Composition and application thereof in improving articular cartilage health

Abstract

The invention relates to the technical field of biology, and particularly discloses a composition and application thereof in improving articular cartilage health. The composition comprises sodium hyaluronate, colostrum basic protein and N-acetylglucosamine, wherein the mass ratio of the sodium hyaluronate to the colostrum basic protein to the N-acetylglucosamine is (1-2): (1-2): (1-8). The composition disclosed by the invention can be used for increasing the bone mineral density, increasing the number of osteoblasts, promoting cartilage growth and increasing the content of II-type collagen in an animal body, so that the bone joint function can be effectively improved, and a new method is provided for joint maintenance.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a composition and its application in improving joint cartilage health. Background Art

[0002] The skeletal system plays a crucial role in the human body. It not only supports the body and protects internal organs, but also participates in various physiological processes. However, with the aggravation of population aging and the change of people's lifestyle, skeletal joint health problems have become increasingly prominent. The incidence of diseases such as osteoarthritis and osteoporosis has been rising continuously, seriously affecting the quality of life of patients. In addition, the popularization of sports and fitness has also made joint problems caused by sports injuries more common.

[0003] At present, there are already products for skeletal joint maintenance on the market, but there are still many deficiencies. For example, traditional products mainly contain ingredients such as glucosamine, chondroitin sulfate, calcium, and vitamin D, and the dosage forms are mainly tablets, capsules, and oral liquids. Although these products can relieve joint discomfort to a certain extent, they have a single composition, limited effects, slow onset, and long treatment courses. Although some products have added other ingredients, the effects on improving bone and joint health still need to be improved. In addition, although some traditional Chinese medicine preparations can also improve joint function as a whole, they cannot supplement cartilage nutrients.

[0004] Therefore, it is still necessary to develop new methods for improving bone and joint function. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a new product for improving the health of bone and joint cartilage.

[0006] The present invention provides a composition, which includes: sodium hyaluronate, colostrum basic protein, and N-acetylglucosamine, and the mass ratio of sodium hyaluronate, colostrum basic protein, and N-acetylglucosamine is (1~2):(1~2):(1~8).

[0007] Preferably, the mass ratio of sodium hyaluronate, colostrum basic protein, and N-acetylglucosamine is (1~1.2):(1.8~2):(4~8).

[0008] More preferably, the mass ratio of sodium hyaluronate, colostrum basic protein, and N-acetylglucosamine is 1:2:(4~8).

[0009] Or preferably, the mass ratio of sodium hyaluronate, colostrum basic protein, and N-acetylglucosamine is 1:1:(1-8).

[0010] The research of the present invention has found that when sodium hyaluronate, colostrum basic protein and N-acetylglucosamine are combined in a specific ratio, the effect of improving bone and joint health can be significantly enhanced.

[0011] Specifically, the research of the present invention has found that when sodium hyaluronate is used alone, there is no effect of increasing bone density and / or the content of type II collagen. When sodium hyaluronate or N-acetylglucosamine is used alone, there is no effect of increasing the number of osteoblasts. When sodium hyaluronate or colostrum basic protein is used alone, there is no effect of promoting cartilage growth. However, when sodium hyaluronate, colostrum basic protein and N-acetylglucosamine are combined in the specific ratio of the present invention, compared with using each component alone or combining these three components with other ratios, the composition under the specific ratio of the present invention unexpectedly simultaneously has the effects of significantly increasing bone density, increasing the number of osteoblasts, promoting cartilage growth, and increasing the content of type II collagen in the animal body, providing a new method for bone and joint protection and treatment.

[0012] The composition of the present invention may further include pharmaceutically acceptable excipients.

[0013] The composition of the present invention can be a liquid preparation or a solid preparation.

[0014] The composition of the present invention may contain a therapeutically effective amount of a pharmacologically active ingredient. The dosage form can be tablets, granules, capsules, injections or oral liquids. The unit content of the active ingredient contained in a single dose in each dosage form does not necessarily need to constitute an effective amount by itself, and the required effective amount can be achieved by administering multiple dose units.

[0015] The present invention also provides the application of the above composition in the preparation of a product for increasing bone density.

[0016] The present invention also provides the application of the above composition in the preparation of a product for increasing the number of osteoblasts.

[0017] The present invention also provides the application of the above composition in the preparation of a product for promoting cartilage growth.

[0018] The present invention also provides the application of the above composition in the preparation of a product for increasing the content of type II collagen in the animal body.

[0019] The present invention also provides the application of the above composition in the preparation of a product for protecting joints.

[0020] The present invention also provides the application of the above composition in the preparation of a product for improving joint cartilage health.

[0021] The beneficial effects of the present invention are at least as follows: The present invention provides a new composition, which can increase bone density, increase the number of osteoblasts, promote cartilage growth, increase the content of type II collagen in animals, and thus can effectively improve the function of articular cartilage, providing a new method for joint maintenance. Description of the Drawings

[0022] Figure 1 It is the results of the fluorescence intensity of the skulls of each experimental group.

[0023] Figure 2 It is the results of the number of osteoblasts (fluorescence intensity) in each experimental group.

[0024] Figure 3 It is the results of the fluorescence intensity of cartilage in each experimental group.

[0025] Figure 4 It is the results of the content of type II collagen in each experimental group.

[0026] In each figure, the data are mean ± SEM; In each figure (if any), compared with the normal control group, #: P < 0.0001; compared with the model control group, : P < 0.05, : P < 0.01, : P < 0.001, : P < 0.0001; compared with the 1:2:8 group (formulation 6), $: P < 0.05, $$: P < 0.01, $$$: P < 0.001, $$$$: P < 0.0001. Detailed Embodiments

[0027] The preferred embodiments of the present invention will be described in detail below in conjunction with the embodiments. It should be understood that the following embodiments are given only for the purpose of illustration and are not used to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0028] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources or prepared by conventional methods in the art unless otherwise specified.

[0029] Example 1 Osteoporosis Model 1.1 Zebrafish strains: Wild-type AB strain and transgenic teleost green fluorescent zebrafish. Bred and provided by the fish breeding center of Hangzhou Huante Biotechnology Co., Ltd., Laboratory Animal Use Permit No. SYXK (Zhejiang) 2022-0004. Animal husbandry and management comply with AAALAC accreditation (certification number: 001458) and IACUC ethics review number: IACUC-2024-10847-01.

[0030] 1.2 Modeling method and principle: Dexamethasone-induced zebrafish osteoporosis model.

[0031] Osteoporosis is a common bone disease, a systemic metabolic bone disease caused by a variety of reasons, which leads to bone loss and reduction, damage to bone tissue microstructure, increased bone brittleness and decreased bone density, making patients prone to fractures. Its clinical manifestations are usually back pain or general bone pain, spinal deformity and fractures, etc. In severe cases, it compresses the heart and lungs, causing abnormalities in the circulatory and respiratory systems.

[0032] The zebrafish's skeletal development process is remarkably similar to that of other vertebrates. The skeleton is composed of cartilage and bone. Cartilage is formed by chondrocytes, while bone is composed of osteoblasts and osteoclasts. Skeletal formation occurs in two ways: intramembranous ossification, in which mesenchymal cells directly differentiate into osteoblasts and generate bone (commonly seen in skull formation); and endochondral ossification, in which mesenchymal cells first differentiate into chondrocytes, which then secrete and form cartilage. This cartilage is then replaced by bone composed of osteoblasts and osteoclasts. Zebrafish have a similar number of vertebrae to humans, and their spinal morphology shares a similar physiological curvature. Furthermore, key genes involved in zebrafish skeletal development, such as runx2a, col2a1a, bmp2a, bmp4, and sp7, share a high degree of homology with mammalian counterparts.

[0033] Glucocorticoids, such as dexamethasone, can directly damage bone cells and their regulatory factors, reducing bone formation and increasing bone resorption, thereby impairing bone remodeling, reducing the body's ability to restore bone microarchitecture, increasing bone brittleness, and causing fractures and osteonecrosis. Glucocorticoids also inhibit gastrointestinal absorption of calcium and phosphorus, lowering blood calcium and triggering a secondary increase in parathyroid hormone, which in turn inhibits osteoblast activity, activates osteoclasts, and promotes bone turnover. Furthermore, long-term glucocorticoid use can affect the hypothalamic-pituitary-gonadal axis, reducing the secretion of growth hormone and other hormones, leading to impaired bone matrix synthesis, inhibiting bone formation, and increasing skeletal muscle protein degradation, exacerbating osteoporosis.

[0034] Bone mineralized matrix deposition is an important indicator of bone formation and is usually evaluated by alizarin red staining. Alizarin red is a dye that can attach to calcium salts and is widely used to observe and measure bone mineralization. The fluorescence intensity collected by fluorescence microscopy after staining the zebrafish bones can reflect the calcified bone content, and bone density is closely related to the calcium content in the bones. Therefore, inducing zebrafish with glucocorticoids will reduce the number of zebrafish bones, weaken bone density, and reduce the bone fluorescence intensity after alizarin red staining.

[0035] 1.3 Determination of the maximum test concentration (MTC) of the sample 1.3.1 Experimental groups: There are a total of 3 samples (N-acetylglucosamine, CBP, sodium hyaluronate), 5 test concentrations (125, 250, 500, 1000, 2000 μg / ml), and a normal control group and a model control group are set up at the same time. There are 30 transgenic osteogreen fluorescent zebrafish in each group.

[0036] 1.3.2 Experimental method: The experimental system is 30 fish / 20 ml. Add the test samples according to the groups and change the liquid every day. Except for the normal control group, dexamethasone 2 μM is additionally dissolved in water to establish a zebrafish osteoporosis model in the other experimental groups. After 4 days of co-treatment, count the number of dead zebrafish and the toxicity in each experimental group to determine the MTC of the sample.

[0037] 1.3.3 Evaluation index: According to the number of dead zebrafish and the toxicity in the experimental group, determine the MTC of the sample, and use the highest sample test concentration without death as the MTC of the sample. The MTC of all three samples in this experiment is 2000 μg / ml.

[0038] 1.4 Determination of the efficacy of increasing bone density 1.4.1 Experimental groups: Except for the normal control group, the model control group, the positive control group (alendronate sodium, 5.00 μg / mL), and the groups of sodium hyaluronate, CBP (colostrum basic protein), and NAG (N-acetylglucosamine) at 500 μg / ml each, including: Formula 1 (500 μg / ml): It includes sodium hyaluronate, CBP (colostrum basic protein), and NAG (N-acetylglucosamine) with a mass ratio of 1:1:1.

[0039] Formula 2 (500 μg / ml): It includes sodium hyaluronate, CBP, and NAG with a mass ratio of 1:1:2.

[0040] Formula 3 (500 μg / ml): It includes sodium hyaluronate, CBP, and NAG with a mass ratio of 1:1:4.

[0041] Formula 4 (500 μg / ml): It includes sodium hyaluronate, CBP, and NAG with a mass ratio of 1:1:8.

[0042] Formulation 5 (500 μg / ml): Comprising sodium hyaluronate, CBP and NAG in a mass ratio of 1:2:4.

[0043] Formulation 6 (500 μg / ml): Comprising sodium hyaluronate, CBP and NAG in a mass ratio of 1:2:8.

[0044] Control Formulation 1 (500 μg / ml): Comprising sodium hyaluronate, CBP and NAG in a mass ratio of 8:1:1.

[0045] Control Formulation 2 (500 μg / ml): Comprising sodium hyaluronate, CBP and NAG in a mass ratio of 1:8:1.

[0046] Control Formulation 3 (500 μg / ml): Comprising sodium hyaluronate, CBP and glucosamine hydrochloride in a mass ratio of 1:2:8.

[0047] Control Formulation 4 (500 μg / ml): Comprising sodium hyaluronate, CBP and glucosamine hydrochloride in a mass ratio of 1:1:2.

[0048] Control Formulation 5 (500 μg / ml): Comprising sodium hyaluronate, CBP and NAG in a mass ratio of 2:2:0.8.

[0049] There are 30 wild-type AB strain zebrafish in each group.

[0050] 1.4.2 Experimental method: The zebrafish osteoporosis model was induced by dexamethasone for testing. According to the groups, the test samples were added (the test concentrations of Formulations 1-6 and Control Formulations 1-5 were all 500 μg / ml), and other treatments were the same as 1.3.2. After 4 days of treatment, the zebrafish in each experimental group were stained with alizarin red. Subsequently, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The NIS-Elements D 3.20 advanced image processing software was used to analyze and collect data, and the fluorescence intensity of the zebrafish skull was analyzed. The efficacy of the sample in increasing the bone density of the skull was evaluated based on the statistical analysis results of this index.

[0051] 1.4.3 Evaluation index: Fluorescence intensity of the skull. The results are shown in Figure 1 .

[0052] 1.5 Determination of the efficacy of increasing the number of osteoblasts 1.5.1 Experimental groups: Refer to 1.4.1. There are 30 transgenic osteocalcin green fluorescent zebrafish in each group.

[0053] 1.5.2 Experimental method: A zebrafish osteoporosis model induced by dexamethasone was used for testing. According to the groups, the test samples were added (the test concentrations of Formulas 1-6 and Comparative Formulas 1-5 were all 500 μg / ml), and other treatments were the same as in 1.3.2. After 4 days of treatment, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The NIS-Elements D 3.20 advanced image processing software was used to analyze and collect data, and the osteogenic fluorescence intensity of zebrafish was analyzed. The statistical analysis results of this index were used to evaluate the efficacy of the sample in increasing the number of osteoblasts.

[0054] 1.5.3 Evaluation index: Number of osteoblasts (fluorescence intensity). The results are shown in Figure 2 .

[0055] Example 2 Joint cartilage protection model 2.1 Zebrafish strain: Transgenic cartilage green fluorescence strain. It is provided by the fish breeding center of Hangzhou Huante Biotechnology Co., Ltd. The experimental animal use license number is: SYXK (Zhe) 2022-0004. The feeding management meets the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethical review number is: IACUC-2024-10847-01.

[0056] 2.2 Modeling method and principle: A zebrafish cartilage injury model induced by dexamethasone. The zebrafish skeleton can be divided into three parts: the head skeleton, the body skeleton, and the fins. Similar to mammals, the bone formation mechanism of zebrafish also includes endochondral ossification and intramembranous ossification. The cartilage in zebrafish is hyaline cartilage, mainly distributed in the head, and is formed by endochondral ossification. The cartilage in zebrafish has basically formed at 72 hpf, and then around 6-7 dpf, the pharyngeal cranial cartilage first develops and matures. According to the development process, the cartilage development of zebrafish includes three stages: cartilage condensation, chondrocyte proliferation and hypertrophy, and endochondral ossification. There are certain differences in the structure of the zebrafish skeleton and that of mammals, but the molecular mechanism of zebrafish bone growth and development is extremely similar to that of mammals. Moreover, in recent years, more and more homologous genes of key genes regulating mammalian bone development have been found in the zebrafish genome. For example, the key genes runx2, sparc (osteonectin), etc. involved in the regulation of zebrafish endochondral ossification process are highly homologous to the related genes in mammals. As one of the main tissue organs carrying collagen, cartilage also expresses various collagens such as Col1a, Col2a1, and Col10a1. Among them, Col2a1 maintains a very high expression throughout the whole process of chondrocyte formation and development, and is one of the specific markers of chondrocytes and cartilage tissue.

[0057] Glucocorticoids are a class of endogenous steroid hormones, and their expression is controlled by the hypothalamic-pituitary-adrenal axis. Glucocorticoid receptors are present in chondrocytes themselves. When the injected hormone binds to the receptor, it will affect cell metabolism. High-dose glucocorticoids can directly induce chondrocyte apoptosis; at the same time, glucocorticoids will affect the metabolism of the cartilage matrix and disrupt the physiological environment of the subchondral bone, indirectly causing ischemic necrosis of chondrocytes. Dexamethasone is a widely used drug in the class of glucocorticoid drugs. It has teratogenic effects and can also inhibit chondrocyte proliferation, causing chondrocyte apoptosis, thus leading to cartilage damage.

[0058] 2.3 Determination of the maximum test concentration (MTC) of the sample 2.3.1 Experimental groups: There are a total of 3 samples (N-acetylglucosamine, CBP, sodium hyaluronate), 5 test concentrations (125, 250, 500, 1000, 2000 μg / ml), and a normal control group and a model control group are set up at the same time. There are 30 transgenic cartilage green fluorescent zebrafish in each group.

[0059] 2.3.2 Experimental method: The experimental system is 30 per 20 ml. Add the samples to be tested according to the groups and change the liquid every day. Except for the normal control group, dexamethasone 25 μM is additionally given in water solution to the rest of the experimental groups to establish a zebrafish osteoporosis model. After 4 days of treatment, count the number of dead zebrafish and the toxicity in each experimental group to determine the MTC of the sample.

[0060] 2.3.3 Evaluation index: The number of dead zebrafish and the toxicity in the experimental group are used to determine the MTC of the sample. The highest sample test concentration without death is used as the MTC of the sample. The MTC of all three samples in this experiment is 2000 μg / ml.

[0061] 2.4 Determination of the efficacy of protecting cartilage damage 2.4.1 Experimental groups: Except for the positive control group (sodium chondroitin sulfate A, 1000 μg / mL), the others are the same as 1.4.1 in Example 1. There are 30 transgenic cartilage green fluorescent zebrafish in each group.

[0062] 2.4.2 Experimental method: Use the dexamethasone-induced zebrafish cartilage damage model for testing. Add the samples to be tested according to the groups (the test concentrations of Formulas 1-6 and Comparative Formulas 1-5 are all 500 μg / ml), and other treatments are the same as 2.3.2. After 4 days of treatment, then randomly select 10 zebrafish from each experimental group to take pictures under a fluorescence microscope, and use the NIS-Elements D 3.20 advanced image processing software to analyze and collect data, analyze the fluorescence intensity of zebrafish cartilage, and evaluate the efficacy of the sample in protecting cartilage damage based on the statistical analysis results of this index.

[0063] 2.4.3 Evaluation Index: Fluorescence Intensity of Cartilage. The results are shown in Figure 3 .

[0064] 2.5 Determination of the Efficacy of Increasing Collagen 2.5.1 Experimental Groups: Refer to 2.4.1. There are 30 transgenic zebrafish with green fluorescent cartilage in each group.

[0065] 2.5.2 Experimental Method: The zebrafish cartilage injury model was induced by dexamethasone for testing. The test samples to be measured were added according to the groups (the test concentrations of Formulas 1-6 and Comparative Formulas 1-5 were all 500 μg / ml), and other treatments were the same as 2.3.2. After 4 days of treatment, zebrafish samples were collected according to the instructions of the Zebrafish Col Ⅱ ELISA KIT, and data were collected using a multifunctional microplate reader to analyze the content of type Ⅱ collagen in zebrafish.

[0066] 2.5.3 Evaluation Index: Content of Type Ⅱ Collagen. The results are shown in Figure 4 .

[0067] From the above results, it can be seen that compared with the non-compounded components, the formula of the present invention has a significant increasing effect on bone density, the number of osteoblasts, fluorescence intensity of cartilage, and content of type Ⅱ collagen at equal doses.

[0068] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A composition, characterized in that, Comprising: Sodium hyaluronate, colostrum basic protein and N-acetylglucosamine, and the mass ratio of sodium hyaluronate, colostrum basic protein and N-acetylglucosamine is (1-2):(1-2):(1-8).

2. The composition according to claim 1, wherein The mass ratio of sodium hyaluronate, colostrum basic protein and N-acetylglucosamine is (1-1.2):(1.8-2):(4-8).

3. The composition according to claim 2, wherein, The mass ratio of sodium hyaluronate, colostrum basic protein and N-acetylglucosamine is 1:2:(4-8).

4. The composition according to claim 1, characterized in that, The mass ratio of sodium hyaluronate, colostrum basic protein and N-acetylglucosamine is 1:1:(1-8).

5. Use of the composition according to any one of claims 1-4 in the preparation of a product for increasing bone density.

6. Use of the composition according to any one of claims 1-4 in the preparation of a product for increasing the number of osteoblasts.

7. Use of the composition according to any one of claims 1-4 in the preparation of a product for promoting cartilage growth.

8. Use of the composition according to any one of claims 1-4 in the preparation of a product for increasing the content of type II collagen in an animal body.

9. Use of the composition according to any one of claims 1-4 in the preparation of a product for protecting joints.

10. Use of the composition according to any one of claims 1-4 in the preparation of a product for improving joint cartilage health.

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