Pharmaceutical composition for promoting cartilage development and repairing cartilage injury as well as preparation method and application of pharmaceutical composition
Through the combination of epimedium polysaccharide and arginine, synergistically promotes cartilage development and repairs cartilage damage, solving the problem of limited cartilage repair effect in the prior art and achieving better therapeutic effects.
Patent Information
- Application Number
- CN202510201250.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-24
- Publication Date
- 2025-05-27
AI Technical Summary
The prior art is difficult to effectively promote cartilage development and repair cartilage damage, and the traditional treatment methods have limited effects and insufficient sources of raw materials, which cannot meet the patients' needs for functional repair.
A combination of epimedium polysaccharide and arginine is used to promote cartilage development and repair cartilage damage through different proportions.
This pharmaceutical composition significantly promotes cartilage development and repairs cartilage injury, has a more significant effect than a single component, and has the potential to use for the treatment of cartilage dysplasia and repairs cartilage injury in human or animal bodies.
Smart Images
Figure CN120037255A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a drug combination for promoting cartilage development and repairing cartilage damage, and a preparation method and application thereof. Background Art
[0002] Cartilage is an indispensable part of the skeletal system and has unique functions such as secreting cartilage matrix, buffering stress concussion, bearing weight, and maintaining normal joint activities. Chondrocytes are the main cells in cartilage tissue, highly differentiated, and have limited proliferation and redifferentiation abilities. It is very difficult for chondrocytes to repair themselves quickly and effectively after injury or degeneration. Therefore, promoting cartilage regeneration and injury repair is an urgent and critical challenge in orthopedic clinical practice. Articular cartilage injury is a common disease, and its incidence gradually increases with the influence of factors such as the age of patients, movement postures, and exercise amounts. There are many methods for repairing cartilage injuries, including surgical treatment and non-surgical treatment. Among them, surgical treatment mainly focuses on microfracture surgery, and also includes autologous or allogeneic cartilage transplantation and chondrocyte transplantation; in addition to traditional treatment methods such as oral anti-inflammatory analgesics and local physical therapy, non-surgical treatment also includes intra-articular injection of sodium hyaluronate, growth factors, stem cells, and hydrogels. However, due to limited repair effects, insufficient raw material sources, and the increasing requirements of patients for functional repair, methods such as microfracture surgery, stem cell transplantation, autologous or allogeneic cartilage transplantation for treating articular cartilage injuries can no longer meet the needs of restoring normal functions.
[0003] The traditional Chinese medicine Epimedium is the dried stems and leaves of the perennial herb Epimedium brevicornu Maxim. of the Berberidaceae family, and has effects such as dispelling cold and dampness, protecting the liver, tonifying the kidney and strengthening yang, antioxidation, and anti-aging, and can treat diseases such as kidney yang deficiency, impotence, wind-cold dampness heat, etc. The active ingredients in Epimedium are complex and diverse, including polysaccharides, glycosides, flavonoids, alkaloids, lignans, trace elements, etc. Epimedium polysaccharide can treat spermatogenic function damage, increase the testosterone content in testicular tissue, maintain sperm density and sperm motility in spermatogenic disorders, and reduce the sperm aberration rate. In addition, it also has pharmacological activities such as antiviral, anti-tumor, anti-radiation, and enhancing the body's immunity.
[0004] Arginine is considered a "semi-essential" or "conditionally essential" amino acid, which mainly catalyzes several bioactive molecules, such as nitric oxide (NO), proline, creatine, and polyamines. At the same time, arginine participates in a variety of biochemical processes, including immune regulation, and the secretion of hormones such as insulin, glucagon, and growth hormone (GH). In recent years, people have paid more and more attention to the physiological functions of arginine, including growth regulation, inflammatory response, oxidative stress, and apoptosis.
[0005] However, so far, no relevant literature reports have been found on the combination of epimedium polysaccharide, arginine, and the combination of epimedium polysaccharide and arginine for their synergistic effect in promoting cartilage development and treating cartilage injury to achieve better therapeutic effects. Providing a drug combination with low cost and good effect for promoting cartilage development and repairing cartilage injury is also one of the technical problems that need to be solved urgently at present. Summary of the Invention
[0006] To solve the above technical problems, the present invention has found through research that epimedium polysaccharide has a significant effect on promoting cartilage development and repairing cartilage injury; in addition, through clinical sample research, the present invention has found that the decrease in blood arginine concentration is directly proportional to knee cartilage loss, indicating that arginine plays an important role in cartilage development and cartilage repair. Therefore, the present invention provides a drug composition for promoting cartilage development and repairing cartilage injury and a preparation method of the drug composition.
[0007] The drug composition is composed of epimedium polysaccharide and arginine, and the weight parts of epimedium polysaccharide are 0 to 100, and the weight parts of arginine are 100 to 0.
[0008] The arginine is preferably L-arginine.
[0009] Preferably, in the drug composition, the mass ratio of epimedium polysaccharide to arginine is 3:2.
[0010] The present invention provides a preparation method of a drug composition for promoting cartilage development and repairing cartilage injury, including the following steps:
[0011] Preparation of epimedium polysaccharide:
[0012] (1) The epimedium medicinal materials or slices are heated with water and extracted several times, each time for at least 1 hour, the decoction is filtered, and the extraction liquids are combined and concentrated into an extract.
[0013] (2) Ethanol with a volume concentration of more than 95% is added to make the alcohol concentration of the mixture reach more than 70%, vortexed, alcohol-precipitated overnight at 4°C, centrifuged, and the precipitate is collected.
[0014] (3) The precipitate is dissolved in water and the operation in step (2) is repeated several times to obtain crude polysaccharide.
[0015] (4) The crude polysaccharide is formulated into a sugar solution with a volume concentration of 5%, 4 times the volume of chloroform-butanol is added, and a mixed solution with a volume ratio of chloroform-butanol to sugar solution of 4:1 is formulated, shaken well, centrifuged, and the aqueous phase and chloroform phase are separated.
[0016] (5) The aqueous phase is repeated in step (4) several times, and nitrogen blowing is performed to obtain epimedium polysaccharide.
[0017] Preparation of the drug composition:
[0018] Mix epimedium polysaccharide and arginine in a mass ratio of 1 - 3:1 - 3.
[0019] In the preparation method of the pharmaceutical composition, the mass ratio of epimedium polysaccharide to arginine is preferably 3:2.
[0020] The present invention also provides the application of the pharmaceutical composition in the preparation of a drug for promoting cartilage development and repairing cartilage damage, which can be used to treat chondrodysplasia and repair cartilage damage in the human body or animal body.
[0021] Based on the above application, the present invention also provides a drug for promoting cartilage development and repairing cartilage damage.
[0022] The present invention also provides the application of the pharmaceutical composition in the preparation of a drug for promoting zebrafish cartilage development and repairing cartilage damage, which can be used to treat zebrafish chondrodysplasia and repair cartilage damage.
[0023] Based on the above application, the present invention also provides a drug for promoting zebrafish cartilage development and repairing cartilage damage. The mass ratio of epimedium polysaccharide to arginine is preferably 3:2.
[0024] The present invention also provides an embryo culture solution for promoting zebrafish cartilage development and repairing cartilage damage. The embryo culture solution contains a drug combination of epimedium polysaccharide and arginine, the concentration of epimedium polysaccharide is not more than 1 mg / ml, and the concentration of arginine is not more than 0.5 mg / ml.
[0025] Preferably, the concentration of epimedium polysaccharide is 0.3 mg / ml, and the concentration of arginine is 0.2 mg / ml.
[0026] Advantages of the present invention:
[0027] The present invention provides a pharmaceutical composition for promoting cartilage development and repairing cartilage damage and the application of the pharmaceutical composition in promoting cartilage development and repairing cartilage damage. The pharmaceutical composition provided by the present invention has a synergistic pharmacological effect. Epimedium polysaccharide and arginine are formulated in combination, and their synergistic effect is used to promote cartilage development and treat cartilage damage to obtain a better therapeutic effect, which has a significant advantage over administering each extract alone and has an application prospect for treating chondrodysplasia and repairing cartilage damage in the human body or animal body. Description of the drawings
[0028] Figure 1 Schematic diagram of the effect of different concentrations of epimedium polysaccharide on the survival rate of zebrafish eggs;
[0029] Figure 2 Schematic diagram of the effect of epimedium polysaccharide on promoting zebrafish cartilage development;
[0030] Figure 3 Schematic diagram of the effect of arginine at different concentrations on the survival rate of zebrafish eggs;
[0031] Figure 4 Schematic diagram of the effect of arginine on promoting cartilage development in zebrafish;
[0032] Figure 5 Schematic diagram of the effect of different ratios of arginine and epimedium polysaccharide on promoting cartilage development in zebrafish;
[0033] Figure 6 Schematic diagram of the effect of epimedium polysaccharide on the repair of OPP-induced cartilage damage in zebrafish;
[0034] Figure 7 Schematic diagram of the repair effect of arginine on OPP-induced cartilage damage in zebrafish;
[0035] Figure 8 Schematic diagram of the repair effect of different ratios of arginine and epimedium polysaccharide on OPP-induced cartilage damage in zebrafish;
[0036] Figure 9 Schematic diagram of the comparison of the drug effects between the composition and single components;
[0037] Note: Compared with the model group and the blank group, #: P < 0.05; ##: P < 0.01; #: P < 0.0001, compared with the model group, *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. Detailed implementation method
[0038] Example 1
[0039] This example provides an animal experiment for promoting cartilage development and repairing cartilage damage, and the method includes the following steps:
[0040] 1.1 Experimental animals
[0041] Zebrafish (AB strain), raised in a breeding system with a water temperature of 28.5 °C and a light-dark cycle of 14h / 10h, and fed 4 times a day.
[0042] 1.2 Drugs
[0043] Drug A (Epimedium polysaccharide):
[0044] The preparation method is as follows:
[0045] (1) Heat the epimedium medicinal materials or slices with hot water and extract 2 times, each time for 1 hour. Filter the decoction, and combine the extracts and concentrate them into an extract;
[0046] (2) Add ethanol with a volume concentration of 95% to make the alcohol concentration reach 70%, vortex, precipitate overnight at 4°C, centrifuge at 10,000 r for 10 min, and collect the precipitate.
[0047] (3) Dissolve the precipitate in water and repeat the operation in step (2) twice to obtain crude polysaccharide.
[0048] (4) Prepare a sugar solution with a volume concentration of 5% of the crude polysaccharide, add 4 times the volume of chloroform-n-butanol, and prepare a mixed solution with a volume ratio of chloroform-n-butanol to sugar solution of 4:1. Place it in a 10 ml EP tube, shake well for 30 min, centrifuge at 10,000 r for 1 min, and separate the aqueous phase and chloroform phase.
[0049] (5) Repeat step (4) twice for the aqueous phase, and blow with nitrogen to obtain epimedium polysaccharide.
[0050] Drug B: L-arginine, sourced from the market or plant extraction.
[0051] Drug C: A mixed solution of epimedium polysaccharide and arginine in different weight part ratios.
[0052] 2.1 Grouping, drug administration and treatment
[0053] 2.1.1 Cartilage development
[0054] Grouping:
[0055] Take healthy fish eggs at 7 hpf and place them in a 12-well plate, with 3 wells in each group and 15 in each well. The zebrafish eggs are divided into a blank group, an epimedium polysaccharide group, an arginine group, and an epimedium polysaccharide + arginine group.
[0056] Drug administration:
[0057] Blank group: 2 ml of zebrafish embryo culture medium was given to each well, and half of the volume of the embryo culture medium was changed each time until 5 dpf. Epimedium polysaccharide group: Epimedium polysaccharide solutions with different ratios of Epimedium polysaccharide were dissolved in zebrafish embryo culture medium. The concentrations of Epimedium polysaccharide in the Epimedium polysaccharide solutions were 0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, 3 mg / ml, and 5 mg / ml respectively. 2 ml of the Epimedium polysaccharide solution was given to each well, and half of the volume of the Epimedium polysaccharide solution was changed each time until 5 dpf. Arginine group: Arginine solutions with different ratios of arginine were dissolved in zebrafish embryo culture medium. The concentrations of arginine in the arginine solutions were 0.1 mg / ml, 0.2 mg / ml, 0.4 mg / ml, 0.8 mg / ml, 1.2 mg / ml, 1.6 mg / ml, and 2 mg / ml respectively. 2 ml of the arginine solution was given to each well, and half of the volume of the arginine solution was changed each time until 5 dpf. Epimedium polysaccharide + arginine group: Mixed solutions with different ratios of Epimedium polysaccharide and arginine were dissolved in zebrafish embryo culture medium. The addition amounts of Epimedium polysaccharide and arginine in the mixed solutions were 0.1 mg / ml:0.1 mg / ml, 0.1 mg / ml:0.3 mg / ml, 0.2 mg / ml:0.3 mg / ml, 0.3 mg / ml:0.1 mg / ml, and 0.3 mg / ml:0.2 mg / ml respectively. 2 ml of the mixed solution was given to each well, and half of the volume of the mixed solution was changed each time until 5 dpf.
[0058] 2.1.2 Zebrafish craniofacial cartilage injury and repair
[0059] Grouping:
[0060] Healthy fish eggs at 7 hpf were taken and placed in a 12-well plate, with 3 wells in each group and 15 fish in each well. The zebrafish eggs were divided into a blank group, a model group, an Epimedium polysaccharide group, an arginine group, and an Epimedium polysaccharide + arginine group.
[0061] Drug administration:
[0062] Blank group: Each well was given 2 ml of zebrafish embryo culture medium containing 0.05% DMSO (dimethyl sulfoxide), and half of the volume of the embryo culture medium was changed each time until 5 dpf. Model group: 0.02 mg of o-phenylphenol was dissolved in 1 ml of DMSO and diluted with zebrafish embryo culture medium to a DMSO concentration of 0.05% to obtain the modeling solution. Each well was given 2 ml of the modeling solution, and half of the volume of the modeling solution was changed each time until 5 dpf. Epimedium polysaccharide group: Epimedium polysaccharide was dissolved in the modeling solution to obtain the epimedium polysaccharide solution, and the concentrations of epimedium polysaccharide were 0.1 mg / ml, 0.5 mg / ml, and 1 mg / ml, respectively. Each well was given 2 ml of the epimedium polysaccharide solution, and half of the volume of the epimedium polysaccharide solution was changed each time until 5 dpf. Arginine group: Arginine was dissolved in the modeling solution to obtain the arginine solution, and the concentrations of arginine were 0.1 mg / ml, 0.2 mg / ml, and 0.4 mg / ml, respectively. Each well was given 2 ml of the arginine solution, and half of the volume of the arginine solution was changed each time until 5 dpf. Epimedium polysaccharide + arginine group: Different proportions of epimedium polysaccharide and arginine were dissolved in the modeling solution to obtain the mixed solution, and the addition amounts of epimedium polysaccharide and arginine were 0.1 mg / ml:0.1 mg / ml, 0.1 mg / ml:0.3 mg / ml, 0.2 mg / ml:0.3 mg / ml, 0.3 mg / ml:0.1 mg / ml, and 0.3 mg / ml:0.2 mg / ml, respectively. Each well was given 2 ml of the mixed solution, and half of the volume of the mixed solution was changed each time until 5 dpf.
[0063] 2.2 Index determination
[0064] For zebrafish larvae at 5 dpf, 1 ml of paraformaldehyde was added and fixed overnight at 4°C. The zebrafish larvae were stained according to the instructions of the zebrafish cartilage staining kit and finally stored in glycerol for microscopic observation. The length of the zebrafish mandibular arch was measured.
[0065] 2.3 Statistical analysis
[0066] GraphPad Prism 8 software was used for statistical analysis. The results of each experiment were expressed as mean ± standard deviation (Mean ± SD), and P < 0.05 was considered to be significantly different.
[0067] 3 Results
[0068] As Figure 1 and Figure 3 shown, epimedium polysaccharide and arginine at different concentrations had a certain effect on the survival rate of zebrafish eggs. When the concentration of epimedium polysaccharide in the single-component solution was greater than 1 mg / ml, or the concentration of arginine was greater than 0.5 mg / ml, the survival rate of zebrafish eggs decreased significantly.
[0069] As Figure 2As shown, the epimedium polysaccharide solutions with concentrations of 0.1 mg / ml, 0.5 mg / ml, and 1 mg / ml have a significant promoting effect on zebrafish cartilage development. For example, Figure 4 As shown, the arginine solutions with concentrations of 0.1 mg / ml, 0.2 mg / ml, and 0.4 mg / ml have an obvious promoting effect on zebrafish cartilage development.
[0070] For example, Figure 5 As shown, different ratios of epimedium polysaccharide and arginine have a certain promoting effect on zebrafish cartilage development. When the concentration ratio of epimedium polysaccharide to arginine is 3:2, it has a significant effect on promoting zebrafish cartilage development.
[0071] For example, Figure 6 As shown, epimedium polysaccharide has a significant promoting effect on the repair of zebrafish cartilage damage induced by OPP. For example, Figure 7 As shown, arginine has a certain promoting effect on the repair of zebrafish cartilage damage induced by OPP.
[0072] For example, Figure 8 As shown, different ratios of epimedium polysaccharide and arginine have a certain promoting effect on the repair of zebrafish cartilage damage induced by OPP. When the concentration ratio of epimedium polysaccharide to arginine is 3:2, it has a significant promoting effect on the repair of zebrafish cartilage damage induced by OPP.
[0073] For example, Figure 9 As shown, the mixture of epimedium polysaccharide and arginine has a more significant effect on the repair of zebrafish cartilage damage induced by OPP than the single component of epimedium polysaccharide or arginine.
[0074] In summary, compared with the blank group, the obvious shortening of the zebrafish mandibular arch in the model group indicates successful modeling. Compared with the model group, the mandibular arch of zebrafish in each administration group is significantly increased, indicating that epimedium polysaccharide, arginine, and the mixture of epimedium polysaccharide and arginine in different ratios can all promote zebrafish cartilage development and inhibit zebrafish cartilage damage caused by OPP. Among them, the mixture of epimedium polysaccharide and arginine has a better effect on promoting cartilage development and inhibiting cartilage damage, indicating that there is a synergistic effect between the two, and the best effect is achieved when the addition amount of epimedium polysaccharide and arginine in each milliliter of solution is 0.3:0.2 mg.
[0075] Example 2,
[0076] This example provides a pharmaceutical composition for promoting cartilage development and repairing cartilage damage. The pharmaceutical composition is composed of epimedium polysaccharide and arginine, and the weight ratio of epimedium polysaccharide to arginine is 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0.
[0077] Example 3
[0078] This example provides a pharmaceutical composition for promoting cartilage development and repairing cartilage damage. The pharmaceutical composition is composed of epimedium polysaccharide and arginine, and the arginine is preferably L-arginine.
[0079] Example 4
[0080] This example provides a drug for promoting cartilage development and repairing cartilage damage. The drug includes the pharmaceutical composition described in Example 2 or 3, and the drug is used for treating chondrodysplasia and repairing cartilage damage in the human body or animal body.
[0081] Example 5
[0082] This example provides a drug for promoting cartilage development and repairing cartilage damage in zebrafish. The drug includes the pharmaceutical composition described in Example 2 or 3, and the drug is used for treating chondrodysplasia and repairing cartilage damage in zebrafish.
[0083] Example 6
[0084] This example provides an embryo culture solution for promoting cartilage development and repairing cartilage damage in zebrafish. The embryo culture solution contains the pharmaceutical composition described in Example 2 or 3.
[0085] Example 7
[0086] This example provides an embryo culture solution for promoting cartilage development and repairing cartilage damage in zebrafish. The embryo culture solution contains epimedium polysaccharide and arginine. The concentration of epimedium polysaccharide is not more than 1 mg / ml, and the concentration of arginine is not more than 0.5 mg / ml.
[0087] Example 8
[0088] This example provides an embryo culture solution for promoting cartilage development and repairing cartilage damage in zebrafish. The embryo culture solution contains epimedium polysaccharide and arginine. The concentration of epimedium polysaccharide is 0.3 mg / ml, and the concentration of arginine is 0.2 mg / ml.
Claims
1. A pharmaceutical composition for promoting cartilage development and repairing cartilage damage, characterized in that: The pharmaceutical composition consists of epimedium polysaccharide and arginine, wherein the weight portion of epimedium polysaccharide is 0-100, and the weight portion of arginine is 100-0.
2. A pharmaceutical composition for promoting cartilage development and repairing cartilage damage according to claim 1, characterized in that: In the pharmaceutical composition, the mass ratio of epimedium polysaccharide to arginine is 3:
2.
3. A pharmaceutical composition for promoting cartilage development and repairing cartilage damage according to claim 1, characterized in that: In the pharmaceutical composition, the arginine is L-arginine.
4. The method for preparing a pharmaceutical composition for promoting cartilage development and repairing cartilage damage according to claim 1, characterized in that: The following steps are involved: Preparation of Epimedium polysaccharide: (1) The herbal medicine or slices of Epimedium are heated and extracted several times, each time for at least 1 hour, the decoction is filtered, and the extracts are combined and concentrated into an extract; (2) adding ethanol with a volume concentration of 95% or more to make the mixture have an alcohol concentration of 70% or more, vortexing, precipitating with alcohol at 4° C. overnight, centrifuging, and collecting the precipitate; (3) dissolving the precipitate in water and repeating step (2) several times to obtain crude polysaccharide; (4) The crude polysaccharide is prepared into a sugar solution with a volume concentration of 5%, and 4 volumes of chloroform-n-butanol are added to prepare a mixed solution with a volume ratio of chloroform-n-butanol to sugar solution of 4:1, and the mixture is shaken thoroughly and centrifuged to separate the aqueous phase and the chloroform phase; (5) Repeat step (4) for several times with the aqueous phase, blow nitrogen, and obtain epimedium polysaccharide; Preparation of pharmaceutical composition: Epimedium polysaccharide and arginine are mixed in a mass ratio of 1-3:1-3.
5. Use of the pharmaceutical composition according to claim 1 in the preparation of a drug for promoting cartilage development and repairing cartilage damage.
6. Use of the pharmaceutical composition according to claim 1 in the preparation of a drug for promoting zebrafish cartilage development and repairing cartilage damage.
7. The use according to claim 6, characterized in that: In the preparation of drugs for promoting zebrafish cartilage development and repairing cartilage damage, the added mass ratio of epimedium polysaccharide and arginine is 3:
2.
8. A drug for promoting cartilage development and repairing cartilage damage, characterized in that: The medicine comprises the pharmaceutical composition according to any one of claims 1 to 3.
9. A drug for promoting cartilage development and repairing cartilage damage in zebrafish, characterized in that: The medicine comprises the pharmaceutical composition according to any one of claims 1 to 3.
10. An embryonic culture medium for promoting cartilage development and repairing cartilage damage in zebrafish, characterized in that: The embryo culture medium comprises the pharmaceutical composition according to any one of claims 1 to 3, wherein the concentration of epimedium polysaccharide is not greater than 1 mg / ml, and the concentration of arginine is not greater than 0.5 mg / ml.