A composition for improving bone health, a formulation thereof, a method for preparing the same, its application, and a product thereof.

Abstract

This invention belongs to the field of nutritional composition technology, specifically relating to a composition, composition formulation, preparation method, application, and product for improving bone health. This invention provides a composition composed of N-acetylglucosamine and 3-O-acetyl-11-keto-β-boswellic acid. The composition and formulation of this invention can promote the differentiation of chondrocyte precursor cells into mature chondrocytes, promote cartilage matrix synthesis, significantly inhibit TNF-α and MMP13 expression, and upregulate COL2A1 expression, with effects superior to single components and traditional combinations. The ingredients are natural and safe, biocompatible, and have no significant toxicity, making them suitable for people with joint noise, joint pain, osteoarthritis, and joint degeneration. Simultaneously, they can improve bone density, promote bone growth and development, and improve bone health. The preparation process is simple and low-cost, and it can be formulated into various dosage forms such as food, health products, and pharmaceuticals, offering flexible applications and combining preventative and therapeutic value.

Description

Technical Field

[0001] This invention belongs to the field of nutritional composition technology, specifically relating to a composition for improving bone health, a composition formulation, its preparation method, application, and product. Background Technology

[0002] Bone health is a crucial foundation for maintaining normal physiological functions in the human body, and its core involves the structural integrity and metabolic homeostasis of bones, cartilage, and joint tissues. Cartilage damage and degeneration are key factors in triggering various bone health problems. If cartilage damage is not addressed promptly, it will gradually progress to joint pain, swelling, limited mobility, and even trigger a chain reaction of osteoporosis.

[0003] Clinical treatments and routine interventions for the aforementioned bone health problems still have many limitations. Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used to relieve pain and inflammation, but long-term use can easily cause side effects such as gastrointestinal ulcers, liver and kidney damage, and increased cardiovascular risk, making them unsuitable for long-term treatment. While steroidal drugs such as glucocorticoids have significant anti-inflammatory effects, long-term use can accelerate cartilage degradation, leading to disease progression. Surgical treatments (such as joint replacement surgery) are only suitable for advanced, severely ill patients and involve significant trauma, long recovery periods, and postoperative complications. In the field of chondroprotective agents, traditional products such as glucosamine sulfate and glucosamine hydrochloride, while providing some raw materials for cartilage matrix synthesis, suffer from low bioavailability, a single mechanism of action, and slow onset of action, only delaying disease progression and unable to fundamentally reverse cartilage degeneration.

[0004] The limitations of single active ingredients are more pronounced: anti-inflammatory components (such as frankincense extract and non-steroidal anti-inflammatory drugs) can inhibit the release of inflammatory factors, but lack the ability to promote cartilage matrix synthesis, making it difficult to repair damaged cartilage tissue; cartilage-nourishing components (such as amino sugars and chondroitin) can replenish raw materials for cartilage synthesis, but their anti-inflammatory activity is weak and cannot effectively control synovial inflammation and cartilage degradation. While there have been attempts to combine amino sugars with anti-inflammatory components in existing technologies, most suffer from problems such as unreasonable component combinations, unclear synergistic mechanisms, and undefined optimal ratios. Some combined products simply mix two components without experimental verification of synergistic effects, making it difficult to achieve the expected cartilage protection effects in practical applications.

[0005] Therefore, developing a composition with natural and safe ingredients that has anti-inflammatory, cartilage repair, bone formation promotion, and bone health improvement effects is of great practical significance and application value for effectively improving bone health, delaying cartilage degeneration, and alleviating joint-related symptoms. Summary of the Invention

[0006] To address the above shortcomings, the present invention provides a composition for improving bone health, its preparation method, application, and product.

[0007] The technical solution of this invention is as follows: On one hand, the present invention provides a composition comprising N-acetylglucosamine and 3-O-acetyl-11-keto-β-boswellic acid.

[0008] Specifically, the composition comprises 200-600 parts of N-acetylglucosamine and 10-100 parts of 3-O-acetyl-11-keto-β-boswellic acid by weight.

[0009] Preferably, the composition contains, by weight, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290, 290-300, 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 390- 400, 400-410, 410-420, 420-430, 430-440, 440-450, 450-460, 460-470, 470-480, 480-490, 490-500, 500-510, 510-520, 520-530, 530-540, 540-550, 550-560, 560-570, 570-580, 580-590 or 590-600 parts of N-acetylglucosamine.

[0010] More preferably, the composition contains 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 390-400, 400-410, 410-420, 420-430, 430-440, and 440 parts by weight. -450, 450-460, 460-470, 470-480, 480-490, 490-500, 500-510, 510-520, 520-530, 530-540, 540-550, 550-560, 560-570, 570-580, 580-590 or 590-600 parts of N-acetylglucosamine.

[0011] More preferably, the composition contains 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 390-400, 400-410, 410-420, 420-430, 430-440, 440-450, 450-460, 460-470, 470-480, 480-490, or 490-500 parts of N-acetylglucosamine by weight.

[0012] More preferably, the composition contains 300 parts by weight of N-acetylglucosamine.

[0013] Preferably, the composition contains 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95 or 95-100 parts of 3-O-acetyl-11-keto-β-boswellic acid by weight.

[0014] More preferably, the composition contains 20-25, 25-30, 30-35, 35-40, 40-45 or 45-50 parts of 3-O-acetyl-11-keto-β-boswellic acid by weight.

[0015] More preferably, the composition contains 20 parts by weight of 3-O-acetyl-11-keto-β-boswellic acid.

[0016] In another aspect, the present invention provides a method for preparing the composition described in any one of the above claims, the method comprising: mixing N-acetylglucosamine and 3-O-acetyl-11-keto-β-boswellic acid in a certain proportion to obtain the composition.

[0017] In this invention, the method of obtaining 3-O-acetyl-11-keto-β-boswellic acid is not limited. It can be obtained directly through commercial purchase, or prepared from frankincense extract or frankincense oil through separation and purification processes, or prepared through microbial fermentation synthesis, chemical synthesis, or other methods. Regardless of the preparation process used, as long as the final active ingredient obtained is 3-O-acetyl-11-keto-β-boswellic acid, it falls within the scope of raw materials that can be used in this invention.

[0018] In some embodiments of the present invention, pure 3-O-acetyl-11-keto-β-boswellic acid (AKBA) is used as the active ingredient and combined with N-acetylglucosamine in a certain weight ratio to obtain the composition described in the present invention.

[0019] In some embodiments of the present invention, a frankincense extract containing 20%-80% wt 3-O-acetyl-11-keto-β-boswellic acid is used to replace the above-mentioned pure AKBA, and combined with N-acetylglucosamine in a weight ratio to prepare the composition formulation described in the present invention. In this composition formulation, the actual weight of AKBA and the weight ratio of AKBA to N-acetylglucosamine are completely consistent with the weight ratio of AKBA to N-acetylglucosamine in the above composition. The amount of frankincense extract is determined according to the purity of the AKBA it contains. For example, if a frankincense extract containing 20% ​​wt AKBA is used, the conversion formula for the weight of frankincense extract is: frankincense extract weight = target weight of AKBA / 20%; and if a frankincense extract containing 80% wt AKBA is used, the conversion formula for the weight of frankincense extract is: frankincense extract weight = target weight of AKBA / 80%.

[0020] The two technical solutions (compositions and formulations) mentioned above have the same core active ingredient ratios. Both achieve the technical effect of improving bone health through the synergistic effect of AKBA and N-acetylglucosamine, and are parallel technical solutions based on the same inventive concept.

[0021] In another aspect, the present invention provides a composition formulation comprising N-acetylglucosamine and frankincense extract, wherein the frankincense extract contains 20%-80%wt of 3-O-acetyl-11-keto-β-boswellic acid.

[0022] Specifically, the frankincense extract contains 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, or 70%-80%wt of 3-O-acetyl-11-keto-β-boswellic acid.

[0023] Preferably, the frankincense extract contains 20%wt of 3-O-acetyl-11-keto-β-boswellic acid.

[0024] Specifically, the composition formulation contains 200-600 parts of N-acetylglucosamine; the weight ratio of N-acetylglucosamine to 3-O-acetyl-11-keto-β-boswellic acid in frankincense extract is 200-600:10-300.

[0025] More specifically, the composition formulation contains, by weight, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290, 290-300, 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 39 0-400, 400-410, 410-420, 420-430, 430-440, 440-450, 450-460, 460-470, 470-480, 480-490, 490-500, 500-510, 510-520, 520-530, 530-540, 540-550, 550-560, 560-570, 570-580, 580-590 or 590-600 parts of N-acetylglucosamine.

[0026] More specifically, in the aforementioned composition formulation, the weight ratio of N-acetylglucosamine to 3-O-acetyl-11-keto-β-boswellic acid from frankincense extract is 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290, 290-300, 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 390-400, 400-410, 410-420, 420-430. 430-440, 440-450, 450-460, 460-470, 470-480, 480-490, 490-500, 500-510, 510-520, 520-530, 530-540, 540-550, 550-560, 560-570, 570-580, 58 0-590 or 590-600: 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95 or 95-100.

[0027] Preferably, the composition formulation contains, by weight, 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 390-400, 400-410, 410-420, 420-430, 430-440, or 440 parts of each other. -450, 450-460, 460-470, 470-480, 480-490, 490-500, 500-510, 510-520, 520-530, 530-540, 540-550, 550-560, 560-570, 570-580, 580-590 or 590-600 parts of N-acetylglucosamine.

[0028] Preferably, in the composition formulation, the weight ratio of N-acetylglucosamine to 3-O-acetyl-11-keto-β-boswellic acid from the frankincense extract is 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370, 370-380, 380-390, 390-400, 400-410, 410-420, 420-430, 430-440, or 440-450. 450-460, 460-470, 470-480, 480-490, 490-500, 500-510, 510-520, 520-530, 530-540, 540-550, 550-560, 560-570, 570-580, 580-590 or 590-600: 20-25, 25-30, 30-35, 35-40, 40-45 or 45-50 parts of 3-O-acetyl-11-keto-β-boswellic acid.

[0029] More preferably, in the composition, the composition formulation contains 300 parts by weight of N-acetylglucosamine, and the weight ratio of N-acetylglucosamine to 3-O-acetyl-11-keto-β-boswellic acid in frankincense extract is 300:20.

[0030] The frankincense extract containing 20%-80%wt of 3-O-acetyl-11-keto-β-boswellic acid is not limited in origin and may be derived from the Boswelliaceae family (…). Burseraceae Frankincense ( ) Boswellia Resin extracts from plants, including but not limited to Boswellia carterii (Boswellia carterii). Boswellia bhaw-dajiana ), toothed frankincense tree ( Boswellia serrata ), Arabian frankincense ( Boswellia sacra Somali frankincense tree ( Boswellia carteriThe frankincense extract can be obtained through commercial procurement or prepared from frankincense raw materials using conventional plant extraction processes (such as solvent extraction, ultrasound-assisted extraction, microwave-assisted extraction, column chromatography purification, etc.). Regardless of the source or preparation process, as long as the purity of 3-O-acetyl-11-keto-β-boswellic acid is within the range of 20%-80%wt and meets the relevant quality standards for food, pharmaceutical, or health food raw materials (such as heavy metal content, microbial limits, residual solvents, etc.), it falls within the scope of frankincense extracts applicable to this invention.

[0031] In another aspect, the present invention provides products comprising any of the compositions or formulations described above, wherein the products include any one or more of food, health products, and pharmaceuticals.

[0032] Specifically, the dosage form of the product includes: solid dosage form, semi-solid dosage form, or liquid dosage form.

[0033] Preferably, the food includes: candy, soy milk, yogurt, canned food, biscuits, chocolate, pastries, cream, cheese, dairy products, milk powder, formula milk powder, ice cream, jam, fruit puree, candied fruit, preserved fruit, dried fruit, bread, egg rolls, protein drinks, solid beverages, lactic acid bacteria beverages, plant protein beverages, carbonated beverages, coffee, or puffed foods.

[0034] Preferably, the food includes human food or animal food.

[0035] Preferably, the dosage form of the health product includes tablets, capsules, soft capsules, granules, pills, gel candies, powders, oral liquids, or drops.

[0036] Specifically, the food or health product also includes nutritionally acceptable nutrients.

[0037] Preferably, the nutrients include any one or more of dietary fiber, polypeptides, food-medicine homologous ingredients, proteins, lipids, minerals, and vitamins.

[0038] Specifically, the food or health product also includes excipients acceptable for use in food or health products.

[0039] Preferably, the excipients include any one or a combination of two or more of the following: wetting agents, emulsifiers, preservatives, antioxidants, buffers, diluents, lubricants, solvents, suspending agents, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0040] Preferably, the food or health product is used to improve bone health, promote bone formation, and / or improve bone density.

[0041] Preferably, the dosage form of the medicine includes any one or more of the following: tablets, pills, powders, suspensions, gels, emulsions, creams, granules, capsules, suppositories, injections, sprays, and injections.

[0042] Specifically, the medicine also includes any one or more pharmaceutically acceptable excipients.

[0043] Preferably, the pharmaceutically acceptable excipients include one or more of the following: wetting agents, emulsifiers, preservatives, antioxidants, buffers, diluents, lubricants, solvents, suspending agents, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

[0044] Preferably, the medicine is used for: preventing or treating osteoporosis, osteoarthritis, cartilage damage, joint degeneration, synovitis, improving joint pain, and promoting cartilage repair and regeneration, among any one or more of these.

[0045] In another aspect, the present invention provides the use of any of the above-described compositions or composition formulations in the preparation of pharmaceuticals, wherein the pharmaceuticals include any one or more of the following: pharmaceuticals for the prevention or treatment of osteoporosis, pharmaceuticals for the prevention or treatment of osteoarthritis, pharmaceuticals for the prevention or treatment of cartilage damage, pharmaceuticals for the prevention or treatment of joint degeneration, pharmaceuticals for the prevention or treatment of synovitis, pharmaceuticals for the improvement of joint pain, and pharmaceuticals for the promotion of cartilage repair and regeneration.

[0046] Preferably, the medicine contains an effective amount of the composition or composition formulation.

[0047] In another aspect, the present invention provides an application of the above-mentioned composition, composition formulation, food, health product or medicine, the application including administering an effective amount of the composition, composition formulation, food, health product or medicine to a subject.

[0048] Specifically, the subjects were mammals.

[0049] Preferably, the subject is a human being.

[0050] In another aspect, the present invention provides a method for preventing or treating osteoporosis, osteoarthritis, cartilage damage, joint degeneration, synovitis, improving joint pain, or promoting cartilage repair and regeneration.

[0051] Specifically, the method includes administering an effective amount of the composition, composition formulation, food, health product, or medicine to the subject.

[0052] Preferably, the subject is a mammal.

[0053] More preferably, the subject is a human being.

[0054] The beneficial effects of this invention are as follows: This invention is the first to discover that the combination of N-acetylglucosamine (GlcNAc) and 3-O-acetyl-11-keto-β-boswellic acid (AKBA) has a significant synergistic effect. It alleviates inflammation by inhibiting the release of inflammatory factors such as TNF-α, IL-6, and COX-2, provides chondrocytes with glycosaminoglycan synthesis precursors and synergistically upregulates the expression of matrix synthesis markers such as COL2A1. At the same time, it strongly inhibits the activity of chondrogenic enzymes such as MMP13, maintains cartilage tissue homeostasis, and the effect is better than that of a single component.

[0055] The composition of this invention is natural and safe, avoiding the side effects of traditional anti-inflammatory drugs. GlcNAc is a natural amino sugar synthesized by the human body, and AKBA is the active ingredient of frankincense extract and frankincense oil. Both have excellent biocompatibility. The selected dosage range has no obvious toxicity to chondrocytes. Long-term use has no side effects such as abnormal liver and kidney function or gastrointestinal irritation. It is suitable for a wide range of people and can meet the dual needs of treatment and prevention for patients with cartilage damage, osteoarthritis, sports injuries, and the middle-aged and elderly. At the same time, it can improve bone density, promote bone growth and development, and improve bone health.

[0056] The composition of this invention has a simple preparation process, requiring only the two components to be mixed evenly in proportion, without the need for complex reactions or precision equipment. It has high production efficiency, low cost, and is easy to scale up industrially. Moreover, the product forms are flexible and diverse, and it can be prepared into various dosage forms such as food, health products, and pharmaceuticals. It supports multiple routes of administration such as oral, topical, and injection, and its application scenarios cover multiple fields such as daily health care and clinical treatment, providing a more efficient and safe new solution for improving bone health. Attached Figure Description

[0057] Figure 1 This is a flowchart of cell experiments in an embodiment of the present invention.

[0058] Figure 2 Cell viability assessment (cell differentiation state); *p<0.05 in the figure, compared with the control group; &p<0.05, compared with comparative examples 1-4, all showed significant differences.

[0059] Figure 3 Images of chondrocytes stained with Alixin Blue under a 20x microscope; *p<0.05 compared to the control group; &p<0.05 compared to comparative examples 1-4.

[0060] Figure 4 The effect of the composition on cartilage synthesis and degradation markers in an ATDC5 chondrocyte progenitor cell inflammation model was shown in the figure. *p<0.05 compared with the control group; #p<0.05 compared with the model group; &p<0.05 compared with comparative examples 1-4.

[0061] Figure 5 The composition was used to demonstrate the anti-inflammatory effect of the composition on the ATDC5 chondrocyte progenitor cell inflammatory model. In the figure, *p<0.05 compared with the control group; #p<0.05 compared with the model group; &p<0.05 compared with comparative examples 1-4, all of which showed significant differences. Detailed Implementation

[0062] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0063] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0064] The N-acetylglucosamine used in this invention has CAS number 7512-17-6, purity 99.20%, and catalog number HY-A0132; the 3-O-acetyl-11-keto-β-boswellic acid used in this invention has CAS number 67416-61-9, purity 99.93%, and catalog number HY-N0892.

[0065] Basic Example 1: Source of Frankincense Extract Frankincense extract (containing 20% ​​wt of 3-O-acetyl-11-keto-β-boswellic acid), purchased from PLT HealthSolutions (USA), catalog number: FBOSE-20A.

[0066] Frankincense extract (containing 80% wt 3-O-acetyl-11-keto-β-boswellic acid), purchased from PLT HealthSolutions (USA), catalog number: FBOSE-80B.

[0067] Example 1 A composition A composition comprising 300 parts of N-acetylglucosamine and 20 parts of 3-O-acetyl-11-keto-β-boswellic acid. The preparation method is as follows: N-acetylglucosamine and 3-O-acetyl-11-keto-β-boswellic acid are mixed evenly to obtain the composition.

[0068] Example 2 A composition A composition comprising 500 parts of N-acetylglucosamine and 50 parts of 3-O-acetyl-11-keto-β-boswellic acid. The preparation method is as follows: N-acetylglucosamine and 3-O-acetyl-11-keto-β-boswellic acid are mixed evenly to obtain the composition.

[0069] Example 3 A composition A composition comprising 600 parts of N-acetylglucosamine and 10 parts of 3-O-acetyl-11-keto-β-boswellic acid. The preparation method is as follows: N-acetylglucosamine and 3-O-acetyl-11-keto-β-boswellic acid are mixed evenly to obtain the composition.

[0070] Example 4 A composition A composition comprising 200 parts of N-acetylglucosamine and 100 parts of 3-O-acetyl-11-keto-β-boswellic acid. The preparation method involves uniformly mixing N-acetylglucosamine and 3-O-acetyl-11-keto-β-boswellic acid to obtain the composition.

[0071] Example 5 A composition formulation A composition formulation comprising 300 parts N-acetylglucosamine and 100 parts frankincense extract. The frankincense extract is the frankincense extract of Basic Example 1 (containing 20% ​​wt of 3-O-acetyl-11-keto-β-boswellic acid). The preparation method is as follows: N-acetylglucosamine and frankincense extract (containing 20% ​​wt of 3-O-acetyl-11-keto-β-boswellic acid) are mixed uniformly to obtain the composition formulation.

[0072] Example 6 A composition formulation A composition formulation comprising 300 parts N-acetylglucosamine and 25 parts frankincense extract. The frankincense extract is the frankincense extract of Basic Example 1 (containing 80% wt of 3-O-acetyl-11-keto-β-boswellic acid). The preparation method is as follows: N-acetylglucosamine and frankincense extract (containing 20% ​​wt of 3-O-acetyl-11-keto-β-boswellic acid) are mixed uniformly to obtain the composition formulation.

[0073] Comparative Example 1: A composition A composition comprising 300 parts glucosamine sulfate and 20 parts 3-O-acetyl-11-keto-β-boswellic acid. The preparation method is as follows: glucosamine sulfate and 3-O-acetyl-11-keto-β-boswellic acid are mixed evenly to obtain the composition.

[0074] Comparative Example 2: A composition A composition comprising 300 parts N-acetylglucosamine and 20 parts β-boswellic acid. The preparation method involves uniformly mixing N-acetylglucosamine and β-boswellic acid to obtain the composition.

[0075] Comparative Example 3: N-acetylglucosamine N-acetylglucosamine was used as a single-component control.

[0076] Comparative Example 4: 3-O-acetyl-11-keto-β-boswellic acid 3-O-acetyl-11-keto-β-boswellic acid was used as a single-component control.

[0077] Experimental Example 1: Effect of the composition or formulation on the differentiation activity of the ATDC5 chondrocyte precursor cell line Mouse-derived ATDC5 chondrocyte precursor cell line was seeded at 5000 cells / well in 96-well plates and Dulbecco modified Eagle / Ham's medium containing 10% fetal bovine serum was added. Incubate overnight in F-12 mixed medium (hereinafter referred to as proliferation medium). The next day, add 1% chondrocyte differentiation inducer (insulin-transferrin-selenium complex) to the ATDC5 chondrocyte proliferation medium and induce for 24 hours. Except for the control group, the other experimental groups were added with 200 μg / mL of the composition samples of Examples 1-4, the composition samples of Examples 5-6, the composition samples of Comparative Examples 1-2, or N-acetylglucosamine of Comparative Example 3, or 3-O-acetyl-11-keto-β-boswellic acid of Comparative Example 4. After 24 hours of intervention, add 10 μL of CCK-8 solution to each well and incubate in a cell culture incubator (37℃, 5% CO2) for 2 hours. The absorbance (OD) at 450 nm was measured using a microplate reader and cell viability (%) was calculated. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 indicated statistical significance.

[0078] The measurement results are shown in Table 1 and Figure 2 As shown.

[0079] Table 1

[0080] Note: *p<0.05 in the table means that there is a significant difference compared with the control group; &p<0.05 means that there is a significant difference compared with comparative examples 1-4.

[0081] ATDC5 chondrocyte precursor cells are core precursor cells for chondrocyte differentiation and endochondral ossification, and their differentiation activity directly determines chondrogenic capacity, bone development, and bone metabolic homeostasis. In the experiment, the cell differentiation activity in Examples 1-6 was significantly higher than that in the control group, demonstrating that the present invention can promote the differentiation of chondrocyte precursor cells into mature chondrocytes.

[0082] Experimental Example 2: Effect of the composition or formulation on cartilage matrix synthesis in the ATDC5 chondrocyte progenitor cell line As a core component of cartilage tissue, the amount and stability of cartilage matrix directly affect the process of endochondrial ossification. Endochondrial ossification is a key step in skeletal development and bone mass accumulation, and an important physiological basis for improving bone health and promoting bone formation. A sufficient and stable cartilage matrix provides a favorable microenvironment for osteoblast proliferation and differentiation, aiding in bone tissue formation and mineralization, and thus playing a crucial supporting role in maintaining bone density homeostasis and improving bone health. This experimental example examines the effect of a composition on cartilage matrix synthesis in the ATDC5 cartilage precursor cell line, not only evaluating its potential role in cartilage repair but also indirectly verifying its application potential in pathways related to improving bone health and promoting bone formation.

[0083] ATDC5 chondrogenic cell line was seeded at 100,000 cells / well in 12-well plates and incubated overnight with proliferation medium. The next day, 1% chondrogenic induction agent (insulin-transferrin-selenium complex) was added to the ATDC5 chondrogenic cell proliferation medium for induction. Simultaneously, except for the control group, the other experimental groups were treated with 200 μg / mL of the compositions from Examples 1-4, Examples 5-6, Comparative Examples 1-2, or N-acetylglucosamine from Comparative Example 3, or 3-O-acetyl-11-keto-β-boswellic acid from Comparative Example 4. The intervention lasted 21 days, with the medium changed 3 times daily. After intervention, the cells were fixed with 4% paraformaldehyde for 20 minutes, washed off the fixative, stained with alexandrite blue for 20 minutes, washed off the staining solution, and photographed under a 20x inverted microscope with PBS buffer. The stained area (%) of cartilage was quantitatively analyzed using ImageJ software. Statistical results are expressed as mean ± SE. Statistical analysis using SPSS software showed that p < 0.05 was statistically significant.

[0084] The measurement results are shown in Table 2 and Figure 3 As shown: Table 2

[0085] Note: *p<0.05 in the table means that there is a significant difference compared with the control group; &p<0.05 means that there is a significant difference compared with comparative examples 1-4.

[0086] The compositions or formulations of Examples 1-6 significantly promoted cartilage matrix synthesis in ATDC5 cells, with effects superior to the single-component or other proportioned compositions in each comparative example. This indicates that the compositions of the present invention have the potential to promote cartilage formation and can improve bone health and promote bone formation.

[0087] Experimental Example 3: Effects of the composition or formulation on markers of cartilage synthesis and degradation in an ATDC5 chondrogenic cell inflammatory model. The ATDC5 chondrogenic cell line was seeded at 100,000 cells / well in 12-well plates and incubated overnight with proliferation medium. The next day, 1% chondrogenic differentiation inducer (insulin-transferrin-selenium complex) was added to the ATDC5 chondrogenic cell proliferation medium for induction. Induction was performed for 21 days, with the medium changed every 3 days during this period. After the ATDC5 cells matured, LPS was added to the culture medium of the other experimental groups (except the control group) to a concentration of 5 μg / mL. At the same time, except for the control group and the model group, the other experimental groups were treated with 200 μg / mL of the composition samples of Examples 1-4, the composition samples of Examples 5-6, the composition samples of Comparative Examples 1-2, or N-acetylglucosamine of Comparative Example 3, or 3-O-acetyl-11-keto-β-lactam acid of Comparative Example 4, respectively, for 1 day. After intervention, total RNA was extracted from cultured cells using the RNA Easy Fast Animal Tissue / Cell Total RNA Extraction Kit (Tiangen Biotech), and reverse transcription was performed using the HiScript IV 1st StrandcDNA Synthesis Kit (Nanjing Novizan Biotechnology). The obtained cDNA was used for quantitative real-time PCR with the SupRealQUltra Hunter SYBR qPCR Master Mix (Nanjing Novizan Biotechnology) to quantify the gene expression levels of chondrocyte matrix synthesis marker COL2A1 (type II collagen) and degradation marker MMP13 (metalloproteinase 13). The expression level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene was used as an internal standard for evaluating gene expression levels. Primer sequences for COL2A1, MMP13, and GAPDH are shown in Table 3. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software; p < 0.05 was considered statistically significant.

[0088] Table 3

[0089] The measurement results are shown in Table 4 and Figure 4 As shown: Table 4

[0090] Note: In the table, *p<0.05 indicates a significant difference compared to the control group; #p<0.05 indicates a significant difference compared to the model group; &p<0.05 indicates a significant difference compared to comparative examples 1-4.

[0091] The compositions or formulations of Examples 1-6 can promote cartilage matrix synthesis (upregulate COL2A1) and inhibit cartilage matrix degradation (downregulate MMP13), effectively reversing LPS-induced cartilage damage. By maintaining the homeostasis of the cartilage matrix under inflammatory conditions, the compositions or formulations of the present invention can reduce the progression of cartilage damage and alleviate osteoarthritis.

[0092] Example 4: Anti-inflammatory effect of the composition or formulation on an ATDC5 chondrocyte progenitor cell inflammatory model The ATDC5 chondrogenic cell line was seeded at 100,000 cells / well in 12-well plates and incubated overnight with proliferation medium. The next day, 1% chondrogenic differentiation inducer (insulin-transferrin-selenium complex) was added to the ATDC5 chondrogenic cell proliferation medium for induction. Induction lasted for 21 days, with the medium changed every 3 days during this period. After the ATDC5 cells matured, LPS was added to the culture medium of the experimental groups other than the control group to a concentration of 5 μg / mL. At the same time, the experimental groups other than the control group and the model group were respectively treated with 200 μg / mL of the composition samples of Examples 1-4, the composition samples of Examples 5-6, the composition samples of Comparative Examples 1-2, or N-acetylglucosamine of Comparative Example 3, or 3-O-acetyl-11-keto-β-lactam acid of Comparative Example 4, for 1 day. The secretion of TNF-α in the supernatant culture medium was detected using a mouse tumor necrosis factor-α (TNF-α) enzyme-linked immunosorbent assay kit (Wuhan Yilairuit Biotechnology Co., Ltd.), and the secretion of IL-6 in the supernatant culture medium was detected using a mouse interleukin-6 (IL-6) enzyme-linked immunosorbent assay kit (Wuhan Yilairuit Biotechnology Co., Ltd.). The absorbance (OD) at 450 nm was measured using an ELISA reader, and the content was calculated according to the standard curve. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 was considered statistically significant.

[0093] The measurement results are shown in Table 5 and Figure 5 As shown: Table 5

[0094] Note: In the table, *p<0.05 indicates a significant difference compared to the control group; #p<0.05 indicates a significant difference compared to the model group; &p<0.05 indicates a significant difference compared to comparative examples 1-4.

[0095] Compared with the control group, the model group showed a significant increase in the secretion of inflammatory factors in the cell supernatant after LPS intervention, proving that LPS successfully constructed an ATDC5 chondrocyte progenitor cell inflammation model. After intervention with the compositions or formulations of Examples 1-6, the secretion of inflammatory factors decreased significantly compared with the model group, demonstrating good anti-inflammatory efficacy.

[0096] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A composition, characterized in that, The composition consists of N-acetylglucosamine and 3-O-acetyl-11-keto-β-boswellic acid.

2. The composition according to claim 1, characterized in that, The composition comprises, by weight, 200-600 parts of N-acetylglucosamine and 10-100 parts of 3-O-acetyl-11-keto-β-boswellic acid.

3. The composition according to claim 1, characterized in that, The composition comprises, by weight, 300-600 parts of N-acetylglucosamine and 20-50 parts of 3-O-acetyl-11-keto-β-boswellic acid.

4. A method for preparing the composition according to any one of claims 1-3, characterized in that, The preparation method includes: mixing N-acetylglucosamine and 3-O-acetyl-11-keto-β-boswellic acid in a certain proportion to obtain a composition.

5. A composition formulation, characterized in that, The composition formulation comprises N-acetylglucosamine and frankincense extract, wherein the frankincense extract contains 20%-80%wt of 3-O-acetyl-11-keto-β-boswellic acid.

6. The composition formulation according to claim 5, characterized in that, The composition formulation contains 200-600 parts of N-acetylglucosamine by weight; The weight ratio of N-acetylglucosamine to 3-O-acetyl-11-keto-β-boswellic acid from frankincense extract is 200-600:10-100.

7. The composition formulation according to claim 6, characterized in that, The composition formulation contains 300-600 parts of N-acetylglucosamine by weight; The weight ratio of N-acetylglucosamine to 3-O-acetyl-11-keto-β-boswellic acid from frankincense extract is 300-600:20-50.

8. A product comprising the composition according to any one of claims 1-3 or the composition formulation according to any one of claims 5-7, characterized in that, The products mentioned include any one or more of the following: food, health products, and pharmaceuticals.

9. The product according to claim 8, characterized in that, The dosage forms of the products include: solid dosage forms, semi-solid dosage forms, or liquid dosage forms.

10. The product according to claim 8, characterized in that, The food or health product mentioned also includes nutritionally acceptable nutrients.

11. The product according to claim 10, characterized in that, The nutrients mentioned include any one or more of the following: dietary fiber, polypeptides, food and medicine homologous ingredients, proteins, lipids, minerals, and vitamins.

12. The product according to claim 8, characterized in that, The food or health product also includes excipients acceptable for use in food or health products.

13. The product according to claim 12, characterized in that, The excipients include any one or a combination of two or more of the following: wetting agents, emulsifiers, preservatives, antioxidants, buffers, diluents, lubricants, solvents, suspending agents, solubilizers, thickeners, stabilizers, sweeteners, and flavorings.

14. The product according to claim 8, characterized in that, The dosage forms of the medicine include any one or more of the following: tablets, pills, powders, suspensions, gels, emulsions, creams, granules, capsules, suppositories, injections, sprays, and injections.

15. The product according to claim 8, characterized in that, The medicine also includes any one or more pharmaceutically acceptable excipients.

16. The product according to claim 8, characterized in that, The medicine is used for: prevention or treatment of osteoporosis, osteoarthritis, cartilage damage, joint degeneration, synovitis, improvement of joint pain, and promotion of cartilage repair and regeneration, or any one or more of these conditions.

17. The use of the composition according to any one of claims 1-3 or the formulation of the composition according to any one of claims 5-7 in the preparation of a pharmaceutical product, characterized in that, The medicines mentioned include any one or more of the following: medicines for preventing or treating osteoporosis, medicines for preventing or treating osteoarthritis, medicines for preventing or treating cartilage damage, medicines for preventing or treating joint degeneration, medicines for preventing or treating synovitis, medicines for relieving joint pain, and medicines for promoting cartilage repair and regeneration.

18. The application according to claim 17, characterized in that, The medicine contains an effective amount of the composition.

Images