Traditional Chinese medicine composition for treating rheumatoid arthritis inflammation and bone destruction as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and relates to a traditional Chinese medicine composition for treating rheumatoid arthritis inflammation and bone destruction as well as a preparation method and application thereof. The traditional Chinese medicine composition is prepared from the following raw materials: sabia japonica, fructus psoraleae, rhizoma drynariae, angelica sinensis and herba epimedii. The preparation method of the traditional Chinese medicine composition comprises the following steps: (1) carrying out alcohol extraction on sabia japonica and rhizoma drynariae to obtain an alcohol extract and medicine residues; (2) performing water extraction on the medicine residues and other raw materials to obtain a water extract; and (3) combining the alcohol extract and the water extract to obtain the traditional Chinese medicine composition. The traditional Chinese medicine composition is prepared by performing alcohol extraction on sabia japonica and rhizoma drynariae, performing water extraction on decoction dregs of the sabia japonica and the rhizoma drynariae and other raw materials, and merging an alcohol extract and a water extract, and experiments prove that compared with the prior art, the traditional Chinese medicine composition has better effects on inhibiting bone mass loss around inflammatory joints, improving osteoporosis and enhancing bone strength, and has a good application prospect. The traditional Chinese medicine composition has a better curative effect on rheumatoid arthritis bone destruction.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and relates to a traditional Chinese medicine composition for treating rheumatoid arthritis inflammation and bone destruction, and a preparation method and use thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a heterogeneous, systemic, autoimmune disease with symmetrical polyarthritis as the main clinical manifestation. The main clinical manifestations are joint swelling and pain caused by small joint synovitis, followed by cartilage destruction and narrowing of joint space. In the late stage, severe bone destruction and absorption lead to joint stiffness, deformity, and dysfunction. It is a chronic invasive, progressive, recurrent, global disease with a high disability rate and poor prognosis. There is currently no good cure.

[0003] Rheumatoid arthritis mainly manifests itself in joint symptoms and multi-system involvement outside joints. The onset is slow and insidious. There may be a low fever for several weeks before obvious joint symptoms appear. A few patients may have high fever, fatigue, general discomfort, weight loss and other symptoms, and then gradually develop typical joint symptoms. A few have a more rapid onset, with multiple joint symptoms appearing within a few days.

[0004] Joint lesions can be divided into synovial inflammation and joint structure destruction. The former is reversible to a certain extent after treatment, but the latter is difficult to reverse once it occurs. The condition and course of the disease vary from individual to individual, ranging from short-term, mild oligoarthritis to rapidly progressive polyarthritis, with morning stiffness, joint pain, joint swelling, joint deformity, special joints, joint dysfunction, etc. X-ray plain film joint lesion staging: In the early stage, swelling of the soft tissue around the joint and osteoporosis at the joint end can be seen (stage I); then the joint space becomes narrower (stage II); worm-like changes appear on the joint surface (stage III); in the late stage, joint subluxation and fibrous and bony ankylosis after joint destruction can be seen (stage IV). The diagnosis should have bone invasion or definite localization or obvious decalcification near the affected joint.

[0005] As the etiology and pathogenesis of this disease are not completely clear, there is currently a lack of effective measures to cure and prevent this disease in clinical practice. The current treatment goals are to alleviate joint symptoms, delay disease progression, prevent and reduce joint damage, protect joint function, and maximize the patient's quality of life.

[0006] CN105687397A discloses a Chinese medicine composition for treating rheumatoid arthritis inflammation and bone destruction and its use, wherein the Chinese medicine composition is made of the following raw materials in parts by weight: 10-20 parts of Qingfengteng, 15-30 parts of psoralea corylifolia, 15-30 parts of bone drynariae, 9-15 parts of angelica sinensis, and 15-20 parts of epimedium. The Chinese medicine composition of the invention has a significant inhibitory effect on the swelling of the toes of the animal model of rheumatoid arthritis, and can significantly reduce the bone loss around the inflammation joints of the animal model of rheumatoid arthritis, improve bone biomechanical parameters, inhibit bone destruction caused by rheumatoid arthritis, and has no toxic side effects. At present, the preparation method of the Chinese medicine composition is traditional decoction, and the loss of effective ingredients is relatively large. How to develop a Chinese medicine composition with better therapeutic effect on this basis is currently very much adjusted. Summary of the invention

[0007] The purpose of the present invention is to provide a Chinese medicine composition with better therapeutic effect for treating rheumatoid arthritis inflammation and bone destruction, as well as a preparation method and use thereof.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a Chinese medicine composition for treating rheumatoid arthritis inflammation and bone destruction, wherein the raw materials of the Chinese medicine composition are: qingfengteng, psoralea corylifolia, drynariae, angelica and epimedium;

[0010] The preparation method of the Chinese medicine composition comprises the following steps:

[0011] (1) extracting the scutellaria baicalensis and rhizoma drynariae with alcohol to obtain an alcohol extract and medicinal residues;

[0012] (2) extracting the medicinal residue and other raw materials with water to obtain a water extract;

[0013] (3) combining the alcohol extract and the water extract to obtain the Chinese medicine composition.

[0014] In some embodiments, the alcohol extraction in step (1) is reflux extraction using alcohol as solvent.

[0015] Preferably, the alcohol is at least one of ethanol, propanol, butanol and pentanol; more preferably, it is ethanol.

[0016] Preferably, the volume fraction of the alcohol is 60-95%, more preferably 70-80%.

[0017] Preferably, the amount of the alcohol used is 8-15 times the total mass of Herba Epimedii and Herba Epimedii, preferably 10 times.

[0018] Preferably, the number of reflux extractions is 1-5 times, more preferably 2-3 times; the time for each reflux extraction is 0.5-3 h, more preferably 1-2 h.

[0019] In some embodiments, the water extraction in step (2) is reflux extraction using water as solvent.

[0020] Preferably, the amount of water used is 6-12 times the total mass of Herba Epimedii, Herba Psoraleae, Herba Drynariae, Herba Angelicae Sinensis and Herba Epimedii, preferably 10 times.

[0021] Preferably, the reflux extraction is performed 1-5 times, more preferably 2-3 times; the time of each reflux extraction is 1-3 hours, more preferably 2 hours.

[0022] In some embodiments, the alcohol extract and the water extract in step (3) are concentrated before being combined;

[0023] The concentration is reduced pressure concentration at a temperature of 50-80°C, preferably 60°C;

[0024] The alcohol extract is concentrated to a relative density of 1.10-1.12 (60° C.); the water extract is concentrated to a relative density of 1.10-1.12 (60° C.).

[0025] Preferably, after the alcohol extract and the water extract described in step (3) are combined, drying is also included.

[0026] The drying is vacuum drying, spray drying or freeze drying.

[0027] In some embodiments, the Chinese medicine composition comprises the following raw materials, by weight: 8-16 parts of Herba Sinomenii Sinensis, 8-12 parts of Psoralea corylifolia, 8-10 parts of Rhizoma Drynariae, 9-15 parts of Radix Angelicae Sinensis, and 10-20 parts of Herba Epimedii.

[0028] In some embodiments, the Chinese medicine composition comprises the following raw materials, by weight: 10-14 parts of Herba Lycopodii, 8-12 parts of Herba Psoraleae, 8-10 parts of Herba Drynariae, 12-14 parts of Herba Angelicae Sinensis, and 16-18 parts of Herba Epimedii.

[0029] In some embodiments, the Chinese medicine composition comprises the following raw materials, by weight: 12 parts of Herba Sinomenii Sinensis, 10 parts of Psoralea corylifolia, 9 parts of Rhizoma Drynariae, 12 parts of Radix Angelicae Sinensis, and 18 parts of Herba Epimedii.

[0030] In a second aspect, the present invention provides a method for preparing a Chinese medicine composition for treating rheumatoid arthritis inflammation and bone destruction, wherein the raw materials of the Chinese medicine composition are: Herba Lycopodii, Herba Psoraleae, Herba Drynariae, Herba Angelicae Sinensis and Herba Epimedii; the method comprises the following steps:

[0031] (1) extracting the scutellaria baicalensis and rhizoma drynariae with alcohol to obtain an alcohol extract and medicinal residues;

[0032] (2) mixing the medicinal residue and the remaining raw materials with water to obtain a water extract;

[0033] (3) combining the alcohol extract and the water extract to obtain the Chinese medicine composition.

[0034] In some embodiments, the alcohol extraction in step (1) is reflux extraction using alcohol as solvent.

[0035] Preferably, the alcohol is at least one of ethanol, propanol, butanol and pentanol; more preferably, it is ethanol.

[0036] Preferably, the volume fraction of the alcohol is 60-95%, more preferably 70-80%.

[0037] Preferably, the amount of the alcohol used is 8-15 times the total mass of Herba Epimedii and Herba Epimedii, preferably 10 times.

[0038] Preferably, the number of reflux extractions is 1-5 times, more preferably 2-3 times; the time for each reflux extraction is 0.5-3 h, more preferably 1-2 h.

[0039] In some embodiments, the water extraction in step (2) is reflux extraction using water as solvent.

[0040] Preferably, the amount of water used is 6-12 times the total mass of Herba Epimedii, Herba Psoraleae, Herba Drynariae, Herba Angelicae Sinensis and Herba Epimedii, preferably 10 times.

[0041] Preferably, the reflux extraction is performed 1-5 times, more preferably 2-3 times; the time of each reflux extraction is 1-3 hours, more preferably 2 hours.

[0042] In some embodiments, the alcohol extract and the water extract in step (3) are concentrated before being combined;

[0043] The concentration is reduced pressure concentration at a temperature of 50-80°C, preferably 60°C;

[0044] The alcohol extract is concentrated to a relative density of 1.10-1.12 (60° C.); the water extract is concentrated to a relative density of 1.10-1.12 (60° C.).

[0045] Preferably, after the alcohol extract and the water extract described in step (3) are combined, drying is also included.

[0046] The drying is vacuum drying, spray drying or freeze drying.

[0047] In a third aspect, the present invention provides use of the aforementioned Chinese medicine composition in the preparation of a medicament for treating rheumatoid arthritis inflammation and / or bone destruction.

[0048] In a fourth aspect, the present invention provides a drug for treating rheumatoid arthritis inflammation and / or bone destruction, comprising the aforementioned Chinese medicine composition and pharmaceutically acceptable excipients.

[0049] The dosage form of the medicine in the present invention is capsule, granule, tablet, mixture or syrup.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The traditional Chinese medicine composition of the present invention is prepared by extracting Radix Psoraleae and Rhizoma Drynariae with alcohol, extracting the residues with water and the remaining raw materials, and combining the alcohol extract and the water extract. Experiments have shown that, compared with the prior art, the present invention has better effects in inhibiting bone loss around inflammatory joints, improving osteoporosis, and enhancing bone strength, and has better therapeutic effect on bone destruction caused by rheumatoid arthritis. DETAILED DESCRIPTION

[0052] The present invention is described below through specific embodiments to make the technical solution of the present invention easier to understand and grasp, but the present invention is not limited thereto, and the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0053] The endpoints and any value of the scope disclosed in this article are not limited to the accurate scope or value, and these scopes or values ​​should be understood to include values ​​close to these scopes or values. For numerical ranges, between the endpoint values ​​of each scope, between the endpoint values ​​of each scope and a separate point value, and between separate point values, one or more new numerical ranges can be combined with each other, and these numerical ranges should be considered as specifically disclosed in this article. Unless the context clearly indicates otherwise, the singular form "one", "a kind of" and "described" as used herein include singular and plural indicators. The numerical range described by the endpoint is included in all numerical values ​​and fractions within the corresponding range, as well as the described endpoint.

[0054] The "medicine" described in the present invention comprises the aforementioned Chinese medicine composition or the aforementioned Chinese medicine extract and pharmaceutically acceptable excipients. In a specific embodiment, the Chinese medicine composition or the aforementioned Chinese medicine extract described in the present invention is provided in the medicine in an effective amount (e.g., a therapeutically effective amount).

[0055] The "pharmaceutically acceptable excipients" described in the present invention include inert diluents, dispersants and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants. Excipients (such as cocoa butter), colorants, coating agents, sweeteners, flavoring agents and aromatics may also be present in the pharmaceutical composition.

[0056] The "drug" described in the present invention can be prepared by any method known in pharmacy. Generally speaking, these preparation methods include associating the aforementioned Chinese medicine composition (i.e., the first active ingredient) with a carrier or excipient and / or one or more other auxiliary ingredients, and then if necessary and / or desired, molding and / or packaging the product into a desired single dose or multiple dose unit.

[0057] The drugs of the present invention can be prepared according to known methods, such as the methods described in the general rules for preparation of the Chinese Pharmacopoeia 2020, the Japanese Pharmacopoeia 16th edition, the United States Pharmacopoeia and the European Pharmacopoeia 9th edition. The specific preparation method depends on the dosage form.

[0058] The first active ingredient and pharmaceutically acceptable excipients in the "medicament" of the present invention will vary according to the identity, body shape and / or condition of the subject to be treated and further according to the route of administration of the composition. The medicament may contain between 0.1% and 100% (w / w) of the first active ingredient.

[0059] "Treatment" as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progression of, or preventing the condition or disorder or one or more symptoms of such condition or disorder to which the term applies, unless otherwise indicated. The term "treat" as used herein refers to the act of treating, as "treat" is defined immediately above.

[0060] The "effective amount" described in the present invention refers to an amount sufficient to induce a desired biological response. The effective amount of the active ingredients of the present invention may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. In certain embodiments, the effective amount is a therapeutically effective amount. The effective amount is the amount of the first active ingredient described in the present invention in a single dose. In certain embodiments, the effective amount is the combined amount of the active ingredients described in the present invention in multiple doses.

[0061] The "therapeutically effective amount" described in the present invention is an amount sufficient to provide a therapeutic benefit in the treatment of a disease or sufficient to delay or minimize one or more symptoms associated with the disease. The therapeutically effective amount of a Chinese medicine composition means the amount of a therapeutic agent that provides a therapeutic benefit in the treatment of a disease alone or in combination with other therapies. The term "therapeutically effective amount" may encompass an amount that improves overall therapy, reduces or avoids symptoms, signs or causes of a disease and / or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount sufficient to treat any disease or disease described.

[0062] The terms "subject" or "patient" as used in the present invention are well known in the art and are used interchangeably herein to refer to mammals, including dogs, cats, rats, mice, monkeys, cows, horses, goats, sheep, pigs, camels, and most preferably humans. The term does not denote a particular age or sex. Thus, both adult and newborn subjects, whether male or female, are encompassed.

[0063] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention. The experimental methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0064] Example 1 Preparation of Chinese medicine composition

[0065] The Chinese medicine composition comprises the following raw materials by weight: 12 parts of Herba Lycopodii, 10 parts of Herba Psoraleae, 9 parts of Herba Drynariae, 12 parts of Herba Angelicae Sinensis, and 18 parts of Herba Epimedii.

[0066] Preparation method:

[0067] (1) Mix the prescribed amount of Qingfengteng and Drynariae Rhizoma, add 10 times the volume of 75% ethanol, reflux and extract three times, each time for 1 hour, filter, and combine the filtrate to obtain an alcohol extract and a medicinal residue;

[0068] (2) mixing the residue obtained in step (1) with the remaining raw materials, adding 10 times the amount of water, extracting at 90-95° C. for 3 times, each time for 2 hours, filtering, and combining the filtrate to obtain a water extract;

[0069] (3) combining the alcohol extract obtained in step (1) and the water extract obtained in step (2), and concentrating under reduced pressure at 60° C. to a relative density of 1.10-1.12 (60° C.) to obtain the Chinese medicine composition.

[0070] Example 2 Preparation of Chinese medicine composition

[0071] The Chinese medicine composition comprises the following raw materials by weight: 8 parts of Herba Lycopodii, 8 parts of Psoralea corylifoliae, 8 parts of Rhizoma Drynariae, 9 parts of Radix Angelicae Sinensis and 10 parts of Herba Epimedii.

[0072] Preparation method:

[0073] (1) Mix the prescribed amount of Qingfengteng and Drynariae Rhizoma, add 15 times the volume fraction of 60% ethanol, reflux and extract three times, each time for 3 hours, filter, and combine the filtrate to obtain an alcohol extract and a medicinal residue;

[0074] (2) mixing the residue obtained in step (1) with the remaining raw materials, adding 6 times the amount of water, extracting at 90-95° C. for 3 times, each time for 1 hour, filtering, combining the filtrate, and obtaining a water extract;

[0075] (3) combining the alcohol extract obtained in step (1) and the water extract obtained in step (2), and concentrating under reduced pressure at 60° C. to a relative density of 1.10-1.12 (60° C.) to obtain the Chinese medicine composition.

[0076] Example 3 Preparation of Chinese medicine composition

[0077] The Chinese medicine composition comprises the following raw materials by weight: 16 parts of Herba Lycopodii, 12 parts of Herba Psoraleae, 10 parts of Herba Drynariae, 15 parts of Herba Angelicae Sinensis, and 20 parts of Herba Epimedii.

[0078] Preparation method:

[0079] (1) Mix the prescribed amount of Qingfengteng and Drynaria fortunei, add 8 times the volume fraction of 95% ethanol, reflux and extract three times, each time for 0.5 h, filter, and combine the filtrate to obtain an alcohol extract and a medicinal residue;

[0080] (2) mixing the residue obtained in step (1) with the remaining raw materials, adding 12 times the amount of water, extracting at 90-95° C. for 3 times, each time for 3 hours, filtering, combining the filtrate, and obtaining a water extract;

[0081] (3) combining the alcohol extract obtained in step (1) and the water extract obtained in step (2), and concentrating under reduced pressure at 60° C. to a relative density of 1.10-1.12 (60° C.) to obtain the Chinese medicine composition.

[0082] Comparative Example 1 Preparation of Chinese medicine composition

[0083] The difference between this comparative example and Example 1 is that the raw materials for water extraction and alcohol extraction in the preparation method are different.

[0084] Preparation method:

[0085] (1) Take the formulated amount of Qingfengteng, add 10 times the volume fraction of 75% ethanol, reflux and extract three times, each time for 1 hour, filter, combine the filtrate, and obtain the alcohol extract and the residue;

[0086] (2) mixing the residue obtained in step (1) with the remaining raw materials, adding 10 times the amount of water, extracting at 90-95° C. for 3 times, each time for 2 hours, filtering, and combining the filtrate to obtain a water extract;

[0087] (3) combining the alcohol extract obtained in step (1) and the water extract obtained in step (2), and concentrating under reduced pressure at 60° C. to a relative density of 1.10-1.12 (60° C.) to obtain the Chinese medicine composition.

[0088] Comparative Example 2 Preparation of Chinese medicine composition

[0089] The difference between this comparative example and Example 1 is that the preparation method is alcohol extraction.

[0090] Preparation method: mix the formulated amounts of Herba Lycopodii, Fructus Psoraleae, Rhizoma Drynariae, Radix Angelicae Sinensis and Herba Epimedii, add 10 times the amount of 75% ethanol by volume, reflux extract for 3 times, each time for 1 hour, filter, combine the filtrate, and concentrate under reduced pressure at 60°C to a relative density of 1.10-1.12 (60°C) to obtain the traditional Chinese medicine composition.

[0091] Comparative Example 3 Preparation of Chinese medicine composition

[0092] The difference between this comparative example and Example 1 is that the prescription of the Chinese medicine composition is different.

[0093] The Chinese medicine composition comprises the following raw materials by weight: 15 parts of Herba Lycopodii, 25 parts of Herba Psoraleae, 25 parts of Herba Drynariae, 12 parts of Herba Angelicae Sinensis and 18 parts of Herba Epimedii.

[0094] Comparative Example 4 Preparation of Chinese medicine composition

[0095] The Chinese medicine composition comprises the following raw materials by weight: 15 parts of Herba Lycopodii, 25 parts of Herba Psoraleae, 25 parts of Herba Drynariae, 12 parts of Herba Angelicae Sinensis and 18 parts of Herba Epimedii.

[0096] Preparation method: mix the formulated amounts of Herba Lycopodii, Herba Psoraleae, Herba Drynariae, Herba Angelicae Sinensis and Herba Epimedii, add 10 times the amount of water, extract for 3 times at 90-95°C, each time for 2 hours, filter, combine the filtrates, concentrate the obtained water extract under reduced pressure at 60°C, and concentrate to a relative density of 1.10-1.12 (60°C) to obtain the traditional Chinese medicine composition.

[0097] Effect example: Effect of Chinese medicine combination on the treatment of bone destruction in rheumatoid arthritis

[0098] 1. Experimental Animals

[0099] Wistar rats, male, weighing 160-180 g.

[0100] 2. Experimental drugs

[0101] The pharmaceutical compositions prepared in Examples 1-3 and Comparative Examples 1-4.

[0102] 3. Experimental methods

[0103] The experimental method of this invention refers to the “Animal experiment on the treatment of bone destruction in rheumatoid arthritis with a Chinese medicine composition” recorded in CN105687397A.

[0104] The details are as follows:

[0105] Establishment of animal model of rheumatoid arthritis

[0106] 3.1 Emulsification of bovine type II collagen

[0107] (1) Prepare acetic acid solution: dissolve anhydrous glacial acetic acid in sterile water to prepare 0.05 mol / L acetic acid;

[0108] (2) Preparation of collagen solution: Add 0.05 mol / L acetic acid solution to bovine type II collagen to make the final concentration of the collagen solution 4 mg / ml, place in a 4°C refrigerator away from light and stir overnight;

[0109] (3) Preparation of collagen emulsion: emulsify equal volumes of 4 mg / ml collagen solution and CFA (syringe method, operate on ice) to a final collagen concentration of 2 mg / ml for primary immunization; emulsify equal volumes of 4 mg / ml collagen solution and IFA to a final collagen concentration of 2 mg / ml for stimulating immunization.

[0110] 3.2 Rat immunization method

[0111] (1) Primary immunization: Rats were anesthetized with 2% sodium pentobarbital solution (0.3 ml / rat) by intraperitoneal injection, and 0.12 ml of collagen emulsion (containing 240 μg collagen) was injected subcutaneously at the base of the tail of each rat;

[0112] (2) Immunization stimulation: On the 7th day after the initial immunization, 0.03 ml of collagen emulsion (containing 120 μg of collagen in total) was injected subcutaneously on both sides of the buttocks and back of the rats.

[0113] 3.3 Model Evaluation

[0114] (1) Observe joint morphology

[0115] The swollen joints of the rats were mainly in the ankle joints and toe joints of both lower limbs. The disease was determined by observing the changes in the joint morphology of the rats. On the 12th day after the first immunization, the ankle joints and interdigital joints of the hind limbs of the modeling rats began to swell, with the swelling centered on the ankle joints. The skin at the swollen part was red or even purple, and the skin temperature of the affected limb was higher than that of the healthy side.

[0116] (2) Joint inflammation score

[0117] The degree of joint inflammation in rats was evaluated by scoring method (0-4 points), and the grading scores and manifestations are shown in Table 1. Rats with inflammation scores ≥ 2 points were considered to have successfully established models and could be randomly enrolled in subsequent experiments.

[0118] Table 1 Inflammation scores and manifestations of rat limb joints

[0119]

[0120] The rats with successful modeling were randomly divided into a model group and a drug administration group (S1-S3 and D1-D4), with 8 rats in each group. The drug administration group was gavaged with the pharmaceutical composition prepared in the corresponding embodiment or comparative example, with a gavage dose of 4 g crude drug / kg, and the model group was gavaged with an equal volume of normal saline, once a day, for 4 consecutive weeks.

[0121] 4. Detection indicators

[0122] 4.1 Method for checking the degree of joint damage

[0123] After 4 weeks of treatment, the rats were killed, the upper tibia was removed to remove the soft tissue, and then fixed in acetone. Micro-CT scanning was performed to compare the bone destruction of the upper tibia and the changes in the secondary cancellous bone trabeculae 1-4 mm below the epiphyseal line of the upper tibia among various groups after treatment.

[0124] 4.2 Histomorphometric examination of cancellous bone tissue in the upper tibia

[0125] (1) Preparation of tissue specimens

[0126] ① Fixation, degreasing, and dehydration: At the end of the 4-week treatment, the rats were anesthetized and killed, and the upper 1 / 3 of the tibia was removed, the soft tissue was removed, and the tibia was immersed in cold acetone solution for fixation, degreasing, and dehydration. The acetone was changed every 12 hours for a total of 6 times;

[0127] ② Permeation: First, prepare the permeation agent, dissolve 2g of BPO (dibenzoyl peroxide) in 30ml of MMA (methyl methacrylate monomer), then add 60ml of HEMA (hydroxyethyl methacrylate monomer), 10ml of HEA (hydroxyethyl acrylate monomer), and 10ml of n-propanol in sequence, stir evenly, immerse the specimen, put it in the ice bath in the vacuum desiccator, and permeate in the vacuum ice bath for 1h;

[0128] ③ Embedding: Dissolve 0.4g of BPO in 40ml of MMA on an ice bath, then add 60ml of HEMA, 10ml of HEA, 10ml of n-propanol, and finally 0.1ml of DMA (xylidine). After stirring evenly, pour the solution into a suitable model for embedding. Finally, drop a thin layer of paraffin oil on the surface of the embedding agent and place it in a 4°C refrigerator to allow it to polymerize.

[0129] ④ Sectioning: After demolding the embedded block, cut 5 μm thick sections on a hard tissue slicer, flatten in a 37°C warm bath, and dry in a 37°C constant temperature incubator;

[0130] ⑤ Staining and sealing: Toluidine blue staining followed by sealing.

[0131] (2) Calculation of cancellous bone tissue morphometric parameters

[0132] ① After the hard tissue sections were prepared and stained with toluidine blue, they were placed on a semi-automatic image analyzer with a special bone tissue morphometric measurement system software. Under a 200x optical microscope, 5 fields of view were randomly selected from each specimen in the secondary cancellous bone area between 1 and 4 mm below the epiphyseal line of the upper tibia and saved for analysis;

[0133] ② On a semi-automatic image analyzer, measure the total area of ​​each field of view, namely the bone tissue area (T.Ar), the total circumference of trabeculae Tb.Pm (Trabecular perimeter), and the total trabecular area Tb.Ar (Trabecular area) in each field of view;

[0134] ③Substitute the above measured parameters into Table 2 to calculate: trabecular area (Tb.Ar), trabecular width (Tb.Wi), trabecular number (Tb.N), and trabecular separation (Tb.Sp).

[0135] Table 2 Static calculation parameters of cancellous bone tissue morphometry

[0136]

[0137] 4.3 Biomechanical examination of the left femur and fifth lumbar vertebra of rats

[0138] (1) After 4 weeks of treatment, the rats were killed and the entire length of the left femur and the fifth lumbar vertebra were removed to remove the soft tissue. Bone biomechanical examination was performed within 2 hours to compare the biomechanical changes of the femur and the fifth lumbar vertebra in each group after treatment.

[0139] (2) The rats were killed and the entire length of the left femur and the fifth lumbar vertebra were removed to remove the soft tissue. The upper and lower articular surfaces of the vertebra were trimmed to keep the upper and lower articular surfaces parallel. After the specimens were taken, they were wrapped with 0.9% saline gauze. Bone biomechanical examination was performed within 2 hours to compare the biomechanical changes of the femur and the fifth lumbar vertebra after treatment in each group. The test room temperature was controlled at 37°C. The femoral shaft parameters were taken as the average of the anterior-posterior diameter and the left-right diameter of the midpoint of the femoral shaft as the diameter. The span was 2 cm and the loading rate was 2 mm / min. The femur was tested in the same direction, with the femoral head facing up. The distal end of the lesser tuberosity of the femur and the upper part of the femoral condyle were placed on the tester, and the midpoint of the femoral shaft was the force point. The lumbar vertebrae parameters were: 1. The average of the anterior-posterior diameter and the left-right diameter of the vertebrae; 2. The height of the vertebrae; the loading rate was 2 mm / min. The femoral three-point bending test took femoral fracture as the experimental end point, and the lumbar compression test took the vertebrae compressed by 1 / 3 as the experimental end point.

[0140] 5. Experimental results

[0141] 5.1 Comparison of bone histomorphometric parameters

[0142] Table 3 Comparison of drug composition on trabecular area percentage (mean ± standard deviation)

[0143]

[0144] Note: Compared with the model group, *P<0.05.

[0145] Table 3 shows that compared with the model group, the percentage of trabecular bone area of ​​rats in the drug-treated groups S1-S3 was significantly increased (P < 0.05), indicating that the Chinese medicine composition prepared by the present invention has the ability to significantly inhibit the loss of bone mass around the inflammatory joints of the model rats.

[0146] Comparison of the medication group D4 and the model group showed that the Chinese medicine composition prepared by the prior art water decoction process in comparative example 4, administered at a low dose of 4 g / kg (compared to CN105687397A), had no ability to inhibit bone loss around the inflammatory joints of the model rats (P>0.05).

[0147] Comparison of the medication group D3 and the model group showed that the Chinese medicine composition prepared in comparative example 3 had no ability to inhibit bone loss around the inflammatory joints of the model rats (P>0.05), indicating that in the technical scheme claimed for protection by the present invention, the adjustment of the dosage relationship of Radix Quadriphyllae, Fructus Psoraleae, Rhizoma Drynariae, Radix Angelicae Sinensis and Herba Epimedii in the Chinese medicine composition had a significant effect on the efficacy of the drug.

[0148] Comparison of the drug-treated groups D1-D2 and the model group showed that the Chinese medicine compositions prepared in comparative examples 1 and 2 had no ability to inhibit bone loss around the inflammatory joints of the model rats (P>0.05), indicating that in the technical scheme claimed to be protected by the present invention, the compound extraction of different raw materials will affect the efficacy of the drugs and have an important effect on inhibiting bone loss around the inflammatory joints.

[0149] Table 4 Comparison of drug compositions on trabecular separation (mean ± standard deviation)

[0150]

[0151] Note: Compared with the model group, *P<0.05.

[0152] Table 4 shows that compared with the model group, the trabecular separation of rats in the drug-treated groups S1-S3 was reduced (P < 0.05), indicating that the Chinese medicine composition prepared by the present invention can prevent osteoporosis.

[0153] Comparison of the medication group D4 and the model group showed that the Chinese medicine composition prepared by the prior art water decoction process in comparative example 4, administered at a low dose of 4 g / kg (compared to CN105687397A), had no effect on improving osteoporosis (P>0.05).

[0154] Comparison of the drug-treated group D3 and the model group showed that the Chinese medicine composition prepared in comparative example 3 had no effect on improving osteoporosis (P>0.05), indicating that in the technical scheme claimed for protection by the present invention, the adjustment of the dosage relationship of Radix Quadriphyllae, Rhizoma Psoraleae, Rhizoma Drynariae, Rhizoma Angelicae Sinensis and Rhizoma Epimedii in the Chinese medicine composition had a significant effect on the efficacy of the drug.

[0155] At the same time, by comparing the drug-dosing groups D1-D2 and the model group, it can be seen that the Chinese medicine compositions prepared in comparative examples 1 and 2 have no effect on improving osteoporosis (P>0.05), indicating that in the technical scheme claimed to be protected by the present invention, the compound extraction of different raw materials will affect the efficacy of the medicine and have an important impact on the improvement of osteoporosis.

[0156] 5.2 Comparison of bone histomorphometric parameters

[0157] Table 5 Comparison of drug composition on rat lumbar vertebrae compressive strength (mean ± standard deviation)

[0158]

[0159] Note: Compared with the model group, *P<0.05.

[0160] Table 5 shows that compared with the model group, the lumbar vertebrae compressive strength of rats in the drug-treated groups S1-S3 was significantly increased (P < 0.05), indicating that the Chinese medicine composition prepared by the present invention can enhance bone strength.

[0161] Comparison of the medication group D4 and the model group showed that the Chinese medicine composition prepared by the prior art water decoction process in comparative example 4, administered at a low dose of 4 g / kg (compared to CN105687397A), had no effect on enhancing bone strength (P>0.05).

[0162] Comparison of the drug-treated group D3 and the model group showed that the Chinese medicine composition prepared in comparative example 3 had no effect on enhancing bone strength (P>0.05), indicating that in the technical scheme claimed for protection by the present invention, the adjustment of the dosage relationship of Radix Quadriphyllae, Fructus Psoraleae, Rhizoma Drynariae, Radix Angelicae Sinensis and Herba Epimedii in the Chinese medicine composition had a significant effect on the efficacy of the drug.

[0163] At the same time, by comparing the drug-treated groups D1-D2 and the model group, it can be seen that the Chinese medicine compositions prepared in comparative examples 1 and 2 have no effect on enhancing bone strength (P>0.05), indicating that in the technical scheme claimed to be protected by the present invention, the compound extraction of different raw materials will affect the efficacy of the medicine and have an important influence on enhancing bone strength.

[0164] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A Chinese medicine composition for treating rheumatoid arthritis inflammation and bone destruction, wherein the raw materials of the Chinese medicine composition are: Herba Lycopodii, Herba Psoraleae, Herba Drynariae, Herba Angelicae Sinensis and Herba Epimedii; characterized in that: The preparation method of the Chinese medicine composition comprises the following steps: (1) extracting the scutellaria baicalensis and rhizoma drynariae with alcohol to obtain an alcohol extract and medicinal residues; (2) extracting the medicinal residue and other raw materials with water to obtain a water extract; (3) combining the alcohol extract and the water extract to obtain the Chinese medicine composition.

2. The Chinese medicine composition according to claim 1, characterized in that: The alcohol extraction in step (1) is performed by reflux extraction using alcohol as solvent.

3. The Chinese medicine composition according to claim 1, characterized in that The alcohol in step (1) is at least one of ethanol, propanol, butanol and pentanol; and / or The volume fraction of the alcohol is 60-95%; and / or The amount of the alcohol is 8-15 times the total mass of Herba Epimedii and Herba Epimedii; and / or The number of reflux extractions is 1-5 times, and the time of each reflux extraction is 0.5-3 hours.

4. The Chinese medicine composition according to claim 1, characterized in that: The water extraction in step (2) is reflux extraction using water as solvent.

5. The Chinese medicine composition according to claim 1, characterized in that: The amount of water used in step (2) is 6-12 times the total mass of Herba Epimedii, Herba Psoraleae, Herba Drynariae, Herba Angelicae Sinensis and Herba Epimedii; and / or The number of reflux extractions is 1-5 times, and the time of each reflux extraction is 1-3 hours.

6. The Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition comprises the following raw materials by weight: 8-16 parts of Herba Lycopodii, 8-12 parts of Herba Psoraleae, 8-10 parts of Herba Drynariae, 9-15 parts of Herba Angelicae Sinensis, and 10-20 parts of Herba Epimedii.

7. A method for preparing a Chinese medicine composition for treating rheumatoid arthritis inflammation and bone destruction, wherein the raw materials of the Chinese medicine composition are: Herba Lycopodii, Herba Psoraleae, Herba Drynariae, Herba Angelicae Sinensis and Herba Epimedii; comprising the following steps: (1) extracting the scutellaria baicalensis and rhizoma drynariae with alcohol to obtain an alcohol extract and medicinal residues; (2) mixing the medicinal residue and the remaining raw materials with water to obtain a water extract; (3) combining the alcohol extract and the water extract to obtain the Chinese medicine composition.

8. The preparation method according to claim 7, characterized in that: The alcohol extraction in step (1) is reflux extraction using alcohol as solvent; and / or The water extraction in step (2) is reflux extraction using water as solvent; and / or The alcohol extract and the water extract in step (3) are concentrated before being combined; and / or After the alcohol extract and the water extract described in step (3) are combined, the step further includes drying.

9. Use of the Chinese medicine composition according to any one of claims 1 to 6 or the preparation method according to any one of claims 7 to 8 in preparing a medicament for treating rheumatoid arthritis inflammation and / or bone destruction.

10. A medicine for treating rheumatoid arthritis inflammation and / or bone destruction, comprising the Chinese medicine composition according to any one of claims 1 to 6 and pharmaceutically acceptable excipients.

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