Application of coix seed ester in medicine for treating RS (Respiratory Syndrome)

Abstract

The invention relates to the field of traditional Chinese medicines, in particular to application of coix seed ester in a medicine for treating rheumatoid sarcopenia. The coix seed ester disclosed by the invention is determined to be capable of effectively treating collagen-induced rat rheumatoid sarcopenia through a large number of practices and repeated verification by taking spleen strengthening, stomach benefiting, dampness clearing and diuresis inducing as treatment principles, is reasonable in medicine flavor and more accurate in dosage, has a better curative effect compared with western medicines in curative effect evaluation of rheumatoid sarcopenia, and can be used for preparing medicines for treating rheumatoid sarcopenia. The experiment has no obvious toxic and side effects and is safe and reliable. The symptoms such as limb tendon and vessel retardation, weakness or amyotrophy can be relieved, and important clinical significance is achieved for relieving the pain of a patient 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 sarcopenia and a preparation method thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic systemic disease mainly characterized by inflammatory synovitis. Its characteristics are polyarticular, symmetrical, and invasive joint inflammations of small joints in the hands and feet, often accompanied by extra-articular organ involvement and positive serum rheumatoid factor. In severe cases, it can lead to joint deformity and loss of function. Sarcopenia is a type of muscle failure mainly characterized by adverse muscle changes. Among them, rheumatoid sarcopenia (RS) is mainly manifested by a decrease in muscle mass, and the fat mass (FM) remains unchanged or increases. Generally, there is rarely a significant weight loss, and most can maintain their normal body mass index (BMI); it is mainly the loss of skeletal muscle mass and strength, which has a negative impact on the body's metabolic rate, function, etc. RS is one of the most common complications during the onset of RA. Studies have shown that about 1 / 3 of RA patients can develop into RS, which further aggravates joint destruction, leading to risks such as falls, fractures, and disabilities, resulting in a decrease in physical activity, and gradually emerging complications such as muscle atrophy and insulin resistance. Therefore, it further aggravates the movement disorder, forming a vicious cycle.

[0003] Among the inflammatory factors leading to RS, the main ones include the overproduction of inflammatory factors, the decline in physical activity, and the reduced utilization of peripheral insulin. Inflammatory factors are also known as "sarcopenia-activating" cytokines. The inflammatory factors causing RS mainly include: tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 6 (IL-6), interferon α (IFN-α), transforming growth factor β (TGF-β), myogenic regulatory protein MYoD, etc. Studies have found that the levels of IL-6 and TNF-α in the elderly with RS are significantly higher than those without sarcopenia, and some studies suggest that IL-6 may be involved in muscle wasting and is an independent risk factor for RA sarcopenia.

[0004] Relevant guidelines point out that the prevention and treatment measures for sarcopenia include exercise therapy, nutritional therapy, and drug treatments such as anabolic hormones, active vitamin D, and β-adrenergic receptor stimulants. However, there is currently no drug indicated for sarcopenia. Therefore, the treatment of RS has gradually attracted attention in recent years, and its effective and scientific treatment methods urgently need to be studied in depth. Summary of the Invention

[0005] The object of the present invention is to provide a traditional Chinese medicine active ingredient for treating rheumatoid sarcopenia - coixenolide and its preparation method. Guided by the principle of strengthening the spleen and stomach and removing dampness and promoting diuresis, through a large amount of practice and repeated verification, it is determined that it has the effect of treating rheumatoid sarcopenia. The combination of herbs is reasonable and the dosage is more accurate. Compared with western medicine in the efficacy evaluation of treating rheumatoid sarcopenia, it has better curative effect, and there are no obvious toxic and side effects in the experiment, which is safe and reliable and has important clinical significance.

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

[0007] The present invention provides the use of coixenolide in the preparation of a medicament for treating rheumatoid sarcopenia.

[0008] The present invention also provides the use of coixenolide in the preparation of a medicament for reducing the degree of joint swelling.

[0009] The present invention also provides the use of coixenolide in the preparation of a medicament for reducing inflammatory indexes.

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

[0011] Further, the above-mentioned medicament is an oral preparation, an injection or an inhalant.

[0012] Further, the preparation method of the above-mentioned traditional Chinese medicine extract coixenolide includes the following steps: (1) Crushing and granulating: Coix seeds that have been shelled and decontaminated are crushed into fine powder of 60 - 200 meshes, poured into a blender, and while stirring, 40% - 80% of the weight of the raw materials of purified water is added, wet granulation is carried out with a 20-mesh sieve, and drying is carried out at 55 - 60 °C, controlling the moisture within 5%, and it is better when the moisture is 3 - 4%; (2) Screening and sizing: The dried and formed coix seed granules are sieved with a 10 - 20-mesh fine powder sieve, and the sieved fine powder is returned for re-granulation; (3) Supercritical CO2 stepwise extraction and fractional collection: The coix seed powder particles that have been screened and sized in (2) are put into the basket of a 24L extraction kettle, and supercritical CO2 stepwise pressure increase and fractional extraction are adopted. The pressure increases step by step at 10, 15, 20, 25, 30 MPa, 1 hour per step, and fractional collection is carried out at 0.5 hour per section; When the extraction pressure rises to about 20 MPa, the dissolution ability of CO2 increases, and coixenolide with relatively large solubility is extracted and the operation is stopped to obtain coixenolide.

[0013] The disease essence of sarcopenia (RS) is the reduction of muscle mass and the relative increase of fat content, which belongs to the category of "flaccidity syndrome" and "muscular flaccidity" in traditional Chinese medicine (TCM), and is closely related to "phlegm-dampness". It is manifested as the relaxation, weakness and inability to move voluntarily of the limb tendons and vessels. Over time, the muscles become paralyzed and useless. "Plain Questions - On Maintaining Health and Communicating with Heaven" emphasizes that damp pathogen is the main external inducing factor for the formation of flaccidity syndrome, describing that "due to dampness, the head is like being wrapped. If the damp-heat is not dispelled, the large tendons will become soft and short, and the small tendons will become flaccid and long. Soft and short means contraction, and flaccid and long means flaccidity." It is considered that damp-heat is one of the causes of flaccidity syndrome. "Plain Questions - Treatise on Flaccidity Syndrome" puts forward the basic principle of "treating flaccidity syndrome by solely regulating Yangming meridian" in treatment. In addition to problems with their own immune systems, most patients with flaccidity syndrome are related to the impairment of internal zang-fu functions. Weakness of the spleen and stomach, deficiency of the liver and kidney, etc. cause the patients to present the conditions of "deficiency" and "flaccidity". Multiple studies on sarcopenia suggest that phlegm-dampness constitution or phlegm-dampness syndrome is the most important constitution or syndrome type. This coincides with the TCM theory that "obese people are mostly with phlegm-dampness". A large number of studies show that sarcopenic obesity is a state of chronic low-grade inflammation, involving the release of various cytokines, the generation of inflammatory mediators and the activation of inflammatory response signaling pathways. Modern medicine believes that the central link of the inflammatory process is the vascular reaction. Blood vessels are equivalent to the category of meridians in TCM. When phlegm-dampness accumulates internally and the meridians are blocked, it may lead to systemic low-grade vascular inflammatory lesions in modern medicine. At the same time, low-grade vascular inflammation will lead to the accumulation of phlegm-dampness and blood stasis. Therefore, in the treatment of RS, the method of resolving phlegm and removing dampness can be used to reduce the body's inflammation level to achieve the purpose of preventing and treating diseases. Our team proposed to use "coix seed" as the representative formula to implement the method of "invigorating the spleen and promoting diuresis to eliminate dampness" in the treatment of RS, and the clinical empirical effect is exact.

[0014] Coix seed: Sweet and light in taste, cool in nature; attributive to the spleen, stomach and lung meridians. It has the effects of promoting diuresis to eliminate dampness, invigorating the spleen and stopping diarrhea, relieving arthralgia, discharging pus, detoxifying and dissipating nodules. It is mainly used to treat edema, beriberi edema, diarrhea due to spleen deficiency, damp arthralgia with contracture, lung abscess and intestinal abscess. "Shennong Ben Cao Jing": "It can relieve spasm and contracture of the tendons, make them unable to stretch and flex, treat wind-damp arthralgia, and lower qi. Long-term use can make the body light and strengthen qi." "Mingyi Bielu": "It can mainly dispel pathogenic factors in the tendons and bones, benefit the intestines and stomach, eliminate edema, and make people able to eat." "Yaopin Huayi": "It is mainly used to treat diarrhea due to spleen deficiency, resulting in edema, and wind-damp sluggishness, resulting in weakness of the hands and feet, unable to stretch and flex. Because when dampness prevails, the earth fails, and when the earth prevails, qi recovers, the edema will subside and strength will be generated. Taking it into the lung to nourish the source of transformation, it is used to treat upper-jiao consumptive thirst, lung abscess and intestinal abscess." Modern pharmacological research shows that coix seed mainly contains triolein, α-monoolein, α-monolinolenin, coixenolide and coixan, and has the effects of regulating the gastrointestinal tract, anti-tumor, hypoglycemic, analgesic, ulcer inhibition, immune regulation, anti-obesity, anti-cancer, etc. As a traditional Chinese medicine for resolving phlegm and removing dampness, coix seed has good anti-inflammatory effects and is an important compatibility traditional Chinese medicine for treating rheumatoid arthritis by the method of invigorating the spleen and promoting diuresis to eliminate dampness. However, there is no report on the research of treating RS.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The active ingredients of traditional Chinese medicine provided by the present invention follow the treatment principles of strengthening the spleen and stomach, and removing dampness and promoting diuresis, and can effectively treat collagen-induced rheumatoid sarcopenia in rats. Moreover, compared with the positive control drug group of methotrexate + testosterone, the present invention has better curative effects and significant differences.

[0016] The active ingredients of traditional Chinese medicine provided by the present invention are used for treating rheumatoid sarcopenia, can relieve symptoms such as slow and weak limb tendons and muscles, and muscle atrophy, and improve the quality of life of patients.

[0017] The present invention has no obvious toxic and side effects, is safe and reliable, has a clear preparation method, and is suitable for large-scale production by grass-roots medical institutions or manufacturers. Description of the Drawings

[0018] Figure 1 The joint swelling conditions of rats in each group. A: The body weights of rats in each group; B: The paw thickness of rats in each group; C: Inter-group comparison of the average body weights of rats in each group on the 35th day; D: Inter-group comparison of the paw thickness of rats in each group on the 35th day. Compared with the blank group, <0.05, <0.01; compared with the CIA sarcopenia group, <0.05, <0.01. Compared with the coixenolide group, <0.05, <0.01; compared with the MTX + testosterone group, <0.05, <0.01.

[0019] Figure 2 The arthritis index scores of rats in each group, A: The arthritis index of rats in each group; B: Inter-group comparison of the arthritis indexes of rats in each group on the 36th day. Compared with the blank group, <0.05, <0.01; compared with the CIA sarcopenia group, <0.05, <0.01; compared with the coixenolide group, <0.05, <0.01; compared with the MTX + testosterone group, <0.05, <0.01.

[0020] Figure 3 The pathological conditions of the ankle joint tissues of rats in each group. A: HE staining pathology; B: Safranin O-fast green staining pathology; C: HE pathology score; D: Safranin O pathology score. Compared with the blank group, <0.05, <0.01; compared with the CIA sarcopenia group, <0.05, < 0.01; compared with the coix seed ester group, < 0.05, < 0.01; compared with the MTX + testosterone group, < 0.05, < 0.01.

[0021] Figure 4 The expression levels of serum inflammatory cytokines in rats of each group. A: The expression level of TNF-α in the serum of rats in each group; B: The expression level of IL-6 in the serum of rats in each group; C: The ratio of the gray value of TNF-α protein to GAPDH in the serum of rats in each group; D: The ratio of the gray value of IL-6 protein to GAPDH in the serum of rats in each group. Compared with the blank group, < 0.05, < 0.01; compared with the CIA sarcopenia group, < 0.05, < 0.01; compared with the coix seed ester group, < 0.05, < 0.01; compared with the MTX + testosterone group, < 0.05, < 0.01.

[0022] Figure 5 Muscle pathological changes in skeletal muscles of each group.

[0023] Figure 6 Changes in the expression of muscle atrophy-related proteins in rats of each group. A: The level of the muscle atrophy index p-FOXO-3A in the skeletal muscle tissues of rats in each group; B: The level of the muscle atrophy index Atrogin-1 in the skeletal muscle tissues of rats in each group; C: The level of the muscle atrophy index MuRF-1 in the skeletal muscle tissues of rats in each group; D: The ratio of the gray value of the muscle atrophy protein p-FOXO-3A to GAPDH; E: The ratio of the gray value of the muscle atrophy protein Atrogin-1 to GAPDH; F: The ratio of the gray value of the muscle atrophy protein MuRF-1 to GAPDH. Compared with the blank group, < 0.05, < 0.01; compared with the CIA sarcopenia group, < 0.05, < 0.01; compared with the coix seed ester group, < 0.05, < 0.01; compared with the MTX + testosterone group, < 0.05, < 0.01.

[0024] Figure 7Changes in the expression of muscle differentiation-related proteins in each group of rats. A: The level of the muscle differentiation index MyoD1 in the skeletal muscle tissues of each group of rats; B: The level of the muscle differentiation index MyoG in the skeletal muscle tissues of each group of rats; C: The level of the muscle differentiation index MHC in the skeletal muscle tissues of each group of rats; D: The ratio of the muscle differentiation protein MyoD1 to GAPDH in each group of rats; E: The ratio of the muscle differentiation protein MyoG to GAPDH in each group of rats; F: The ratio of the muscle differentiation protein MHC to GAPDH in each group of rats. Compared with the blank group, <0.05, <0.01; compared with the CIA sarcopenia group, <0.05, <0.01; compared with the coixenolide group, <0.05, <0.01; compared with the MTX + testosterone group, <0.05, <0.01. Specific implementation manners

[0025] The present invention will be described in detail below in conjunction with specific embodiments. The following are only the preferred embodiments of the present invention and are not used to limit the present invention. Any equivalent replacement of some technical features, and any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

[0026] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0027] Example 1 Preparation of coixenolide.

[0028] (1) Crushing and granulation: The coix seeds that have been shelled and decontaminated are crushed into fine powder with a mesh size of 60 - 200, poured into a blender, and while stirring, add pure water accounting for 40% - 80% of the raw material weight. Wet granulation is carried out with a 20-mesh sieve and dried at 55 - 60 °C, controlling the moisture within 5%, and it is better when the moisture is 3 - 4%; (2) Granule sizing and screening: The dried and formed coix seed granules are sized with a 10 - 20-mesh sieve and then the fine powder is screened out with a 50-mesh sieve. The screened fine powder is returned for re-granulation; (3)Stepwise extraction and fractional collection with supercritical CO2: A carbon dioxide cylinder, a carbon dioxide storage tank, an extraction kettle, and a separation kettle are connected in sequence. There are pipelines above them for CO2, coix seed oil, and coix seed ester to pass through. Carbon dioxide goes from the carbon dioxide cylinder to the carbon dioxide storage tank, passes through heat exchanger I, high-pressure pump, and heat exchanger II in sequence, enters the extraction kettle and the separation kettle, and then returns to the carbon dioxide storage tank to complete a working cycle. The coix seed powder particles that have been whole-grained and sieved are placed in the material basket of a 24L extraction kettle. The pressure of supercritical CO2 introduced into the kettle is increased step by step between 10 and 30 MPa, and the extraction temperature is between 40 and 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. Fractional collection is carried out in segments of 0.5 - 1.0 hr / segment according to the extraction time. The valve below the separation kettle is for collection. The total time for stepwise extraction and fractional collection is 5 - 5.5 hr.

[0029] Specifically, for example, the coix seed powder particles that have been whole-grained and sieved as in (2) are placed in the material basket of a 24L extraction kettle. Supercritical CO2 is used for stepwise pressure increase and fractional extraction. The pressures for pressure increase are increased step by step to 10, 15, 20, 25, and 30 MPa respectively, with 1 hr for each step, and fractional collection is carried out in segments of 0.5 hr / segment. Stepwise pressure increase is used to improve 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. Then, as the pressure further increases to 25 MPa, the coix seed oil with relatively low solubility is also extracted. When continuing to extract 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 fractional collection, the online separation of moisture, coix seed ester, coix seed oil, and other impurities in coix seed powder particles can be achieved. When obtaining two products of coix seed ester and coix seed oil, moisture and some impurities are separated simultaneously, and yellowish-white coix seed ester, light yellow coix seed oil, moisture, and other impurity components can be separated.

[0030] Pathological expression of rats in each group of Example 2.

[0031] 1. Animal experiment data and methods.

[0032] 1.1 Experimental animals.

[0033] From December 2019 to April 2021, in the laboratory of the Department of Traditional Chinese Medicine, the First Affiliated Hospital of China Medical University, 50 6-week-old SPF-grade male SD rats, weighing 200-220 g, were housed in the Animal Center of China Medical University at a temperature of 25±2°C with a 12-hour light-dark cycle. The rats were maintained under specific pathogen-free conditions and given standard food and water.

[0034] 1.2 Experimental methods.

[0035] Establish a CIA sarcopenia rat model: Under sterile and light-proof conditions, chicken type II collagen powder was fully dissolved in glacial acetic acid. 2 mg / ml of chicken type II collagen was thoroughly ground and emulsified with an equal volume of complete Freund's adjuvant CFA on ice until it did not disperse when dropped into water. The white collagen emulsion (CII emulsion) was taken. Rats in the model group were injected at the base of the tail and 3-5 points on the back at a dose of 0.1 mL / 100 g on day 0. Seven days after the primary immunization, avoiding the primary injection site, the above CII emulsion was used for booster injection on the back and tail. At the same time, a splint fixation experiment for sarcopenia modeling was carried out, and the rats were anesthetized before the operation. The hind limbs were fixed by suturing the feet to the limbs using a surgical stapler (automatic suture Royal 35W stapler) and fixed for 21 days. Rats in the blank group only received anesthesia.

[0036] Grouping and drug administration: 50 rats were first randomly divided into a 10-rat blank group and a 40-rat model group. After the model group was successfully modeled, it was randomly and evenly divided into the CIA sarcopenia group, coixenolide group, MTX + testosterone group, and coix seed oil group. The coixenolide group and the MTX + testosterone group started intragastric administration 21 days after modeling. The coixenolide group and the coix seed oil group were intragastrically administered 4.2 g / kg twice a day, and the MTX + testosterone group was intragastrically administered 0.5 mg / kg every 3 days once. The blank group and the CIA sarcopenia group were intragastrically administered an equal amount of normal saline, and the drug administration continued for 4 weeks. The 500 μg / ml coixenolide was obtained according to the preparation method described in Example 1.

[0037] 1.3 Animal sampling.

[0038] After 7 weeks, the above rats in each group were anesthetized and abdominal aortic blood was collected. After standing for 1 hour, it was centrifuged at 3000 rpm for 10 minutes at 4°C, and the serum was collected and stored in a -80°C refrigerator. The knee joint synovium was collected, and the tissue 1 cm above the ankle joint was intercepted. Half was frozen at -80°C, and half was fixed in 4% paraformaldehyde for 48 hours. After being washed clean with the fixing solution, the hind limb ankle joint was placed in an EDTA decalcifying solution for decalcification, and the decalcifying solution was changed twice a week until decalcification was complete.

[0039] 2. Observation indicators.

[0040] 2.1 General indicators: Mental state, diet and water intake, sleep state, activity level, hair condition, etc.

[0041] 2.2 Efficacy observation indicators: (1) Effects of coixenolide on joint swelling in CIA sarcopenia rats; (2) Effects of coixenolide on arthritis index scores in CIA sarcopenia rats; (3) Effects of coixenolide on ankle joint histopathology in CIA sarcopenia rats; (4) Effects of coixenolide on serum inflammatory cytokine levels in CIA sarcopenia rats; (5) Comparison of skeletal muscle pathological changes in each group; (6) Comparison of changes in the expression of muscle atrophy-related proteins in each group of rats; (7) Comparison of changes in the expression of muscle differentiation-related proteins in each group of rats.

[0042] 3. Statistical analysis.

[0043] Data analysis was performed using SPSS 23.0 statistical software. Analysis of variance was used for measurement data that conformed to a normal distribution, and non-parametric tests were used for those that did not. Repeated measures analysis of variance was used for VAS scores, the rank sum test was used for ranked data, and the X 2 test was used for comparison of count data.

[0044] 4. Results.

[0045] 4.1 General efficacy observation in each group.

[0046] During the experiment, the general conditions of the rats were observed: mental state, food and water intake, sleep state, activity level, hair condition, etc. The rats in the blank group were in good spirits, had normal food and water intake, good sleep, moved freely, and had shiny and smooth hair. The rats in the CIA sarcopenia group were listless, had reduced food and water intake, decreased activity, dull and yellowish hair, hunched backs and huddled together, and could not put their affected limbs on the ground. The mental state, food and water intake, activity level, hair condition, etc. of the rats in the coixenolide group, coix oil group, and MTX + testosterone group were improved.

[0047] 4.2 Conditions of joint swelling in each group of rats (as Figure 1 shown).

[0048] The body weights of the rats in the blank group increased steadily, the thickness of the paw did not increase, and the joints were not swollen. Compared with the blank group, the body weights of the rats in the CIA sarcopenia group decreased, the paws thickened, and the arthritis gradually swelled after modeling. After 21 days, the body weights gradually recovered, and the paw thickness and joint swelling gradually alleviated (P < 0.01). Compared with the CIA sarcopenia group, coixenolide, coix seed oil, and MTX + testosterone groups could alleviate the body weight loss of the CIA sarcopenia rats, and the paw thickness and joint swelling were also alleviated (P < 0.01). Among them, the effect of the coixenolide group on body weight recovery was equivalent to that of the MTX + testosterone group. On the 35th day, compared with the coixenolide group, the MTX + testosterone group had a better effect on body weight recovery, and the paw thickness decreased significantly (P < 0.01). The coix seed oil group had a poor effect on body weight recovery, and the effect of reducing paw thickness was not significant (P < 0.01). Compared with the MTX + testosterone group, the coix seed oil group had a poor effect on body weight recovery and less reduction in paw thickness (P < 0.01). The above results indicate that compared with coix seed oil, coixenolide has a significant effect on the recovery of body weight and paw thickness in CIA sarcopenia rats.

[0049] 4.3 The arthritis index scores of rats in each group (as Figure 2 shown).

[0050] The paws of the rats in the blank group were not swollen, and the arthritis index score was 0. Compared with the blank group, joint swelling began to appear after modeling in the CIA sarcopenia group, and the arthritis index score increased (P < 0.01). Compared with the CIA sarcopenia group, the arthritis index of the rats in the coixenolide group, coix seed oil group, and MTX + testosterone group gradually decreased (P < 0.01). Among them, the MTX + testosterone group had the most significant effect on reducing the inflammation score, the coixenolide group was the second, and the coix seed oil group had a poor effect on reducing inflammation. On the 35th day, compared with the coixenolide group, the MTX + testosterone group had the most significant effect on reducing the arthritis index, and the coix seed oil group was not as effective as the coixenolide group (P < 0.01). Compared with the MTX + testosterone group, the coix seed oil group had a poor effect on reducing the arthritis index (P < 0.01).

[0051] 4.4 The pathological conditions of the ankle joints of rats in each group (as Figure 3 shown).

[0052] 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 the rats in the blank group was intact, the articular cartilage was flat and smooth, there was no infiltration of inflammatory cells in the space, and no bone destruction was seen, and the pathological score was 0; compared with the blank group, the ankle joint structure of the rats in the CIA sarcopenia group was disordered, the joint surface was rough, the synovium hyperplasia was present, the space was narrow and there was a large amount of inflammatory cell infiltration, pannus formation was visible, and obvious bone and cartilage destruction was seen, and the pathological score was increased compared with the normal group (P < 0.01); compared with the CIA sarcopenia group, the pathological score of the ankle joint in the coixenolide group was decreased compared with the CIA sarcopenia group (P < 0.01); the safranin O score in the coix seed oil group was slightly decreased, but there was no statistical significance; the pathological score of the ankle joint of the rats in the MTX + testosterone group was significantly decreased, showing that the joint surface was slightly smooth, with a small amount of inflammatory cell infiltration, no obvious synovium hyperplasia, and no obvious cartilage and bone destruction (P < 0.01). Compared with the coixenolide group, the joint surface of the rats in the MTX + testosterone group was smoother, with less inflammatory cell infiltration, less synovium hyperplasia, and lower HE staining and safranin O staining scores; the joint surface of the coix seed oil group was rougher, with more synovium hyperplasia and more inflammatory cell infiltration, and higher HE staining and safranin O staining scores. Compared with the MTX + testosterone group, the HE staining and safranin O staining scores of the coix seed oil group were higher, the joint surface was rougher, and the cartilage destruction was more significant. The above shows that the pathological score of the ankle joint of the rats in the coixenolide group was roughly equivalent to that of the positive drug MTX + testosterone group, with less inflammatory cell infiltration and synovium hyperplasia, and less bone destruction, while the pathological score of the ankle joint of the rats in the coix seed oil group was higher, and the reduction of joint inflammation was worse.

[0053] 4.5 Expression of serum inflammatory cytokine levels in rats of each group (as Figure 4 shown).

[0054] Compared with the blank group, the levels of TNF-α and IL-6 in the serum of the rats in the CIA sarcopenia group were significantly increased, and the results were statistically significant (P < 0.01); compared with the CIA sarcopenia group, the levels of TNF-α and IL-6 in the serum of the rats in the coixenolide group and the MTX + testosterone group were significantly decreased (P < 0.01); the level of TNF-α in the coix seed oil group was slightly decreased, but there was no statistical significance, and the level of IL-6 was slightly decreased (P < 0.01). Compared with the coixenolide group, the levels of TNF-α and IL-6 in the serum of the rats in the MTX + testosterone group were significantly decreased, and the levels of TNF-α and IL-6 in the coix seed oil group were significantly increased. Compared with the MTX + testosterone group, the levels of TNF-α and IL-6 in the serum of the rats in the coix seed oil group were significantly decreased. The above shows that the coixenolide group is more effective in reducing the levels of TNF-α and IL-6 than the coix seed oil group.

[0055] 4.6 Comparison of pathological changes of skeletal muscle in each group (as Figure 5 shown).

[0056] HE staining showed that the skeletal muscle tissue structure of the blank group was intact, the muscle fibers were arranged tightly and orderly, without rupture or edema. Compared with the blank group, the muscle fibers in the CIA sarcopenia group were arranged disorderly, and the inter-fiber space was increased. Compared with the CIA sarcopenia group, the skeletal muscle tissue structure and the disordered arrangement of muscle fibers in the coixenolide group and the MTX+testosterone group were improved. The coix seed oil group showed a slight improvement, but the effect was not as obvious as that of the coixenolide group. Compared with the coixenolide group, the skeletal muscle tissue structure of the MTX+testosterone group was more complete and the arrangement was more orderly. The coix seed oil group was slightly disordered and the space was slightly increased. Compared with the MTX+testosterone group, the improvement of the skeletal muscle tissue structure and arrangement in the coix seed oil group was poor. The above results indicate that coixenolide can significantly improve the tightness of muscle fiber arrangement, and the effect is more significant than that of the coix seed oil group.

[0057] 4.7 Compare the changes in the expression of muscle atrophy-related proteins in rats of each group (as Figure 6 shown).

[0058] The expression levels of muscle atrophy indexes p-FOXO-3A, Atrogin-1, and MuRF-1 in skeletal muscle tissues were detected by Western blotting. Compared with the blank group, the expressions of p-FOXO-3A and Atrogin-1 in the RS group were significantly up-regulated (P<0.05, P<0.01). Compared with the CIA sarcopenia group, the expression of p-FOXO-3A in the coixenolide group and the MTX+testosterone group of rats was significantly down-regulated (P<0.01), and the expression of Atrogin-1 was significantly up-regulated (P<0.01). Compared with the MTX+testosterone group, the level of muscle atrophy index p-FOXO-3A in the coixenolide group decreased more, and the level of Atrogin-1 increased significantly. The coix seed oil group did not show an obvious decreasing trend in the expression of muscle atrophy proteins p-FOXO-3A, Atrogin-1, and MuRF-1. Each drug administration group did not show a therapeutic effect on the expression of MuRF-1.

[0059] 4.8 Compare the changes in the expression of muscle differentiation-related proteins in rats of each group (as Figure 7 shown).

[0060] The expression levels of muscle differentiation indexes MyoD1, MyoG, and MHC in skeletal muscle tissues were detected by Western blotting. Compared with the blank group, the expressions of MyoD1, MyoG, and MHC in the CIA sarcopenia group were significantly decreased (P<0.01). Compared with the CIA sarcopenia group, the expressions of MyoD1 and MHC in the coixenolide group and the MTX+testosterone group of rats were significantly up-regulated (P<0.05, P<0.01). Compared with the MTX+testosterone group, the coixenolide group had a better up-regulation effect on MyoD1 and MyoG, and the up-regulation effect of MHC was roughly the same in both groups. The coix seed oil group did not show an obvious up-regulation trend for muscle differentiation-related proteins.

[0061] In summary, the coixenolide of the present invention has the advantages of good curative effect, few adverse reactions, and low cost in the treatment of traditional Chinese medicine flaccidity syndromes such as rheumatoid cachexia (sarcopenia), etc., can effectively improve the symptoms of limb muscle relaxation, weakness, muscle atrophy, etc. of patients, improve the quality of life of patients, and is worthy of promotion in clinical practice.

Claims

1. Use of coixenolide in the preparation of a medicament for treating rheumatoid sarcopenia.

2. Use of coixenolide in the preparation of a medicament for reducing the degree of joint swelling.

3. Use of coixenolide in the preparation of a medicament for reducing inflammatory indexes.

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

5. The application according to claims 1 to 3, characterized in that, The medicament 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 coixenolide of the traditional Chinese medicine extract comprises the following steps: (1) Crushing and granulating: Crushing the coix seeds with the husk removed and impurities removed into fine powder of 60-200 mesh, pouring it into a blender, adding pure water accounting for 40%-80% of the raw material weight while stirring, performing wet granulation with a 20-mesh sieve, drying at 55-60 °C, controlling the moisture within 5%, and it is better when the moisture is 3%-4%; (2) Granule sizing and screening: Sizing the dried and formed 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 fine powder for re-granulation; (3) Stepwise extraction and fractional collection with supercritical CO2: Putting the coix seed powder granules after (2) granule sizing and screening into the material basket of a 24L extraction kettle, adopting stepwise pressure increase and fractional extraction with supercritical CO2, increasing the pressure step by step to 10, 15, 20, 25, 30 MPa respectively, 1 hour per step, collecting fractionally at 0.5 hour per section. When the extraction pressure rises to about 20 MPa, the dissolution ability of CO2 increases, and the coixenolide with relatively large solubility is extracted.

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