid glycosides and polysaccharides and is readily soluble in water, so water decoction is preferred. White Peony Root: It primarily contains various glycosides such as paeoniflorin, hydroxypeoniflorin, and benzoylpeoniflorin, as well as tannins. Paeoniflorin has the effects of inhibiting platelet aggregation, relieving pain, and combating inflammation. Its extraction rate is high in ethanol. Based on the solvent investigation results, 70% ethanol was used for extraction. Myrrh: It primarily contains volatile oils, resins, and gums.Myrrh in the formula is used to promote blood circulation and relieve pain. Modern research shows that both its volatile oil and decoction prolong blood clotting time. Myrrh volatile oil also has anti-inflammatory properties, so steam distillation is used to extract the volatile oil. The distilled aqueous solution is then combined with the decoction. Cyperus rotundus primarily contains volatile oil, which primarily contains sesquiterpenes such as α-cyperone and its oxides. The volatile oil and decoction also have analgesic properties, so steam distillation is used to extract the volatile oil. The aqueous solution is then combined with the decoction. Polygonum cuspidatum primarily contains polydatin, anthraquinones, and tannins. Polydatin and anthraquinones have a high extraction rate in ethanol. Based on solvent research, 70% ethanol was used for extraction. Cinnamon twigs primarily contain volatile oils and water-soluble components such as trans-cinnamic acid and protocatechuic aldehyde. The main component of the oil is cinnamaldehyde, which has anti-inflammatory properties. Combining cinnamon twigs with astragalus root decoction significantly enhances the anti-inflammatory and analgesic effects of astragalus root. Therefore, steam distillation is used to extract the volatile oils, and the distilled aqueous solution is then combined with the decoction. Cyathulae root contains sterols. The decoction significantly improves microcirculation and has anti-inflammatory effects. Therefore, decoction is preferred.

[0069] The extraction of volatile oil in the prescription was investigated as follows:

[0070] 1. Investigation on extraction of volatile oil from cassia twig: Weigh 200 g of each of 3 pieces of cassia twig slices, add 8 times the amount of water, and extract the volatile oil by steam distillation. Record the volume of volatile oil every 1 hour and observe the oil production.

[0071] Oil yield: Light yellow oil, slowly separating, with a white emulsion evaporating. The average oil yield was 0.79%. The volatile oil content stopped increasing after 6 hours of extraction, indicating that extraction was essentially complete.

[0072] 2. Investigation of Cyperus rotundus volatile oil extraction: Take an appropriate amount of Cyperus rotundus slices and coarsely crush them into 3-5 mm particles and particles smaller than 3 mm. Weigh 200 g of each of the Cyperus rotundus slices, 3-5 mm particles, and particles smaller than 3 mm in triplicate. Add 8 times the volume of water and extract the volatile oil by steam distillation. Record the volatile oil volume every hour and observe the oil yield.

[0073] Oil yield: Light brown-yellow oil that easily separates. Direct extraction from slices yields a low yield, averaging 0.34%. Particles 3-5 mm and smaller than 3 mm show similar yields, averaging 0.54%. Therefore, coarsely crushing the Cyperus rotundus into particles smaller than 5 mm is optimal for oil extraction. The volatile oil content of Cyperus rotundus of varying particle sizes remained stable after 7 hours of extraction, indicating near-complete extraction.

[0074] 3. Investigation of the extraction of volatile oil from vinegar myrrh: Weigh 200 g of each of 3 portions of vinegar myrrh, add 8 times the amount of water, and extract the volatile oil by steam distillation. Record the volume of volatile oil every hour and observe the oil production phenomenon.

[0075] Oil yield: Light green oil that easily separates. Average oil yield is 1.35%. Volatile oil content stops increasing after 7 hours of extraction, indicating near-complete extraction.

[0076] In order to illustrate the efficacy of the pharmaceutical composition of the present invention, functional tests were conducted on samples prepared in the examples at the Beijing University of Chinese Medicine.

[0077] Test example

[0078] 1. Materials and methods

[0079] 1.1 General Information

[0080] In order to identify the efficacy of the pharmaceutical composition of the present invention, the granules before tableting of Example 1 and the granules before capsule filling of Example 2 were used as test samples, as well as the granules before tableting prepared in a comparative example with the same medicinal materials outside the scope of protection of the claims, and the Beijing University of Chinese Medicine Health Analysis and Testing Center was commissioned to conduct an efficacy evaluation experiment.

[0081] The pharmaceutical composition tablets of the present invention are clinically indicated for the treatment of rheumatoid arthritis. This study investigated the efficacy of the pharmaceutical composition tablets of the present invention using rat models of adjuvant arthritis (AA) and type II collagen-induced arthritis (CIA). AA is a relatively ideal arthritis model that closely resembles human rheumatoid arthritis, primarily mediated by cellular immunity. Its symptoms and pathological manifestations are similar to those of human rheumatoid arthritis, but their pathogenesis differs. The pathogenesis of the type II collagen-induced arthritis animal model is closer to that of human RA. Therefore, this study selected these two models for research. The AA model primarily evaluated the efficacy of the pharmaceutical composition of the present invention when administered prophylactically. The CIA model also evaluated the efficacy of both prophylactic and therapeutic administration, and preliminary studies were conducted on the therapeutic efficacy mechanism of the pharmaceutical composition of the present invention, providing experimental evidence for the clinical treatment of rheumatoid arthritis. The research results were summarized in accordance with the "Regulations on the Approval of New Drugs," the "Guidelines for the Research of New Traditional Chinese Medicines," and the "Regulations on Drug Registration and Administration."

[0082] Experiment 1: Preventive and therapeutic effects of the pharmaceutical composition of the present invention on adjuvant-induced arthritis in rats

[0083] In the first experiment, SD rats were used to prepare adjuvants to induce rat arthritis, and the preventive and therapeutic effects of the pharmaceutical composition of the present invention on this model were evaluated mainly by observing the volume changes of the contralateral toes of the immunized toes and the changes in joint histology.

[0084] Experimental Materials

[0085] 1. Animals: SD rats, both male and female, weighing 180-200 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. License No. SCXK (Beijing) 2006-0009.

[0086] 2. Drugs and reagents:

[0087] (1) Test Drug: The pharmaceutical composition of the present invention was provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd., with an adult dosage of 17 g of crude drug per day. Pharmaceutical compositions 1 and 2 of the present invention and the control drug group were yellow-brown granules, each gram equivalent to 3.5 g of crude drug. The pharmaceutical composition of the present invention was prepared with 0.5% sodium carboxymethylcellulose to the desired concentration.

[0088] (2) Positive control drug: Dexamethasone tablets, Tianjin Lisheng Pharmaceutical Co., Ltd., batch number 20090105. Specification: 0.75 mg / tablet. Oral administration: The starting dose for adults is 0.75-3.00 mg (1-4 tablets) once, 2-4 times a day. The maintenance dose is approximately 0.75 mg (1 tablet) per day, depending on the condition.

[0089] (3) Incomplete Freund's adjuvant (IFA) was a Sigma product, batch number 62K8928.

[0090] (4) BCG was purchased from the National Institute for the Control of Pharmaceutical and Biological Products, batch number 20110130.

[0091] (5) PRMI1640 culture medium: GIBCO product, batch number 1024455. Before use, add 100u / ml penicillin, 100u / ml streptomycin, and 2mM glutamine to the 1640 culture medium, and add 10% inactivated fetal bovine serum to the complete 1640 culture medium.

[0092] (6) ConA (lot number 71K7024) and MTT (lot number 66H5033) are both products of Sigma.

[0093] (7) Sodium dodecyl sulfate (SDS): Serva product, batch number 20760.

[0094] (8) NN-dimethylformamide (DMF): a product of Amresco.

[0095] 3. Instruments:

[0096] (1) The YLS-7B toe volume measuring instrument is a product of Huaibei Zhenghua Biological Instrument Co., Ltd.

[0097] (2) Calaxy S CO2 incubator, RS Biotech.

[0098] (3) SCR20BA refrigerated centrifuge, Hitachi, Japan.

[0099] (4) Biological microscope, Olympus, Japan.

[0100] (5) DNM microplate reader, Beijing Prolon New Technology Co., Ltd.

[0101] (6) Sakura push-pull slicer, Japan.

[0102] Methods and Results

[0103] (I) Effect of the pharmaceutical composition of the present invention on the toe volume of adjuvant-induced arthritis in rats

[0104] 1. Preparation of Freund's Complete Adjuvant (CFA): Add BCG to incomplete Freund's adjuvant (IFA) to a final concentration of 10 mg / ml and repeatedly pump to form an emulsion.

[0105] 2. Preparation of rat adjuvant arthritis (AA) model: 100 μl of the above emulsion was injected subcutaneously into the left plantar of each rat to induce adjuvant arthritis. The normal control group received no treatment.

[0106] 3. Animal Grouping and Dosing: SD rats were randomly divided into six groups. The normal control group received an equal volume of vehicle; the adjuvant arthritis model group received an equal volume of vehicle; the present invention's Pharmaceutical Composition 1, the present invention's Pharmaceutical Composition 2, and the comparative drug group received oral administration of 2.26 g / kg, equivalent to 8 times the adult daily dose, starting the day after model preparation; and the positive control group received 0.1 mg / kg of dexamethasone, equivalent to 8 times the adult daily dose. The dosing volume for each group was 1 ml / 100 g body weight.

[0107] 4. Effect of the pharmaceutical composition of the present invention on the paw volume of rats with adjuvant arthritis: Before inflammation was induced, the paw volume of each rat was measured using a paw volume meter. Starting after immunization, the volume of the paw was measured every 4 days until the 30th day. Changes in paw volume were observed in each group of rats. Data were analyzed using SPSS 10.0 software, and all data are expressed as mean ± standard deviation. The significance of the difference was analyzed using one-way analysis of variance (One-Way ANOVA), and the LSD test was used for pairwise comparison between groups. P<0.05 was considered statistically significant. The results showed that from 11 days after immunization to the end of the experiment, the contralateral paws of rats in the adjuvant model began to swell gradually. The maximum toe volume occurred on the 23rd day after immunization. Thereafter, as the inflammation subsided, the toe volume gradually decreased. From 15 days after immunization to the end of the experiment, the toe volume of the model group was significantly greater than that of the normal control group (p<0.05 or p<0.01). From 15 days after immunization to the end of the experiment, the toe volume of the dexamethasone group was significantly smaller than that of the model control group (p<0.01). The present invention's pharmaceutical composition group 1 significantly reduced paw volume compared to the model group starting 15 days after immunization, and this reduction persisted through 30 days (p < 0.05). The present invention's pharmaceutical composition group 2 showed significant reductions compared to the model control group at 15, 19, 27, and 30 days after immunization (p < 0.05). The control group showed a trend toward reduced paw swelling, but this trend was not statistically significant. The results are shown in Tables 1-1 and 1-2.

[0108] Table 1-1: Effects of the pharmaceutical composition of the present invention on contralateral paw swelling in rats with adjuvant arthritis

[0109]

[0110] Note: Compared with the control group, △ p<0.05, △△ Compared with the model group, *p<0.05, **p<0.01, the drug composition groups 1 and 2, and the dexamethasone group showed significant differences compared with the model group.

[0111] Table 1-2: Effects of the pharmaceutical composition of the present invention on contralateral paw swelling in rats with adjuvant arthritis

[0112]

[0113] Note: Compared with the control group, △ p<0.05, △△ Compared with the model group, *p<0.05, **p<0.01, the drug composition groups 1 and 2, and the dexamethasone group showed significant differences compared with the model group.

[0114] (II) Effect of the pharmaceutical composition of the present invention on the proliferation of spleen T lymphocytes in rats with adjuvant-induced arthritis

[0115] 1. Preparation of adjuvant arthritis model, animal grouping and drug administration: same as experiment 1 (I).

[0116] 2. Effect of the pharmaceutical composition of the present invention on the proliferation of splenic T lymphocytes in rats with adjuvant arthritis: At the end of the experiment, rats in each group were killed, and the spleens were aseptically removed and separated into single cells. The red blood cells were destroyed by hypotonic method, and the splenic lymphocyte count was adjusted to 5×109 / L. The cells were added to a 96-well culture plate, 100 μl per well, and then 1640 complete culture medium containing ConA (final concentration of 5 μg / ml) was added, 100 μl / well, and cultured for 72 hours. 20 μl of 5 mg / ml MTT solution was added to each well 4 hours before the end of the culture. After the end of the culture, 50 μl of triple dissolution solution (20% SDS-45% DMF, HCl adjusted to pH 2.0) was added to each well and incubated at room temperature overnight. After the formazan particles were dissolved, the colorimetry was performed at a wavelength of 570 nm. SPSS 10.0 software was used for data analysis, and all data results are expressed as mean ± standard deviation. The significance of differences was determined using one-way analysis of variance (ANOVA). Pairwise comparisons between groups were performed using the LSD test, with P < 0.05 considered statistically significant. The results showed that splenic T lymphocyte proliferation in the adjuvant model group was significantly increased compared to the normal group (P < 0.01). Dexamethasone significantly inhibited splenic T lymphocyte proliferation in the adjuvant model rats (P < 0.01). Pharmaceutical compositions 1 and 2 of the present invention also significantly inhibited splenic T lymphocyte proliferation in rats with adjuvant arthritis (P < 0.01 and P < 0.05, respectively). See Table 2.

[0117] Table 2: Effect of the pharmaceutical composition of the present invention on lymphocyte proliferation in rats with adjuvant arthritis

[0118]

[0119] Note: Compared with the control group, △ p<0.05, △△ Compared with the model group, *p<0.05, **p<0.01, the drug composition groups 1 and 2, and the dexamethasone group showed significant differences compared with the model group.

[0120] (III) Effect of the pharmaceutical composition of the present invention on the pathomorphology of joint tissues in rats with adjuvant arthritis

[0121] 1. Preparation of adjuvant arthritis model, animal grouping and drug administration: same as experiment 1 (I).

[0122] 2. Effect of the pharmaceutical composition of the present invention on the pathomorphological changes of joint tissue in rats with adjuvant arthritis: Rats with AA were decapitated 30 days after modeling, and the right ankle joints were removed. The rats were fixed in formalin, embedded in paraffin, and sectioned for HE staining. Results showed that the matrix of the joint capsule connective tissue in the normal group was colloidal, primarily composed of collagen fibers, with few small blood vessels, and a small number of macrophages, plasma cells, mast cells, and lymphocytes. The synovial tissue in the joints contained one to two layers of synovial cells, with mature synovial tissue and a few small blood vessels visible beneath the synovium. The synovial cells were neatly arranged and flattened, the joint surface was smooth, and the joint cavity was free of exudate. Skeletal muscle cells in the joints of the normal group were strip-like or clumpy, with minimal connective tissue between the muscle cells, and occasional fibroblasts and macrophages were observed within the connective tissue. In the model group, the connective tissue in the joint capsule of the rats contained macrophages, plasma cells, and mast cells, with inflammatory cells present in clusters, occupying a significant portion of the tissue space. In the model group, the synovial tissue was significantly thickened, with marked synovial cell proliferation, a significant increase in the number of cell layers, and cellular swelling. Infiltration by numerous lymphocytes, plasma cells, macrophages, and a small number of polymorphonuclear leukocytes was observed. Fibrous tissue proliferated, and small blood vessels increased significantly. The synovial layer adhered to the cartilage, leading to cartilage degeneration. Fibrous exudates were observed within the joint cavity, and synovium invaded the cartilage, causing destruction of both cartilage and bone, often manifesting as proliferative inflammation. Connective tissue between skeletal muscle cells was less abundant, but more abundant between muscle bundles. Fibroblasts and macrophages were occasionally seen within the interfascicular connective tissue, but inflammatory cells were often present in clusters, indicating exudative inflammation. In the dexamethasone group, a small number of inflammatory cells were scattered within the connective tissue matrix of the joint capsule; connective tissue between skeletal muscle cells was less abundant, with no prominent fibroblasts or macrophages, indicating normal tissue morphology. Normal synovial cells within the synovial tissue consisted of approximately one to three layers, arranged neatly with regular morphology. Infiltration of inflammatory cells and pannus formation were absent. In Group 1, the joint capsule connective tissue was essentially normal, with few inflammatory cells and less intermuscular connective tissue. The cartilage tissue on both sides of the joint was smooth, with approximately 1-3 layers of synovial cells, similar to the normal group, and a small infiltration of inflammatory cells. In Group 2, the joint capsule connective tissue had scattered inflammatory cells and small blood vessels, with few macrophages, plasma cells, and mast cells. Connective tissue between skeletal muscle cells was less abundant, but more abundant between muscle bundles, with occasional fibroblasts and macrophages in the connective tissue. A small amount of fibrous tissue was attached to the surface of the articular cartilage tissue, and the intercellular matrix contained numerous bundled collagen fibers. In the control group, the joint capsule connective tissue had macrophages, plasma cells, and mast cells, with inflammatory cells present in clusters, occupying some tissue spaces. Connective tissue between skeletal muscle cells was less abundant, with occasional fibroblasts and macrophages in the connective tissue. The articular cartilage tissue was thin in some areas, stained darkly, and had unclear boundaries at the junction with the osteoblastic interstitium. Synovial cells were hyperplastic, and fibrous tissue was present between the cartilage.

[0123] summary

[0124] (1) The contralateral paws of rats with adjuvant arthritis began to swell gradually from 11 days after modeling until the end of the experiment. The maximum toe volume occurred on the 23rd day after immunization. Thereafter, as the inflammation subsided, the toe volume gradually decreased. From 15 days after modeling until the end of the experiment, the toe volume of the model group was significantly greater than that of the normal control group (p < 0.05 or p < 0.01). The proliferation of spleen T lymphocytes in the adjuvant model group was significantly increased compared with the normal group (p < 0.01), indicating that the T cell immunity of the model rats was enhanced. Histology showed that there were a large number of inflammatory cells in the connective tissue of the joint capsule of rats with adjuvant arthritis, and the synovial cells of the joints proliferated significantly. Infiltration of a large number of lymphocytes, plasma cells, macrophages and a small number of polymorphonuclear leukocytes was also observed, fibrous tissue proliferated, and small blood vessels increased significantly.

[0125] (2) The toe volume of the positive control group, dexamethasone, was significantly smaller than that of the model control group from 15 days after immunization until the end of the experiment (p < 0.01). It also significantly inhibited the proliferation of splenic T lymphocytes in the adjuvant model rats (p < 0.01). Histology showed that there were a small number of inflammatory cells scattered in the connective tissue of the joint capsule, and the synovial cells were approximately 1-3 layers, arranged neatly, with regular cell morphology, and no obvious inflammatory cell infiltration was observed.

[0126] (3) The pharmaceutical composition group 1 of the present invention was able to significantly reduce the toe volume of rats compared with the model group starting from 15 days after immunization and continued to 30 days (p < 0.05). The pharmaceutical composition group 2 of the present invention was significantly reduced compared with the model control group at 15 days, 19 days, 27 days and 30 days after immunization (p < 0.05). The pharmaceutical composition groups 1 and 2 of the present invention were also able to significantly inhibit the proliferation of splenic T lymphocytes in rats with adjuvant arthritis model (p < 0.01 and p < 0.05). Histological results showed that the connective tissue of the joint capsule of the pharmaceutical composition groups 1 and 2 of the present invention was basically normal, with a small amount of inflammatory cells, and the surface of the articular cartilage tissue was basically normal or slightly damaged, with a small amount of inflammatory cell infiltration and fibrous tissue.

[0127] Experiment 2: Effect of preventive administration of the pharmaceutical composition of the present invention on type II collagen-induced arthritis in rats

[0128] In the second experiment, SD rats were used to prepare a type II collagen-induced rat arthritis model, and the preventive and therapeutic effects of the pharmaceutical composition of the present invention on this model were evaluated mainly by observing the immune arthritis index, changes in the right toe volume, and joint histological changes.

[0129] Experimental Materials

[0130] 1. Animals: SD rats, both male and female, weighing 180-200 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. License No. SCXK (Beijing) 2006-0009.

[0131] 2. Drugs and reagents:

[0132] (1) Test Drug: The pharmaceutical composition of the present invention was provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd., with an adult dosage of 17 g / crude drug / day. Compositions 1 and 2 of the present invention and the control drug group were yellow-brown granules, each gram equivalent to 3.5 g of crude drug. Batch number: 110301. The pharmaceutical composition of the present invention was prepared with 0.5% sodium carboxymethylcellulose to the desired concentration.

[0133] (2) Positive control drug: Dexamethasone tablets, Tianjin Lisheng Pharmaceutical Co., Ltd., batch number 20090105. Specification: 0.75 mg / tablet.

[0134] (3) Incomplete Freund's adjuvant (IFA) (batch number 62K8928) and acid-soluble type II collagen (collagen II, 3806) (batch number 130435) are both Sigma products.

[0135] (4) BCG was purchased from the National Institute for the Control of Pharmaceutical and Biological Products, batch number 20110130.

[0136] 3. Instruments:

[0137] (1) The YLS-7B toe volume measuring instrument is a product of Huaibei Zhenghua Biological Instrument Co., Ltd.

[0138] (2) Sakura push-pull slicer, Japan.

[0139] Methods and Results

[0140] (1) Effect of the pharmaceutical composition of the present invention on the joint index of the rat arthritis model induced by type II collagen

[0141] 1. Preparation of Freund's Complete Adjuvant (CFA): Add BCG to incomplete Freund's adjuvant (IFA) to a final concentration of 7.5 mg / ml and repeatedly pump to form an emulsion.

[0142] 2. Preparation of Rat Collagen Arthritis Model: Bovine type II collagen was dissolved in 0.1 M glacial acetic acid at 4°C overnight. The mixture was then ground with CFA to form an emulsion containing 2 mg / ml of type II collagen. This emulsion was injected subcutaneously at multiple points in the left foot and tail base of the model group and the immunized rats in each treatment group (150 μl / rat) (equivalent to 150 μg of type II collagen per rat). On day 10, each rat received a booster injection of 150 μl of the same emulsion at the tail base. The normal control group received no treatment.

[0143] 3. Animal Grouping and Dosing: SD rats were randomly divided into six groups: a normal control group, which received an equal volume of vehicle; a collagen arthritis model group, which received an equal volume of vehicle; and a control group, which received oral administration of 2.26 g / kg of Pharmaceutical Composition 1, Pharmaceutical Composition 2, and dexamethasone starting the day after model preparation, at a dose of 8 times the adult daily dose. A positive control group received 0.1 mg / kg of dexamethasone, 8 times the adult daily dose. The dosing volume for each group was 1 ml / 100 g body weight.

[0144] 4. Effect of the pharmaceutical composition of the present invention on the arthritis index of rats with collagen arthritis: Starting from the 14th day after inflammation, each rat was scored using the joint scoring method. Ear: nodules and redness symptoms (0 points for absence, 1 point for presence); nose: redness and swelling (0 points for absence, 1 point for presence); tail: nodules (0 points for absence, 1 point for presence); front paw: inflammation of at least one joint (0 points for absence, 1 point for presence); right hind paw: swelling (0 points for absence, 1 point for mild, 2 points for moderate, 3 points for severe). The average score of each group of rats was the arthritis index. SPSS10.0 software was used for data analysis, and all data results were expressed as mean ± standard deviation. The significance of the difference was analyzed using one-way analysis of variance (One-Way ANOVA), and the LSD test was used for pairwise comparison between groups. P<0.05 was considered statistically significant. The results showed that about 14 days after the first immunization, the type II collagen model rats and the rats in each drug-treated group began to experience redness and swelling of the ears and joints. The redness and swelling of the hind toe joints spread to the forefoot and tail, and became increasingly severe. Obvious nodules appeared on the tails of some rats. It reached a peak around the 35th day, and then the joint swelling was slightly relieved and continued until the 50th day. The arthritis index showed that the type II collagen model group had significant differences compared with the normal control group from the 14th day to the end of the 50th day experiment, indicating that the model was successfully prepared. There were no significant differences between the groups administered with the pharmaceutical composition of the present invention and the model group from the start of immunization to 31 days. From 31 days to 43 days, the arthritis index in group 1 of the present invention was significantly reduced compared with the model group (p < 0.05 and p < 0.01); and from 35 days to 43 days, the arthritis index in group 2 of the present invention was significantly reduced compared with the model group (p < 0.05). This indicates that pharmaceutical compositions 1 and 2 of the present invention can inhibit the late-stage peak of inflammation in rats caused by type II collagen immunization. However, as the inflammation in the model group gradually subsided, there was no significant difference between the groups administered with the pharmaceutical composition of the present invention and the model group. From 18 days after immunization until the end of the experiment, the arthritis index in the dexamethasone group was significantly lower than that in the model group (p < 0.01). The results are shown in Tables 3-1 and 3-2.

[0145] Table 3-1: Effects of preventive administration of the pharmaceutical composition of the present invention on the arthritis index of collagen-induced arthritis in rats

[0146]

[0147] Note: Compared with the control group, △ p<0.05, △△ Compared with the model group, *p<0.05, **p<0.01, the drug composition groups 1 and 2, and the dexamethasone group showed significant differences compared with the model group.

[0148] Table 3-2: Effects of preventive administration of the pharmaceutical composition of the present invention on the arthritis index of collagen-induced arthritis in rats

[0149]

[0150] Note: Compared with the control group, △ p<0.05, △△ Compared with the model group, *p<0.05, **p<0.01, the drug composition groups 1 and 2, and the dexamethasone group showed significant differences compared with the model group.

[0151] (II) Effect of the pharmaceutical composition of the present invention on the toe volume of type II collagen-induced arthritis rat model

[0152] 1. Preparation of collagen-induced rat arthritis model, animal grouping and drug administration: same as Experiment 2 (I).

[0153] 2. Effect of the pharmaceutical composition of the present invention on the paw volume of rats with collagen-induced arthritis: Before induction of inflammation, the paw volume of each rat's right hind paw was measured using a paw volume meter. Starting from the 11th day after the first immunization, the volume of the paw was measured every 4 days until the 50th day. Changes in paw volume were observed in each group of rats. Data were analyzed using SPSS 10.0 software, and all data are expressed as mean ± standard deviation. The significance of the difference was analyzed using one-way analysis of variance (One-Way ANOVA), and the LSD test was used for pairwise comparisons between groups. P < 0.05 was considered statistically significant. The results showed that there was no significant difference in the paw volume among the groups from 11 days to 14 days after the first immunization (data not shown). From 18 days after the first immunization to the end of the experiment, the toe volume of the rats in the collagen arthritis model group was significantly higher than that of the rats in the control group, indicating that the model rats had an inflammatory reaction in the toes. The maximum toe volume occurred 35-39 days after immunization. Thereafter, as the inflammation subsided, the toe volume gradually decreased. From 18 days after the first immunization to the end of the experiment, the toe volume of the dexamethasone group was significantly smaller than that of the collagen arthritis model group (p < 0.01). Due to long-term use of dexamethasone, the weight of the rats in this group decreased, so that the toe volume was even smaller than that of the rats in the normal control group. Pharmaceutical composition group 1 of the present invention significantly reduced paw volume compared to the model group starting 22 days after immunization and this effect persisted until 43 days (p < 0.05). Pharmaceutical composition group 2 of the present invention significantly reduced paw volume compared to the model group starting 26 days after immunization and this effect persisted until 39 days (p < 0.05). However, as inflammation subsided in the model group, the inhibitory effects of pharmaceutical compositions 1 and 2 of the present invention on paw swelling were not significantly different from those in the model group. The inhibitory effects of the control drug groups on paw swelling were not significant. The results are shown in Tables 4-1 and 4-2.

[0154] Table 4-1: Effects of preventive administration of the pharmaceutical composition of the present invention on the volume of the right toes of rats with collagen-induced arthritis

[0155]

[0156] Note: Compared with the control group, △△ Compared with the model group, *p<0.05, **p<0.01, the drug composition groups 1 and 2, and the dexamethasone group showed significant differences compared with the model group.

[0157] Table 4-2: Effects of preventive administration of the pharmaceutical composition of the present invention on the volume of the right toes of rats with collagen-induced arthritis

[0158]

[0159] Note: Compared with the control group, △△ Compared with the model group, *p<0.05, **p<0.01, the drug composition groups 1 and 2, and the dexamethasone group showed significant differences compared with the model group.

[0160] (III) Effect of the pharmaceutical composition of the present invention on joint tissue pathology in rat arthritis model induced by type II collagen

[0161] 1. Preparation of collagen-induced rat arthritis model, animal grouping and drug administration: same as Experiment 2 (I).

[0162] 2. Effects of the pharmaceutical composition of the present invention on the pathomorphological changes of joint tissue in rats with collagen-induced arthritis: Rats in each experimental group were decapitated on day 50 after modeling, and the right ankle joints were removed. The rats were fixed in formalin, embedded in paraffin, and sectioned for HE staining. Results showed that in the normal group, connective tissue contained a small number of macrophages, plasma cells, mast cells, and lymphocytes, with minimal connective tissue between muscle cells. One to two layers of synovial cells were visible in the articular cartilage, the joint surface was smooth, and the joint cavity was free of exudate. In the collagen model group, inflammatory cell infiltration was observed in the joint connective tissue and muscle tissue, and neutrophils, monocytes, and especially lymphocytes were infiltrated into the synovial tissue of the articular cartilage. There was also increased vascularity, hyperplasia of the vascular intima, narrowing or obstruction of the lumen, and pannus formation. There was also extensive cellulose exudation beneath the synovial cells and collagen fiber deposition. In the dexamethasone group, the joint connective tissue and muscle tissue were essentially normal, with slight hyperplasia of synovial cells in the articular cartilage and no significant inflammatory cell infiltration. In Groups 1 and 2 of the present invention's drug combination, there was a small amount of inflammatory cell infiltration in the joint connective tissue, while muscle tissue was essentially normal. In the joint cavity, synovial cells showed mild hyperplasia, with visible inflammatory cell infiltration and a small amount of fibrosis, as well as mild vascular hyperplasia. In the control drug group, there was a greater infiltration of inflammatory cells in the joint connective tissue and muscle tissue, with synovial cell hyperplasia accompanied by inflammatory cell infiltration. The present drug combination in Groups 1 and 2 demonstrated a significant improvement in the tissue damage of collagen arthritis.

[0163] summary

[0164] (1) The arthritis index of the collagen-induced arthritis model rats began to be significantly higher than that of the normal control group around the 14th day after the first immunization, and the toe volume began to be significantly higher than that of the normal control group around the 18th day after the first immunization. The significant difference was maintained until the end of the experiment (p < 0.01). The arthritis index and toe volume of the model group reached a peak around the 35th to 39th day after the first immunization. Thereafter, as the inflammation subsided, the arthritis index and toe volume decreased. Pathological observation showed that the model rats had inflammatory cell infiltration in the connective tissue and muscle tissue, hyperplasia and inflammatory cell infiltration of the synovial tissue, increased blood vessels accompanied by pannus formation, large amounts of cellulose exudation under the synovial cells, and collagen fiber deposition. This indicated that the model was successful.

[0165] (2) The positive control drug dexamethasone significantly inhibited the arthritis index and reduced the toe volume starting from the 18th day after the first immunization, and its inhibitory effect was maintained until the end of the experiment. There were significant differences compared with the model group (p < 0.01). Pathological observation showed that the joint connective tissue and muscle tissue in the dexamethasone group were basically normal, the synovial cells of the articular cartilage were slightly proliferative, and the inflammatory cell infiltration was not obvious.

[0166] (3) The arthritis index of arthritis rats was significantly reduced in group 1 of the present invention's pharmaceutical composition from 31 days to 43 days after the first immunization, and in group 2 of the present invention from 35 days to 43 days after the first immunization (p < 0.05 and p < 0.01). Group 1 of the present invention was able to significantly reduce the toe volume of rats compared with the model group from 22 days after the first immunization, and the effect lasted until 43 days (p < 0.05). Group 2 of the present invention was able to significantly reduce the toe volume of rats compared with the model group from 26 days after the first immunization, and the effect lasted until 39 days (p < 0.05). Thereafter, as the inflammation of the model gradually subsided, there was no significant difference between the control drug group and the model group. Pathological results showed that there was a small amount of inflammatory cell infiltration in the joint connective tissue of groups 1 and 2 of the present invention's pharmaceutical composition, the muscle tissue was basically normal, the synovial cells in the joint cavity were slightly proliferative, inflammatory cell infiltration and a small amount of fibrosis were visible, and blood vessels were slightly proliferative.

[0167] Experiment 3 Effects and mechanisms of therapeutic administration of the pharmaceutical composition of the present invention on type II collagen-induced arthritis in rats

[0168] In Experiment 3, pure Lewis rats were used to prepare type II collagen-induced arthritis. The therapeutic effect of the pharmaceutical composition of the present invention on this model was evaluated by observing the arthritis index and right toe volume change, and its therapeutic mechanism was preliminarily studied through observation.

[0169] Experimental Materials

[0170] 1. Animals: Lewis rats, male, weighing 170-190 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Animal qualification certificate number: License No. SCXK (Beijing) 2006-0009.

[0171] 2. Drugs and reagents:

[0172] (1) Test Drug: The pharmaceutical composition of the present invention was provided by Shijiazhuang Yiling Pharmaceutical Co., Ltd., with an adult dosage of 17 g / crude drug / day. Compositions 1 and 2 of the present invention and the control drug group were yellow-brown granules, each gram equivalent to 3.5 g of crude drug. Batch number: 110301. The pharmaceutical composition of the present invention was prepared with 0.5% sodium carboxymethylcellulose to the desired concentration.

[0173] (2) Positive control drug: Dexamethasone tablets, Tianjin Lisheng Pharmaceutical Co., Ltd., batch number 20090105. Specification: 0.75 mg / tablet.

[0174] (3) Incomplete Freund's adjuvant (IFA) was a Sigma product, batch number 62K8928.

[0175] (4) Acid-soluble type II collagen (collagen II, 3806) (lot number 130435) and MTT (66H5033) were products of Sigma.

[0176] (5) BCG was purchased from the National Institute for the Control of Pharmaceutical and Biological Products, batch number 20100130.

[0177] (6) PRMI1640 culture medium: GIBCO product, batch number 1024455. Before use, add 100u / ml penicillin, 100u / ml streptomycin, and 2mM glutamine to the 1640 culture medium, and add 10% inactivated fetal bovine serum to the complete 1640 culture medium.

[0178] (7) Sodium dodecyl sulfate (SDS): Serva product, batch number 20760.

[0179] (8) NN-dimethylformamide (DMF): a product of Amresco.

[0180] (9) Rat IL-1 cytokine ELISA kit (batch number 1209145), TNF-α cytokine ELISA kit (batch number 1210132), and IL-10 cytokine ELISA kit (batch number 1210162) were purchased from Beijing Bondingtech Biotechnology Co., Ltd.

[0181] (10) Horseradish peroxidase (HRP)-labeled goat anti-rat IgG antibody (batch number 20111002) and goat anti-rat IgM antibody (batch number 20111015) were purchased from Beijing Boya Xiangda Biotechnology Co., Ltd.

[0182] 3. Instruments:

[0183] (1) The YLS-7B toe volume measuring instrument is a product of Huaibei Zhenghua Biological Instrument Co., Ltd.

[0184] (2) Calaxy S CO2 incubator, RS Biotech.

[0185] (3) SCR20BA refrigerated centrifuge, Hitachi, Japan.

[0186] (4) Biological microscope, Olympus, Japan.

[0187] (5) DNM microplate reader, Beijing Prolon New Technology Co., Ltd.

[0188] Methods and Results

[0189] (1) Effect of therapeutic administration of the pharmaceutical composition of the present invention on the joint index of collagen-induced arthritis rat model

[0190] 1. Preparation of rat type II collagen-induced arthritis model: same as experiment 2 (1).

[0191] 2. Animal Grouping and Dosing: Lewis rats were randomly divided into six groups: a normal control group, which received an equal volume of vehicle; a collagen-induced arthritis model group, which received an equal volume of vehicle; and a control group, which received pharmaceutical compositions 1 and 2 of the present invention by oral gavage starting on day 14 after model establishment, at a dose of 2.26 g / kg, equivalent to 8 times the adult daily dose; and a positive control group, dexamethasone, which received 0.1 mg / kg of dexamethasone, equivalent to 8 times the adult daily dose. The dosing volume for each group was 1 ml / 100 g body weight.

[0192] 3. Effect of the pharmaceutical composition of the present invention on the arthritis index of rats with collagen arthritis: Starting from the 14th day after inflammation, each rat was scored using the joint scoring method. Ear: nodules and redness symptoms (0 points for absence, 1 point for presence); nose: redness and swelling (0 points for absence, 1 point for presence); tail: nodules (0 points for absence, 1 point for presence); front paw: inflammation of at least one joint (0 points for absence, 1 point for presence); right hind paw: swelling (0 points for absence, 1 point for mild, 2 points for moderate, 3 points for severe). The average score of each group of rats was the arthritis index. SPSS10.0 software was used for data analysis, and all data results were expressed as mean ± standard deviation. The significance of the difference was expressed using one-way analysis of variance (One-Way ANOVA), and the LSD test was used for pairwise comparison between groups. P<0.05 was considered statistically significant. The results showed that the rats with type II collagen-induced arthritis model began to develop arthritis symptoms about 14 days after the first immunization, reaching a peak around the 35th day, and then the joint swelling was slightly relieved and continued until the 50th day. The arthritis index showed that the model group had significant differences compared with the normal control group from the 14th day to the end of the 50th day of the experiment, indicating that the model was successfully prepared. From the 22nd day after immunization with dexamethasone until the end of the experiment, its arthritis index was significantly lower than that of the model group (p < 0.05 or p < 0.01). The arthritis index of the pharmaceutical composition group 1 of the present invention was significantly reduced compared with the model group from the 35th day (21st day of administration) (p < 0.05 and p < 0.01) until the 47th day. From day 39 (25 days of dosing) to day 43, the severity of the disease in Group 2, a significant reduction was observed compared to the model group (p < 0.05). The control group also showed a decreasing trend, but the difference was not significant. This indicates that Groups 1 and 2 can inhibit the late peak of inflammation in rats induced by type II collagen immunization. However, as the inflammation in the model gradually subsided, no significant difference was observed between Groups 1 and 2 and the model group. The results are shown in Tables 5-1 and 5-2.

[0193] Table 5-1: Effects of the therapeutic administration of the pharmaceutical composition of the present invention on the arthritis index of rats with type II collagen-induced arthritis model

[0194]

[0195] Note: Compared with the control group, △△ Compared with the model group, *p<0.05, **p<0.01, the drug composition groups 1 and 2, and the dexamethasone group showed significant differences compared with the model group.

[0196] Table 5-2: Effects of the therapeutic administration of the pharmaceutical composition of the present invention on the arthritis index of rats with type II collagen-induced arthritis model

[0197]

[0198] Note: Compared with the control group, △△ Compared with the model group, *p<0.05, **p<0.01, the drug composition groups 1 and 2, and the dexamethasone group showed significant differences compared with the model group.

[0199] (II) Effect of the pharmaceutical composition of the present invention on the toe volume of type II collagen-induced arthritis rat model

[0200] 1. Preparation of collagen-induced rat arthritis model, animal grouping and drug administration: same as Experiment 3 (I).

[0201] 2. Effect of the pharmaceutical composition of the present invention on paw volume in a collagen-induced arthritis rat model: Before induction of inflammation, the paw volume of each rat's right hind paw was measured using a paw volume meter. Starting from the 14th day after the first immunization, the volume of the paw was measured every 4 days until the 50th day. Changes in paw volume were observed in each group of rats. Data were analyzed using SPSS 10.0 software, and all data are expressed as mean ± standard deviation. The significance of the difference was analyzed using one-way analysis of variance (One-Way ANOVA), and the LSD test was used for pairwise comparison between groups. P < 0.05 was considered statistically significant. The results showed that there was no significant difference in the paw volume of each group 14 days after immunization (data not shown). From 18 days after immunization to the end of the experiment, the toe volume of the model group rats was significantly higher than that of the control group rats, indicating that the rats in the type II collagen-induced arthritis model had an inflammatory response in the toes. The maximum toe volume occurred 31-39 days after immunization. Thereafter, as the inflammation subsided, the toe volume decreased. From 22 days after immunization (8 days after administration) to the end of the experiment, the toe volume of the dexamethasone group was significantly smaller than that of the arthritis model group (p < 0.05 or p < 0.01). The present invention's pharmaceutical composition group 1 significantly reduced the paw volume of rats compared to the model group starting 35 days after immunization (21 days after administration) and this effect persisted until 47 days (p < 0.05). The present invention's pharmaceutical composition group 2 significantly reduced the paw volume of rats compared to the model group starting 39 days after immunization (25 days after administration) and this effect persisted until 50 days (p < 0.05). The control group did not show a significant inhibitory effect on paw swelling. The results are shown in Tables 6-1 and 6-2.

[0202] Table 6-1: Effects of the pharmaceutical composition of the present invention on the volume of the right toes in rats with type II collagen-induced arthritis

[0203]

[0204] Note: Compared with the control group, △△ Compared with the model group, *p<0.05, **p<0.01, the drug composition groups 1 and 2, and the dexamethasone group showed significant differences compared with the model group.

[0205] Table 6-2: Effects of the pharmaceutical composition of the present invention on the volume of the right toes in rats with type II collagen-induced arthritis

[0206]

[0207] Note: Compared with the control group, △△ Compared with the model group, *p<0.05, **p<0.01, the drug composition groups 1 and 2, and the dexamethasone group showed significant differences compared with the model group.

[0208] (III) Effect of the pharmaceutical composition of the present invention on the proliferation of type II collagen-specific splenic lymphocytes in the collagen-induced arthritis model 1. Preparation of collagen-induced arthritis model, animal grouping and drug administration: Same as experiment 3 (I).

[0209] 2. Effect of the pharmaceutical composition of the present invention on the proliferation of splenic lymphocytes in a collagen-induced rat arthritis model: At the end of the reference experiment, rats in each group were sacrificed, and the spleens were aseptically removed and separated into single cells. Red blood cells were destroyed by hypotonic method, and the splenic lymphocyte concentration was adjusted to 5×109 / L. The cells were added to a 96-well culture plate, 100 μl per well, and 100 μl / well of complete culture medium containing type II collagen was added to make the final concentration of type II collagen 2.5 μg / ml. The cells were cultured for 72 hours. 4 hours before the end of the culture, 20 μl of 5 mg / ml MTT solution was added to each well. After the culture was completed, 50 μl of triple lysis solution (20% SDS-45% DMF, HCl adjusted to pH 2.0) was added to each well and incubated at room temperature overnight. After the formazan particles were dissolved, the colorimetric analysis was performed at a wavelength of 570 nm. SPSS 10.0 software was used for data analysis, and all data results are expressed as mean ± standard deviation. The significance of the difference was expressed using one-way analysis of variance (One-Way ANOVA), and the LSD test was used for pairwise comparison between groups. P < 0.05 was considered statistically significant. The results showed that the proliferation capacity of rat spleen lymphocytes stimulated by type II collagen was significantly higher than that of the normal group (p < 0.01), indicating that type II collagen-specific lymphocytes were activated in the model mice and that type II collagen-specific cellular immune responses existed in the model mice. Dexamethasone significantly inhibited the proliferation response of spleen lymphocytes induced by type II collagen (p < 0.01). The drug combination of the present invention, groups 1 and 2 significantly inhibited the proliferation response of type II collagen-specific lymphocytes in the spleen of collagen model rats (p < 0.05 or p < 0.01).

[0210] Table 7: Effect of the pharmaceutical composition of the present invention on the proliferation of spleen lymphocytes in rats with collagen arthritis

[0211]

[0212] Note: Compared with the control group, △△ Compared with the model group, the group consisting of the pharmaceutical composition of the present invention and the dexamethasone group, *p<0.05, **p<0.01.

[0213] (IV) Effect of the pharmaceutical composition of the present invention on serum anti-type II collagen antibodies in rats with collagen-induced arthritis model

[0214] 1. Preparation of collagen-induced arthritis model, animal grouping and drug administration: same as experiment 3 (I).

[0215] 2. Effect of the pharmaceutical composition of the present invention on type II collagen antibodies in the serum of collagen-induced rat arthritis model: At the end of the experiment, blood was collected from the abdominal aorta of the rats after anesthesia, and the serum was separated. The anti-type II collagen antibodies in the serum were determined by ELISA. The serum was heat-inactivated and the antibody content was detected by ELISA. A microplate with 2.5 μg / ml type II collagen was coated at 4°C overnight. The plate was blocked with 1% BSA, and the serum was diluted 1:100 with PBS and added to the wells. The plate was incubated at 37°C for 2 hours. A 1:8000 diluted horseradish peroxidase (HRP)-labeled goat anti-rat IgG antibody was added and incubated at 37°C for 1 hour. The OPD reaction substrate was added, and the reaction was stopped with 2N sulfuric acid after 30 minutes. The optical density (OD) value was read at 492 nm by a microplate reader. SPSS10.0 software was used for data analysis, and all data results are expressed as mean ± standard deviation. The significance of the differences was determined using one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant. The results showed that the level of type II collagen antibodies in the serum of rats in the collagen model group was significantly higher than that in the normal group (p < 0.01). Dexamethasone significantly inhibited the production of type II collagen antibodies in the collagen model rats (p < 0.01). Pharmaceutical compositions 1 and 2 of the present invention, as well as the control drug group, were able to significantly inhibit the production of type II collagen antibodies in the serum of rats with collagen-induced arthritis (p < 0.05 or p < 0.01). See Table 8.

[0216] Table 8: Effects of the pharmaceutical composition of the present invention on type II collagen antibodies in the serum of rats with collagen arthritis

[0217]

[0218] Note: Compared with the control group, △△ Compared with the model group, *p<0.05, **p<0.01, the drug composition groups 1 and 2, and the dexamethasone group showed significant differences compared with the model group.

[0219] (V) Effect of the pharmaceutical composition of the present invention on cytokines in serum of rats with collagen-induced arthritis model

[0220] Tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) play a crucial role in the pathogenesis and joint damage of rheumatoid arthritis. TNF-α and IL-1 contribute to the pathogenesis and progression of rheumatoid arthritis through multiple pathways, including activation of endothelial cells and promotion of the synthesis and release of inflammatory factors. IL-10 is a key inhibitory cytokine that downregulates immune responses and improves the inflammatory response in arthritis.

[0221] 1. Preparation of collagen-induced arthritis model, animal grouping and drug administration: same as experiment 3 (I).

[0222] 2. Effect of the pharmaceutical composition of the present invention on cytokines in serum of rats with collagen-induced arthritis: At the end of the experiment, blood was collected from the abdominal aorta of the rats after anesthesia, and serum was separated. The levels of cytokines IL-1, TNF-α, and IL-10 in the serum were determined according to the instructions of the ELISA kit. Data analysis was performed using SPSS 10.0 software, and all data results are expressed as mean ± standard deviation. The significance of the difference was expressed using one-way analysis of variance (One-Way ANOVA), and the LSD test was used for pairwise comparison between groups. P < 0.05 was considered statistically significant. The results showed that the levels of inflammatory cytokines IL-1 and TNF-α in the serum of the model group were significantly higher than those in the normal control group (p < 0.01), and dexamethasone significantly reduced the levels of IL-1 and TNF-α in the serum (p < 0.01). The pharmaceutical composition groups 1 and 2 of the present invention were also able to significantly reduce the levels of IL-1 and TNF-α in the serum (p < 0.05). The inhibitory cytokine IL-10 in the serum of the model group was significantly lower than that of the control group (p < 0.01), but the pharmaceutical composition groups of the present invention and dexamethasone had no significant improvement effect on the reduction of IL-10 in the serum of the model rats (p > 0.05). See Table 9.

[0223] Table 9: Effects of the pharmaceutical composition of the present invention on serum cytokines in rats with collagen arthritis model

[0224]

[0225] Note: Compared with the control group, △△ Compared with the model group, *p<0.05, **p<0.01, the drug composition groups 1 and 2, and the dexamethasone group showed significant differences compared with the model group.

[0226] (VI) Effect of the pharmaceutical composition of the present invention on serum IgG and IgM rheumatoid factors in collagen-induced arthritis rat models

[0227] Rheumatoid factor (RF), the most common autoantibody produced in patients with rheumatoid arthritis, is an autoantibody targeting the Fc region of IgG. RF can form immune complexes, activate complement, and lead to vasculitis. RF consists of two main types: IgG and IgM. IgM RF is the first to appear in the course of rheumatoid arthritis, followed by IgG RF later in the course. The levels of these two types of RF are closely correlated with the pathogenesis of rheumatoid arthritis.

[0228] 1. Preparation of collagen-induced arthritis model, animal grouping and drug administration: same as experiment 3 (I).

[0229] 2. Effect of the pharmaceutical composition of the present invention on IgG and IgM rheumatoid factor levels in the serum of rats with collagen-induced arthritis: At the end of the experiment, blood was collected from the abdominal aorta of anesthetized rats. Serum was separated, and purified rabbit IgG was dissolved in PBS, heat-denatured at 63°C for 20 minutes, and precipitated by adding 0.36 mol / L Na2SO4 saline solution. The plate was centrifuged at 10,000 rpm for 20 minutes, and the supernatant was discarded. The precipitate was dissolved in coating solution (20 μg / ml) and coated on a polystyrene plate overnight at 4°C. The plate was blocked with 1% BSA, and the rat serum to be tested was diluted 1:100 in PBS and added to the wells. The plate was incubated at 37°C for 2 hours. A 1:8000 dilution of horseradish peroxidase (HRP)-labeled goat anti-rat IgG or goat anti-rat IgM antibody was then added, and the plate was incubated at 37°C for 1 hour. OPD substrate was added, and the reaction was terminated with 2N sulfuric acid after 30 minutes. The optical density (OD) was read at 492 nm on a microplate reader. Data analysis was performed using SPSS 10.0 software, and all data were expressed as mean ± standard deviation. One-way ANOVA was used to determine the significance of differences, and pairwise comparisons between groups were performed using the LSD test. P < 0.05 was considered statistically significant. The results showed that serum IgG and IgM rheumatoid factor levels were significantly elevated in rats with collagen-induced arthritis, while dexamethasone significantly inhibited their production (p < 0.01). The drug combination of the present invention, Groups 1 and 2, also significantly inhibited the production of IgG and IgM rheumatoid factor in the serum of rats with collagen-induced arthritis (p < 0.05 or p < 0.01, respectively).

[0230] Table 10: Effects of the pharmaceutical composition of the present invention on serum IgG and IgM rheumatoid factor in rats with collagen arthritis

[0231]

[0232] Note: Compared with the control group, △△ Compared with the model group, the group consisting of the pharmaceutical composition of the present invention and the dexamethasone group, *p<0.05, **p<0.01.

[0233] summary

[0234] (1) Lewis rats were used to establish a type II collagen-induced rat arthritis model. It was found that the model rats began to develop arthritis symptoms about 14 days after the first immunization and continued until the 50th day. The arthritis index and toe volume were significantly higher than those in the control rats. The proliferation of type II collagen-specific spleen lymphocytes in the model rats was significantly higher than that in the control group; the serum levels of type II collagen-specific antibodies, IgG and IgM rheumatoid factors, inflammatory factors IL-1 and TNF-α were significantly higher than those in the control group (p < 0.01), and the regulatory cytokine IL-10 in the serum was significantly lower than that in the control group. The above results show that the type II collagen-induced rat joint model was successfully established, and the model rats had significant inflammatory responses and specific immune responses to type II collagen and inflammatory responses.

[0235] (2) The positive control drug dexamethasone showed that the arthritis index of the rats was significantly lower than that of the model group from 22 days after immunization until the end of the experiment (p < 0.05 or p < 0.01). The toe volume of the rats was significantly smaller than that of the arthritis model group from 22 days after immunization (8 days after administration) until the end of the experiment (p < 0.05 or p < 0.01). Dexamethasone also significantly inhibited the proliferation of spleen lymphocytes induced by type II collagen (p < 0.01), the production of type II collagen antibodies in serum (p < 0.01), the production of inflammatory cytokines IL-1 and TNF-α in serum (p < 0.01), and the production of rheumatoid factor IgG and IgM (p < 0.01), but had no significant effect on the regulatory cytokine IL-10.

[0236] (3) The arthritis index of the pharmaceutical composition group 1 of the present invention was significantly reduced from 35 days (21 days after administration) to 47 days, and the pharmaceutical composition group 2 of the present invention was significantly reduced from 39 days (25 days after administration) to 43 days compared with the model group (p < 0.05 and p < 0.01). The toe volume of the rats in the pharmaceutical composition group 1 of the present invention was significantly reduced from 35 days after immunization (21 days after administration) to 47 days, and from 39 days after immunization (25 days after administration) to 50 days compared with the model group (p < 0.05). The pharmaceutical compositions of the present invention in groups 1 and 2 significantly inhibited the proliferation of type II collagen-specific lymphocytes in the spleen of collagen model rats (p < 0.05 or p < 0.01), the production of type II collagen antibodies in the serum (p < 0.05 or p < 0.01), the levels of inflammatory cytokines IL-1 and TNF-α in the serum (p < 0.05), and the production of rheumatoid factors IgG and IgM (p < 0.05 or p < 0.01). However, it had no significant effect on reducing the level of regulatory cytokine IL-10 in the serum of model rats.

[0237] in conclusion

[0238] Rheumatoid arthritis (RA) is a multisystem inflammatory autoimmune disease that primarily affects peripheral joints. RA is characterized by joint inflammation and synovial neovascularization, leading to chronic synovial hyperplasia, which further affects cartilage and bone, resulting in joint deformity and loss of function. Rat adjuvant arthritis (AA) is an ideal model of arthritis that closely resembles human RA, essentially reproducing the pathological changes of human RA. The primary mechanism of AA is T cell activation by Mycobacterium tuberculosis, which contributes to RA pathogenesis through cytokine release and macrophage activation. Therefore, AA is primarily mediated by cell-mediated immunity. This model is simple and inexpensive to operate. The AA model exhibits rapid onset of disease after induction of inflammation, with secondary inflammatory reactions in the contralateral paw appearing 10 days after antigen challenge. Peak inflammation typically occurs between 20 and 25 days after infection, after which the inflammatory response gradually subsides. Although the symptoms and pathological manifestations of the AA model are similar to those of human rheumatoid arthritis, their pathogenesis differs. The collagen-induced arthritis (CIA) animal model is currently recognized as an ideal animal model for studying the pathogenesis of RA and developing new therapeutics for RA. This model is induced by type II collagen (CII), a major component of cartilage and an autoantigen involved in the pathogenesis of rheumatoid arthritis. Antibodies to type II collagen can be detected in the serum and synovial fluid of clinical rheumatoid arthritis patients. Because the pathogenesis of the CIA model more closely resembles that of human RA, it has become the preferred model for rheumatoid arthritis research in recent years. CIA arthritis develops more slowly in rats than in the AA model, beginning with joint swelling approximately 2-3 weeks after the initial immunization. Pathological manifestations include progressive synovitis and synovial hyperplasia, inflammatory cell infiltration, cartilage destruction, and ultimately joint damage and stiffness. Once CIA is established, joint inflammation persists for a prolonged period in rats, with clinical, histological, and immunological changes similar to those in human RA. Furthermore, the CIA model showed essentially no significant pathological changes before the booster immunization. However, after the booster immunization, paw swelling, arthritis scores, and serum autoantibody levels increased significantly, suggesting pathological changes similar to those seen in clinical RA. Therefore, when evaluating the pharmacodynamics of the pharmaceutical composition of the present invention using this model, preventive administration was performed before the booster immunization (the day after the initial immunization) and therapeutic administration was performed after the booster immunization (four days after the booster immunization).

[0239] We used the above two models to study the preventive and therapeutic effects of the pharmaceutical composition of the present invention on experimental arthritis and conducted preliminary observations on its mechanism of action. The results showed that the pharmaceutical composition of the present invention can significantly reduce the toe volume that appears after immunization of adjuvant-induced arthritis model rats, and can significantly inhibit the proliferation of splenic T lymphocytes in model rats, and improve joint pathological changes. Studies on type II collagen-induced arthritis showed that both preventive and therapeutic administration of the pharmaceutical composition of the present invention can significantly improve the joint index and toe swelling of model rats. In addition, the pharmaceutical composition of the present invention can also significantly improve the joint histological changes in model rats. Studies on its mechanism of action showed that the pharmaceutical composition of the present invention can reduce the content of type II collagen antibodies and rheumatoid factors in the serum of model rats, inhibit the proliferation of type II collagen-specific splenic lymphocytes, and inhibit the production of inflammatory cytokines IL-1 and TNF-α in serum.

[0240] In summary, the pharmaceutical composition of the present invention has a good preventive and therapeutic effect on adjuvant arthritis and type II collagen-induced arthritis. Its mechanism of action may be achieved by inhibiting excessive lymphocyte response, inhibiting the specific immune response to type II collagen and reducing inflammatory cytokines. DETAILED DESCRIPTION

[0241] Example 1:

[0242] The raw material formula is: Astragalus 156g, Gentiana macrophylla 156g, Stephania tetrandra 63g, Heishun Pian 63g, Polygonum cuspidatum 146g, Millettia reticulata 146g, Clemati 146g, Mustard 31g, White Peony 63g, Rehmannia glutinosa 156g, Angelica sinensis 63g, Myrrh 52g, Cyperus rotundus 52g, Cinnamon twig 63g, Cyathula officinalis 63g

[0243] Preparation method:

[0244] A. Grind 60 portions of Stephania tetrandra into fine powder, sterilize by gamma irradiation, and set aside.

[0245] B. Take cinnamon twig, vinegar myrrh, and fragrant spice with 6 times the amount of water, and extract the volatile oil by steam distillation for 8 hours continuously. Collect the volatile oil, filter the distilled water solution, collect it in another container, and set aside;

[0246] C. Take Gentiana macrophylla, Millettia reticulata, Chinese Angelica sinensis, Mustard seeds, White Peony Root, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, each time for 1 hour, filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear paste with a relative density of 1.15±0.05, and set aside;

[0247] D. Take Astragalus, Heishun Pian, Clematidis, Rehmannia glutinosa, and Cyathula officinalis, add 11 times the amount of water, decoct 3 times, each time for 1.5 hours, filter the decoction, and concentrate under reduced pressure to a clear paste with a relative density of 1.10±0.05. Then combine it with the aqueous solution after distillation of volatile oil, and concentrate under reduced pressure to a clear paste with a relative density of 1.15±0.05, and set aside;

[0248] E. Combine the clear pastes obtained in step C and step D, mix well, dry, and grind to obtain a mixed clear paste powder;

[0249] F. Take the volatile oil obtained in step B and carry out inclusion complexation, and the inclusion complex is ready for use;

[0250] G. Granulate the sterilized powder obtained in step A and the clear paste powder obtained in step E, add the inclusion compound obtained in step F, and compress the tablets according to conventional methods.

[0251] Example 2:

[0252] The raw material formula is: 146g of Astragalus, 139g of Gentiana macrophylla, 58g of Stephania tetrandra, 75g of Heishun Pian, 120g of Polygonum cuspidatum, 125g of Millettia reticulata, 135g of Clematidis, 28g of Mustard, 60g of White Peony, 150g of Rehmannia, 70g of Angelica sinensis, 60g of Myrrh, 60g of Cyperus rotundus, 70g of Cassia twig, and 70g of Cyathula capitata.

[0253] Preparation method:

[0254] A. Grind 60 portions of Stephania tetrandra into fine powder, sterilize by gamma irradiation, and set aside.

[0255] B. Take cinnamon twig, vinegar myrrh, and fragrant root with 8 times the amount of water, and extract the volatile oil by steam distillation for 10 hours. Collect the volatile oil, filter the distilled water solution, collect it in another container, and set aside;

[0256] C. Take Gentiana macrophylla, Millettia reticulata, Chinese Angelica sinensis, Mustard seeds, White Peony Root, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, each time for 1 hour, filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear paste with a relative density of 1.15±0.05, and set aside;

[0257] D. Take Astragalus, Heishun Pian, Clematidis, Rehmannia glutinosa, and Cyathula officinalis, add 8 times the amount of water, decoct 4 times, each time for 1.5 hours, filter the decoction, and concentrate under reduced pressure to a clear paste with a relative density of 1.10±0.05. Then combine it with the aqueous solution after distillation of volatile oil, and concentrate under reduced pressure to a clear paste with a relative density of 1.15±0.05, and set aside;

[0258] E. Combine the clear pastes obtained in step C and step D, mix them evenly, dry them, and grind them to obtain a mixed clear paste powder. Spray the volatile oil obtained in step B into it, add the sterilized powder in step A, spray granulate it, shape the granules, and fill them into capsules.

[0259] Example 3:

[0260] The formula of raw materials is: 150g of Astragalus, 160g of Gentiana macrophylla, 60g of Stephania tetrandra, 65g of Heishunpian, 120g of Polygonum cuspidatum, 130g of Millettia reticulata, 135g of Clematidis, 35g of Mustard seed, 50g of White Peony Root, 160g of Rehmannia glutinosa, 60g of Angelica sinensis, 55g of Myrrh, 45g of Cyperus rotundus, 60g of Cassia twig, and 60g of Cyathula capitata.

[0261] Preparation method:

[0262] A. Grind 60 portions of Stephania tetrandra into fine powder, sterilize by gamma irradiation, and set aside.

[0263] B. Take cinnamon twig, vinegar myrrh, and fragrant root with 8 times the amount of water, and extract the volatile oil by steam distillation for 10 hours. Collect the volatile oil, filter the distilled water solution, collect it in another container, and set aside;

[0264] C. Take Gentiana macrophylla, Millettia reticulata, Chinese Angelica sinensis, Mustard seeds, White Peony Root, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, each time for 1 hour, filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear paste with a relative density of 1.15±0.05, and set aside;

[0265] D. Take Astragalus, Heishun Pian, Clematidis, Rehmannia glutinosa, and Cyathula officinalis, add 8 times the amount of water, decoct 4 times, each time for 1.5 hours, filter the decoction, and concentrate under reduced pressure to a clear paste with a relative density of 1.10±0.05. Then combine it with the aqueous solution after distillation of volatile oil, and concentrate under reduced pressure to a clear paste with a relative density of 1.15±0.05, and set aside;

[0266] E. Combine the clear pastes obtained in step C and step D, mix them evenly, dry them, and grind them to obtain a mixed clear paste powder. Spray the volatile oil obtained in step B into it, add the sterilized powder in step A, spray granulate it, and shape the particles to obtain granules.

[0267] Example 4:

[0268] The raw material formula is: 120g of Astragalus, 190g of Gentiana macrophylla, 45g of Heishunpian, 80g of Aconitum, 110g of Polygonum cuspidatum, 180g of Millettia reticulata, 110g of Clematidis, 40g of Mustard, 45g of White Peony Root, 190g of Rehmannia glutinosa, 45g of Angelica sinensis, 65g of Myrrh, 40g of Cyperus rotundus, 80g of Cassia twig, and 45g of Cyathula capitata.

[0269] Preparation method:

[0270] A. Grind 60 portions of Stephania tetrandra into fine powder, sterilize by gamma irradiation, and set aside.

[0271] B. Take cinnamon twig, vinegar myrrh, and fragrant root with 8 times the amount of water, and extract the volatile oil by steam distillation for 10 hours. Collect the volatile oil, filter the distilled water solution, collect it in another container, and set aside;

[0272] C. Take Gentiana macrophylla, Millettia reticulata, Chinese Angelica sinensis, Mustard seeds, White Peony Root, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, each time for 1 hour, filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear paste with a relative density of 1.15±0.05, and set aside;

[0273] D. Take Astragalus, Heishun Pian, Clematidis, Rehmannia glutinosa, and Cyathula officinalis, add 8 times the amount of water, decoct 4 times, each time for 1.5 hours, filter the decoction, and concentrate under reduced pressure to a clear paste with a relative density of 1.10±0.05. Then combine it with the aqueous solution after distillation of volatile oil, and concentrate under reduced pressure to a clear paste with a relative density of 1.15±0.05, and set aside;

[0274] E. Combine the clear pastes obtained in step C and step D, mix well, dry, and grind to obtain a mixed clear paste powder, add the fine powder obtained in step A, spray the volatile oil obtained in step B, and prepare pills according to conventional methods.

[0275] Example 5:

[0276] 190g of Astragalus, 120g of Gentiana macrophylla, 80g of Stephania tetrandra, 45g of Heishun Pian, 180g of Polygonum cuspidatum, 110g of Millettia reticulata, 180g of Clematidis, 20g of Mustard seeds, 80g of White Peony Root, 120g of Rehmannia glutinosa, 80g of Angelica sinensis, 40g of Myrrh, 65g of Cyperus rotundus, 45g of Cassia twig, and 80g of Cyathula capitata.

[0277] Preparation method:

[0278] A. Grind 60 portions of Stephania tetrandra into fine powder, sterilize by gamma irradiation, and set aside.

[0279] B. Take cinnamon twig, vinegar myrrh, and fragrant root with 8 times the amount of water, and extract the volatile oil by steam distillation for 10 hours. Collect the volatile oil, filter the distilled water solution, collect it in another container, and set aside;

[0280] C. Take Gentiana macrophylla, Millettia reticulata, Chinese Angelica sinensis, Mustard seeds, White Peony Root, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, each time for 1 hour, filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear paste with a relative density of 1.15±0.05, and set aside;

[0281] D. Take Astragalus, Heishun Pian, Clematidis, Rehmannia glutinosa, and Cyathula officinalis, add 8 times the amount of water, decoct 4 times, each time for 1.5 hours, filter the decoction, and concentrate under reduced pressure to a clear paste with a relative density of 1.10±0.05. Then combine it with the aqueous solution after distillation of volatile oil, and concentrate under reduced pressure to a clear paste with a relative density of 1.15±0.05, and set aside;

[0282] E. Combine the clear pastes obtained in step C and step D, mix them evenly, dry them, and grind them to obtain a mixed clear paste powder. Spray the volatile oil obtained in step B into it, add the sterilized powder in step A, spray granulate it, and shape the particles to obtain granules.

[0283] Comparative Example 1:

[0284] The raw material formula is: Astragalus 220g, Gentiana macrophylla 220g, Stephania tetrandra 95g, Aconite 85g, Polygonum cuspidatum 192g, Millettia reticulata 80g, Clemati 90g, Mustard 60g, White Peony 50g, Rehmannia glutinosa 100g, Angelica 100g, Myrrh 35g, Cyperus rotundus 80g, Cinnamon twig 35g, Achyranthes bidentata 90g

[0285] Preparation method:

[0286] A. Grind 60 portions of Stephania tetrandra into fine powder, sterilize by gamma irradiation, and set aside.

[0287] B. Take cinnamon twig, myrrh, and angelica root and add 6 times the amount of water, and extract the volatile oil by steam distillation for 8 hours. Collect the volatile oil, filter the distilled water solution, and collect it in another container for later use;

[0288] C. Take Gentiana macrophylla, Millettia reticulata, Chinese Angelica sinensis, Mustard seeds, White Peony Root, and Polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract three times, each time for 1 hour, filter the extract, recover the ethanol under reduced pressure, and concentrate to a clear paste with a relative density of 1.15±0.05, and set aside;

[0289] D. Take Astragalus, Aconite, Clematidis, Rehmannia, and Achyranthes, add 11 times the amount of water, decoct three times, each time for 1.5 hours, filter the decoction, and concentrate under reduced pressure to a clear paste with a relative density of 1.10±0.05. Combine the decoction with the aqueous solution after distillation of volatile oil, and concentrate under reduced pressure to a clear paste with a relative density of 1.15±0.05, and set aside;

[0290] E. Combine the clear pastes obtained in step C and step D, mix well, dry, and grind to obtain a mixed clear paste powder;

[0291] F. Take the volatile oil obtained in step B and carry out inclusion complexation, and the inclusion complex is ready for use;

[0292] The sterilized powder obtained in step A and the clear paste powder obtained in step E are granulated, the granules are shaped, the inclusion compound obtained in step F is added, and tablets are obtained by conventional methods.

Claims

1. A composition for treating rheumatoid arthritis, characterized in that the composition comprises the following components in parts by weight: 120 - 190 parts of Astragalus membranaceus, 120 - 190 parts of Gentiana macrophylla, 45 - 80 parts of Stephania tetrandra, 45 - 80 parts of Aconitum carmichaelii Debx., 110 - 180 parts of Polygonum cuspidatum, 110 - 180 parts of Spatholobus suberectus, 110 - 180 parts of Clematis chinensis, 20 - 40 parts of Brassica juncea, 45 - 80 parts of Paeonia lactiflora, 120 - 190 parts of Rehmannia glutinosa, 45 - 80 parts of Angelica sinensis, 40 - 65 parts of Myrrha, 40 - 65 parts of Cyperus rotundus, 45 - 80 parts of Cinnamomum cassia, 45 - 80 parts of Achyranthes bidentata.

2. The composition according to claim 1, characterized in that the composition comprises the following components in parts by weight: 120 parts of Astragalus membranaceus, 190 parts of Gentiana macrophylla, 45 parts of Stephania tetrandra, 80 parts of Aconitum carmichaelii Debx., 110 parts of Polygonum cuspidatum, 180 parts of Spatholobus suberectus, 110 parts of Clematis chinensis, 40 parts of Brassica juncea, 45 parts of Paeonia lactiflora, 190 parts of Rehmannia glutinosa, 45 parts of Angelica sinensis, 65 parts of Myrrha, 40 parts of Cyperus rotundus, 80 parts of Cinnamomum cassia, 45 parts of Achyranthes bidentata.

3. The composition according to claim 1, characterized in that the composition comprises the following components in parts by weight: 150 parts of Astragalus membranaceus, 160 parts of Gentiana macrophylla, 60 parts of Stephania tetrandra, 65 parts of Aconitum carmichaelii Debx., 120 parts of Polygonum cuspidatum, 130 parts of Spatholobus suberectus, 135 parts of Clematis chinensis, 35 parts of Brassica juncea, 50 parts of Paeonia lactiflora, 160 parts of Rehmannia glutinosa, 60 parts of Angelica sinensis, 55 parts of Myrrha, 45 parts of Cyperus rotundus, 60 parts of Cinnamomum cassia, 60 parts of Achyranthes bidentata.

4. The composition according to claim 1, characterized in that the composition comprises the following components in parts by weight: 146 parts of Astragalus membranaceus, 139 parts of Gentiana macrophylla, 58 parts of Stephania tetrandra, 75 parts of Aconitum carmichaelii Debx., 120 parts of Polygonum cuspidatum, 125 parts of Spatholobus suberectus, 135 parts of Clematis chinensis, 28 parts of Brassica juncea, 60 parts of Paeonia lactiflora, 150 parts of Rehmannia glutinosa, 70 parts of Angelica sinensis, 60 parts of Myrrha, 60 parts of Cyperus rotundus, 70 parts of Cinnamomum cassia, 70 parts of Achyranthes bidentata.

5. The composition according to claim 1, characterized in that the composition comprises the following components in parts by weight: 156 parts of Astragalus membranaceus, 156 parts of Gentiana macrophylla, 63 parts of Stephania tetrandra, 63 parts of Aconitum carmichaelii Debx., 146 parts of Polygonum cuspidatum, 146 parts of Spatholobus suberectus, 146 parts of Clematis chinensis, 31 parts of Brassica juncea, 63 parts of Paeonia lactiflora, 156 parts of Rehmannia glutinosa, 63 parts of Angelica sinensis, 52 parts of Myrrha, 52 parts of Cyperus rotundus, 63 parts of Cinnamomum cassia, 63 parts of Achyranthes bidentata.

6. The composition according to any one of claims 1 - 5, characterized in that among the components of the composition, Aconitum carmichaelii Debx. is preferably Radix Aconiti Lateralis Preparata, Myrrha is preferably Myrrha carbonisata, and Achyranthes bidentata is preferably Radix Achyranthis Bidentatae.

7. The composition according to any one of claims 1 - 5, characterized in that the dosage form of the composition is capsule, tablet, pill, oral liquid, granule or powder.

8. The composition according to any one of claims 1 - 5, characterized in that the active components of the composition are prepared by the following steps: A. Take Stephania tetrandra and crush it into fine powder, sterilize 60 parts with o - γ ray irradiation, and set aside; B. Take Cinnamomum cassia, Myrrha, and Cyperus rotundus, add 5 - 8 times the amount of water, and continuously extract volatile oil by steam distillation for 5 - 10 hours, collect the volatile oil, filter the aqueous solution after distillation, and collect it separately for standby; C. Take gentiana macrophylla, millettia reticulata, angelica sinensis, mustard seed, white peony root, and polygonum cuspidatum, add 8 - 12 times the amount of 50 - 80% ethanol, reflux and extract 1 - 3 times, 1 hour each time. Filter the extract, recover ethanol under reduced pressure, and concentrate to a clear paste with a relative density of 1.15 ± 0.05 for standby; D. Take astragalus membranaceus, aconite carmichaeli, clematis chinensis, rehmannia glutinosa, and achyranthes bidentata, add 8 - 12 times the amount of water, decoct 2 - 5 times, 1.5 hours each time. Filter the decoction, concentrate under reduced pressure to a clear paste with a relative density of 1.10 ± 0.05, then combine it with the aqueous solution after distilling the volatile oil, and concentrate under reduced pressure to a clear paste with a relative density of 1.15 ± 0.05 for standby; E. Combine the clear pastes obtained in steps C and D, mix well, dry, and pulverize to obtain a mixed clear paste powder; The fine powder obtained in step A, the volatile oil obtained in step B, and the clear paste powder obtained in step E together constitute the active components of the pharmaceutical composition of the present invention.

9. According to the composition described in claim 7, wherein the preparation process of the capsule is as follows: A. Take stephania tetrandra and pulverize it into fine powder, sterilize 60 parts by o - γ ray irradiation for standby; B. Take cinnamon twig, myrrh, and cyperus rotundus, add 8 times the amount of water, continuously extract the volatile oil by steam distillation for 10 hours, collect the volatile oil, filter the aqueous solution after distillation, and collect it separately for standby; C. Take gentiana macrophylla, millettia reticulata, angelica sinensis, mustard seed, white peony root, and polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, 1 hour each time. Filter the extract, recover ethanol under reduced pressure, and concentrate to a clear paste with a relative density of 1.15 ± 0.05 for standby; D. Take astragalus membranaceus, aconite carmichaeli, clematis chinensis, rehmannia glutinosa, and achyranthes bidentata, add 8 times the amount of water, decoct 4 times, 1.5 hours each time. Filter the decoction, concentrate under reduced pressure to a clear paste with a relative density of 1.10 ± 0.05, then combine it with the aqueous solution after distilling the volatile oil, and concentrate under reduced pressure to a clear paste with a relative density of 1.15 ± 0.05 for standby; E. Combine the clear pastes obtained in steps C and D, mix well, dry, and pulverize to obtain a mixed clear paste powder. Spray the volatile oil obtained in step B into it, add the sterilized powder obtained in step A, spray - granulate, size - screen, and fill into capsules to obtain the product.

10. According to the composition described in claim 7, wherein the preparation process of the tablet is as follows: A. Take stephania tetrandra and pulverize it into fine powder, sterilize 60 parts by o - γ ray irradiation for standby; B. Take cinnamon twig, myrrh, and cyperus rotundus, add 6 times the amount of water, continuously extract the volatile oil by steam distillation for 8 hours, collect the volatile oil, filter the aqueous solution after distillation, and collect it separately for standby; C. Take gentiana macrophylla, millettia reticulata, angelica sinensis, mustard seed, white peony root, and polygonum cuspidatum, add 10 times the amount of 70% ethanol, reflux and extract 3 times, 1 hour each time. Filter the extract, recover ethanol under reduced pressure, and concentrate to a clear paste with a relative density of 1.15 ± 0.05 for standby; D. Take astragalus membranaceus, aconite carmichaeli, clematis chinensis, rehmannia glutinosa, and achyranthes bidentata, add 11 times the amount of water, decoct 3 times, 1.5 hours each time. Filter the decoction, concentrate under reduced pressure to a clear paste with a relative density of 1.10 ± 0.05, then combine it with the aqueous solution after distilling the volatile oil, and concentrate under reduced pressure to a clear paste with a relative density of 1.15 ± 0.05 for standby; E. Combine the clear pastes obtained in steps C and D, mix well, dry, and pulverize to obtain a mixed clear paste powder; F. After performing inclusion on the volatile oil obtained in step B, the inclusion compound is reserved for use; Portion, granulate the sterilized powder obtained in step A and the clear paste powder obtained in step E, size the granules, add the inclusion compound obtained in step F, and press tablets by a conventional method to obtain the tablets.

11. The composition according to claim 9, characterized in that The inclusion process in step F is as follows: Take cyclodextrin in an amount 6 - 10 times that of the volatile oil, add water in an amount 6 - 10 times that of the cyclodextrin, grind, then add a mixture of the volatile oil and ethanol with the same volume of 90 - 95%, continue to grind evenly, and then dry to obtain.

12. The composition according to any one of claims 1 - 5, characterized in that The use of the pharmaceutical composition in the preparation of an anti - inflammatory drug.

13. The composition according to any one of claims 1 - 5, characterized in that The use of the pharmaceutical composition in the preparation of a drug for inhibiting the increase in capillary permeability.

14. The composition according to any one of claims 1 - 5, characterized in that The use of the pharmaceutical composition in the preparation of an analgesic drug.

15. The composition according to any one of claims 1 - 5, characterized in that The use of the pharmaceutical composition in the preparation of a drug for inhibiting the proliferation of splenic lymphocytes and the production of serum type II collagen antibodies, reducing the contents of inflammatory cytokines IL - 1 and TNF - α, and inhibiting the production of rheumatoid factors IgG and IgM.

16. The composition according to any one of claims 1 - 5, characterized in that The rheumatoid arthritis is preferably adjuvant arthritis or type II collagen - induced arthritis.