A compound nutritional composition beneficial for joint function improvement of sports population and application thereof

Abstract

This invention relates to a compound nutritional composition and its application. The compound nutritional composition comprises the following components: 1 part by weight of turmeric; 0.002-5 parts by weight of enzymatically hydrolyzed bone powder; and 0.02-0.2 parts by weight of cartilage powder containing type II collagen or 0.02-10 parts by weight of collagen peptide powder. The compound nutritional composition provided in this application addresses the specific osteoarthritis issues of athletes. By constructing an articular cartilage cell inflammation model and conducting population testing, the intervention effects of the compound nutritional composition provided in this application on exercise-related oxidative stress, inflammatory response, and pain perception are systematically evaluated. The aim is to overcome the limitations of traditional broad-based formulations and develop precise nutritional solutions that combine component synergy with exercise physiological adaptability, providing athletes with more scientific and effective joint health protection.

Description

Technical Field

[0001] This invention relates to a compound nutritional composition that is beneficial to improving joint function in athletes and its application. Background Technology

[0002] With the rise of the global fitness craze and the continuous expansion of the sports population, the incidence of sports-related bone and joint injuries (such as cartilage wear and joint inflammation) has increased significantly. This population, due to joint micro-damage caused by exercise load, increased oxidative stress, and chronic inflammatory responses, has a greater need for nutritional intervention.

[0003] Currently, there are various products and technologies on the market for joint health maintenance. However, most mainstream joint health products on the market are designed for a broad population and lack reasonable formulas tailored to the physiological characteristics of athletes (such as the surge in free radicals and cartilage metabolic imbalance caused by high-intensity exercise). In addition, existing products generally suffer from two major problems: First, the mechanisms of action of single ingredients (such as glucosamine and chondroitin sulfate) are limited, making it difficult to simultaneously cover the multi-dimensional needs of anti-inflammation, anti-oxidation, and cartilage repair; second, the impact of the special metabolic environment of athletes (such as high ROS levels and active inflammatory factors) on the synergistic effect of ingredients is not fully considered, resulting in insufficient formula effectiveness. Summary of the Invention

[0004] To address the aforementioned issues, this invention, taking into account the specific characteristics of osteoarthritis in athletes, systematically evaluates the intervention effects of the compound nutritional composition provided in this application on exercise-related oxidative stress, inflammatory response, and pain perception by constructing an articular chondrocyte inflammation model and conducting population testing. The aim is to overcome the limitations of traditional broad-based formulations and develop precise nutritional solutions that combine synergistic component effects with physiological adaptation to exercise, providing athletes with more scientific and effective joint health protection.

[0005] To achieve the above-mentioned technical effects, the first aspect of the present invention provides a compound nutritional composition comprising the following components: 1 part by weight of turmeric; 0.002 to 5 parts by weight of enzymatically hydrolyzed bone powder; and 0.02 to 0.2 parts by weight of cartilage powder containing type II collagen or 0.02 to 10 parts by weight of collagen peptide powder.

[0006] Optionally, the cartilage powder contains 4% or more of non-denatured type II collagen by weight, the collagen peptide powder contains 90% or more of collagen peptides by weight, and the enzymatically hydrolyzed bone powder contains 80% or more of chondroitin sulfate and protein by weight.

[0007] Optionally, the collagen peptide powder contains at least 55% by weight of peptides with a weight of less than 1000 Da.

[0008] Optionally, the weight percentage of chondroitin sulfate in the enzymatically hydrolyzed bone powder is above 60%.

[0009] Optionally, the type II collagen-containing cartilage powder is obtained by washing, disinfecting, and pulverizing chicken breast cartilage, adding potassium chloride, and drying it at low temperature; the collagen peptide powder is obtained by enzymatic hydrolysis of bovine cartilage.

[0010] Optionally, the curcumin in the turmeric shall account for no less than 18% by weight.

[0011] Optionally, the enzymatically hydrolyzed bone powder is made from bovine bones through steaming, pulverizing, enzymatic hydrolysis, and drying.

[0012] Optionally, the protease is derived from Bacillus subtilis.

[0013] Optionally, the enzymatically hydrolyzed bone powder is 0.01 to 4 parts by weight, preferably 0.1 to 3 parts by weight.

[0014] Optionally, the type II collagen-containing cartilage powder is 0.025 to 0.15 parts by weight, preferably 0.03 to 0.1 parts by weight.

[0015] Optionally, the collagen peptide powder is 1 to 8 parts by weight, preferably 2 to 7 parts by weight.

[0016] This application also provides a second aspect of the technical solution, namely, the use of any of the aforementioned compositions as active ingredients in the preparation of medicaments for improving joint function, repairing cartilage damage and / or treating joint inflammation.

[0017] Optionally, the drug is targeted at athletes.

[0018] All the raw materials described in this application can be obtained commercially.

[0019] Unless otherwise specified, all units of measurement used in this application are by weight or weight percentage.

[0020] The turmeric, enzymatically hydrolyzed bone powder, and cartilage powder used in this application (since the type II collagen-containing cartilage powder and collagen peptide powder described in this invention are both prepared from cartilage, they are collectively referred to as cartilage powder) can also be prepared by oneself. For example, turmeric is obtained from the rhizome of the ginger family plant turmeric; enzymatically hydrolyzed bone powder is obtained from bovine bone through enzymatic hydrolysis; and cartilage powder is obtained from chicken breast cartilage or bovine cartilage.

[0021] The enzymatically hydrolyzed bone powder described in this application is a nationally approved common raw material, prepared from bovine bones. The enzymatic hydrolysis process employs conventional steps, such as washing, steaming, crushing, enzymatic hydrolysis with protease (derived from Bacillus subtilis), and drying of animal bones. When 1 part by weight of turmeric is used, the content of enzymatically hydrolyzed bone powder can be selected from 0.002 to 5 parts by weight, preferably 0.002 to 4.5 parts by weight, more preferably 0.01 to 4 parts by weight, and even more preferably 0.1 to 3 parts by weight.

[0022] The type II collagen-containing cartilage powder described in this application is a nationally approved common raw material, which can be prepared from chicken breast cartilage. The preparation process can be a conventional process, such as washing, disinfecting, and pulverizing the chicken breast cartilage, adding potassium chloride, and drying at low temperature. With 1 part by weight of turmeric, the type II collagen-containing cartilage powder is 0.02~0.2 parts by weight, preferably 0.025~0.15 parts by weight, and more preferably 0.03~0.1 parts by weight. The collagen peptide powder described in this application is a nationally approved common raw material, which can be prepared from bovine cartilage. The preparation process can be a conventional enzymatic hydrolysis process, such as washing, pulverizing, filtering, enzymatically hydrolyzing, inactivating enzymes, and centrifuging the animal cartilage. The collagen peptide powder contains more than 90% collagen peptides by weight, and more than 55% peptides below 1000 Da. When 1 part by weight of turmeric is used, the amount of collagen peptide powder is 0.02 to 10 parts by weight, preferably 1 to 8 parts by weight, and more preferably 2 to 7 parts by weight.

[0023] In this invention, the main differences between undenatured type II collagen and collagen peptides are as follows: In terms of composition, undenatured type II collagen is a complete macromolecular protein retaining its natural triple helix structure, containing immunologically active epitopes and a natural conformation that binds to the cartilage matrix; collagen peptides, on the other hand, are small peptide segments formed after enzymatic hydrolysis and other treatments, mostly consisting of short-chain amino acid sequences, lacking a complete triple helix structure. In terms of mechanism of action, undenatured type II collagen interacts with the intestinal immune system through its natural conformation, inducing immune tolerance and reducing immune attack against articular cartilage; collagen peptides mainly function through direct absorption by the human body, providing raw materials for cartilage synthesis, or participating in the regulation of cell metabolism and promoting collagen synthesis. The two have significantly different pathways of action and biological effects.

[0024] The turmeric described in this application is a powder made from the rhizome of the turmeric plant (Zingiberaceae family), and its curcumin content must be no less than 18% by weight.

[0025] This application uses a compound nutritional composition obtained by combining turmeric, enzymatically hydrolyzed bone meal, and cartilage meal, which can more effectively exert the synergistic effect between the components, and is more effective in targeting athletes, especially those with joint injuries and inflammation. Attached Figure Description

[0026] Figures 1-4 These are the results of cell viability assays;

[0027] Figures 5-10 These are the results of GSH concentration measurement;

[0028] Figures 11-12 This is the result of SOD measurement;

[0029] Figures 13-18 These are the results of inflammatory factor assays;

[0030] Figure 19 These are ROS measurement results;

[0031] Figure 20 This is the VAS score result. Detailed Implementation

[0032] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0033] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0034] Preparation of the compound nutrient composition provided in this application

[0035] According to conventional preparation methods, turmeric, enzymatically hydrolyzed bone powder, type II collagen-containing cartilage powder, or collagen peptide powder are prepared or purchased directly from the market. These are then compounded according to Tables 1 and 2 below to obtain a partial ratio of the compound nutritional composition described in this application, illustrating the efficacy of the compound nutritional components. For example, the conventional preparation methods used are as follows: turmeric is obtained by grinding the rhizome of the ginger plant (Curcuma longa) into powder; enzymatically hydrolyzed bone powder is obtained by washing, crushing, enzymatically hydrolyzing, and drying bovine bones; type II collagen-containing cartilage powder is obtained by processing chicken breast cartilage; and collagen peptide powder is prepared from bovine cartilage. In addition to using different cartilage powders, Tables 1 and 2 also include blank groups and several control groups. In Table 1, the cartilage powder is type II collagen-containing cartilage powder (UCII is used as an example in the attached figure), and in Table 2, the cartilage powder is collagen peptide powder (collagen peptide is used as an example in the attached figure). The cartilage powder used in Table 1 (i.e., cartilage powder containing type II collagen) contains 4% or more of non-denatured type II collagen by weight. The cartilage powder (i.e., collagen peptide powder) in Table 2 contains over 90% collagen peptides by weight, with peptides below 1000 Da comprising over 55% by weight. The enzymatically hydrolyzed bone powder used in Tables 1 and 2 contains over 80% chondroitin sulfate and protein by weight, with chondroitin sulfate comprising over 60% by weight. The turmeric used in Tables 1 and 2 contains at least 18% curcumin by weight.

[0036] Table 1 - Cartilage powder containing type II collagen.

[0037]

[0038] Table 2 - Cartilage powder using collagen peptide powder

[0039]

[0040] Experiment 1: Cell viability detection

[0041] Cell proliferation and viability were determined using CCK-8 assays after intervention with the compound composition and different concentrations of single compounds. Cells were first seeded and seven single compound concentration gradients were set up (0.1, 0.5, 1, 10, 30, 50, 100 μg / mL). Figures 1-4 (x-axis) Add 10 μL of LCK-8 reagent to every 100 μL of culture medium, incubate in an incubator for 1–4 h, measure OD values ​​at different time points, and calculate cell viability for each study group after a certain culture time. Figures 1-4(Vertical axis). The results showed that turmeric and cartilage powder containing type II collagen had no significant cytotoxicity at the studied dose, and cell viability increased in a dose-dependent manner with increasing concentration of the intervention, demonstrating a promoting effect on cell viability. Enzymatically hydrolyzed bone powder and collagen peptide powder showed some cytotoxicity at the set concentrations, but had little effect on cell viability. At the highest concentration within the study range of 100 μg / mL, the cells still maintained about 90% cell viability.

[0042] For detailed results from this experiment, please refer to [link / reference]. Figures 1-4 , Figure 1 This is cell viability measured by turmeric. Figure 2 The cell viability measured was based on type II collagen chondroitin powder. Figure 3 It is cell viability measured by collagen peptide powder. Figure 4 This is the cell viability assay of enzymatically hydrolyzed bone meal. In the attached figure, UC II (i.e., non-denatured type II collagen) represents cartilage meal containing type II collagen, collagen peptides represent collagen peptide meal, and the control represents the blank control group. All subsequent attached figures are the same.

[0043] Experiment 2: Synergistic Promotion Effect on GSH Expression

[0044] The antioxidant and anti-inflammatory effects were verified using the ratios obtained from Tables 1 and 2 above.

[0045] Elevated expression of the antioxidant factor GSH (reduced glutathione) signifies enhanced antioxidant capacity, which can alleviate oxidative stress damage to articular chondrocytes and synovial tissue. Specifically, elevated GSH indicates that it alleviates the imbalance between intra-articular oxidation and inflammation caused by exercise or inflammation through mechanisms such as directly scavenging free radicals (e.g., superoxide anion, hydrogen peroxide), enhancing antioxidant enzyme activity as a coenzyme for glutathione peroxidase, and inhibiting the NF-κB inflammatory pathway, while simultaneously creating a favorable microenvironment for cartilage repair. A significant increase in GSH levels after intervention reflects the substance's ability to effectively activate endogenous antioxidant defenses and block the vicious cycle of oxidation-inflammation, making it an important positive indicator for evaluating the product's antioxidant efficacy and joint protection potential.

[0046] The GSH detection method is as follows:

[0047] For the GSH kit, first store the cell supernatant collected under different intervention conditions at 4°C. Simultaneously, remove all reagents from the refrigerator and allow them to equilibrate to room temperature for 10 minutes. Prepare clean centrifuge tubes, labeling them as blank, standard, and assay tubes. Add reagents according to the table below, mix thoroughly, and incubate in a 37°C water bath for 5-10 minutes to allow the reaction to fully occur. After incubation, immediately measure the absorbance of each tube at 405nm using a spectrophotometer. Calculate the GSH content using the formula, paying attention to the actual dilution factor of the sample during calculation. Throughout the entire process, careful control of the reagent addition order and water bath temperature is crucial to ensure accurate results.

[0048]

[0049] Table 1 compares the proportions for proportion series 1, 2, and 3 respectively. Proportion series 1 includes proportions S1-1, K1, A1, B1, C1, D1-1, E1-1, and F1-1; proportion series 2 includes proportions S1-2, K1, A1, B1, C1, D1-2, E1-2, and F1-2; and proportion series 3 includes proportions S1-3, K1, A1, B1, C1, D1-3, E1-3, and F1-3. The GSH concentrations obtained from the proportions in Table 1 are used as references. Figures 5-7 , Figure 5 It is a partial formulation of the 1 series. Figure 6 It is a partial formulation of the 2 series. Figure 7 It is a partial formulation of the 3 series. Figure 8 These are examples of GSH concentrations obtained from the series of ratios 1 in Table 2. Figure 8 The examples in Table 2 are only partial examples of the proportions in series 1, namely S2-1, K2, A2, B2, C2, D2-1, E2-1, and F2-1.

[0050] In addition, existing technologies often use N-acetylglucosamine as the main component for joint repair. It is an amino derivative of glucose and an important raw material for the synthesis of proteoglycans and glycosaminoglycans (hereinafter referred to as glucosamine). Chondrocytes take up glucosamine and, through a series of enzymatic reactions in the Golgi apparatus, generate glycosaminoglycan chains, which then combine with core proteins to form proteoglycan aggregates. These aggregates fill the collagen fiber network of the cartilage matrix, giving the cartilage compressive strength and elasticity, thus promoting joint health. In this application, glucosamine is used to replace certain components in Tables 1 and 2, for example, replacing the ratio 2 series in Table 1 and the ratio 1 series in Table 2, while keeping the rest unchanged, to form a new control group for comparative explanation. See the measurement results for details. Figure 9 , Figure 10 . Figure 9 This is a comparison chart of the formulation series 1 in Table 2 and the control group obtained by replacing cartilage powder with glucosamine. Figure 10This is a comparison chart of the formulation series 2 in Table 1 and the control group obtained after replacing enzymatically hydrolyzed bone meal with glucosamine.

[0051] Compared with the blank group and the control group, the compound of cartilage powder, turmeric, and enzymatically hydrolyzed bone powder promoted GSH expression and exhibited antioxidant effects. In addition, among the different compound combinations mentioned above, the GSH expression level of the "cartilage powder + turmeric + enzymatically hydrolyzed bone powder" group was higher than that of any other group, indicating that the compound's promoting effect on GSH expression was synergistic.

[0052] To further verify the synergistic effect of cartilage powder, turmeric, and enzymatically hydrolyzed bone powder, glucosamine was added to the cartilage powder, turmeric, and enzymatically hydrolyzed bone powder series based on the ratio series 1 in Table 1 and Table 2. The results are shown in the table below:

[0053]

[0054] This application uses cartilage powder, turmeric, and enzymatically hydrolyzed bone powder as active ingredients to prepare corresponding oral products to improve joint function, repair cartilage damage, and / or treat joint inflammation, especially for oral pharmaceutical products targeting athletes.

[0055] Experiment 3: Verification of Synergistic Antioxidant Efficacy

[0056] Elevated expression of the antioxidant SOD (superoxide dismutase) signifies a significantly enhanced ability of the body to scavenge free radicals, a key positive signal for joints to resist oxidative stress damage. Direct antioxidant mechanism: SOD is the only metalloenzyme in the body that can specifically scavenge superoxide anions (O2⁻). Increased SOD expression accelerates the conversion of O2⁻ to hydrogen peroxide (H2O2), which is then cleared by catalase (CAT) or glutathione peroxidase (GPx), reducing the production of lipid peroxidation products (such as MDA) and alleviating damage to chondrocyte membranes and organelles. Inflammation regulation linkage: High concentrations of O2⁻ can activate the NF-κB inflammatory pathway, inducing the release of pro-inflammatory factors such as IL-6 and TNF-α. Elevated SOD levels indirectly inhibit inflammatory signal transduction by reducing O2⁻ levels, forming a synergistic "antioxidant-anti-inflammatory" effect, alleviating synovial inflammation and cartilage matrix degradation. Joint repair promotion: During exercise or inflammation, excessive free radicals can inhibit chondrocyte proliferation and induce apoptosis (e.g., by activating Caspase-3 via the mitochondrial pathway). Increased SOD expression can improve the intracellular oxidative microenvironment, enhance chondrocyte viability, and promote the synthesis of type II collagen and proteoglycans, thus accelerating damage repair.

[0057] Decreased MDA expression is one of the core positive indicators of antioxidant efficacy, with the following specific significance: MDA is the end product of lipid peroxidation, and its level directly reflects the degree of free radical attack on cells and tissues. When joints are under stress, such as exercise load or inflammation, excessive free radicals (such as superoxide anions) attack polyunsaturated fatty acids in cell membrane phospholipids, triggering a lipid peroxidation chain reaction, leading to a significant increase in MDA content (e.g., MDA levels in the synovial fluid of osteoarthritis patients can be 30% to 50% higher than in healthy individuals), thereby damaging cell membrane structure, interfering with chondrocyte function, and exacerbating the inflammatory response by activating the NF-κB pathway. If MDA expression decreases after intervention, it indicates that the lipid peroxidation process has been effectively inhibited.

[0058] The detection methods for SOD and MDA are as follows:

[0059] SOD Kit: First, thaw the cell supernatants collected under different intervention conditions at 4°C. Simultaneously, remove all reagents from the refrigerator and allow them to equilibrate to room temperature for 10 minutes. Prepare the reaction solution according to the instructions (e.g., mix the chromogenic agent, enzyme working solution, and buffer according to the proportions in the table). Prepare test tubes and label them as blank tubes, standard tubes, and assay tubes. Add distilled water to the blank tubes, add SOD standard solution of known concentration to the standard tubes, and add supernatant to the assay tubes according to the amounts listed in the table. Then add the prepared reaction solution to each tube, mix gently, and incubate in a 37°C water bath for 20-30 minutes. After incubation, add stop solution to each tube, mix thoroughly, and measure the absorbance of each tube at 550 nm using a spectrophotometer. Calculate the SOD activity using the formula after measurement.

[0060]

[0061] MDA Reagent Kit: First, collect cell supernatants from different intervention conditions and store them at 4°C. Simultaneously, remove all reagents from the refrigerator and allow them to equilibrate to room temperature for 10 minutes. Prepare centrifuge tubes, labeling blank tubes, standard tubes, and assay tubes. Add double-distilled water to the blank tubes, add MDA standard to the standard tubes, and add an appropriate amount of supernatant to the assay tubes. Then, add reagents to each tube according to the table below, mix thoroughly, and heat in a 95°C water bath in the dark for 40 minutes to ensure the reaction proceeds completely. After the water bath, rapidly cool the centrifuge tubes to room temperature and centrifuge at 4000 rpm for 10 minutes. Collect the supernatant. Measure the absorbance of each tube at 530 nm using a spectrophotometer. First, scan the reading of the empty plate, and then calculate the MDA content in the sample according to the formula.

[0062]

[0063] Reagent 1: It solidifies easily and needs to be heated in a water bath until it becomes transparent before use.

[0064] Reagent 2: Add 340mL of double-distilled water to each bottle and mix well.

[0065] Reagent 3: The ratio of each reagent is Reagent 1: Reagent 2: Reagent 3 = 0.2:3:1

[0066] In this part of the experiment, the series with ratio 3 in Table 1 was selected to evaluate the effect on SOD, and the series with ratio 2 in Table 1 was selected to evaluate the effect on MDA. For example... Figure 11 and Figure 12 As shown, Figure 11 These are the test results for series 3 of the formula in Table 1. Figure 12 The results are the determination results of the series with ratio 2 in Table 1.

[0067] Compared with the blank group and the control group, the compound of cartilage powder, turmeric, and enzymatically hydrolyzed bone powder showed that it could promote SOD expression and exhibited antioxidant effects. In addition, among the different compound combinations mentioned above, the SOD expression level of the "cartilage powder + turmeric + enzymatically hydrolyzed bone powder" group was higher than that of any other group, that is, the promoting effect of the compound on SOD expression was synergistic.

[0068] Compared with the blank group and the control group, the compound of cartilage powder, turmeric, and enzymatically hydrolyzed bone powder showed that it could inhibit MDA expression and exhibited antioxidant effects. The results show that the MDA expression level of the "cartilage powder + turmeric + enzymatically hydrolyzed bone powder" group was lower than that of any other group, indicating that the combination of the three can produce an antioxidant effect superior to that of the individual raw materials, that is, the compound exhibits antioxidant synergy.

[0069] Experiment 4: Verification of anti-inflammatory efficacy - Inhibition of inflammatory factors

[0070] Decreased expression of inflammatory factors IL-18, IL-6, and TNF-α is a core manifestation of anti-inflammatory efficacy. IL-6: Decreased levels indicate suppression of the intra-articular inflammatory cascade. IL-6 can induce hepatocytes to produce acute inflammatory proteins and, in conjunction with other factors (such as TNF-α), amplify synovial inflammation and promote chondrocyte apoptosis. Lowering IL-6 can alleviate joint swelling, pain, and cartilage matrix degradation. TNF-α: As a "core initiator" of pro-inflammatory signals, decreased expression directly reduces synovial macrophage activation and osteoclast formation, inhibits the NF-κB pathway-mediated inflammatory factor network (such as IL-1β and MMPs), thereby delaying the cartilage degeneration process. IL-18: Decreased levels signify inhibition of NLRP3 inflammasome activation, which can block the vicious cycle of "inflammation-oxidative stress," while simultaneously relieving the inhibition of chondrocyte synthesis function and accelerating damage repair. The combined reduction of all three indicators suggests that the key pathological process of "excessive secretion of pro-inflammatory factors - activation of inflammasomes - imbalance of cartilage metabolism" within the joint has been effectively intervened. This can significantly alleviate exercise-induced joint inflammation, reduce cartilage damage, and create a favorable microenvironment for antioxidant and repair mechanisms to play their role. These indicators are the core quantitative indicators for evaluating the anti-inflammatory efficacy and joint protection potential of the product.

[0071] Inflammatory factor detection steps (IL-6, IL-18, TNF-α kit)

[0072] Using the IL-6, IL-18, and TNF-α kit, cell supernatants collected under different intervention conditions were first stored at 4°C. The strips from the kit were removed and allowed to equilibrate to room temperature for 20 min. Microplates were prepared, with standard wells, sample wells, and blank wells set up according to the number of samples. 50 μL of five different concentrations of standard were added to each standard well. 10 μL of the test sample and 40 μL of sample diluent were added to each sample well, and the mixture was gently vortexed to mix. 100 μL of horseradish peroxidase-labeled detection antibody was added to all wells except the blank wells. The microplates were sealed with sealing film and incubated at 37°C for 60 min. After incubation, the liquid in the wells was discarded, and each well was filled with washing buffer. After standing for 2 min, the buffer was discarded. This washing process was repeated 5 times, and the microplates were patted dry. 50 μL each of substrate chromogenic solutions A and B were added, and the plates were sealed and incubated at 37°C for 15 min in the dark. Once a clear color gradient change appeared in the standard wells, 50 μL of stop solution was added to each well to terminate the reaction. Immediately measure the absorbance of each well at 450 nm using a microplate reader. Plot a standard curve based on the absorbance values ​​of the standards, and then calculate the concentrations of IL-6, IL-18, and TNF-α in the samples from the standard curve based on the absorbance values ​​of the samples. Throughout the entire detection process, it is essential to strictly control conditions such as incubation time at room temperature and the number of washes to avoid affecting the accuracy of the test results due to operational errors.

[0073] like Figures 13-17 As shown, Figure 13 The series of formulations in Table 1 are used to evaluate the effect on IL-6. Figure 14 The effect of formulation 2 in Table 1 on IL-18 is evaluated. Figure 15 The effect of the 3-series formulation in Table 1 on TNF-α is evaluated. Figure 16 The series of formulations in Table 2, specifically formulation 1, were used to assess the effects on TNF-α. Figure 17 The series of ratios 1 in Table 2 are used to evaluate the effect on IL-18.

[0074] In this application, glucosamine is used to replace the cartilage powder component in Table 2, for example, replacing the series of ratios 1 in Table 2, while keeping the rest unchanged, to form a new control group for corresponding comparative explanation. See the test results for details. Figure 18 . Figure 18 This is a comparison chart of the formulation series 1 in Table 2 and the control group obtained by replacing cartilage powder with glucosamine.

[0075] Compared with the blank group and the control group, the compound of cartilage powder, turmeric and enzymatically hydrolyzed bone powder showed that it could inhibit the expression of inflammatory factors such as IL-18, IL-6 and TNF-α, and exhibited anti-inflammatory effects.

[0076] Experiment 5: Verification of Antioxidant Efficacy - Inhibition of ROS

[0077] The reduced expression of ROS (reactive oxygen species) indicates that the product effectively enhances the intra-articular antioxidant defense system, reduces oxidative stress damage to chondrocyte DNA, lipids, and proteins, inhibits the amplification effect of the "oxidation-inflammation" interaction, alleviates cartilage matrix degradation and synovial inflammation, and promotes chondrocyte anabolic metabolism (such as the production of type II collagen and proteoglycans). This is a direct manifestation of its antioxidant, anti-inflammatory, and anti-degenerative joint effects.

[0078] The ROS detection method is as follows:

[0079] ROS Kit: When using the ROS kit, first remove the kit from the refrigerator and allow it to equilibrate at room temperature for 10 minutes. Simultaneously, wash cells treated under different intervention conditions for 24 hours twice using DMEM. Prepare centrifuge tubes, and pipette an appropriate amount of detection buffer according to the sample quantity. Add the corresponding probe working solution (e.g., DCFH-DA needs to be dissolved and diluted with DMEM at a ratio of 1:1000), and mix well to prepare the reaction working solution. Add a certain amount of sample to the reaction working solution. Simultaneously, set up a blank control group (containing only detection buffer and probe working solution) and a positive control group (containing ROS inducer). Gently mix and incubate at 37°C in the dark for 20-30 minutes. During incubation, wash the sample with PBS as needed to remove fluorescent probes that have not entered the cells. After incubation, immediately measure the fluorescence intensity using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. During measurement, avoid light exposure to prevent probe fluorescence quenching. Finally, calculate the relative level or concentration of ROS in the sample using the formula.

[0080] The measurement results are as follows Figure 19 As shown, Figure 19 These are the test results for the series of proportions 1 in Table 2.

[0081] Compared with the blank group and the control group, the three dosage groups of cartilage powder, turmeric, and enzymatically hydrolyzed bone powder compound (in the intervention concentration setting, the concentration of the medium dose group was 20 times that of the low dose group; while the concentration of the high dose group was 5 times that of the medium dose group) showed that they could inhibit ROS expression and exhibited antioxidant effects.

[0082] Experiment 6: Co-promoting effect on Aggrecan expression

[0083] Aggrecan (proteoglycan polymers) are key components of the extracellular matrix of chondrocytes, composed of a core protein covalently linked to numerous glycosaminoglycans (such as chondroitin sulfate and keratin sulfate). Through the binding of linking proteins with hyaluronic acid, they form large polymers, endowing cartilage with strong hydrophilicity and compressive elasticity. Aggrecan plays a central role in maintaining cartilage structural integrity, buffering mechanical stress, and regulating cell signal transduction. Decreased synthesis or increased degradation of aggrecan is closely related to the occurrence and development of degenerative cartilage diseases such as osteoarthritis. Aggrecan expression levels are often used as a key indicator for assessing cartilage repair efficacy. An increase in aggrecan expression indicates that exogenous intervention has successfully induced the generation of cartilage-specific matrix, which helps to counteract matrix degradation under pathological conditions and block the vicious cycle of "inflammation-degradation-cartilage damage," thus playing a significant role in cartilage repair and disease prevention.

[0084] Aggrecan gene expression was detected using RT-PCR, and the steps are as follows:

[0085] First, RNA extraction was performed. The cells from the wells were pipetted into RNase-free centrifuge tubes containing 1 ml of Trizol reagent and vortexed to mix. After standing at room temperature for 5 minutes, 0.2 ml of chloroform was added to each tube, and the mixture was vigorously vortexed for 15 seconds. The cells were incubated at room temperature for 2-3 minutes, and then centrifuged at 12000 g for 15 minutes at 4°C. The colorless aqueous phase was transferred to a new tube, and an equal volume of isopropanol was added. The mixture was inverted to mix, and the cells were stood at room temperature for 10 minutes. After centrifugation at 12000 g for 10 minutes at 4°C, the supernatant was discarded. The precipitate was washed with 1 ml of 75% ethanol prepared with DEPC water, and centrifuged at 7500 g for 5 minutes at 4°C. The supernatant was discarded, and the precipitate was air-dried at room temperature, but complete drying was avoided as it would make the RNA less soluble. An appropriate amount of DEPC water was added to dissolve the RNA. The RNA concentration and purity were measured using Nanodrop, ensuring that the A260 / A280 ratio was between 1.8 and 2.0.

[0086] Next, cDNA synthesis was performed: Based on the RNA concentration, 1000 ng of total RNA was transferred to an RNase-free centrifuge tube, 1 μl of Oligo(dT) primer was added, and DEPC water was added to bring the volume to 10 μl. The tube was incubated at 65°C for 5 minutes and then immediately placed on ice for 2 minutes. 4 μl of 5×Reaction Buffer, 2 μl of dNTP Mix (10 mM), 1 μl of RNase inhibitor, and 1 μl of reverse transcriptase were added sequentially, and the mixture was gently mixed and centrifuged. PCR procedures typically begin with pre-denaturation at 94–95°C for 3–5 minutes to completely unwind the template DNA double strand and activate thermostable DNA polymerase. This is followed by a cyclical process consisting of denaturation, annealing, and extension, usually 30–40 cycles. Denaturation occurs at 94–96°C to unwind the DNA into single strands for 10–30 seconds, followed by primer-template specific binding for 10–30 seconds. Extension occurs at 72°C using dNTPs to synthesize new strands, with the time adjusted according to the amplified fragment length. After cycling, final extension is performed at 72°C for 5–10 minutes to ensure complete extension of all DNA strands. Finally, the product is maintained at 4–10°C to prevent degradation or non-specific reactions. The resulting cDNA can be used immediately or stored at -20°C for later use. The entire process requires strict RNase-free operation, and the synthesized cDNA should be immediately used for PCR experiments or aliquoted for storage to prevent repeated freeze-thaw cycles from affecting quality.

[0087] Quantitative PCR was performed using the prepared cDNA as a template. Target primers and SYBR Green Mix were added to label the target gene fragment. The target gene was synthesized using a programmed procedure. Then, the transcriptional level of the target gene was analyzed based on the Cq value.

[0088] Table 3. Synergistic promoting effect of the composition on Aggrecan expression (Table 1 Composition series)

[0089]

[0090] Compared with the blank group and the control group, the compound containing type II collagen cartilage powder, turmeric, and enzymatically hydrolyzed bone powder promoted Aggrecan expression and showed a cartilage repair effect. In addition, among the three compound compositions with different proportions, the Aggrecan expression level in the "cartilage powder + turmeric + enzymatically hydrolyzed bone powder" group was higher than that in any other group, indicating that the compound composition had a synergistic effect on promoting Aggrecan expression.

[0091] The synergistic effect of the compositions in this application, as shown in the accompanying figures, is demonstrated by the superior effect of the compositions compared to individual components or other proportions when using the same amount but different ratios. Figure 5 For example, the ordinate of the GSH concentration achieved by the other individual components or other proportions is between 6 and 13. However, after replacing the corresponding components in equal amounts, the ordinate of the GSH concentration achieved by the resulting composition is greater than 15, which is significantly greater than the actual effective value of each component (reference). Figure 5 The ordinate of the composition (GSH) and the theoretical combined effect (theoretically, after equal combinations, the GSH ordinate should not exceed 13); therefore, the composition of this application can play a significant synergistic effect relative to each component.

[0092] Experiment 7: Efficacy Experiment in Promoting Type II Collagen Expression (COL2A)

[0093] 1) Synergistic promoting effect on type II collagen expression (COL2A)

[0094] COL2A (type II collagen) is a major structural protein of the extracellular matrix of chondrocytes and a skeletal component of the cartilage matrix, providing cartilage with tensile strength and structural stability. Its synthesis and degradation balance maintains the normal physiological function of cartilage and plays a central role in joint homeostasis. In degenerative diseases such as osteoarthritis, its content is often reduced due to increased degradation by proteases such as MMP13. Increased COL2A expression after intervention usually indicates that the intervention may have promoted the production of this protein, contributing to the repair or improvement of tissue structure and function. Specifically, this manifests as: enhanced chondrocyte synthesis function, which can significantly improve the structural support capacity of articular cartilage and delay the process of cartilage wear and degeneration; simultaneously, high expression of type II collagen can improve cartilage elasticity and impact resistance, enhancing the joint's tolerance to exercise loads. Especially for athletes, it can effectively address exercise-induced cartilage micro-damage and is a core positive indicator for evaluating cartilage repair efficacy.

[0095] COL2A gene expression was detected using RT-PCR, following the same procedure as Aggrecan assay.

[0096] Table 4. Synergistic promoting effect of the composition on COL2A expression (Table 2 series of ratios 1)

[0097]

[0098] Note: In the setting of intervention concentration, the concentration of dose 2 group is 5 times that of dose 1 group.

[0099] Compared with the blank group and the control group, the compound of collagen peptide powder, turmeric, and enzymatically hydrolyzed bone powder promoted COL2A expression, demonstrating the effect of promoting type II collagen expression. In addition, among the compound combinations of different dosage groups, the COL2A expression level of the "cartilage powder + turmeric + enzymatically hydrolyzed bone powder" group was higher than that of any other group, indicating that the compound's promoting effect on COL2A expression was synergistic.

[0100] 2) Comparative experiment on the efficacy of promoting type II collagen expression (COL2A)

[0101] To further verify the synergistic effect of turmeric, enzymatically hydrolyzed bone powder, and collagen peptide powder in promoting type II collagen expression, the following experimental scheme was designed: First, a raw material combination comparison experimental group was set up, and the target component combination was compared with the "glucosamine + enzymatically hydrolyzed bone powder + cartilage powder" group, the "glucosamine + turmeric + cartilage powder" group, and the "glucosamine + turmeric + enzymatically hydrolyzed bone powder" group; Second, for the "cartilage powder + turmeric + enzymatically hydrolyzed bone powder" combination, the differences between the ratio series 2-3 in Table 2 and other different ratios were mainly compared.

[0102] Table 5 Comparative Experiments on the Efficacy of Promoting Type II Collagen Expression

[0103]

[0104] In this part of the experiment, the results showed that when the dosages of turmeric, enzymatically hydrolyzed bone powder, and collagen peptide powder were 0.1 μg / mL, 0.5 μg / mL, and 1 μg / mL (ratio of 1:5:10, Table 2, series 3) or 10 μg / mL, 0.1 μg / mL, and 50 μg / mL (ratio of 1:0.01:5, Table 2, series 2), the promoting effect on type II collagen expression was significantly better than that of other raw material combinations and ratios.

[0105] The experimental conclusions are as follows:

[0106] 1) Among the four raw materials, turmeric, enzymatically hydrolyzed bone meal, collagen peptide powder and glucosamine, the combination of turmeric, enzymatically hydrolyzed bone meal and collagen peptide powder performed better. Its promoting effect on type II collagen expression was significantly better than other combinations (including ① turmeric + enzymatically hydrolyzed bone meal + glucosamine, ② turmeric + cartilage meal + glucosamine, ③ cartilage meal + glucosamine + enzymatically hydrolyzed bone meal).

[0107] 2) Within a specific ratio range, specifically, turmeric: hydrolyzed bone powder: collagen peptide powder = 1: (0.002~5): (0.02~10), the combination of turmeric, hydrolyzed bone powder and collagen peptide powder can produce a synergistic effect, which is manifested in anti-inflammatory, antioxidant and type II collagen expression promotion effects.

[0108] Experiment 8: Human Verification

[0109] This study investigated endurance athletes who self-reported musculoskeletal discomfort. A total of 12 endurance athletes were recruited. Inclusion criteria for volunteers were: 1) age between 20 and 60 years; 2) monthly running distance of at least 150 kilometers; 3) formal registration for marathon or trail running events; 4) significant discomfort symptoms in joints such as the knee and ankle; 5) no use of joint medications or nutritional supplements (such as glucosamine products) in the past two weeks; 6) no participation in any other form of exercise or nutritional intervention studies in the past month; and 7) willingness to comply with the trial protocol and plan.

[0110] The protocol is as follows: Starting 4 days before the competition, volunteers will take one dose of the combination daily (turmeric: enzymatically hydrolyzed bone powder: cartilage powder containing type II collagen = 1:3.2:0.16, specifically 250 doses of turmeric, 800 doses of enzymatically hydrolyzed bone powder, and 40 doses of cartilage powder); on the day of the competition, one dose will be taken immediately after the competition; after the competition, volunteers will continue to take one dose of the combination daily for 3 consecutive days. The entire course of treatment will last 8 days. Throughout the testing period, volunteers must maintain their original dietary habits and training plan, while avoiding alcohol intake to ensure the accuracy and reliability of the research results.

[0111] Assessment Metric: Visual Analogue Scale / Score (VAS). The VAS is a 10-centimeter straight line marked with numbers 0 and 10 at each end. 0 represents "no pain," and 10 represents "most intense pain." Volunteers mark their perceived pain levels on the line; the corresponding number is their pain score. This method converts subjective pain perception into quantifiable numerical values, facilitating evaluation and comparison by researchers.

[0112] like Figure 20Experimental data showed that before taking the composition, the average VAS score of the subjects was 3.2. After 4 days of composition intervention, the average VAS score significantly decreased to 1.8, a reduction of approximately 43.8%. More notably, after continuing to take the composition for 3 days after the competition, the average VAS score further decreased to 1.5, representing an overall reduction of 53.1% compared to before taking it. This series of data clearly demonstrates that the composition exhibits good sustainability and effectiveness in alleviating the subjects' related symptoms.

[0113] All the experiments above demonstrate that the compound nutritional composition provided in this application has a positive and significant effect on joint function repair, cartilage damage relief, and joint inflammation relief for athletes.

[0114] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A compound nutritional composition beneficial for improving joint function in athletes, characterized in that, It is composed of the following ingredients, 1 part by weight of turmeric, 0.002-5 parts by weight of enzymatically hydrolyzed bone meal, and 0.02 parts by weight of cartilage powder containing type II collagen; or, 1 part by weight of turmeric, 0.002 parts by weight of enzymatically hydrolyzed bone meal and 0.02 parts by weight of collagen peptide powder; The curcumin content in the turmeric shall be no less than 18% by weight; The cartilage powder contains 4% or more of type II collagen by weight. The enzymatically hydrolyzed bone powder contains chondroitin sulfate at a weight ratio of over 60%, and the enzymatically hydrolyzed bone powder contains chondroitin sulfate and protein at a weight ratio of over 80%. The collagen peptide powder contains at least 55% by weight of peptides with a weight of less than 1000 Da, and at least 90% by weight of collagen peptides.

2. The use of the compound nutritional composition of claim 1 as an active ingredient in the preparation of a medicament for improving joint function, repairing cartilage damage and / or treating joint inflammation.

3. The application according to claim 2, characterized in that, The drug is targeted at athletes.

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