Application of pharmaceutical composition containing tortoise-plastron glue, deer-horn glue and chondroitin sulfate to prevention and treatment of bone and joint diseases of pets

Abstract

The invention discloses a pharmaceutical composition containing tortoise-plastron glue, deer-horn glue and chondroitin sulfate. The pharmaceutical composition comprises a traditional Chinese medicine compound and chondroitin sulfate, wherein the traditional Chinese medicine compound comprises deer-horn glue, tortoise-plastron glue, a ginseng extract and a fructus lycii extract; wherein the mass ratio of the traditional Chinese medicine compound to the chondroitin sulfate is (1.5-2.5): about 1. A traditional Chinese medicine compound is prepared from tortoise-plastron glue, deer-horn glue, a ginseng extract and a fructus lycii extract according to a certain proportion, the traditional Chinese medicine compound is added into rat basal feed, and the anti-inflammatory and analgesic effects of the traditional Chinese medicine compound on osteoarthritis (OA) rats are observed; meanwhile, the traditional Chinese medicine compound and chondroitin sulfate are combined to generate a synergistic effect, and reference is provided for clinical treatment of animal OA.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to use of a pharmaceutical composition comprising tortoise shell glue, deer antler glue and chondroitin sulfate for preventing and treating bone and joint diseases of pets. Background Art

[0002] Osteoarthritis (OA) is a common degenerative joint disease, which is mainly manifested by the degeneration and wear of cartilage tissue around the joints, leading to joint pain, stiffness, swelling and limited movement. The pathogenesis of OA is complex, involving multiple factors such as mechanics, inflammation and metabolism. Modern research has found that abnormal expression of inflammatory cytokines and matrix components may lead to an imbalance in the proliferation and apoptosis of articular chondrocytes, ultimately causing the disease. A survey shows that about 20% of dogs will have varying degrees of OA, which will not only affect the dog's mobility, but also significantly reduce its quality of life; and the dysfunction caused by OA is one of the main causes of disability in elderly dogs, especially police dogs. At present, Western medicines used clinically to treat animal OA mainly include non-steroidal anti-inflammatory drugs, glucocorticoids and chondroitin sulfate. Long-term use of anti-inflammatory analgesics may lead to greater adverse reactions, and although chondroitin sulfate can improve joint pain and promote cartilage regeneration, its onset is usually slow.

[0003] The use of the pharmaceutical composition comprising tortoise shell glue, deer horn glue and chondroitin sulfate claimed in the present invention for preventing and treating bone and joint diseases of pets has not been found in the prior art. Summary of the invention

[0004] Based on this, the present invention provides a pharmaceutical composition comprising tortoise shell glue, deer horn glue and chondroitin sulfate, wherein the pharmaceutical composition comprises a traditional Chinese medicine compound and chondroitin sulfate;

[0005] The Chinese medicine complex includes deer antler glue, tortoise shell glue, ginseng extract and wolfberry extract;

[0006] The mass ratio between the traditional Chinese medicine complex and the chondroitin sulfate is (1.5-2.5):about 1.

[0007] Furthermore, the mass ratio between the traditional Chinese medicine complex and the chondroitin sulfate is (1.8-2.2):about 1.

[0008] Furthermore, the mass ratio between the traditional Chinese medicine complex and the chondroitin sulfate is about 2: about 1.

[0009] Furthermore, in the traditional Chinese medicine composite, the mass ratio of the deer antler glue, the tortoise shell glue, the ginseng extract and the wolfberry extract is (30-70): (10-40): (1-10): (5-20).

[0010] Furthermore, in the traditional Chinese medicine composite, the mass ratio of the deer antler glue, the tortoise shell glue, the ginseng extract and the wolfberry extract is (40-60): (20-30): (4-6): (8-12).

[0011] Furthermore, in the traditional Chinese medicine composite, the mass ratio of the deer antler glue, the tortoise shell glue, the ginseng extract and the wolfberry extract is about 50: about 25: about 4.5: about 9.

[0012] Furthermore, the traditional Chinese medicine compound contains ginsenoside Re, ginsenoside Rb1 and ginsenoside Rd components.

[0013] Furthermore, in the traditional Chinese medicine complex, the concentration of ginsenoside Re is 50-200 μg / g, for example, about 96.94 μg / g.

[0014] Furthermore, in the traditional Chinese medicine compound, the ginsenoside Rb 1 The concentration is 1 to 10 μg / g, for example, about 6.98 μg / g.

[0015] Furthermore, in the traditional Chinese medicine complex, the concentration of ginsenoside Rd is 50-200 μg / g, for example, about 81.78 μg / g.

[0016] Furthermore, in the chondroitin sulfate, the content ratios of total sugar, glucuronic acid, protein and sulfate group in the chondroitin sulfate are (10% to 25%): (20% to 40%): (0.1% to 5%): (10% to 30%).

[0017] Furthermore, in the chondroitin sulfate, the content ratios of total sugar, glucuronic acid, protein and sulfate group in the chondroitin sulfate are (15% to 20%): (25% to 35%): (0.5% to 2%): (15% to 25%).

[0018] Furthermore, in the chondroitin sulfate, the content ratios of total sugar, glucuronic acid, protein, and sulfate group in the chondroitin sulfate are about 17.3%: about 29.3%: about 0.88%: about 20.5%.

[0019] Furthermore, in the chondroitin sulfate, the molar ratio of glucuronic acid, galacturonic acid, glucose and galactosamine is (0.5-5): (0.1-1): (0.05-1): (0.5-5).

[0020] Furthermore, in the chondroitin sulfate, the molar ratio of glucuronic acid, galacturonic acid, glucose and galactosamine is (1-2): (0.2-0.3): (0.1-0.2): (1-2).

[0021] Furthermore, in the chondroitin sulfate, the molar ratio of glucuronic acid, galacturonic acid, glucose, and galactosamine is about 1.63: about 0.24: about 0.13: about 1.84.

[0022] Furthermore, the relative molecular mass of the chondroitin sulfate is 100-200 kDa.

[0023] Furthermore, the relative molecular mass of the chondroitin sulfate is about 160 kDa.

[0024] Furthermore, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0025] Furthermore, the auxiliary material is selected from one or more of the following: antioxidants, preservatives, sweeteners and pH regulators.

[0026] Furthermore, the dosage form of the pharmaceutical composition is an oral solution, emulsion, ointment or plaster.

[0027] According to another aspect of the present invention, there is provided a use of the above-mentioned pharmaceutical composition in the preparation of medicines, foods and / or health products for preventing and / or treating animal bone and joint diseases or for improving limb weight distribution or for improving abnormal joint tissue structure.

[0028] Furthermore, the animal is a mammal.

[0029] Furthermore, the bone and joint disease is osteoarthritis.

[0030] Furthermore, the animal is a pet.

[0031] Furthermore, the pet is a dog, a cat, a mouse or a rabbit.

[0032] Furthermore, the pharmaceutical composition prevents and / or treats animal bone and joint diseases in one or more of the following ways: increasing the mechanical stimulation foot withdrawal threshold, improving limb weight distribution, improving joint tissue structural abnormalities, increasing cartilage type II collagen expression, and reducing synovial inflammatory cytokine expression.

[0033] Furthermore, the improved limb weight distribution increases the load-bearing capacity of the lower limbs.

[0034] Furthermore, the abnormal joint tissue structure includes one or more of the following: redness and swelling of the joint, rough surface of the articular cartilage, articular cartilage defect, thickening of the joint capsule, synovial hyperplasia and synovial muscle adhesion.

[0035] Furthermore, the inflammatory cytokine is IL-1β.

[0036] Beneficial effects of the present invention:

[0037] The invention prepares a Chinese medicine composite by mixing tortoise shell glue, deer horn glue, ginseng and wolfberry in a certain proportion, and adds the composite into rat basic feed to observe the anti-inflammatory and analgesic effects on rats with osteoarthritis (OA). It is also found that the composite Chinese herbal medicine produces a synergistic effect when used in combination with chondroitin sulfate, which provides a reference for the clinical treatment of animal OA. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0039] Figure 1 Schematic diagram of gross observation and scoring results of knee joints of OA rats in each group.

[0040] Figure 2 Schematic diagram of the effects of different additives on type II collagen in cartilage of OA rats.

[0041] Figure 3 Schematic diagram of the effects of different additives on the expression of IL-1β in the synovium of OA rats. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0043] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0044] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0045] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0046] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0047] As described in the background technology section, long-term use of anti-inflammatory analgesics may lead to greater adverse reactions, and although chondroitin sulfate can improve joint pain and promote cartilage regeneration, its onset is usually slow, and the prior art does not find the use of a pharmaceutical composition comprising tortoise shell glue, deer horn glue and chondroitin sulfate for the prevention and treatment of pet bone and joint diseases as claimed in the present invention. In order to solve the above problems, the present invention provides a pharmaceutical composition comprising tortoise shell glue, deer horn glue and chondroitin sulfate, the pharmaceutical composition comprising a traditional Chinese medicine compound and chondroitin sulfate;

[0048] The Chinese medicine complex includes deer antler glue, tortoise shell glue, ginseng extract and wolfberry extract;

[0049] The mass ratio between the traditional Chinese medicine complex and the chondroitin sulfate is (1.5-2.5):about 1.

[0050] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0051] In the present invention, when mass, concentration, percentage, molar ratio, relative molecular mass, or other values ​​or parameters are expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when the range "1.5-2.5" is disclosed, the described range should be interpreted as including the range "1.5-2.5", "1.5-2.3", "1.5-2.1", "1.5-1.9", "1.5-1.7", "1.7-2.5", "1.7-2.3", "1.7-2.1", "1.7-1.9", "1.9-2.5", "1.9-2.3", "1.9-2.1", "2.1-2.5", "2.1-2.3", "2.3-2.5", etc. When a numerical range is described herein, unless otherwise indicated, that range is intended to include the endpoints and all integers and fractions within that range.

[0052] In a preferred embodiment, the mass ratio between the traditional Chinese medicine complex and the chondroitin sulfate is (1.8-2.2):about 1.

[0053] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0054] In a preferred embodiment, the mass ratio between the traditional Chinese medicine complex and the chondroitin sulfate is about 2: about 1.

[0055] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 2" includes ±5% of 2, or from 1.9 to 2.1; "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0056] In a preferred embodiment, in the traditional Chinese medicine composite, the mass ratio of the deer antler glue, the tortoise shell glue, the ginseng extract and the wolfberry extract is (30-70): (10-40): (1-10): (5-20).

[0057] In a preferred embodiment, in the traditional Chinese medicine composite, the mass ratio of the deer antler glue, the tortoise shell glue, the ginseng extract and the wolfberry extract is (40-60): (20-30): (4-6): (8-12).

[0058] In a preferred embodiment, in the traditional Chinese medicine composite, the mass ratio of the deer antler glue, the tortoise shell glue, the ginseng extract and the wolfberry extract is about 50: about 25: about 4.5: about 9.

[0059] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50" includes ±5% of 50, or from 47.5 to 52.5; "about 25" includes ±5% of 25, or from 23.75 to 26.25; "about 4.5" includes ±5% of 4.5, or from 4.275 to 4.725; "about 9" includes ±5% of 9, or from 8.55 to 9.45.

[0060] In a preferred embodiment, the traditional Chinese medicine compound comprises ginsenoside Re, ginsenoside Rb1 and ginsenoside Rd.

[0061] In a preferred embodiment, in the traditional Chinese medicine complex, the concentration of ginsenoside Re is 50-200 μg / g, for example, about 96.94 μg / g.

[0062] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 96.94" includes ±5% of 96.94, or from 92.093 to 101.787.

[0063] In a preferred embodiment, in the traditional Chinese medicine compound, the ginsenoside Rb 1 The concentration is 1 to 10 μg / g, for example, about 6.98 μg / g.

[0064] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 6.98" includes ±5% of 6.98, or from 6.631 to 7.329.

[0065] In a preferred embodiment, in the traditional Chinese medicine complex, the concentration of ginsenoside Rd is 50-200 μg / g, for example, about 81.78 μg / g.

[0066] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 81.78" includes ±5% of 81.78, or from 77.691 to 85.869.

[0067] In a preferred embodiment, in the chondroitin sulfate, the content ratio of total sugar, glucuronic acid, protein and sulfate group in chondroitin sulfate is (10% to 25%): (20% to 40%): (0.1% to 5%): (10% to 30%).

[0068] In a preferred embodiment, in the chondroitin sulfate, the content ratio of total sugar, glucuronic acid, protein and sulfate group in chondroitin sulfate is (15% to 20%): (25% to 35%): (0.5% to 2%): (15% to 25%).

[0069] In a preferred embodiment, in the chondroitin sulfate, the content ratio of total sugar, glucuronic acid, protein, and sulfate group in the chondroitin sulfate is about 17.3%: about 29.3%: about 0.88%: about 20.5%.

[0070] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 17.3%" includes ±5% of 17.3%, or from 16.435% to 18.165%; "about 29.3%" includes ±5% of 29.3%, or from 27.835% to 30.765%; "about 0.88%" includes ±5% of 0.88%, or from 0.836% to 0.924%; "about 20.5%" includes ±5% of 20.5%, or from 19.475% to 21.525%.

[0071] In a preferred embodiment, in the chondroitin sulfate, the molar ratio of glucuronic acid, galacturonic acid, glucose and galactosamine is (0.5-5): (0.1-1): (0.05-1): (0.5-5).

[0072] In a preferred embodiment, in the chondroitin sulfate, the molar ratio of glucuronic acid, galacturonic acid, glucose and galactosamine is (1-2): (0.2-0.3): (0.1-0.2): (1-2).

[0073] In a preferred embodiment, in the chondroitin sulfate, the molar ratio of glucuronic acid, galacturonic acid, glucose, and galactosamine is about 1.63: about 0.24: about 0.13: about 1.84.

[0074] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.63" includes ±5% of 1.63, or from 1.5485 to 1.7115; "about 0.24" includes ±5% of 0.24, or from 0.228 to 0.252; "about 0.13" includes ±5% of 0.13, or from 0.1235 to 0.1365; "about 1.84" includes ±5% of 1.84, or from 1.748 to 1.932.

[0075] In a preferred embodiment, the relative molecular mass of the chondroitin sulfate is 100-200 kDa.

[0076] In a preferred embodiment, the relative molecular mass of the chondroitin sulfate is about 160 kDa.

[0077] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 160" includes ±5% of 160, or from 152 to 168.

[0078] In a preferred embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0079] In a preferred embodiment, the auxiliary material is selected from one or more of the following: antioxidants, preservatives, sweeteners and pH adjusters.

[0080] In a preferred embodiment, the pharmaceutical composition is in the form of an oral solution, emulsion, ointment or plaster.

[0081] In a preferred embodiment, the Chinese medicine complex is Guiluerxian Paste, which can be a finished product sold on the market, or a finished product made according to the method of the prior art (refer to relevant patents and journal documents).

[0082] According to another aspect of the present invention, there is provided a use of the above-mentioned pharmaceutical composition in the preparation of medicines, foods and / or health products for preventing and / or treating animal bone and joint diseases or for improving limb weight distribution or for improving abnormal joint tissue structure.

[0083] In a preferred embodiment, the animal is a mammal.

[0084] In a preferred embodiment, the bone and joint disease is osteoarthritis.

[0085] In a preferred embodiment, the animal is a pet.

[0086] In a preferred embodiment, the pet is a dog, a cat, a mouse or a rabbit.

[0087] In a preferred embodiment, the pharmaceutical composition prevents and / or treats animal bone and joint diseases in one or more of the following ways: increasing the mechanical stimulation foot withdrawal threshold, improving limb weight distribution, improving joint tissue structural abnormalities, increasing cartilage type II collagen expression, and reducing synovial inflammatory cytokine expression.

[0088] In a preferred embodiment, the improved limb weight distribution is an increase in the weight-bearing capacity of the lower limbs.

[0089] In a preferred embodiment, the abnormal joint tissue structure includes one or more of the following: redness and swelling of the joint, rough surface of the articular cartilage, articular cartilage defect, thickening of the joint capsule, synovial hyperplasia and synovial muscle adhesion.

[0090] In a preferred embodiment, the inflammatory cytokine is IL-1β.

[0091] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0092] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0093] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0094] Example

[0095] 1. Materials and methods

[0096] 1.1 Test Materials

[0097] 1.1.1. Experimental drugs

[0098] Chondroitin sulfate (Batch No.: 2024011303) was purchased from Meitai Technology (Qingdao) Co., Ltd.

[0099] The Chinese medicine compound is a compound formed by mixing tortoise shell glue, deer antler glue, ginseng extract and wolfberry extract in a mass ratio of about 50: about 25: about 4.5: about 9, wherein tortoise shell glue (batch number 2023121103), deer antler glue (batch number 2023121106), ginseng extract (batch number 2024021601) and wolfberry extract (batch number 2023121201) are all purchased from Hanzhong Natural Valley Biotechnology Co., Ltd. The preparation method of ginseng extract or wolfberry extract is: select Chinese herbal medicine slices that meet the quality standards, use 70% ethanol solution to extract three times at 80°C, and each extraction time is 1.5 hours. During the extraction process, the ratio of the slices to the extraction solvent is 1:7 for the first time, 1:5 for the second time, and 1:4 for the third time. After each extraction, the extract needs to be filtered while hot. The filtered liquid is concentrated to a specific gravity of 1.10-1.20, and the concentration temperature is maintained in the range of 70-80°C. After the concentrated paste is homogenized, it is dried by spray drying, with an inlet air temperature of 130-160°C and an outlet air temperature of 60-90°C. The dried coarse powder is crushed through an 80-mesh sieve, mixed for 60 minutes, and finally subjected to quality control (QC) inspection. The ginseng extract or wolfberry extract was qualitatively and quantitatively analyzed for the main quality control indicators by the Biomedical Innovation Institute of China Pharmaceutical City, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, and the above extracts were confirmed to be qualified and authentic. The main quality control indicators of chondroitin sulfate, tortoise shell glue, deer antler glue, ginseng extract and wolfberry extract are shown in Table 1.

[0100] Table 1 Main quality control indicators of chondroitin sulfate, tortoise shell glue, deer antler glue, ginseng extract and wolfberry extract

[0101]

[0102] 1.1.2 Reagents and instruments

[0103] Main reagents: Sodium iodoacetate (MIA, batch number: S817623) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; Immunohistochemistry kit (batch number: BA05265207) was purchased from Beijing Bioson Biotechnology Co., Ltd.; Safranin fast green stain (batch number: G1053), hematoxylin stain (batch number: G1004), eosin stain (batch number: G1001), EDTA decalcification solution (batch number: G1105) were all purchased from Wuhan Saiweier Biotechnology Co., Ltd.; anti-CollagenⅡ (batch number: AF0135) and anti-IL-1β (batch number: AF5103) were purchased from Affinity Biotechnology Co., Ltd.; Ginsenoside Re (batch number: RS02511120), Ginsenoside Rb1 (batch number: RS02481120), and Ginsenoside Rd (batch number: RS05971120) were all purchased from Shanghai Standard Standard Technology Service Co., Ltd.;

[0104] Main instruments: AniView600 small animal X-ray imaging system (Guangzhou Boluteng Biotechnology Co., Ltd.); BIO-EVF4-POIGN Von Frey analgesia instrument, BIO-SWB bipedal balance instrument, R583S small animal respiratory anesthesia machine (Shenzhen Ruiwode Co., Ltd.); CKX-41-32 inverted microscope (Japan OLYMPUS Company); LEICA DM6 fluorescence microscope (Leica Microsystems); ACQUITY UPLC I-CLASS ultra-high performance liquid chromatograph (Waters Company, USA).

[0105] 1.2. Drug content determination

[0106] 1.2.1. Preparation and content determination of compound Chinese herbal medicine

[0107] The appropriate amount of deer horn, tortoise shell, ginseng and wolfberry was added in a ratio of 50:25:4.5:9. According to the extract yield provided by the raw material supplier, the actual ratio of each component (i.e., deer horn glue, tortoise shell glue, ginseng extract and wolfberry extract) was calculated, and finally a Chinese medicine complex was prepared. The complex was placed in a centrifuge tube, 60% ethanol solution was added, and after ultrasonic treatment for 30 minutes, it was shaken and centrifuged at 12700r / min for 10 minutes. The supernatant was taken for liquid chromatography analysis to detect the content of each component. The chromatographic analysis conditions were as follows: the chromatographic column used was ACQUITY UPLC HSS T3 (2.1mm×100mm, 1.7μm); the mobile phase A was acetonitrile, and the mobile phase B was an aqueous solution containing 0.1% phosphoric acid; the flow rate was set to 0.3mL / min.

[0108] 1.2.2 Characterization of Chondroitin Sulfate

[0109] The total sugar content in chondroitin sulfate was determined by the phenol-sulfuric acid method, the glucuronic acid content was determined by the sulfuric acid-carbazole method, the protein content was determined by the Folin phenol method, and the BaCl 2 -The sulfate content was determined by gelatin turbidimetry, the monosaccharide composition was determined by PMP derivatization, and the relative molecular mass was determined by high performance liquid chromatography.

[0110] 1.3 Experimental Design

[0111] 64 healthy SPF male SD rats, weighing (180±20) g, were purchased from Guangzhou Ruige Biotechnology Co., Ltd. The experimental animal unit license number is SCXK (Guangdong) 2023-0059, and animal experiments were carried out in accordance with the regulations of the Experimental Animal Management Committee of Guangzhou University of Chinese Medicine. Rats were randomly divided into 9 groups, 6 in each group, namely sham operation group, model group and 7 experimental groups, as shown in Table 2. After 1 week of adaptive feeding, the rats in the sham operation group were injected with 0.9% saline into the joint cavity, and the rats in the other groups were injected with MIA to establish the OA model. The specific operation was as follows: after the rats were anesthetized, the hair in the 1 cm area around the knee joint of the right hind limb was shaved, disinfected, the knee joint was flexed 90°, and the joint cavity was punctured from the outside of the inferior patellar ligament. The needle insertion angle was 90° with the tibia, and 50 μL of MIA was injected. After the right hind limbs of the rats injected with MIA were extended and bent 5 times, they were returned to the cage for free activity for 14 days. The sham operation group was injected with 50 μL of 0.9% saline in the same way.

[0112] After the model was successfully established on the 14th day, drug intervention began. The rats in the sham operation group and the model group continued to be fed with the basic diet, and the seven experimental groups added different proportions of the traditional Chinese medicine complex and the chondroitin sulfate drug composition to the basic diet for 4 weeks.

[0113] Table 2 Experimental groups

[0114] Group Add ingredients Blank group / Model Group / Experimental Group 1 Chondroitin Sulfate Experimental Group 2 Chinese medicine complex Experimental Group 3 Chinese medicine complex: Chondroitin sulfate 3:1 Experimental Group 4 Chinese medicine complex: Chondroitin sulfate 2:1 Experimental Group 5 Chinese medicine complex: Chondroitin sulfate 1:1 Experimental Group 6 Chinese medicine complex: Chondroitin sulfate 1:2 Experimental Group 7 Chinese medicine complex: Chondroitin sulfate 1:3

[0115] 1.4. Measurement indicators and methods

[0116] 1.4.1. Evaluation of OA efficacy

[0117] 1.4.1.1 Mechanical pain threshold measurement

[0118] The mechanical pain threshold of the right lower limb of the rats was measured using an electronic VonFrey analgesic instrument 1 day before modeling and on days 14, 28, 35, and 42 after modeling. VonFrey fibers were used to stimulate the center of the plantar surface of the rat's right hind limb paw, and pressure was applied vertically and evenly upward. When the rat's hind limb showed a reaction of foot withdrawal, foot lifting, or foot licking, the measurement was completed. The analgesic instrument automatically recorded the maximum value detected, and the measurement was repeated 3 times, and the average value was taken as the mechanical stimulation paw withdrawal threshold (PWT).

[0119] 1.4.1.2 Load-bearing test

[0120] The weight bearing of both feet was tested using a bipedal balance tester 1 day before modeling and on days 14, 28, 35, and 42 after modeling. The pain level of the rat limbs was evaluated by testing the weight bearing distribution of the hind limbs on the modeling side and the hind limbs on the blank control side. The specific operation steps were to stand the rat's forelimbs on the fixator partition, and the lower limbs on two highly sensitive weight testing plates, keeping the bilateral positions consistent, recording the average weight bearing of the left and right hind limbs, performing 3 measurements and taking the average value, and calculating the weight bearing percentage of the right hind limb.

[0121] Right hind limb weight-bearing percentage = right hind limb weight / (left hind limb weight + right hind limb weight) × 100%

[0122] 1.4.2. Morphological structure of knee joint tissue

[0123] 1.4.2.1 X-ray examination

[0124] The rats in each group were examined by X-ray at 14, 28, 35 and 42 days after modeling. After anesthesia, the rats were fixed in the center of the stage in prone position, with the femur and tibia of the right knee joint fixed at 45-60 degrees, and the conversion screen was placed above the rats for X-ray shooting.

[0125] 1.4.2.2. Gross Observation of Knee Joint

[0126] After the administration, the rats were killed, and the left knee joint was dissected to fully expose the distal femoral articular surface, articular groove, and medial and lateral condyles. The articular cartilage, synovial tissue, and joint capsule were observed, recorded, and scored. The scoring criteria were as follows: smooth joint surface, light blue and translucent, scored as 0 points; slightly rough joint surface or small cracks, grayish color, scored as 1 point; rough joint surface, cartilage defects can be seen and deep into the middle layer of cartilage, scored as 2 points; extremely rough joint surface, ulceration, cartilage defects deep into the deep layer of cartilage, scored as 3 points; large defects and exfoliation of articular cartilage, and exposure of subchondral bone, scored as 4 points.

[0127] 1.4.2.3 Knee joint histopathology

[0128] The rat knee joint tissue was fixed with 4% paraformaldehyde for 24 hours, and then decalcified with EDTA decalcification solution. After decalcification, the joint tissue was dehydrated with gradient ethanol, embedded in paraffin, and then cut into tissue sections with a thickness of 4 μm and dried. The tissue sections were then stained with conventional HE and safranin fast green, observed under an optical microscope, and the severity of pathological injury of the rat knee joint tissue was evaluated according to the modified Mankin score.

[0129] 1.4.3 Expression of type II collagen and interleukin-1β (IL-1β)

[0130] Immunohistochemical staining was performed on the rat knee joint and synovial tissues according to the method of MAGAKI et al. (MAGAKI S, HOJAT SA, WEI B, et al. An introduction to the performance of immunohistochemistry [J]. Biobanking: Methods and Protocols, 2019, 1897: 289-298.), and the expression of type II collagen in the knee joint tissue and IL-1β in the synovium of each group was observed under a microscope.

[0131] 1.5 Data Statistics and Analysis

[0132] IBM SPSS Statistics 24 software was used for statistical analysis of the experimental data. One-way analysis of variance was used for comparison among multiple groups. LSD test was used for homogeneous variances, and Dunnett's T3 test was used for unequal variances. Shapiro-Wilk test was used for normal distribution. Data that met normal distribution were expressed as "mean ± standard deviation", and P < 0.05 indicated significant differences.

[0133] 2. Results and Analysis

[0134] 2.1. Drug content determination results

[0135] 2.1.1. Results of determination of compound Chinese herbal medicine content

[0136] The test results showed that the contents of ginsenoside Re, ginsenoside Rb1 and ginsenoside Rd in the compound Chinese herbal medicine were 96.94, 6.98 and 81.78 μg / g, respectively.

[0137] 2.1.2. Chondroitin sulfate characterization results

[0138] The test results show that the total sugar, glucuronic acid, protein and sulfate content of chondroitin sulfate are 17.3%, 29.3%, 0.88% and 20.5% respectively; it contains glucuronic acid, galacturonic acid, glucose and aminogalactose with a molar ratio of 1.63:0.24:0.13:1.84; the relative molecular mass is 160kDa.

[0139] 2.2 Effects of different additives on pain in OA rats

[0140] 2.2.1 Effects of different additives on PWT of OA rats (as shown in Table 3)

[0141] As shown in Table 3, 1 day before modeling, there was no significant difference in PWT of rats in each group (P>0.05). At different time points (14d, 28d, 35d, 42d), the PWT of the model group was significantly reduced compared with the sham operation group (P<0.05), indicating that the modeling was successful. Compared with the model group, the PWT of the Chinese medicine complex 2:1 chondroitin group (experimental group 4) was significantly higher than that of the model group at 14d, 28d, 35d, and 42d (P<0.05 or P<0.01). Specifically, on the 14th day, the PWT of experimental group 4 was significantly higher than that of the model group (P<0.05), and on the 28th, 35th, and 42nd days, the PWT of experimental group 4 was extremely significantly higher than that of the model group (P<0.01). Compared with other experimental groups, it had the fastest onset of effect, showed significant differences at different time points, and showed a gradual increase with the passage of time. The PWT of experimental group 1 increased significantly on the 35th day (P<0.05), but no effect was observed on the 14th, 28th and 42nd days; the PWT of experimental group 2 increased significantly on the 28th and 35th days (P<0.05), but no effect was observed on the 14th and 42nd days; the PWT of experimental group 3 and experimental group 5 increased significantly on the 28th and 35th days (P<0.05), but no effect was observed on the 14th and 42nd days; there was no significant difference in the PWT of experimental group 6 and experimental group 7 compared with the model group. At the same time, on the 28th day, the PWT value of experimental group 4 increased significantly compared with experimental groups 1, 2, 3, 5, 6, and 7 (P<0.01).

[0142] Table 3 Effects of different additives on PWT of OA rats (unit: g)

[0143] Group No.-1d 14d No. 28d No. 35d Chapter 42d Sham operation group 49.29±3.42 55.42±3.98 54.36±3.71 54.29±3.36 53.82±2.09 Model Group 49.39±3.86 33.25±2.98* 30.66±1.44* 29.99±0.81* 32.01±1.72* Experimental Group 1 49.13±4.29 33.25±2.98 33.13±2.38 <![CDATA[32.85±2.66 # ]]> 33.89±4.06 Experimental Group 2 49.25±3.56 34.81±2.92 <![CDATA[33.52±3.13 # ]]> <![CDATA[32.75±2.85 # ]]> 34.05±3.86 Experimental Group 3 49.28±3.99 36.16±2.66 <![CDATA[34.38±2.56 # ]]> <![CDATA[33.08±3.10 # ]]> 34.95±5.78 Experimental Group 4 49.43±3.26 <![CDATA[37.11±3.23 # ]]> <![CDATA[36.05±2.48 ## ]]> <![CDATA[42.13±2.47 ## ]]> <![CDATA[45.7±2.43 ## ]]> Experimental Group 5 49.61±3.08 34.32±3.71 <![CDATA[33.43±3.33 # ]]> <![CDATA[33.18±3.48 # ]]> 34.53±2.73 Experimental Group 6 49.60±3.95 33.63±3.31 33.17±3.03 31.21±2.51 33.54±3.97 Experimental Group 7 49.96±4.05 33.51±3.01 32.28±2.52 32.28±2.52 33.45±5.70

[0144] Note: 1. The day of modeling is taken as day 0. The situation in Table 4 is the same as that in Table 3.

[0145] 2. Statistical significance between the groups was defined as *P<0.05, **P<0.01, vs sham operation group; # P<0.05, ## P<0.01, vs model group; the situation in Table 4 is the same as that in Table 3.

[0146] 2.2.2 Effects of different additives on the weight distribution of the right foot of OA rats (as shown in Table 4)

[0147] As shown in Table 4, 1 day before modeling, the weight of the lower limbs of rats in each group was basically the same (P>0.05). At different time points (14d, 28d, 35d, 42d), the weight-bearing percentage of the right hind limb of rats in the model group was significantly reduced compared with that in the sham operation group (P<0.01), indicating that the modeling was successful. Compared with the model group, the weight-bearing percentage of the right hind limb of experimental group 1 increased significantly on the 35th day (P<0.05), but no effect was observed on the 28th and 42nd days; the weight-bearing percentage of the right hind limb of experimental group 2 increased significantly on the 35th day (P<0.05), but no effect was observed on the 28th and 42nd days; the weight-bearing percentage of the right lower limb of experimental group 3 increased significantly on the 28th and 35th days (P<0.05), but no effect was observed on the 42nd day; the weight-bearing percentage of the right hind limb of experimental group 5 increased significantly on the 28th day (P<0.05), but no effect was observed on the 35th and 42nd days; there was no significant difference in the weight-bearing percentage of the right hind limb of experimental groups 6 and 7 compared with the model group. On the 28th, 35th, and 42nd days, the right hind limb weight-bearing percentage of experimental group 4 was significantly higher than that of the model group (P<0.01). At the same time, on the 32nd day, the right hind limb weight-bearing percentage of experimental group 4 was significantly higher than that of experimental groups 1, 2, 3, 5, 6, and 7 (P<0.01). The right hind limb weight-bearing percentage of experimental group 4 increased significantly on the 28th day, and the effect lasted until 42 days, indicating that experimental group 4 had a positive effect on improving the weight-bearing capacity of the right hind limb of rats with osteoarthritis.

[0148] Table 4 Effects of different additives on the weight-bearing percentage of the right hind limb of OA rats (unit: %)

[0149] Group No.-1d 14d No. 28d No. 35d Chapter 42d Sham operation group 49.93±1.54 49.46±1.66 50.11±1.05 49.62±1.04 49.60±0.67 Model Group 50.44±2.27 40.15±3.06** 37.68±3.78** 40.47±0.82** 42.56±1.26** Experimental Group 1 50.65±1.54 37.38±2.73 38.34±3.08 <![CDATA[42.56±1.84 # ]]> 43.87±1.43 Experimental Group 2 50.58±1.88 38.00±2.29 39.99±3.02 <![CDATA[42.93±1.91 # ]]> 43.32±4.05 Experimental Group 3 50.40±1.59 39.20±2.63 <![CDATA[41.06±2.35 # ]]> <![CDATA[42.87±1.70 # ]]> 43.11±3.12 Experimental Group 4 50.35±1.56 39.68±3.68 <![CDATA[43.98±1.72 ## ]]> <![CDATA[43.83±1.67 ## ]]> <![CDATA[44.82±2.28 # ]]> Experimental Group 5 50.98±1.12 38.64±4.62 <![CDATA[41.11±2.42 # ]]> 39.24±1.86 42.59±3.07 Experimental Group 6 50.72±1.48 38.13±2.82 39.54±2.48 40.34±2.68 41.65±2.39 Experimental Group 7 50.20±1.73 37.58±3.36 37.92±1.89 39.54±2.48 41.47±4.36

[0150] 2.3 Effects of different additives on the morphology and structure of joint tissues in OA rats

[0151] 2.3.1. Gross observation and scoring of knee joints of OA rats in each group (e.g. Figure 1 (shown)

[0152] Depend on Figure 1 It can be seen that the right knee joint in the sham operation group had no redness and swelling, the surface of the articular cartilage was smooth and translucent, neat and without defects, and the joint capsule had no hyperplasia and hypertrophy. The left knee joint in the model group was obviously red and swollen, the articular cartilage was dull and dull, the defects were serious, the joint capsule was thickened, and the synovium proliferated and adhered to the muscle tissue. Compared with the model group, the cartilage surface of experimental group 4 was smoother, the defect repair was more complete, and the improvement of the knee joint tissue of OA rats was most obvious, while no obvious changes were found in other experimental groups.

[0153] 2.4 Effects of different additives on the expression of type II collagen in cartilage and IL-1β in synovium of OA rats (e.g. Figure 2 and Figure 3 (shown)

[0154] Depend on Figure 2 It can be seen that the cartilage matrix of the rats in the sham operation group was intact, and the type II collagen content was rich and evenly distributed. The cartilage matrix of the rats in the model group was severely defective, with a small amount of cartilage matrix remaining, the normal cartilage structure disappeared, and the type II collagen staining almost disappeared. The cartilage structure of the rats in the experimental group 4 was still intact, and the type II collagen content was increased compared with the model group, with a more even distribution and a better effect. The results showed that the 2:1 ratio of the traditional Chinese medicine complex and chondroitin sulfate significantly increased the expression of type II collagen in rat cartilage, while no significant changes were observed in the other experimental groups. Figure 3 It can be seen that there was basically no expression of IL-1β in the synovium of rats in the sham operation group, and the expression of IL-1β in the synovium of rats in the model group increased significantly. After treatment, the expression of IL-1β in experimental group 4 was significantly reduced compared with the model group, and the effect was obvious. The results showed that the ratio of Chinese medicine complex and chondroitin sulfate 2:1 significantly reduced the expression of inflammatory factor IL-1β in rat synovium, while no significant changes were observed in other experimental groups.

[0155] 3. Conclusion

[0156] OA is a multifactorial whole-joint disease, and its main pathological features include articular cartilage damage, synovial inflammation, subchondral bone changes, and osteophyte formation. Among the many osteoarthritis models, MIA is one of the most commonly used modeling drugs. The morphological changes in rat joint tissues caused by MIA are similar to those in mammals and can simulate OA pain. Therefore, this study selected the knee joint cavity injection MIA modeling method to study the anti-inflammatory and analgesic effects of compound Chinese herbal medicine on OA and its protective effect on articular cartilage.

[0157] The 2:1 ratio of the Chinese medicine compound and chondroitin sulfate of the present invention has a good therapeutic effect on rat OA, and may play a protective role by inhibiting the inflammatory factor IL-1β and promoting the expression of type II collagen; the effect of the Chinese medicine compound and chondroitin sulfate used in combination is better than that of the two used alone. The results of this study can provide a certain reference for the development of feed to alleviate canine OA in the future, and by using it as a feed additive for nutritional supplementation in advance, it is expected to play a role in avoiding disability in elderly dogs and delaying the early retirement of police dogs.

[0158] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A pharmaceutical composition comprising tortoise shell glue, deer horn glue and chondroitin sulfate, characterized in that: The pharmaceutical composition comprises a traditional Chinese medicine compound and chondroitin sulfate; The Chinese medicine compound comprises deer antler glue, tortoise shell glue, ginseng extract and wolfberry extract; Wherein, the mass ratio between the traditional Chinese medicine complex and the chondroitin sulfate is (1.5-2.5): about 1.

2. The pharmaceutical composition according to claim 1, characterized in that The mass ratio between the traditional Chinese medicine complex and the chondroitin sulfate is (1.8-2.2): about 1; Preferably, the mass ratio between the traditional Chinese medicine complex and the chondroitin sulfate is about 2: about 1.

3. The pharmaceutical composition according to claim 1, characterized in that In the traditional Chinese medicine composite, the mass ratio of the deer antler glue, the tortoise shell glue, the ginseng extract and the wolfberry extract is (30-70): (10-40): (1-10): (5-20); Preferably, in the traditional Chinese medicine composite, the mass ratio of the deer antler glue, the tortoise shell glue, the ginseng extract and the wolfberry extract is (40-60): (20-30): (4-6): (8-12); More preferably, in the traditional Chinese medicine composite, the mass ratio of the deer antler glue, the tortoise shell glue, the ginseng extract and the wolfberry extract is about 50: about 25: about 4.5: about 9.

4. The pharmaceutical composition according to claim 1, characterized in that The traditional Chinese medicine compound comprises ginsenoside Re, ginsenoside Rb1 and ginsenoside Rd components; Preferably, in the traditional Chinese medicine composite, the concentration of ginsenoside Re is 50-200 μg / g, for example, about 96.94 μg / g; Preferably, in the traditional Chinese medicine complex, the concentration of ginsenoside Rb1 is 1 to 10 μg / g, for example, about 6.98 μg / g; Preferably, in the traditional Chinese medicine complex, the concentration of ginsenoside Rd is 50-200 μg / g, for example, about 81.78 μg / g.

5. The pharmaceutical composition according to claim 1, characterized in that In the chondroitin sulfate, the content ratios of total sugar, glucuronic acid, protein and sulfate group in the chondroitin sulfate are (10% to 25%): (20% to 40%): (0.1% to 5%): (10% to 30%); Preferably, in the chondroitin sulfate, the content ratio of total sugar, glucuronic acid, protein and sulfate group of chondroitin sulfate is (15% to 20%): (25% to 35%): (0.5% to 2%): (15% to 25%); More preferably, in the chondroitin sulfate, the content ratio of total sugar, glucuronic acid, protein, and sulfate group in the chondroitin sulfate is about 17.3%: about 29.3%: about 0.88%: about 20.5%; Preferably, in the chondroitin sulfate, the molar ratio of glucuronic acid, galacturonic acid, glucose and galactosamine is (0.5-5): (0.1-1): (0.05-1): (0.5-5); More preferably, in the chondroitin sulfate, the molar ratio of glucuronic acid, galacturonic acid, glucose and galactosamine is (1-2): (0.2-0.3): (0.1-0.2): (1-2); Particularly preferably, in the chondroitin sulfate, the molar ratio of glucuronic acid, galacturonic acid, glucose, and galactosamine is about 1.63: about 0.24: about 0.13: about 1.84; Particularly preferably, the relative molecular mass of the chondroitin sulfate is 100 to 200 kDa; Most preferably, the relative molecular mass of the chondroitin sulfate is about 160 kDa.

6. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients; Preferably, the auxiliary material is selected from one or more of the following: antioxidants, preservatives, sweeteners and pH adjusters; Preferably, the pharmaceutical composition is in the form of an oral solution, emulsion, ointment or plaster.

7. Use of the pharmaceutical composition according to any one of claims 1 to 6 in the preparation of medicines, foods and / or health products for preventing and / or treating animal bone and joint diseases or for improving limb weight distribution or for improving abnormal joint tissue structure.

8. The use according to claim 7, characterized in that The animal is a mammal; Preferably, the bone and joint disease is osteoarthritis; Preferably, the animal is a pet; More preferably, the pet is a dog, a cat, a mouse or a rabbit.

9. The use according to claim 7, characterized in that: The pharmaceutical composition prevents and / or treats animal bone and joint diseases in one or more of the following ways: increasing the mechanical stimulation foot withdrawal threshold, improving limb weight distribution, improving joint tissue structural abnormalities, increasing cartilage type II collagen expression, and reducing synovial inflammatory cytokine expression.

10. The use according to claim 9, characterized in that The improved limb weight distribution refers to increased load-bearing capacity of the lower limbs; Preferably, the abnormal joint tissue structure includes one or more of the following: redness and swelling of the joint, rough surface of the articular cartilage, articular cartilage defect, thickening of the joint capsule, synovial hyperplasia and synovial muscle adhesion; Preferably, the inflammatory cytokine is IL-1β.

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