Composite nutritional composition beneficial to improvement of joint function of sports people and application of composite nutritional composition

Abstract

The invention relates to a composite nutritional composition and application thereof. The composite nutritional composition comprises the following components in parts by weight: 1 part of turmeric; 0.002 to 5 parts by weight of enzymatically hydrolyzed bone meal; and 0.02 to 0.2 part by weight of cartilage powder containing type II collagen or 0.02 to 10 parts by weight of collagen peptide powder. According to the compound nutritional composition provided by the invention, aiming at the particularity of bone and joint problems of sports people, the intervention effect of the compound nutritional composition provided by the invention on oxidative stress, inflammatory response and pain feeling related to sports is systematically evaluated by constructing an articular cartilage cell inflammation model and testing people; the invention aims to break through the limitation of a traditional general population formula, develop a precise nutrition scheme with component synergy and exercise physiology adaptability, and provide more scientific and effective joint health protection for exercise crowds.

Description

Technical Field The present invention relates to a composite nutritional composition and application thereof, which is beneficial to improving joint function of athletes. Background Art

[0001] With the rise of the global fitness craze and the continued expansion of the sports population, the incidence of sports-related bone and joint injuries (such as cartilage wear and joint inflammation) has increased significantly. The micro-injuries to joints, increased oxidative stress, and chronic inflammatory responses caused by exercise loads in this population have placed a higher demand on nutritional intervention.

[0002] Currently, there are a variety of products and technologies on the market for joint health maintenance. However, the mainstream joint health products on the market are mostly designed for a wide range of people, and lack a reasonable formula tailored to the physiological characteristics of athletes (such as the surge in free radicals and imbalanced cartilage metabolism caused by high-intensity exercise). In addition, existing products generally have two core problems: first, the mechanism of action of a single ingredient (such as glucosamine and chondroitin sulfate) is limited, making it difficult to simultaneously cover multi-dimensional needs such as anti-inflammatory, antioxidant, 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 effects of ingredients is not fully considered, resulting in insufficient effectiveness of the formula. Summary of the Invention

[0003] In response to the above problems, the present invention targets the particularity of bone and joint problems among athletes. By constructing an articular chondrocyte inflammation model and population testing, it systematically evaluates the intervention effect of the compound nutritional composition provided in this application on exercise-related oxidative stress, inflammatory response and pain perception. It aims to break through the limitations of traditional broad population formulas and develop a precise nutritional plan with both ingredient synergy and exercise physiological adaptability, providing more scientific and effective joint health protection for athletes.

[0004] In order to achieve the above technical effects, the first aspect of the present invention provides a compound nutritional composition comprising the following ingredients: 1 part by weight of turmeric; 0.002 to 5 parts by weight of enzymatic 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.

[0005] Optionally, the weight proportion of non-denatured type II collagen in the cartilage powder is 4% or more, the weight proportion of collagen peptides in the collagen peptide powder is more than 90%; and the weight proportion of chondroitin sulfate and protein in the enzymatic bone powder is more than 80%.

[0006] Optionally, the weight proportion of peptide segments below 1000 Da in the collagen peptide powder is greater than 55%.

[0007] Optionally, the weight proportion of chondroitin sulfate in the enzymatically hydrolyzed bone powder is greater than 60%.

[0008] Optionally, the cartilage powder containing type II collagen is made from chicken sternal cartilage, which is cleaned, disinfected, crushed, and then potassium chloride is added and dried under low temperature conditions to obtain the cartilage powder containing collagen; the collagen peptide powder is made from bovine cartilage, and the cartilage powder is obtained by enzymatic hydrolysis.

[0009] Optionally, the weight proportion of curcumin in the turmeric is not less than 18%.

[0010] Optionally, the enzymatic bone powder is made from cattle bones through steaming, crushing, enzymatic hydrolysis with protease, and drying.

[0011] Optionally, the protease is derived from Bacillus subtilis.

[0012] Optionally, the enzymatically hydrolyzed bone powder is 0.01 to 4 parts by weight, preferably 0.1 to 3 parts by weight.

[0013] Optionally, the cartilage powder containing type II collagen is 0.025 to 0.15 parts by weight, preferably 0.03 to 0.1 parts by weight.

[0014] Optionally, the collagen peptide powder is 1 to 8 parts by weight, preferably 2 to 7 parts by weight.

[0015] The present application also provides a second technical solution, namely, the use of any of the aforementioned compositions as an active ingredient in the preparation of an oral finished product, wherein the oral finished product includes a medicine, a health product or a food.

[0016] Optionally, the oral product can improve joint function, repair cartilage damage and / or treat joint inflammation.

[0017] Optionally, the oral product is targeted at athletes.

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

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

[0020] The turmeric, enzymatic bone meal, and cartilage powder used in this application (because the type II collagen cartilage powder and collagen peptide powder described in this application are all prepared from cartilage, they are collectively referred to as cartilage powder) can also be prepared independently. For example, turmeric is prepared from the rhizome of the ginger plant turmeric; enzymatic bone meal is prepared from cattle bones through protease hydrolysis; and cartilage powder is prepared from chicken sternum cartilage or cattle cartilage.

[0021] The enzymatic bone powder described in this application is a common food raw material approved by the state and is prepared using cattle bones. The enzymatic hydrolysis process adopts conventional enzymatic hydrolysis steps, for example, animal bones are made by steps such as cleaning, boiling, crushing, enzymatic hydrolysis with protease (derived from Bacillus subtilis) and drying. When turmeric is used in the case of 1 weight part, the content of enzymatic bone powder can be selected from 0.002 to 5 weight parts, preferably 0.002 to 4.5 weight parts, more preferably 0.01 to 4 weight parts, and more preferably 0.1 to 3 weight parts.

[0022] The cartilage powder containing type II collagen described in this application is a common food raw material approved by the state, and can be prepared from chicken sternal cartilage. The preparation process can be a conventional process step, for example, after cleaning, disinfecting, and crushing the chicken sternal cartilage, potassium chloride is added and the powder is dried under low temperature conditions to obtain the result. When turmeric is used in 1 part by weight, the cartilage powder containing type II collagen is 0.02 to 0.2 parts by weight, preferably 0.025 to 0.15 parts by weight, and more preferably 0.03 to 0.1 parts by weight. The collagen peptide powder described in this application is a common food raw material approved by the state, and can be prepared from bovine cartilage. The preparation process can be a conventional enzymatic process step, for example, after cleaning, crushing, filtering, enzymatic hydrolysis, enzyme inactivation, and centrifugation of animal cartilage to obtain the result. The weight proportion of collagen peptides in the collagen peptide powder is more than 90%, and the content of peptides below 1000Da is more than 55%. When turmeric is used in 1 part by weight, 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 the present invention, the main differences between non-denatured type II collagen and collagen peptides are: in terms of composition, non-denatured type II collagen is a complete macromolecular protein that retains the natural triple helical structure, contains immune active epitopes and a natural conformation that binds to the cartilage matrix; collagen peptides are small molecule peptides formed after enzymatic hydrolysis and other treatments, mostly short-chain amino acid sequences, without a complete triple helical structure. In terms of mechanism of action, non-denatured type II collagen interacts with the intestinal immune system through its natural conformation, inducing immune tolerance and reducing immune attacks against articular cartilage; collagen peptides mainly play a role by being directly absorbed by the human body, providing raw materials for cartilage synthesis, or participating in regulating cell metabolism, promoting collagen synthesis, etc. There are significant differences in the pathways of action and biological effects of the two.

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

[0025] The present application adopts a compound nutritional composition obtained by compounding turmeric, enzymatic bone powder and cartilage powder, which can more effectively exert the synergistic effect between the various ingredients to more effectively target athletic people, especially those with joint injuries, inflammation, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figures 1 to 4 is the result of cell viability assay; Figures 5 to 10 is the result of GSH concentration determination; Figures 11 and 12 It is the result of SOD determination; Figures 13 to 18 It is the result of inflammatory factor measurement; Figure 19 is the ROS measurement result; Figure 20 It is the VAS score result. DETAILED DESCRIPTION

[0027] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0028] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0029] Preparation of the composite nutritional composition provided by this application According to the conventional preparation method, turmeric, enzymatic bone powder, cartilage powder containing type II collagen or collagen peptide powder are prepared or directly purchased from the market, and they are compounded according to Table 1 and Table 2 below to prepare some of the proportions of the composite nutritional composition described in this application to illustrate the effectiveness of the composite nutritional components of this application. For example, the conventional preparation method used is as follows: turmeric is made by grinding the rhizomes of the ginger plant turmeric into powder; enzymatic bone powder is obtained by washing, crushing, proteolysis and drying cattle bones; cartilage powder containing type II collagen is obtained by processing chicken breast cartilage; collagen peptide powder is prepared from cattle cartilage. In addition to using different cartilage powders, the proportions in Tables 1 and 2 below also use blank groups and several control groups using the proportions of each component. The cartilage powder in Table 1 is cartilage powder containing type II collagen (UCII is used as an example in the accompanying drawings), and the cartilage powder in Table 2 is collagen peptide powder (collagen peptide is used as an example in the accompanying drawings). The cartilage powder (i.e., cartilage powder containing type II collagen) used in Table 1 contains 4% or more of non-denatured type II collagen by weight. The cartilage powder (i.e., collagen peptide powder) in Table 2 contains 90% or more of collagen peptides by weight, and peptides below 1000 Da by weight account for 55% or more. The enzymatic bone powder used in Tables 1 and 2 contains 80% or more of chondroitin sulfate and protein by weight, and 60% or more of chondroitin sulfate by weight. The turmeric used in Tables 1 and 2 contains no less than 18% of curcumin by weight.

[0030] Table 1- Cartilage powder containing type II collagen

[0031] Table 2- Cartilage powder uses collagen peptide powder

[0032] Experiment 1: Cell viability assay The cell proliferation and viability of cells after intervention with the compound composition and different concentrations of the single compound were determined based on CCK-8. Cells were first inoculated and 7 single compound concentration gradients (0.1, 0.5, 1, 10, 30, 50, 100 μg / mL, Figures 1 to 4 10 μL of culture medium was added with 10 μL of CCK-8 reagent, and the cells were incubated in an incubator for 1 to 4 h. The OD values ​​were measured at different time points, and the cell viability of each study group was calculated after a certain period of culture ( Figures 1 to 4The results showed that turmeric and cartilage powder containing type II collagen had no significant cytotoxicity at the studied doses, and cell viability increased in a dose-dependent manner with increasing intervention concentrations, demonstrating a promoting effect on cell viability. Enzymatic bone powder and collagen peptide powder exhibited some cytotoxicity at the set concentrations, but had little effect on cell viability. At the highest dose of 100 μg / mL within the studied range, cells still maintained approximately 90% cell viability.

[0033] The results of this experiment can be found in Figures 1 to 4 , Figure 1 is the cell viability measured by turmeric, Figure 2 It is the cell viability determined by cartilage powder containing type II collagen. Figure 3 It is the cell viability measured by collagen peptide powder, Figure 4 The cell viability is determined by enzymatically hydrolyzing bone powder. In the accompanying figures, UC II (i.e., non-denatured type II collagen) represents cartilage powder containing type II collagen, collagen peptide represents collagen peptide powder, and control represents a blank control group. The following figures all use the same method.

[0034] Experiment 2: Synergistic promotion of GSH expression The antioxidant and anti-inflammatory efficacy verification experiments were carried out using the ratios obtained in Tables 1 and 2 above.

[0035] Increased expression of the antioxidant factor GSH (reduced glutathione) means that the body's antioxidant capacity is enhanced, which can reduce oxidative stress damage to articular chondrocytes and synovial tissues. Specifically, increased GSH indicates that it alleviates the imbalance of oxidation and inflammation in the joints caused by exercise or inflammation through mechanisms such as directly scavenging free radicals (such as superoxide anions, hydrogen peroxide), enhancing the activity of antioxidant enzymes as a coenzyme of glutathione peroxidase, and inhibiting the NF-κB inflammatory pathway, while creating a favorable microenvironment for cartilage repair. If the GSH level is significantly increased after intervention, it can reflect that the substance has the ability to effectively activate endogenous antioxidant defenses and block the vicious cycle of "oxidation-inflammation". It is an important positive indicator for evaluating the antioxidant efficacy and joint protection potential of the product.

[0036] GSH detection method is as follows: For the GSH test kit, first collect the cell supernatant under different intervention conditions and store it at 4°C. At the same time, take out the reagents of the test kit from the refrigerator and equilibrate them at room temperature for 10 minutes. Prepare clean centrifuge tubes, label the blank tube, standard tube and assay tube respectively, add the reagents according to the table below, mix thoroughly, and incubate in a 37°C constant temperature water bath for 5-10 minutes to allow the reaction system to fully function. After incubation, immediately use a spectrophotometer to measure the absorbance value of each tube at a wavelength of 405nm, and calculate the GSH content according to the formula. Pay attention to the actual dilution multiple of the sample when calculating. And pay attention to the precise control of the order of reagent addition and the water bath temperature throughout the operation process to ensure the accuracy of the test results.

[0037] The ratios in Table 1 are compared with those in Ratio 1 series, Ratio 2 series and Ratio 3 series respectively, that is, Ratio 1 series includes Ratio S1-1, K1, A1, B1, C1, D1-1, E1-1, F1-1; Ratio 2 series includes Ratio S1-2, K1, A1, B1, C1, D1-2, E1-2, F1-2; Ratio 3 series includes S1-3, K1, A1, B1, C1, D1-3, E1-3, F1-3; The GSH concentrations obtained by measuring the ratios in Table 1 are respectively referred to Figures 5 to 7 , Figure 5 It is a series of ratio 1 (partial ratio), Figure 6 It is a 2-series ratio (partial ratio), Figure 7 It is a 3-series ratio (partial ratio). Figure 8 This is an example of the GSH concentration obtained by the series 1 in Table 2. Figure 8 Only the ratio 1 series (partial ratio) in Table 2 is illustrated, namely S2-1, K2, A2, B2, C2, D2-1, E2-1, and F2-1.

[0038] In addition, N-acetylglucosamine is often used in the prior art as the main component for joint repair. It is an amino derivative of glucose and an important raw material for synthesizing proteoglycans and glycosaminoglycans (hereinafter referred to as glucosamine). Chondrocytes take in glucosamine and generate glycosaminoglycan chains in the Golgi apparatus through a series of enzymatic reactions, which then combine with core proteins to form proteoglycan aggregates, which are filled in the collagen fiber network of the cartilage matrix, giving the cartilage compressive resistance and elasticity, which is beneficial to joint health. In this application, it is substituted for a component in Table 1 or Table 2, for example, the ratio 2 series in Table 1 is substituted, and the ratio 1 series in Table 2 is substituted, and the rest remain unchanged to form a new control group for corresponding comparative description, see the measurement results for details. Figure 9 、 Figure 10 . Figure 9 This is a comparison chart of the series 1 in Table 2 and the control group obtained by replacing cartilage powder with glucosamine. Figure 10 This is a comparison chart of the second series of ratios in Table 1 and the control group obtained by replacing enzymatic bone powder with glucosamine.

[0039] Compared to the blank and control groups, the combination of cartilage powder, turmeric, and enzymatically hydrolyzed bone meal promoted GSH expression, demonstrating antioxidant efficacy. Furthermore, among the various combinations described above, the "cartilage powder + turmeric + enzymatically hydrolyzed bone meal" group showed higher GSH expression levels than any of the other groups, indicating a synergistic effect among the combinations on GSH expression.

[0040] To further verify the synergistic effect of cartilage powder, turmeric and enzymatic bone meal, based on the ratio series 1 in Table 1 and the ratio series 1 in Table 2, glucosamine was additionally added to the cartilage powder, turmeric and enzymatic bone meal. The results are as follows:

[0041] This application uses cartilage powder + turmeric + enzymatic bone powder as active ingredients to prepare corresponding oral finished products to improve joint function, repair cartilage damage and / or treat joint inflammation, especially oral finished products such as medicines, health products or foods for athletes.

[0042] Experiment 3: Verification of synergistic antioxidant efficacy The increased expression of the antioxidant factor SOD (superoxide dismutase) means that the body's ability to scavenge free radicals is significantly enhanced, which is a key positive signal for joints to resist oxidative stress damage. Direct antioxidant mechanism: SOD is the only enzyme in the body that can specifically scavenge superoxide anions (O2 - ) metalloenzyme, whose increased expression can accelerate O2 - It is converted into hydrogen peroxide (H2O2), which is then cleared by catalase (CAT) or glutathione peroxidase (GPx), reducing the generation of lipid peroxidation products (such as MDA) and alleviating damage to chondrocyte membranes and organelles. - It can activate the NF-κB inflammatory pathway and induce the release of pro-inflammatory factors such as IL-6 and TNF-α. - Levels of free radicals indirectly inhibit inflammatory signaling, creating a synergistic "antioxidant-anti-inflammatory" effect, alleviating synovial inflammation and cartilage matrix degradation. Joint repair promotion: During exercise or inflammation, excessive free radicals inhibit chondrocyte proliferation and induce apoptosis (e.g., through mitochondrial activation of Caspase-3). Increased SOD expression improves the intracellular oxidative microenvironment, enhances chondrocyte viability, and promotes the synthesis of type II collagen and proteoglycans, accelerating injury repair.

[0043] Reduced MDA expression is one of the core positive indicators of antioxidant efficacy. Its specific significance is as follows: MDA is the end product of lipid peroxidation, and its level directly reflects the extent of free radical attack on cells and tissues. When joints are exposed to 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 and significantly increasing MDA levels (for example, MDA levels in the synovial fluid of osteoarthritis patients can be 30% to 50% higher than those in healthy controls). This in turn damages cell membrane structure, interferes with chondrocyte function, and exacerbates the inflammatory response by activating the NF-κB pathway. Reduced MDA expression after intervention indicates effective inhibition of lipid peroxidation.

[0044] The detection methods of SOD and MDA are as follows: SOD kit: First, thaw the cell supernatant collected under different intervention conditions at 4°C. At the same time, take out the reagents of the kit from the refrigerator and equilibrate at room temperature for 10 minutes. Prepare the reaction solution according to the instructions (such as mixing the color developer, enzyme working solution, and buffer according to the proportions in the table below), prepare test tubes and label blank tubes, standard tubes, and assay tubes. Add distilled water to the blank tube, add a known concentration of SOD standard solution to the standard tube, add the supernatant to the assay tube according to the amount added in the table, and then add the prepared reaction solution to each tube. After gently mixing, incubate in a constant temperature water bath at 37°C for 20-30 minutes. After the incubation is completed, add the stop solution to each tube, mix thoroughly, and use a spectrophotometer to measure the absorbance value of each tube at a wavelength of 550nm. After the measurement, calculate the SOD activity according to the formula. Unit: μL Control wells Control blank well Measurement well Determination of blank wells Samples to be tested 20 20 Double distilled water 20 20 Enzyme working solution 20 20 Enzyme dilution 20 20 Substrate application liquid 200 200 200 200

[0045] MDA kit: First, collect the cell supernatant collected under different intervention conditions and store it at 4°C. Simultaneously, remove the reagents from the refrigerator and allow them to equilibrate at room temperature for 10 minutes. Prepare centrifuge tubes, label blank tubes, standard tubes, and assay tubes. Add double-distilled water to the blank tube, MDA standard to the standard tube, and an appropriate amount of supernatant to the assay tube. Then, add the reagents to each tube according to the table below. After thorough mixing, heat in a 95°C water bath in the dark for 40 minutes to allow the reaction to proceed. After the water bath, quickly cool the centrifuge tube to room temperature, centrifuge at 4000 rpm for 10 minutes, and collect the supernatant. Measure the absorbance of each tube at a wavelength of 530 nm using a spectrophotometer. Scan the reading of the blank plate first and calculate the MDA content in the sample according to the formula.

[0046] Reagent 1: Easy to solidify, need to be heated in a water bath until transparent before use Reagent 2: Add 340 mL of double distilled water to each bottle and mix well Reagent 3: Reagent ratio, Reagent 1: Reagent 2: Reagent 3 = 0.2:3:1 In this part of the experiment, the series 3 of the mixture in Table 1 was selected to evaluate the effect on SOD, and the series 2 of the mixture in Table 1 was selected to evaluate the effect on MDA. Figure 11 and Figure 12 As shown, Figure 11 This is the measurement result of the 3 series of ratios in Table 1. Figure 12 These are the results of the measurement of the 2nd series of mixes in Table 1.

[0047] Compared to the blank and control groups, the combination of cartilage powder, turmeric, and enzymatically hydrolyzed bone meal promoted SOD expression, demonstrating antioxidant efficacy. Furthermore, among the various combinations described above, the "cartilage powder + turmeric + enzymatically hydrolyzed bone meal" group showed higher SOD expression levels than any of the other groups, indicating a synergistic effect among the combinations in promoting SOD expression.

[0048] Compared to the blank and control groups, the combination of cartilage powder, turmeric, and enzymatically hydrolyzed bone meal inhibited MDA expression and exhibited antioxidant efficacy. The results showed that the MDA expression level in the "cartilage powder + turmeric + enzymatically hydrolyzed bone meal" group was lower than in any of the other groups, indicating that the combination of the three ingredients produces an antioxidant effect superior to that of each individual ingredient, indicating that the combination exhibits antioxidant synergy.

[0049] Experiment 4: Anti-inflammatory efficacy verification - inhibition of inflammatory factors The reduced expression of inflammatory factors IL-18, IL-6, and TNF-α is the core manifestation of anti-inflammatory efficacy. IL-6: Its reduced level indicates that the inflammatory cascade in the joint is suppressed. IL-6 can induce hepatocytes to produce acute inflammatory proteins, and cooperate with other factors (such as TNF-α) to amplify synovial inflammation and promote chondrocyte apoptosis. Reducing IL-6 can reduce joint swelling, pain, and cartilage matrix degradation. TNF-α: As the "core initiator" of proinflammatory signals, its reduced expression directly reduces synovial macrophage activation and osteoclast formation, inhibits the inflammatory factor network mediated by the NF-κB pathway (such as IL-1β, MMPs), and thus delays the process of cartilage degeneration. IL-18: Its decreased level means that NLRP3 inflammasome activation is suppressed, which can block the vicious cycle of "inflammation-oxidative stress" while relieving the inhibition of chondrocyte synthesis function and accelerating damage repair. The combined reduction in the expression of the three indicates effective intervention in the key pathological links of "excessive secretion of pro-inflammatory factors-activation of inflammasomes-imbalance of cartilage metabolism" in the joints. It can significantly alleviate exercise-induced joint inflammation, reduce cartilage damage, and create a favorable microenvironment for the antioxidant and repair mechanisms to take effect. It is a core quantitative indicator for evaluating the product's anti-inflammatory efficacy and joint protection potential.

[0050] Inflammatory factor detection steps (IL-6, IL-18, TNF-α kit) Using an IL-6, IL-18, and TNF-α assay kit, cell supernatants collected under different intervention conditions were first stored at 4°C. The strips were removed from the kit and equilibrated to room temperature for 20 minutes. A microplate was prepared, with standard wells, sample wells, and blank wells assigned according to the number of samples. Five standards of varying concentrations (50 μL each) were added to the standard wells. 10 μL of the test sample and 40 μL of sample diluent were added to the sample wells. Mix thoroughly by gently vortexing. 100 μL of horseradish peroxidase-conjugated detection antibody was added to all wells except the blank wells. The microplate was sealed with a film sealer and incubated in a 37°C incubator for 60 minutes. After incubation, the liquid in the wells was discarded, and each well was filled with wash buffer. The wash buffer was allowed to stand for 2 minutes before being discarded. The wash process was repeated five times, and the microplate was patted dry. 50 μL each of substrate and colorimetric solutions A and B were added. After sealing the plate, the reaction was incubated in the dark at 37°C for 15 minutes. After a clear color gradient was observed 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. Draw a standard curve based on the absorbance of the standard. Calculate the concentration of IL-6, IL-18, and TNF-α in the sample from the standard curve based on the absorbance of the sample. Throughout the assay, strictly control the incubation time at room temperature and the number of washes to avoid operational errors that could affect the accuracy of the test results.

[0051] like Figures 13 to 17 As shown, Figure 13 The effect of the 1st series of the ratio in Table 1 on IL-6 was evaluated. Figure 14 The effect of the 2nd series of the ratio in Table 1 on IL-18 was evaluated. Figure 15 The effects of the three series of combinations in Table 1 on TNF-α were evaluated. Figure 16 The effect of the series 1 in Table 2 on TNF-α was evaluated. Figure 17 The effects of the combination 1 series in Table 2 on IL-18 were evaluated.

[0052] In this application, glucosamine is substituted for the cartilage powder component in Table 2, for example, replacing the ratio 1 series in Table 2, and the rest remain unchanged to form a new control group for corresponding comparative description. See the test results for details. Figure 18 . Figure 18 This is a comparison chart of the Ratio 1 series in Table 2 and the control group obtained by replacing cartilage powder with glucosamine.

[0053] Compared with the blank group and the control group, the combination of cartilage powder, turmeric and enzymatic bone powder showed the ability to inhibit the expression of inflammatory factors IL-18, IL-6 and TNF-α, showing anti-inflammatory effect.

[0054] Experiment 5: Antioxidant efficacy verification - inhibition of ROS The reduced expression of ROS (reactive oxygen species) means that the product effectively enhances the antioxidant defense system in the joint, reduces the damage of oxidative stress to chondrocyte DNA, lipids and proteins, inhibits the "oxidation-inflammation" interactive amplification effect, alleviates cartilage matrix degradation and synovial inflammatory response, and at the same time promotes chondrocyte anabolism (such as type II collagen and proteoglycan production), which is a direct manifestation of the antioxidant, anti-inflammatory and delayed joint degeneration effects.

[0055] The ROS detection method is as follows: ROS Kit: When using the ROS kit for detection, first remove the kit from the refrigerator and equilibrate at room temperature for 10 minutes. Simultaneously, wash both sides of the cells, which have been treated with different treatments for 24 hours, with DMEM. Prepare a centrifuge tube and pipette an appropriate amount of assay buffer based on the number of samples. Add the corresponding probe working solution (e.g., DCFH-DA should be dissolved in DMEM at a ratio of 1:1000) and mix thoroughly to prepare the reaction working solution. Add a predetermined amount of sample to the reaction working solution. Simultaneously, set up a blank control group containing only assay buffer and probe working solution and a positive control group containing ROS inducer as a control. After gentle mixing, incubate at 37°C in the dark for 20-30 minutes. During the incubation period, wash the sample with PBS as needed to remove any fluorescent probe that has not entered the cells. After the incubation period, immediately measure the fluorescence intensity using a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Carefully protect the tube from light to avoid quenching the probe fluorescence. Calculate the relative level or concentration of ROS in the sample according to the formula.

[0056] The results of the test are as follows Figure 19 As shown, Figure 19 These are the results of the measurement of the series 1 in Table 2.

[0057] Compared with the blank group and the control group, the three dosage groups of cartilage powder, turmeric, and enzymatic bone powder compound (in the intervention concentration setting, the concentration of the medium dose group was 20 times that of the low dose group; and the concentration of the high dose group was 5 times that of the medium dose group) showed the ability to inhibit ROS expression and exhibit antioxidant efficacy.

[0058] Experiment 6: Synergistic promotion of Aggrecan expression Aggrecan (proteoglycan aggregates) are key components of the extracellular matrix of cartilage, composed of a core protein covalently linked to numerous glycosaminoglycans (such as chondroitin sulfate and keratan sulfate). Aggrecans, formed by linking proteins to hyaluronic acid, form large aggregates that impart strong hydrophilicity and compressive elasticity to cartilage. Aggrecans play a central role in maintaining cartilage structural integrity, buffering mechanical stress, and regulating cell signaling. Reduced synthesis or increased degradation of aggrecans is closely associated with the development and progression of cartilage degenerative diseases such as osteoarthritis. Aggrecan expression levels are often used as a key indicator for evaluating cartilage repair efficacy. Increased aggrecan expression indicates that exogenous intervention has successfully induced the production of a cartilage-specific matrix, which helps offset matrix degradation in pathological conditions and interrupt the vicious cycle of inflammation-degradation-cartilage damage, thus having important implications for cartilage repair and disease prevention.

[0059] Aggrecan gene expression was detected using RT-PCR assay, and the steps were as follows: First, RNA extraction was performed. The cells in the wells were pipetted into an RNase-free centrifuge tube with 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 tubes were vigorously shaken for 15 seconds. The tubes were incubated at room temperature for 2-3 minutes, and centrifuged at 12000 g for 15 minutes at 4°C. The upper colorless aqueous phase was transferred to a new tube, and an equal volume of isopropanol was added. The tubes were mixed by inversion and allowed to stand at room temperature for 10 minutes. The tubes were centrifuged 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. The tubes were centrifuged at 7500 g for 5 minutes at 4°C. The supernatant was discarded. The precipitate was dried at room temperature, but complete drying should be avoided, as this will cause the RNA to become difficult to dissolve. An appropriate amount of DEPC water was added to dissolve the RNA. The RNA concentration and purity were determined using Nanodrop, so that the A260 / A280 ratio should be between 1.8 and 2.0.

[0060] Next, perform cDNA synthesis: Based on the RNA concentration, take 1000 ng of total RNA into an RNase-free centrifuge tube, add 1 μl of Oligo (dT) primer, make up to 10 μl with DEPC water, incubate at 65°C for 5 minutes, and then immediately ice bath for 2 minutes; add 4 μl of 5× Reaction Buffer, 2 μl of dNTP Mix (10 mM), 1 μl of RNase inhibitor, and 1 μl of reverse transcriptase in sequence, mix gently, and centrifuge. PCR procedures typically begin with a pre-denaturation step at 94–95°C for 3–5 minutes to completely unwind the double-stranded template DNA and activate the thermostable DNA polymerase. This is followed by a cycling phase consisting of denaturation, annealing, and extension, typically performed for 30–40 cycles. The denaturation step involves unwinding the DNA into single strands at 94–96°C for 10–30 seconds, allowing primers to bind specifically to the template for 10–30 seconds. The extension step involves synthesizing new strands using dNTPs at 72°C, with the duration adjusted according to the length of the amplified fragment. After the cycling is complete, a final extension step 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 and nonspecific reactions. The resulting cDNA can be used immediately or stored at -20°C until further use. The entire process must be strictly RNase-free, and cDNA synthesis must be performed immediately after PCR or stored in aliquots to prevent freeze-thaw cycles that may affect quality.

[0061] Quantitative PCR was performed using the prepared cDNA as a template. Target primers and SYBR Green Mix were added to label the target gene fragments. The program was set to synthesize the target gene, and then the target gene transcription level was analyzed based on the Cq value.

[0062] Table 3. Synergistic effect of the composition on Aggrecan expression (composition series in Table 1)

[0063] Compared with the blank and control groups, the combination of cartilage powder containing type II collagen, turmeric, and enzymatically hydrolyzed bone powder promoted Aggrecan expression, demonstrating cartilage repair efficacy. Furthermore, among the three different proportions of the combined compositions, the "cartilage powder + turmeric + enzymatically hydrolyzed bone powder" group showed higher Aggrecan expression than any of the other groups, indicating a synergistic effect of the combination on Aggrecan expression.

[0064] The synergistic effect of the composition of the present application in each figure is reflected in the fact that under the conditions of using the same amount of different ratios, the composition of the present application has a better effect than the individual components or other ratios. Figure 5For example, the vertical coordinates of the GSH concentrations achieved by the other individual components or other proportions are all between 6 and 13. However, after the corresponding components are replaced in equal amounts in the present application, the vertical coordinate of the GSH concentration achieved by the obtained composition is greater than 15, which is significantly greater than the actual effective values ​​of each component (refer to Figure 5 ordinate) and theoretical combination efficacy value (theoretically, after equal combination, the GSH ordinate should not exceed 13); therefore, the composition of the present application can play an obvious synergistic role relative to each component.

[0065] Experiment 7: Efficacy experiment on promoting type II collagen expression (COL2A) 1) Synergistic effect on the expression of type II collagen (COL2A) COL2A (type II collagen) is the primary structural protein in the extracellular matrix of cartilage and a skeletal component of the cartilage matrix, endowing cartilage with tensile strength and structural stability. The balance between its synthesis and degradation maintains the normal physiological function of cartilage and plays a central role in joint homeostasis. Its content is often reduced in degenerative diseases such as osteoarthritis due to increased degradation by proteases such as MMP13. Increased COL2A expression after intervention generally indicates that the intervention may have promoted the production of this protein, contributing to the repair or improvement of tissue structure and function. Specifically, enhanced chondrocyte synthesis can significantly improve the structural support capacity of articular cartilage and slow the progression of cartilage wear and degeneration. Furthermore, high expression of type II collagen can improve cartilage elasticity and impact resistance, enhancing joint tolerance to exercise loads. This is particularly effective for athletes, effectively addressing exercise-induced cartilage microdamage and serving as a key indicator for evaluating cartilage repair efficacy.

[0066] COL2A gene expression was detected by RT-PCR assay, with the same procedure as Aggrecan assay.

[0067] Table 4. Synergistic effect of the composition on COL2A expression (Table 2, ratio 1 series)

[0068] Note: In the setting of intervention concentration, the concentration of dose group 2 was 5 times that of dose group 1.

[0069] Compared to the blank and control groups, the combination of collagen peptide powder, turmeric, and enzymatically hydrolyzed bone meal promoted COL2A expression, demonstrating the efficacy of promoting type II collagen expression. Furthermore, among the above-mentioned different dosage groups of the combined composition, the "cartilage powder + turmeric + enzymatically hydrolyzed bone meal" group showed higher COL2A expression levels than any other group, indicating that the combination exhibited a synergistic effect on promoting COL2A expression.

[0070] 2) Comparative experiment on the efficacy of promoting type II collagen expression (COL2A) To further verify the synergistic effect of turmeric, enzymatic bone meal and collagen peptide powder in promoting the expression of type II collagen, the following experimental plan was designed: First, a raw material combination comparison experimental group was set up, and the target ingredient combination was compared with the "glucosamine + enzymatic bone meal + cartilage powder" group, "glucosamine + turmeric + cartilage powder" group, and "glucosamine + turmeric + enzymatic bone meal" group; second, for the "cartilage powder + turmeric + enzymatic bone meal" combination, the differences between the ratio series 2-3 in Table 2 and other different ratios were compared.

[0071] Table 5 Comparative experiment on promoting the expression of type II collagen

[0072] In this part of the experiment, the results showed that when the dosages of turmeric, enzymatic bone powder and collagen peptide powder were 0.1μg / mL, 0.5μg / mL, 1μg / mL (ratio of 1:5:10, Table 2 Ratio 3 series) or 10μg / mL, 0.1μg / mL, 50μg / mL (ratio of 1:0.01:5, Table 2 Ratio 2 series), the promoting effect on type II collagen expression was significantly better than the compound effects of other raw material combinations and ratios.

[0073] The experimental conclusions are as follows: 1) Among the four raw materials, turmeric, enzymatic bone meal, collagen peptide powder and glucosamine, the combination of turmeric, enzymatic bone meal and collagen peptide powder performed better, and its promoting effect on type II collagen expression was significantly better than other combination combinations (including ① turmeric + enzymatic bone meal + glucosamine, ② turmeric + cartilage powder + glucosamine, ③ cartilage powder + glucosamine + enzymatic bone meal).

[0074] 2) Within a specific ratio range, specifically, turmeric: enzymatic bone powder: collagen peptide powder = 1: (0.002-5): (0.02-10), the combination of turmeric, enzymatic bone powder and collagen peptide powder can produce a synergistic effect, which is manifested in anti-inflammatory, antioxidant, and promotion of type II collagen expression.

[0075] Experiment 8: Human Verification This study involved endurance athletes who self-reported joint discomfort. Twelve endurance athletes were recruited, and inclusion criteria included: 1) age range 20 to 60; 2) monthly running mileage of at least 150 kilometers; 3) official registration for a marathon or cross-country race; 4) significant joint discomfort in the knees, ankles, and other joints; 5) no joint medication or nutritional supplements (such as glucosamine) in the past two weeks; 6) no participation in any other exercise or nutritional intervention studies in the past month; and 7) voluntary agreement to adhere to the trial protocol and plan.

[0076] The protocol was as follows: Starting four days before the competition, volunteers were to take one serving of the composition daily (turmeric: enzymatic bone meal: cartilage powder containing type II collagen = 1:3.2:0.16, specifically 250 servings of turmeric, 800 servings of enzymatic bone meal, and 40 servings of cartilage powder). On the day of the competition, volunteers were to take one serving of the composition immediately after the competition. Following the competition, volunteers were to continue taking one serving of the composition daily for three consecutive days. The entire dosing cycle lasted eight days. Throughout the testing period, volunteers were required to maintain their original diet and training schedule and avoid alcohol to ensure the accuracy and reliability of the research results.

[0077] Assessment Indicator: Visual Analogue Scale / Score (VAS). The VAS uses a 10-cm straight line with the numbers 0 and 10 marked at either end, with 0 representing "no pain" and 10 representing "worst possible pain." Volunteers mark the corresponding position on the line according to their perceived pain intensity, and the number corresponding to that mark represents the patient's pain score. This method converts subjective pain perception into a quantifiable value, making it easier for researchers to evaluate and compare.

[0078] like Figure 20 Experimental data showed that before taking the composition, the average VAS score of the subjects was 3.2 points. After 4 days of intervention with the composition, the average VAS score dropped significantly to 1.8 points, a decrease of approximately 43.8%. More noteworthy is that after continuing to take the composition for 3 days after completing the competition, the average VAS score further decreased to 1.5 points, an overall decrease of 53.1% compared to before taking the composition. This series of data intuitively shows that the composition has shown good persistence and effectiveness in alleviating the relevant symptoms of the subjects.

[0079] It can be seen from the above experiments that the compound nutritional composition provided by the present application has positive and significant effects in repairing joint function, alleviating cartilage damage, and alleviating joint inflammation for athletes.

[0080] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A composite nutritional composition that is beneficial to improving joint function in sports people, characterized in that: Contains the following ingredients, 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.

2. The complex nutritional composition according to claim 1, characterized in that The weight proportion of type II collagen in the cartilage powder is 4% or more, the weight proportion of collagen peptide in the collagen peptide powder is more than 90%, and the weight proportion of chondroitin sulfate and protein in the enzymatic bone powder is more than 80%.

3. The complex nutritional composition according to claim 1, characterized in that The weight proportion of peptide segments below 1000 Da in the collagen peptide powder is more than 55%.

4. The complex nutritional composition according to claim 1, characterized in that The weight proportion of chondroitin sulfate in the enzymatic bone powder is more than 60%.

5. The complex nutritional composition according to claim 1, characterized in that The weight proportion of curcumin in the turmeric is not less than 18%.

6. The complex nutritional composition according to claim 1, characterized in that The amount of the cartilage powder containing type II collagen is 0.025 to 0.15 parts by weight; the amount of the collagen peptide powder is 1 to 8 parts by weight; and the amount of the enzymatically hydrolyzed bone powder is 0.01 to 4 parts by weight.

7. The complex nutritional composition according to claim 1, characterized in that The amount of the cartilage powder containing type II collagen is 0.03 to 0.1 parts by weight; the amount of the collagen peptide powder is 2 to 7 parts by weight; and the amount of the enzymatically hydrolyzed bone powder is 0.1 to 3 parts by weight.

8. Use of the composite nutritional composition according to claim 1 as an active ingredient in preparing an oral finished product, characterized in that: The oral finished product includes medicine, health care product or food.

9. The use according to claim 8, characterized in that The oral product can improve joint function, repair cartilage damage and / or treat joint inflammation.

10. The use according to claim 8, characterized in that The oral product is targeted at athletes.

Images