Traditional Chinese medicine extract for preventing and treating rheumatoid arthritis

Abstract

The invention relates to the field of traditional Chinese medicines, in particular to an active pharmaceutical ingredient for preventing and treating rheumatoid arthritis and a preparation method thereof. The traditional Chinese medicine extract, namely coix seed ester, disclosed by the invention has the effects of preventing and treating pain, numbness and soreness of limbs, joints, muscles and the like due to arthralgia syndromes as well as adverse flexion and extension, swelling and deformation of the joints and the like. Animal experiment research proves that the coix seed ester has a good improvement effect on treating a rat model with arthritis induced by collagen, and the coix seed ester can obviously inhibit synovial hyperplasia and formation of vascular nebula and inhibit inflammatory response of RA (rheumatoid arthritis). The idea of treating the RA by using the coix seed ester is based on the pathogenesis of phlegm-dampness formation in the traditional Chinese medicine theory and the inflammation mechanism of modern research, the traditional Chinese medicine and the western medicine are combined, and the important clinical significance is achieved for relieving the pain of patients and improving the life quality.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine extract for preventing and treating rheumatoid arthritis and a preparation method thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a systemic chronic inflammatory autoimmune disease, which is characterized by the formation of chronic inflammation of the synovium, progressive bone erosion and joint destruction. The occurrence of RA is a continuous, progressive and systemic pathological process. In the early stage of the disease, various autoantibodies can be detected in the serum, including rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA). With the interaction between various immune cells, fibroblasts and cytokines, the synovial tissue gradually produces chronic inflammation of bone erosion and destruction, resulting in various clinical symptoms and joint damage, seriously reducing the quality of life of RA patients and even increasing the mortality rate of RA.

[0003] The disease progression of RA is affected by the crosstalk of various immune cells such as adipocytes, macrophages, fibroblast-like synoviocytes and osteoclasts in the synovial tissue of joints. These immune cells show inflammation in the disease microenvironment and plasticity in their functions according to the disease background. Immune processes occur in the joints and synovium of RA, in which synovial macrophages release pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6), which jointly stimulate the inflammation of osteoclasts and the activity of fibroblast-like synoviocytes (FLS), thus leading to the progression of bone erosion. In addition, activated FLS can produce matrix metalloproteinases (MMP), resulting in cartilage degeneration. Nuclear factor-κ-light chain enhancer of activated B cells (NF-κB) is involved in the pathogenesis of chronic inflammatory diseases. FLS stimulates the NF-κB signaling pathway, enabling T cells to bind to proteins on the surface of osteoclasts, which also leads to the further development of bone erosion. The interaction between macrophages and adipocytes in RA patients leads to the release of adipokines such as leptin, and the inflammatory response is more obvious in RA patients with obesity. Therefore, RA is closely related to cellular inflammatory responses.

[0004] Current guidelines recommend that the medications for RA mainly include conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), biological DMARDs (bDMARDs), targeted synthetic DMARDs (tsDMARDs), and glucocorticoids. However, more and more evidence supports that long-term use of non-steroidal anti-inflammatory drugs may pose a risk of cardiovascular diseases. Long-term application of glucocorticoids can exacerbate hypertension or cause abnormalities in blood lipid levels, glucose tolerance, insulin resistance, and obesity, and promote the occurrence and development of CVD. In traditional Chinese medicine theory, RA belongs to the category of "Bi syndrome", and it is considered that feeling damp pathogen is the key etiological factor for the onset of RA. Clinically, the methods of removing dampness and clearing heat, dredging collaterals and relieving pain are commonly used for treatment. Traditional Chinese medicine shows good effects in the treatment of RA, but most of the commonly used traditional Chinese medicine compounds contain toxic traditional Chinese medicines marked in the Chinese Pharmacopoeia, such as vine medicines (Sinomenium acutum, Tripterygium wilfordii), aconite medicines (Aconitum carmichaelii, Aconitum kusnezoffii), insect medicines (Zaocys dhumnades, Pheretima aspergillum), etc., which have been proven to have obvious reproductive toxicity, cardiac and nerve toxicity. At the same time, liver damage and bone marrow suppression caused by high-dose use also limit the clinical application to a certain extent. Summary of the Invention

[0005] The object of the present invention is to provide a traditional Chinese medicine extract for treating rheumatoid arthritis - coixenolide and its preparation method. Animal experimental studies have proved that coixenolide has a good improvement effect on the collagen-induced arthritis rat model. Coixenolide can significantly inhibit synovial hyperplasia and pannus formation, and inhibit the inflammatory response of RA. The idea of treating RA with coixenolide starts from the etiological factor of phlegm-dampness formation in traditional Chinese medicine theory and the inflammatory mechanism of modern research, integrating traditional Chinese and Western medicine, which has important clinical significance for reducing the pain of patients and improving the quality of life.

[0006] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions.

[0007] The present invention discloses an application of coix seed extract in the preparation of a drug for treating rheumatoid arthritis, characterized in that the coix seed extract is coixenolide.

[0008] The present invention also discloses an application of coix seed extract in the preparation of a drug for reducing the degree of joint swelling, characterized in that the coix seed extract is coixenolide.

[0009] The present invention also discloses an application of coix seed extract in the preparation of a drug for reducing inflammatory indexes, characterized in that the coix seed extract is coixenolide.

[0010] Furthermore, the above-mentioned drug includes coixenolide and a pharmaceutically acceptable carrier.

[0011] Furthermore, the above-mentioned drug includes oral preparations, injections or inhalants.

[0012] Further, the preparation method of coixenolide, an extract of coix seed as described above, comprises the following steps: (1) Crushing and granulating: Crushing the coix seeds with the hulls removed and impurities removed into fine powder of 60 - 200 meshes, pouring it into a blender, adding 40% - 80% of pure water by weight of the raw materials while stirring, performing wet granulation with a 20 - mesh sieve, drying at 55 - 60°C, controlling the moisture content within 5%, and preferably at 3 - 4%; (2) Granule sizing: Sizing the dried coix seed granules with a 10 - 20 - mesh sieve and then screening out the fine powder with a 50 - mesh sieve, and returning the screened - out fine powder for re - granulation; (3) Supercritical CO2 step - by - step extraction and fractionated collection: Placing the coix seed powder granules that have been sized in step (2) into the basket of a 24L extraction kettle, performing supercritical CO2 step - by - step pressure - increasing and fractionated extraction, with the pressure increased step - by - step to 10, 15, 20, 25, 30 MPa respectively, 1 hour per step, and fractionated collection at 0.5 hour per fraction; When the extraction pressure rises to about 20 MPa, the dissolution ability of CO2 increases, and the coixenolide with a relatively large solubility is extracted and the operation is stopped to obtain coixenolide.

[0013] The applicant has observed through clinical research that RA patients are often accompanied by obesity and have abnormal manifestations of excessive lipid metabolism. Traditional Chinese medicine theory believes that obesity or abnormal lipid metabolism belongs to the category of "phlegm - dampness". "Huangdi Neijing" refers to people with a plump body shape as "fat people, lipid people, grease people, and fleshy people", which is the origin and basis of the theory that "fat people are more likely to have phlegm - dampness" in later generations. Traditional Chinese medicine theory believes that the normal metabolism of phlegm, drink, water, and dampness depends on the normal functions of the spleen and lungs, so that the essence of water is distributed everywhere and the five meridians function normally, and finally it is excreted downward into the bladder to complete. Modern medicine believes that the central link of the inflammatory process is the vascular reaction. Blood vessels are equivalent to the meridians in traditional Chinese medicine. If phlegm and fluid accumulate and the meridians are blocked, it may lead to systemic low - grade inflammatory lesions. Therefore, it is inferred that people with the phlegm - dampness constitution may be in a chronic low - grade inflammatory state for a long time. In the treatment concept, the inflammatory level of the body can be reduced by the method of eliminating phlegm and removing dampness, effectively avoiding or slowing down the onset and development of RA. At the same time, a large number of modern studies have proved that obesity is a state of chronic low - grade inflammation. In RA, adipocytes proliferate and hypertrophy, secrete inflammatory mediators, resulting in an increase in serum levels. RA patients with obesity or abnormal lipid metabolism further exacerbate the inflammatory reaction of RA. At the same time, research shows that the factor of "phlegm - dampness" will further aggravate the degree of inflammation in RA.

[0014] Coix seed is a commonly used traditional Chinese medicine in the prescriptions for treating arthralgia in traditional Chinese medicine clinically. During the Spring and Autumn Period and the Warring States Period, "Plain Questions of Huangdi Neijing" set a special chapter on "arthralgia syndrome", with a clear understanding of the causes and syndrome classifications of arthralgia syndrome. It is believed that the occurrence of this disease is related to the invasion of wind, cold, and damp pathogens. As stated in "Plain Questions - Treatise on Arthralgia": "The so-called arthralgia means that at each corresponding season, one is severely affected by wind, cold, and damp qi." In the Eastern Han Dynasty, Zhang Zhongjing recorded the name of "severe arthralgia" in "Synopsis of Prescriptions of the Golden Chamber - Treatise on Apoplexy and Severe Arthralgia, Pulse Syndromes and Their Treatments", summarized the characteristics of severe arthralgia as "severe arthralgia pain and inability to flex and extend", and used Guizhi Shaoyao Zhimu Decoction and Aconite Decoction as treatment formulas. During the Jin and Yuan Dynasties, Zhu Danxi first proposed the disease name of "gout" in "On Gout in the Treatise of Investigation and Extension of Knowledge", believing that the occurrence of this disease is related to the living environment. The external evils of wind, cold, dampness, and heat in the external environment are the external conditions for the onset of this disease. Due to long-term residence in damp places, wading through water, being exposed to rain, sleeping in the wind, working in water, etc., damp pathogens invade the human body, block the qi mechanism, resulting in spleen deficiency and the production of dampness, stagnation of water and dampness, and impaired transportation and transformation; or if wind-cold-damp arthralgia persists for a long time and turns into heat due to stagnation, it may also be due to a yang-deficient constitution, resulting in the invasion of wind-cold-damp-heat pathogens taking advantage of the deficiency to invade the human body and lodging in the meridians to form arthralgia syndrome. Just as stated in "Plain Questions - Treatise on Arthralgia": "The combination of wind, cold, and dampness leads to arthralgia." Therefore, being affected by damp pathogens is most likely to trap the spleen. External dampness damages the spleen and stomach, resulting in spleen-yang deficiency and weakness in transportation, and the production of internal dampness. This further causes stasis and obstruction in the limb meridians, forming phlegm coagulation and qi aggregation in the limb joints, and then causing symptoms such as inflammatory pain. According to traditional Chinese medicine theory, insufficient congenital endowment, physical weakness, weak external defense, or abnormal spleen transportation and transformation, the source of qi and blood production is weak, and one is susceptible to external pathogens. As stated in "Treatise on the Causes and Symptoms of Various Diseases - Syndrome of Wind-Damp Arthralgia": "Due to qi and blood deficiency, one is affected by wind-damp and develops this disease." Therefore, coix seed is sweet and light in taste, cool in nature, enters the spleen, stomach, and lung meridians, and has the effects of promoting diuresis to eliminate dampness, strengthening the spleen to stop diarrhea, dispelling dampness and relieving arthralgia, etc. For patients with arthralgia syndrome of spleen deficiency and dampness accumulation, it plays the role of strengthening the spleen and removing dampness, thus achieving the effect of strengthening the spleen and promoting diuresis.

[0015] Based on the theory of "phlegm-dampness" in RA, animal experimental studies have proven that coixenolide has a good improvement effect on the collagen-induced arthritis rat model. Coixenolide can significantly inhibit synovial hyperplasia and pannus formation, and inhibit the inflammatory response of RA. The idea of treating RA with coixenolide starts from the causes of phlegm-dampness formation in traditional Chinese medicine theory and the inflammatory mechanism of modern research, integrating traditional Chinese and Western medicine, which has important clinical significance for reducing the pain of patients and improving the quality of life.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0017] The traditional Chinese medicine component complex provided by the present invention takes strengthening the spleen and stomach and promoting diuresis to remove dampness as the treatment principle, can effectively treat the inflammatory response of collagen-induced rats, and compared with the positive control group of methotrexate, the present invention has better curative effects and significant differences.

[0018] The traditional Chinese medicine active ingredients provided by the present invention are used for treating RA, can relieve symptoms such as limb joint and muscle soreness, numbness and heaviness, difficulty in flexion and extension, and joint swelling, and improve the quality of life of patients.

[0019] The present invention has no obvious toxic and side effects, is safe and reliable, and has a clear preparation method, which is suitable for large-scale production by grass-roots medical institutions or manufacturers. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Swelling conditions of joints of rats in each group. A: Body weights of rats in each group; B: Paw thickness of rats in each group; C: Inter-group comparison of average body weights of rats in each group on the 35th day; D: Inter-group comparison of paw thickness of rats in each group on the 35th day. Compared with the blank group, <0.05, <0.01; compared with the CIA group, <0.05, <0.01; compared with the coixenolide group, <0.05, <0.01; compared with the MTX group, <0.05, <0.01; compared with the coix seed oil group, <0.05, <0.01; compared with the MTX + coix seed oil group, <0.05, <0.01.

[0021] Figure 2 Arthritis index scores of rats in each group. A: Arthritis index of rats in each group; B: Inter-group comparison of arthritis index of rats in each group on the 36th day. Compared with the blank group, <0.05, <0.01; compared with the CIA group, <0.05, <0.01; compared with the coixenolide group, <0.05, <0.01; compared with the MTX group, <0.05, <0.01; compared with the coix seed oil group, <0.05, <0.01; compared with the MTX + coix seed oil group, <0.05, <0.01.

[0022] Figure 3The pathological conditions of the ankle joints of rats in each group. A: HE staining pathological images of the ankle joints and the formation of new blood vessels in synovial tissues; B: Safranin O-fast green staining pathology of the ankle joints; C: HE staining pathological score; D: Safranin O-fast green staining pathological score. Compared with the blank group, <0.05, <0.01; compared with the CIA group, <0.05, <0.01; compared with the coixenolide group, <0.05, <0.01; compared with the MTX group, <0.05, <0.01; compared with the coix seed oil group, <0.05, <0.01; compared with the MTX + coix seed oil group, <0.05, <0.01.

[0023] Figure 4 The effects on the levels of serum inflammatory cytokines in rats in each group. A: The levels of TNF-α in the sera of rats in each group; B: The levels of IL-6 in the sera of rats in each group. Compared with the blank group, <0.05, <0.01; compared with the CIA group, <0.05, <0.01; compared with the coixenolide group, <0.05, <0.01; compared with the MTX group, <0.05, <0.01; compared with the coix seed oil group, <0.05, <0.01; compared with the MTX + coix seed oil group, <0.05, <0.01. Specific implementation manners

[0024] The present invention will be described in detail below with reference to specific embodiments. The following are only the preferred embodiments of the present invention and are not used to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0025] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0026] Example 1 Preparation of coixenolide.

[0027] (1) Crushing and granulation: The coix seeds that have been shelled and de - impurities are crushed into fine powder with a mesh size of 60 - 200. Pour it into a blender, and while stirring, add pure water accounting for 40% - 80% of the raw material weight. Perform wet granulation with a 20 - mesh sieve, and dry it at 55 - 60 °C, controlling the moisture content within 5%, and it is better when the moisture content is 3% - 4%. (2) Granule sizing: The dried and formed coix seed granules are sized with a 10 - 20 - mesh sieve, and then the fine powder is sieved out with a 50 - mesh sieve. The sieved - out fine powder is returned for re - granulation. (3) Supercritical CO₂ step - by - step extraction and fractionated collection: The carbon dioxide cylinder, carbon dioxide storage tank, extraction kettle, and separation kettle are connected in sequence. There are pipes above them for CO₂, coix seed oil, and coix seed ester to pass through. After carbon dioxide goes from the carbon dioxide cylinder to the carbon dioxide storage tank, through heat exchanger I, high - pressure pump, and heat exchanger II in sequence, it enters the extraction kettle and separation kettle, and then returns to the carbon dioxide storage tank to complete a working cycle. Put the sized coix seed powder into the basket of the 24L extraction kettle. The pressure of supercritical CO₂ introduced into the kettle is increased step - by - step between 10 - 30 MPa, and the extraction temperature is 40 - 60 °C. There is a valve below the extraction kettle to control the introduction of CO₂. The supercritical CO₂ dissolved with coix seed ester and coix seed oil enters the separation kettle from the extraction kettle through valve pressure reduction. The pressure in the kettle is 5 - 7 MPa, the temperature is 40 - 60 °C, and the CO₂ flow rate is 100 - 130 L / hr. Collect fractionally according to the extraction time at 0.5 - 1.0 hr / section. The lower part of the separation kettle is the collection valve. The total time for step - by - step extraction and fractionated collection is 5 - 5.5 hr.

[0028] Specifically as described below: Put the coix seed powder particles that have been sieved through whole grains in the material basket of a 24L extraction kettle, and use supercritical CO2 for stepwise pressure increase and segmented extraction. The pressures for pressure increase are increased step by step to 10, 15, 20, 25, and 30 MPa respectively, with 1 hour for each step, and segmented collection is carried out at 0.5 hour per segment. Stepwise pressure increase is used to increase the density and dissolution ability of supercritical CO2. When the extraction pressure is 10 MPa or 15 MPa, the dissolution ability of supercritical CO2 is low, and only the fat-soluble impurity components and moisture with high solubility in this CO2 can be extracted. As the extraction pressure increases to about 20 MPa, the dissolution ability of CO2 increases, and the coix seed ester with relatively high solubility is extracted; as the pressure further increases to 25 MPa, the coix seed oil with relatively low solubility is also extracted. When continuing the extraction at a high pressure of 30 MPa, some low-polarity impurities with even lower solubility will finally be extracted, making the color of the oil darker. Therefore, by stepwise increasing the extraction pressure of supercritical CO2, components with different solubilities are extracted step by step and successively, and through the method of segmented collection, the moisture, coix seed ester, coix seed oil, and other impurities in the coix seed powder particles can be separated online. When obtaining two products, coix seed ester and coix seed oil, the moisture and some impurities are separated simultaneously, and a yellowish-white coix seed ester, light yellow coix seed oil, moisture, and other impurity components can be separated out.

[0029] Example 2 Effect of coix seed ester on a collagen-induced arthritis rat model.

[0030] 1. Animal experiment data and methods.

[0031] 1.1 Experimental animals.

[0032] 56 SPF-grade 6-week-old male SD rats, weighing 200 ± 20 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd., and the animal production license number is: SCXK (Jing) 2019~0008. All experimental rats were raised in the Experimental Animal Department of China Medical University, with a temperature of 24 ± 2 °C, a humidity of 50 ± 10%, and a light-dark cycle every 12 hours. They had free access to drinking water and food. This experiment has been approved by the Ethics Committee of the First Affiliated Hospital of China Medical University.

[0033] 1.2 Experimental methods.

[0034] Collagen-induced arthritis (CIA) model establishment: SD rats were adaptively fed for one week to avoid stress. According to the previous experience of the research group, chicken type II collagen powder was fully mixed with 0.05 mmol / L glacial acetic acid one day in advance to finally form a 2 mg / mL collagen mixture, which was stored in the refrigerator at 4 °C overnight. The next day, BCG was inactivated in an 80 °C water bath for 1 h. Then, under a sterile and light-shielded ultra-clean bench, BCG and paraffin solution were fully mixed and ground vigorously in a mortar for 2 - 3 h to finally obtain a 8 mg / mL complete Freund's adjuvant (CFA). Subsequently, the collagen mixture was fully mixed with an equal volume of complete Freund's adjuvant for 2 - 3 h to form a collagen emulsion. The collagen emulsion was subcutaneously injected at multiple positions at the root of the rat's tail and back (rat injection standard: 100 μL / 100 g) for the first immunization, which was recorded as Day 0 at this time. Seven days after the first immunization, a second immunization was carried out, and the operation was the same as the first immunization, which was recorded as Day 7 at this time. At the same time, during the first immunization, the same positions of the blank group rats without modeling were treated with an equal amount of normal saline as a control to obtain a blank control model.

[0035] Grouping and drug administration: Before modeling, SD rats were randomly divided into 8 blank groups and 48 model groups according to body weight. After modeling, they were randomly divided into a CIA group, a coixenolide group (4.2 g / kg), a coix seed oil group (4.2 g / kg), a methotrexate (MTX) (0.5 mg / kg) group, an MTX + coix seed oil group, and an MTX + coixenolide group, with 8 experimental rats in each group. Immediately after the second immunization, the rats in the drug administration groups were given intragastric administration. The coixenolide and coix seed oil groups were given 4.2 g / kg twice a day; the MTX group was given 0.5 mg / kg once every 3 days by intragastric administration; the MTX + coix seed oil group was given 4.2 g / kg of coix seed oil twice a day, and at the same time, 0.5 mg / kg of MTX was given once every 3 days by intragastric administration; the MTX + coixenolide group was given 4.2 g / kg of coixenolide twice a day, and at the same time, 0.5 mg / kg of MTX was given once every 3 days by intragastric administration; at the same time, the blank group and the CIA group were given intragastric administration with an equal dose of normal saline, and the drug administration continued for 4 weeks. During this period, the arthritis index score and the systemic inflammation score were observed and recorded. The 500 μg / mL coixenolide was obtained according to the preparation method described in Example 1.

[0036] 1.3 Animal sampling.

[0037] After 4 weeks of drug administration, each rat was euthanized. Using a 5 mL vacuum blood collection tube, 2 - 3 mL of abdominal aortic blood was drawn from the rats and placed in a 4℃ refrigerator for about one hour, and then centrifuged (centrifugation conditions: 4℃, 3000 rpm, 10 min). The serum was carefully aspirated with a pipette tip and placed in the refrigerator for future use. The synovial tissues of both knees of the rats were collected. The synovial tissues of the left knees of each rat were fixed with 4% fixative for 24 - 48 h for subsequent paraffin embedding. The synovial tissues of the right knees were quickly frozen in liquid nitrogen and stored in a -80℃ ultra-low temperature refrigerator for later use.

[0038] 2. Observation indicators.

[0039] 2.1 General indicators: mental state, diet and water intake, sleep state, activity level, hair condition, etc.

[0040] 2.2 Efficacy observation indicators: (1) Effect of coixenolide on joint swelling in CIA rats; (2) Improvement of pathological morphology of synovial tissues in CIA rats by coixenolide; (3) Effect of coixenolide on serum inflammatory cytokine levels in CIA rats.

[0041] 3. Statistical analysis.

[0042] All data in this study were analyzed using GraphPad Prism 8.4.2 software and the corresponding graphs were drawn, and were expressed as mean ± standard deviation (SD). The differences between multiple groups were analyzed using one-way analysis of variance (One-Way ANOVA). If the variances were not homogeneous, the Kruskal Wallis test method was used for statistical analysis. Bonferroni test was used for multiple comparisons. P < 0.05 was considered statistically significant, and P < 0.01 was considered to have significant differences.

[0043] 4. Results.

[0044] 4.1 General efficacy observation of each group.

[0045] During the experiment, the general conditions of the rats were observed: mental state, diet and water intake, sleep state, activity level, hair condition, etc. The rats in the blank group were in good spirits, had good diet and water intake, good sleep, moved freely, and had bright and smooth hair; the rats in the CIA group were listless, had reduced diet and water intake, reduced activity, dull and yellowish hair, hunched backs and huddled together, and the affected limbs could not touch the ground; the mental state, diet and water intake, activity level, hair condition, etc. of the rats in the drug administration group were all improved, and among them, the rats in the MTX + coixenolide group had the best improvement in mental state, diet and water intake, activity level, hair condition, etc.

[0046] 4.2 The body weights and joint swelling conditions of each group of rats (as Figure 1 shown).

[0047] The body weights of the rats in the blank group increased steadily, the paw thickness did not increase, and the joints were not swollen. Compared with the blank group, the body weights of the rats in the CIA group decreased, the paw thickness increased, and the arthritis gradually swelled after modeling. The body weights gradually recovered 21 days later, and the paw thickness and joint swelling gradually alleviated ( P < 0.01); on the 35th day, the body weight loss of the CIA rats could be alleviated in each drug administration group, and the paw thickness and joint swelling were also alleviated. Compared with the CIA group, the body weight increase effect was the most significant in the MTX + coixenolide group of rats, and the recovery effects of the paw thickness and swelling were also significant ( P < 0.01); compared with the CIA group, the body weight increase effect was the second in the MTX + coix seed oil group of rats, and the recovery effects of the paw thickness and swelling were the second ( P < 0.01); compared with the CIA group, the body weight increase effects were equivalent in the MTX group and the coixenolide group, and the recovery effects of the paw thickness and swelling were also good ( P < 0.01); compared with the CIA group, the body weight increase effect was less obvious in the coix seed oil group than in other drug administration groups, and the recovery effects of the paw thickness and swelling were also poor (see Figure 1 A, B). Statistical analysis of the average body weights and paw thicknesses of the rats in each group on the 35th day found (see Figure 1 C, D) that compared with the coixenolide group, the body weight recovery effects were slightly worse in the MTX group and the coix seed oil group of rats, and the paw thickness recovery effects were poor. However, the body weight recovery effects were excellent in the MTX + coix seed oil group and the MTX + coixenolide group of rats, and the paw thickness recovery effects were good. Among them, the body weight recovery effect was the most significant in the MTX + coixenolide group, and the paw thickness recovery effect was the best ( P < 0.01). Compared with the MTX group, the body weight recovery and increase were less in the coix seed oil group, and the paw thickness recovery effect was not obvious. However, the body weight recovery effects were more obvious in the MTX + coix seed oil group and the MTX + coixenolide group, and the paw thickness recovery effects were also better ( P < 0.01). Compared with the coix seed oil group, the body weight recovery effect was more obvious in the MTX + coix seed oil group, and the reduction of the paw thickness was also more obvious. However, the body weight recovery effect was the most obvious in the MTX + coixenolide group, and the reduction of the paw thickness was the most significant ( P < 0.01). Compared with the MTX + coix seed oil group, the body weight recovery effect was more significant in the MTX + coixenolide group, and the paw thickness recovery effect was better ( P < 0.01).

[0048] 4.3 The arthritis index scores of the CIA rats in each group (as Figure 2 shown).

[0049] The paws of rats in the blank group did not show swelling, and the arthritis index score was 0. Compared with the blank group, joint swelling began to appear in the CIA group after modeling, and the arthritis index score increased ( P <0.01); compared with the CIA group, the arthritis index of rats in the drug administration groups gradually decreased, and the arthritis index of rats in the MTX + coixenolide group decreased most significantly ( P <0.01) (see Figure 2 A). Statistical analysis of the arthritis index of rats in each group on the 36th day found that (see Figure 2 B), compared with the coixenolide group, the MTX group, the MTX + coixenol oil group, and the MTX + coixenolide group had more obvious effects on improving the joint index, and the coixenol oil group had a relatively poor effect on improving joint swelling ( P <0.01). Compared with the MTX group, the MTX + coixenol oil group and the MTX + coixenolide group had better effects on reducing the arthritis index, and the coixenol oil group had a relatively less obvious reducing effect ( P <0.01). Compared with the coixenol oil group, the MTX + coixenol oil group and the MTX + coixenolide group had better effects on reducing the arthritis index ( P <0.01). Compared with the MTX + coixenol oil group, the MTX + coixenolide group had a more significant reducing effect ( P <0.01).

[0050] 4.4 Pathological conditions of the ankle joints of CIA rats in each group (as Figure 3 shown).

[0051] HE staining and safranin O staining were used to observe the pathological changes of the ankle joint tissues of rats in each group. The ankle joint tissue structure of rats in the blank group was intact, the articular cartilage was flat and smooth, there was no infiltration of inflammatory cells in the gap, and no bone destruction was seen, and the pathological score was 0; the ankle joint structure of rats in the CIA group was disordered, the joint surface was rough, the synovium proliferated, the gap was narrow and there was a large amount of inflammatory cell infiltration, pannus formation, obvious bone and cartilage destruction were seen, and the pathological score was higher than that of the normal group ( P <0.01); compared with the CIA group, the MTX + coixenolide group of rats had the best recovery effect on the pathological conditions of the joints, the joint surface was smooth, there was no obvious synovial hyperplasia, and no bone destruction, etc. ( P <0.01); compared with the CIA group, the MTX + coixenol oil group had slight damage to the joint tissue structure, the articular cartilage was relatively smooth, there was a small amount of cell infiltration in the gap, and there was slight bone destruction ( P <0.01); compared with the CIA group, the pathological score of the ankle joints of rats in the MTX group was significantly reduced, showing that the joint surface was slightly smooth, there was a small amount of inflammatory cell infiltration, no obvious synovial hyperplasia, and no obvious cartilage and bone destruction ( P<0.01); Compared with the CIA group, the pathological score of the ankle joint in the coixenolide group decreased, and it was higher than that in the MTX + coixenolide group. The joint structure was relatively disordered, the joint surface was rougher, and there was slight hyperplasia in the synovial cavity ( P <0.01); Compared with the CIA group, the pathological score of the ankle joint in the coix seed oil group decreased significantly. Slight damage to the joint structure was visible, cell infiltration was visible in the space, and obvious bone destruction was present ( P <0.01). Compared with the coixenolide group, the HE score and safranin O score of the rats in the MTX group, MTX + coix seed oil group, and MTX + coixenolide group decreased more significantly. Among them, the MTX + coixenolide group had the most significant decrease, and the coix seed oil group had a slightly worse effect on reducing the pathological score ( P <0.01). Compared with the MTX group, the coix seed oil group had a higher pathological score, and the MTX + coix seed oil group and MTX + coixenolide group had a more obvious effect on reducing the pathological score ( P <0.01). Compared with the coix seed oil group, the MTX + coix seed oil group had a more obvious decrease in the pathological score, and the MTX + coixenolide group had a more significant effect on reducing the pathological score ( P <0.01). Compared with the MTX + coix seed oil group, the MTX + coixenolide group had a better effect on reducing the pathological score ( P <0.01).

[0052] 4.5 The levels of serum inflammatory cytokines in CIA rats in each group (as Figure 4 shown).

[0053] Compared with the blank group, the levels of TNF-α and IL-6 in the serum of rats in the CIA group increased significantly, and the results were statistically significant ( P <0.01); Compared with the CIA group, the levels of TNF-α and IL-6 in the serum of the rats in the drug administration groups decreased significantly. Among them, the MTX + coixenolide group had the most significant decrease in TNF-α and IL-6, and the inflammatory indicators could even approach the blank group level ( P <0.01); The MTX + coix seed oil group had a significant decrease in TNF-α and IL-6 ( P <0.01); Compared with the CIA group, the levels of TNF-α and IL-6 in the coixenolide group and MTX group decreased significantly ( P <0.01); Compared with the CIA group, the coix seed oil group had a relatively significant decrease in the levels of TNF-α and IL-6 ( P <0.01). Compared with the coixenolide group, the rats in the MTX group, MTX + coix seed oil group, and MTX + coixenolide group had a more significant decrease in TNF-α and IL-6. Among them, the MTX + coixenolide group had the most significant decrease, and the coix seed oil group had a slightly worse effect on reducing TNF-α and IL-6 ( P<0.01). Compared with the MTX group, the reduction of TNF-α and IL-6 in the coixenolide group was not significant, while the reduction of TNF-α and IL-6 in the MTX + coixenolide group and the MTX + coixenolide acetate group was more significant ( P <0.01). Compared with the coixenolide group, the reduction of TNF-α and IL-6 in the MTX + coixenolide group was more obvious, and the reduction effect in the MTX + coixenolide acetate group was more significant ( P <0.01). Compared with the MTX + coixenolide group, the MTX + coixenolide acetate group had a better effect in reducing TNF-α and IL-6 ( P <0.01).

[0054] In summary, the present invention has an obvious improvement effect on the rats with rheumatoid arthritis model, can improve their general condition, reduce the degree of joint swelling and lower the inflammatory indexes, and has few adverse reactions, low cost and excellent treatment effect, which is worthy of clinical promotion.

Claims

1. Use of coix seed extract in the preparation of a medicament for treating rheumatoid arthritis, characterized in that, The coix seed extract is coixenolide.

2. Use of an extract of Coix lacryma-jobi L. in the preparation of a drug for reducing the degree of joint swelling, characterized in that, The coix seed extract is coixenolide.

3. Use of coix seed extract in a medicament for reducing inflammatory indexes, characterized in that, The coix seed extract is coixenolide.

4. The application according to claims 1 to 3, characterized in that, The drug includes coixenolide and a pharmaceutically acceptable carrier.

5. The application according to claims 1 to 3, characterized in that, The drug is an oral preparation, an injection or an inhalant.

6. The application according to claims 1 to 3, characterized in that, The preparation method of the coix seed extract coixenolide of traditional Chinese medicine includes the following steps: (1) Crushing and granulating: Crushing the coix seeds with the hulls removed and impurities removed into fine powder of 60-200 meshes, pouring it into a blender, adding 40%-80% of pure water by weight of the raw materials while stirring, carrying out wet granulation with a 20-mesh sieve, drying at 55-60°C, controlling the moisture content within 5%, and it is better when the moisture content is 3-4%; (2) Granule sizing and sieving: Sizing the dried and formed coix seed granules with a 10-20-mesh sieve and then sieving out the fine powder with a 50-mesh sieve, and returning the sieved fine powder for re-granulation; (3) Supercritical CO2 stepwise extraction and fractional collection: Putting the coix seed powder particles that have been sized and sieved in step (2) into the material basket of a 24L extraction kettle, adopting supercritical CO2 stepwise pressure increase and fractional extraction, increasing the pressure step by step to 10, 15, 20, 25, 30 MPa respectively, 1hr / each step, and collecting fractionally at 0.5hr / section; When the extraction pressure rises to about 20 MPa, the dissolution ability of CO2 increases, and coixenolide with a relatively large solubility is extracted.

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