Composition for improving and / or treating exercise-induced osteoarthritis and preparation method and application thereof

Abstract

The present invention relates to a composition for improving and / or treating exercise-induced osteoarthritis and a preparation method and application thereof, the composition comprises a protein complex, the protein complex is prepared from a CP complex and a non-denatured type-II collagen (UC-II), and the CP complex is prepared from curcumin and a black pepper extract. The curcumin and the black pepper extract form the CP composition, the CP composition and the non-denatured type II collagen form a protein compound, the gastrointestinal tract digestion retention rate of the non-denatured type II collagen (UC-II) is improved through the formation of the protein compound, and therefore the effect of the non-denatured type II collagen (UC-II) on the immune oral tolerance mechanism is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a composition for improving and / or treating sports osteoarthritis and its preparation method and application. BACKGROUND

[0002] Sports osteoarthritis (OA) is a common degenerative joint disease characterized by joint cartilage degeneration, inflammatory response and pain, which seriously affects the quality of life of patients. Although its exact cause has not been fully elucidated, a large number of studies have shown that the occurrence of sports osteoarthritis is closely related to multiple risk factors and complex pathophysiological mechanisms, involving biomechanics, inflammation, abnormal cell metabolism and other aspects. One of the pathophysiological characteristics of sports osteoarthritis (OA) is the elevation of pro-inflammatory cytokines (such as IL-1β, TNF-α, IL-6 and IL-17) in joint synovial membrane and cartilage. Non-denatured type II collagen can inhibit the production of these pro-inflammatory cytokines, thereby reducing joint inflammation.

[0003] Although existing treatment methods and nutritional supplements can alleviate the symptoms of osteoarthritis to some extent, there are still some deficiencies. First, the bioavailability of the active ingredients of many products in the body is low, resulting in unsatisfactory therapeutic effect. Second, existing compositions often lack a multi-target integrated mechanism, which cannot comprehensively regulate immunity and bone metabolism. In addition, existing researches are mostly focused on the role of single ingredient, lacking in-depth exploration of the synergistic effect of multiple ingredients. Especially when using non-denatured type II collagen (such as UC-II) as a dietary supplement to improve and / or relieve osteoarthritis, since non-denatured type II collagen (such as UC-II) works by immune oral tolerance mechanism, it is easily broken down by pepsin in the stomach during taking, reducing its efficacy, which urgently needs to be further improved.

[0004] Therefore, how to develop a new type of non-denatured type II collagen (such as UC-II) that maintains its stability in gastric juice, improves its immune oral tolerance, and effectively integrates the advantages of multiple ingredients to improve the effect of improving and / or treating osteoarthritis has become a technical problem to be solved. SUMMARY

[0005] The present application provides a composition for improving and / or treating sports osteoarthritis and its preparation method and application.

[0006] To solve the above technical problems, the present application provides the use of a composition in the preparation of a product for improving and / or treating sports osteoarthritis, the composition comprising a protein complex prepared from non-denatured type II collagen and a pre-prepared CP complex, wherein the CP complex is prepared from curcumin and black pepper extract;

[0007] The preparation method of the composition comprises the following steps:

[0008] forming the CP complex;

[0009] forming a non-denatured type II collagen solution with a pH value of 4-6;

[0010] adding the CP complex into the non-denatured type II collagen solution at 35-39 DEG C, slowly mixing to form a mixed solution;

[0011] freeze-drying the mixed solution at -10 DEG C to -80 DEG C to obtain the protein complex.

[0012] In one embodiment of the present application, the composition is prepared from the following raw materials by weight: 10.5-32 parts of the CP complex and 2-64 parts of the non-denatured type II collagen;

[0013] The CP complex is prepared from the following raw materials by weight: 10-30 parts of curcumin and 0.5-2 parts of black pepper extract.

[0014] In the present application, curcumin can be turmeric or turmeric extract with a curcumin content of more than 5%, and the curcumin content meets the formula composition, that is, if turmeric or turmeric extract is used, the curcumin content is 5%, 6%, 7%, 8%, 15%, 30%, 50%, 70%, 80% or more.

[0015] In one embodiment of the present application, the composition is prepared from the following active ingredients by weight: 11-31 parts of the CP complex and 3-63 parts of the non-denatured type II collagen.

[0016] In one embodiment of the present application, the preparation method of the CP complex comprises the following steps:

[0017] adding curcumin into a 4-10% ethanol solution to form a curcumin ethanol solution;

[0018] adding black pepper extract into the curcumin ethanol solution, uniformly mixing for 3-5 min to form a CP mixture solution;

[0019] drying the CP mixture solution at 30-40 DEG C to obtain the CP complex.

[0020] In one embodiment of the present application, the CP complex is added into the non-denatured type II collagen solution, and the incubation time is 30-40 min to form a mixed solution.

[0021] In one embodiment of the present application, the pH value of the non-denatured type II collagen solution is 4.5-5.5.

[0022] In one embodiment of the present invention, the concentration of the non-mutated type II collagen solution is 1.0-1.5 mg / mL.

[0023] The present invention also provides a product comprising the composition for any of the above-described uses, the product further comprising one or more of the following: hydrolyzed type II collagen, vitamin B1, vitamin B2, vitamin D, milk mineral salts, maltitol powder, microcrystalline cellulose, magnesium stearate, silicon dioxide, compound coloring agents, and compound food additives.

[0024] In one embodiment of the present invention, the product comprises the following components in parts by weight: 18-36 parts of hydrolyzed type II collagen, 0.4-1.2 parts of vitamin B1, 0.2-0.8 parts of vitamin B2, and 2×10 parts of vitamin D. -3 - 4×10 -3 share.

[0025] The present invention also provides the application of the above-described product in any of the following a)-c): a) improving the gastrointestinal digestibility and retention rate of non-denatured type II collagen; b) enhancing immunity; c) improving osteoarthritis.

[0026] In this invention, compound colorant refers to a colorant product made by mixing two or more food colorings together through physical methods. It is used to color film coatings, sugar coatings, or tablet cores to make tablets more aesthetically pleasing and easier to identify (e.g., different sizes of tablets are distinguished by different colors), to mask the color of the raw materials themselves, or to improve patient compliance with medication.

[0027] In this invention, compound food additives refer to composite products made by mixing two or more single-variety food additives (which may include excipients) through physical methods for ease of application.

[0028] In another aspect, the present invention also provides the use of the protein complex prepared by any of the above methods in the preparation of any of the following a)-c): a) a product for improving the gastrointestinal digestibility and retention of non-denatured type II collagen; b) a product for improving immunity; c) a product for improving osteoarthritis.

[0029] In this invention, black pepper extract, the main component of which is piperine, is an N-acylpiperidine alkaloid, such as... Figure 2 The diagram shows the structural formula of a piperine molecule, where A represents the three-dimensional structure of the piperine molecule, B represents the two-dimensional structure of the piperine molecule, and its chemical formula is C. 17 H 19NO3. Its molecular weight is 285.34 g / mol. The structural features of piperine include benzodioxole, N-acylpiperidine, and tertiary carboxamide.

[0030] In the present application, raw turmeric or turmeric extract can be used in the preparation of the composition, and the amount added should meet the content requirements of curcumin in the product. Curcumin is a natural polyphenolic compound extracted from the rhizomes of Curcuma longa. For example, Figure 1 As shown in the figure, the three-dimensional spatial structure of curcumin molecule, A is the three-dimensional spatial structure of curcumin molecule, B is the two-dimensional structure of curcumin molecule, and its chemical formula is C 21 H 20 O6, with a molecular weight of 368.38 g / mol. Curcumin is a diarylheptanoid compound, with two ortho-substituted aryloxy groups connected by a seven-carbon chain (heptadienedione). The structure contains multiple functional groups, including phenolic hydroxyl and β-diketone structures, which are key to its multiple biological activities.

[0031] Curcumin and piperine can form a stable complex through hydrogen bonding, hydrophobic interaction, van der Waals force and π-π stacking non-covalent interaction. This stable complex is the CP complex. By uniformly mixing curcumin and black pepper extract at the molecular level, a uniform CP complex matrix is formed, which provides a basis for further and full binding with non-denatured type II collagen. Among them, the amino group of piperine molecule forms a hydrogen bond with the phenolic hydroxyl group of curcumin, and the hydrophobic interaction between the two further enhances the stability of the complex. The theoretical adsorption energy of the complex is -12.6 kcal / mol, indicating that the formation process is an exothermic reaction. Electronegativity analysis shows that the electronegativity of the CP complex (4.312 eV) is lower than that of the single component, indicating that there is a charge transfer between curcumin and piperine, which affects the overall electronic structure of the complex. In addition, piperine not only acts as a solubilizer for curcumin, but also enhances the binding affinity of curcumin and protein by binding to the allosteric site of the protein, stabilizing the interaction.

[0032] Undenatured type-II collagen (UC-II) can self-assemble into nanofiber structures with a diameter of 50–200 nm at 37℃ and pH 4.5-5.5. The self-assembly process is a non-spontaneous endothermic reaction (ΔG > 0) with an activation energy of 58.19 kJ / mol and a half-life of 4.79 minutes, indicating that the process requires external energy driving and has limited kinetic stability. At physiological temperature of 37℃, the self-assembly rate of non-denatured type-II collagen (undenatured type-II collagen (UC-II)) is the fastest, which helps to form a dense network structure.

[0033] The formation of the protein complex can be that curcumin and piperine first form a stable CP complex, and then bind with undenatured type-II collagen (UC-II). The main driving force for complexation includes hydrophobic interaction and hydrogen bonding. The hydrophobic groups in the CP complex can bind with the hydrophobic regions exposed after the denaturation of undenatured type-II collagen, and hydrogen bond networks can be formed between the amide bond and ether bond of piperine and the hydroxyl proline, lysine and other residues of undenatured type-II collagen.

[0034] Although undenatured type-II collagen (UC-II) is prone to aggregation near the isoelectric point (Zeta potential is -5 to +5 mV) in the pH range of 4.5-5.5, the CP complex can regulate the surface charge distribution by electrostatic interaction between its phenolic hydroxyl and carbonyl groups and the amino acid residues (such as lysine and glutamic acid) on the surface of undenatured type-II collagen, thereby increasing the absolute value of Zeta potential and inhibiting aggregation and stabilizing the system.

[0035] Thermodynamically, the exothermic binding (ΔG < 0) of the CP complex can compensate for the energy required for the self-assembly of undenatured type-II collagen (UC-II), thereby enhancing the stability of the overall complex. Structurally, it is speculated that the CP complex is attached to the surface of the undenatured type-II collagen fiber through hydrogen bonding or hydrophobic interaction, forming a "fiber-small molecule" complex structure. In addition, the hydrophobic side chains of undenatured type-II collagen and the aromatic ring structure of the CP complex can further stabilize the ternary complex system of the protein complex through van der Waals forces.

[0036] In the present application, curcumin and piperine as small molecules form a stable complex with undenatured type-II collagen (UC-II), and the CP complex formed by curcumin and piperine is cross-linked with undenatured type-II collagen, which confers high thermal stability to collagen. At the same time, the complex physically protects the triple helix structure of collagen from attack by gastric acid and pepsin. This protective effect can reduce the dissociation and degradation of collagen in the stomach, thereby improving the integrity of undenatured type-II collagen (UC-II) and allowing it to enter the intestinal tract in a more complete form for absorption.

[0037] In the present application, the black pepper extract is prepared as follows: the black pepper is pulverized, extracted twice with 95% ethanol by refluxing for 1.5 hours each time, filtered, and the filtrates are combined and concentrated to a paste under vacuum to obtain a paste-like crude extract, which is freeze-dried at-45℃, then pulverized with a ball mill to obtain a crude extract powder, then the paste-like crude extract powder is dissolved with 95% ethanol at 25-60℃, filtered with activated carbon, the filtrate is concentrated under vacuum, and then the concentrated filtrate is freeze-dried again at-15℃, and ground at-15℃ to obtain a black pepper extract with an average particle size of 15-30 μm.

[0038] Advantages of the present application:

[0039] In the present application, curcumin and black pepper extract are first combined to form a CP composition, and the CP composition is combined with undenatured type-II collagen (UC-II) to form a protein complex, which improves the stability of the undenatured type-II collagen, increases the retention rate of the undenatured type-II collagen in the gastrointestinal tract, ensures that more complete undenatured type-II collagen enters the intestinal tract and is completely absorbed, and thus improves the effect of the undenatured type-II collagen (UC-II) acting through the immune oral tolerance mechanism.

[0040] Meanwhile, the protein complex of the present application is also combined with hydrolyzed type-II collagen, undenatured type-II collagen, vitamin B group, vitamin D and other components to improve calcium ion absorption, and significantly improves the effect of improving and / or treating sports osteoarthritis.

[0041] The anti-inflammatory effect of curcumin in the composition of the present application can effectively reduce joint inflammation, the combination of hydrolyzed type-II collagen and undenatured type-II collagen (UC-II) promotes the regeneration and repair of articular cartilage, and the addition of vitamin B group and vitamin D enhances bone health, and overall realizes the synergistic effect of multiple targets, improves the bioavailability, and ultimately improves the joint function and quality of life of patients with sports osteoarthritis. BRIEF DESCRIPTION OF DRAWINGS

[0042] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0043] Figure 1A three-dimensional space structure diagram of a curcumin molecule of the present application, A is a three-dimensional space structure of the curcumin molecule, and B is a two-dimensional structure of the curcumin molecule;

[0044] Figure 2 A structural formula of the piperine molecule of the present application, wherein A is a three-dimensional space structure of the piperine molecule, and B is a two-dimensional structure of the piperine molecule;

[0045] Figure 3 A circular dichroism ellipticity curve diagram at 222 nm of each time point, wherein T: protein complex group; B: collagen binary complex group; C: control group. DETAILED DESCRIPTION

[0046] Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. Before the description, it should be understood that the term used in the specification and the appended claims should not be construed as limited to general and dictionary meanings, but interpreted based on the meanings and concepts corresponding to technical aspects of the present application on the basis of the principle that the inventor is allowed to define appropriate terms in order to best explain the application. Therefore, the description herein is merely a preferred example of the application, and not intended to limit the scope of the application, and it should be understood that other equivalent embodiments and modifications can be made thereto without departing from the spirit and scope of the application.

[0047] Example 1, preparation of a protein complex

[0048] The protein complex of the present example is composed of a CP complex and non-denatured type II collagen, wherein the mass of the CP complex is 11.0 g, which is made of curcumin 10.0 g and black pepper extract 1.0 g; the mass of the non-denatured type II collagen is 16.0 g.

[0049] The preparation method of the protein complex comprises the following steps:

[0050] Preparation of the CP complex: curcumin is added to a 4% ethanol solution to form a curcumin ethanol solution;

[0051] The black pepper extract is added to the curcumin ethanol solution and uniformly mixed for 3-5 min to form a CP mixture solution, and the mass concentration of the CP mixture solution is 5 g / L;

[0052] The CP mixture solution is dried at a temperature of 30°C to obtain the CP complex.

[0053] A 1.0 mg / mL non-denatured type II collagen solution is prepared, and the pH value of the non-denatured type II collagen solution is 4.5-5.5. The concentration of the non-denatured type II collagen has a significant influence on the uniformity of the complex, so as to balance the integrity of the fiber network and the dispersion stability.

[0054] The CP compound is added to the non-denatured type II collagen protein solution at 35°C, and the incubation time is 30 min. After mixing, a protein mixture solution is formed;

[0055] The protein mixture solution is freeze-dried at -10°C to -80°C to obtain a protein compound.

[0056] Example 2, composition for treating sports osteoarthritis

[0057] The protein compound of this example is composed of a CP compound and non-denatured type II collagen protein, wherein: the mass of the CP compound is 11.0 g, which is made of curcumin 10.0 g and black pepper extract 1.0 g; the mass of the non-denatured type II collagen protein is 16.0 g.

[0058] The preparation method of the protein compound comprises the following steps:

[0059] Preparation of CP compound: curcumin is added to a 4% ethanol solution to form a curcumin ethanol solution;

[0060] The black pepper extract is added to the curcumin ethanol solution and uniformly mixed for 3-5 min to form a CP mixture solution with a mass concentration of 5 g / L;

[0061] The CP mixture solution is dried at 30°C to obtain the CP compound.

[0062] A 1.0 mg / mL non-denatured type II collagen protein solution is prepared, and the pH value of the non-denatured type II collagen protein solution is 4.5-5.5. The concentration of non-denatured type II collagen protein has a significant effect on the uniformity of the compound, so as to balance the integrity of the fiber network and the dispersion stability.

[0063] The CP compound is added to the non-denatured type II collagen protein solution at 35°C, and the incubation time is 30 min. After mixing, a protein mixture solution is formed;

[0064] The protein mixture solution is freeze-dried at -10°C to -80°C to obtain a protein compound.

[0065] The composition for treating sports osteoarthritis of this example comprises the above-mentioned protein compound 27.0 g, hydrolyzed type II collagen protein 24 g, vitamin B1 0.5 g, vitamin B2 0.3 g, vitamin D 2.5×10 -3 g, and a suitable amount of milk mineral salt, maltitol powder, microcrystalline cellulose, magnesium stearate, silicon dioxide, compounded colorant and compounded food additive are added to prepare tablets, each weighing 0.5 g.

[0066] Example 3, composition for treating sports osteoarthritis

[0067] The protein complex of the present embodiment is composed of a CP complex and non-denatured type II collagen, wherein:

[0068] The CP complex has a mass of 32 g, which is made of curcumin 30 g and black pepper extract 2 g; the non-denatured type II collagen has a mass of 64 g.

[0069] The preparation method of the protein complex comprises the following steps:

[0070] The CP complex preparation method comprises the following steps: adding curcumin into a 7% ethanol solution to form a curcumin ethanol solution;

[0071] The black pepper extract is added to the curcumin ethanol solution and uniformly mixed for 3-5 min to form a CP mixture solution, and the mass concentration of the CP mixture solution is 8 g / L;

[0072] The CP mixture solution is dried to obtain the CP complex, and the drying temperature of the CP mixture solution is 38°C.

[0073] A 1.2 mg / mL non-denatured type II collagen solution is prepared, and the pH value of the non-denatured type II collagen solution is 4.5-5.5. The concentration of non-denatured type II collagen has a significant effect on the uniformity of the complex, so as to balance the integrity of the fiber network and the dispersion stability.

[0074] The CP complex is added to the 38°C non-denatured type II collagen solution, and the incubation time is 35 min. After mixing, a protein mixture solution is formed;

[0075] The protein mixture solution is freeze-dried at -10°C to -80°C to obtain the protein complex.

[0076] The composition for treating sports osteoarthritis of the present embodiment comprises the above-mentioned protein complex 96 g, hydrolyzed type II collagen 36 g, vitamin B1 1.2 g, vitamin B2 0.8 g, vitamin D 4×10 -3 g, and a suitable amount of milk mineral salt, maltitol powder, microcrystalline cellulose, magnesium stearate, silicon dioxide, compounded colorant and compounded food additive are added to prepare tablets, each weighing 0.5 g.

[0077] Example 4, composition for treating sports osteoarthritis

[0078] The protein complex of the embodiment is composed of a CP complex and non-denatured type II collagen, wherein: the mass of the CP complex is 10.5 g, which is made of curcumin 10.0 g and black pepper extract 0.5 g; the mass of the non-denatured type II collagen is 2.0 g.

[0079] The preparation method of the protein complex comprises the following steps:

[0080] The CP complex preparation method comprises the following steps: adding curcumin into a 10% ethanol solution to form a curcumin ethanol solution;

[0081] Adding black pepper extract into the curcumin ethanol solution, uniformly mixing for 3-5 min to form a CP mixture solution, and the mass concentration of the CP mixture solution is 10 g / L;

[0082] Drying the CP mixture solution to obtain the CP complex, and the drying temperature of the CP mixture solution is 30-40°C.

[0083] Preparation of a 1.5 mg / mL non-denatured type II collagen solution, and the pH value of the non-denatured type II collagen solution is 4.5-5.5, and the non-denatured type II collagen concentration has a significant effect on the uniformity of the complex, so as to balance the integrity of the fiber network and the dispersion stability.

[0084] Adding the CP complex into the 39°C non-denatured type II collagen solution, and incubating for 30 min, and after mixing, a protein mixture solution is formed;

[0085] Freeze-drying the protein mixture solution at -10°C to -80°C to obtain the protein complex.

[0086] The composition for treating sports bone and joint injury of the embodiment comprises the above-mentioned protein complex 12.5 g, hydrolyzed type II collagen 18 g, vitamin B1 0.4 g, vitamin B2 0.2 g, vitamin D 2×10 -3 g, and adding appropriate amounts of maltitol powder, microcrystalline cellulose, magnesium stearate, silicon dioxide, compound colorant and compound food additive to prepare tablets, each tablet 0.5 g.

[0087] As a changeable embodiment, other nutrients can be added to the protein complex to prepare nutritional supplements in other forms such as powder, liquid, jelly and capsule.

[0088] Test Example 1: Protein complex improves the retention rate of non-denatured type II collagen in the gastrointestinal tract

[0089] 1 Research method

[0090] 1.1 Preparation of experimental samples

[0091] Protein complex group (Group T): The composition prepared in Example 1 was used as the experimental sample, which was dissolved with deionized water to obtain a protein complex mixed solution containing 5 mg / mL of non-denatured type II collagen.

[0092] Curcumin-non-denatured type II collagen binary complex group (Group B): A 5 mg / mL non-denatured type II collagen solution was prepared and the pH value was adjusted to 5.0, and 28 g of curcumin powder was added to the solution. The mixture was stirred at a speed of 500 rpm for 5 h, and then centrifuged at 25°C and 8000 x g for 10 min. The supernatant was collected as the collagen binary complex sample.

[0093] Control group (Group C): The non-denatured type II collagen powder was used as the control sample, which was dissolved with deionized water to prepare a solution with a non-denatured type II collagen concentration of 5 mg / mL.

[0094] 1.2 Simulated gastric digestion

[0095] First, 1.25 times the concentration of simulated gastric juice was preheated to 37°C, and then added to the experimental sample solution at a ratio of 1:1 (v / v). The pH value of the resulting mixture was adjusted to 1.2 with 1 mol / L HCl, and the pH value was stable for 5 min before adding calcium chloride dihydrate solution. Then porcine pepsin (final system concentration 2000 U / mL) was added, and the 1.25 times the concentration of simulated gastric juice was diluted to 1 times the concentration with deionized water. The mixture was incubated in a 37°C constant temperature shaker for 0.5 h or 1.0 h. After the simulated digestion was completed, the sample was taken out and immediately placed in the refrigerator to terminate the activity of pepsin.

[0096] 1.3 Simulated intestinal digestion

[0097] A 1.25 times the concentration of simulated intestinal juice was prepared and preheated to 37°C, and added to the above-mentioned gastric digested chyme at a ratio of 1:1 (v / v). The pH value of the system was adjusted to 7.0 with 1 mol / L NaOH, and bile and calcium chloride dihydrate solution were added. Then trypsin (final system concentration 100 U / mL) was added, and the 1.25 times the concentration of simulated intestinal juice was diluted to 1 times the concentration with deionized water. The mixture was incubated in a 37°C constant temperature shaker for 0.5 h, 1 h, 2 h, 4 h and 6 h, respectively. After the simulated digestion was completed, the sample was taken out and immediately placed in the refrigerator to terminate the activity of trypsin.

[0098] 1.4 Circular dichroism analysis

[0099] The simulated gastric juice and intestinal juice prepared in 1.2 and 1.3 were diluted 20 times and 10 times with phosphate buffer (0.1 mol / L, pH 7.0), respectively. The diluted solutions were placed in a 1 mm quartz cuvette, and the absorbance in the wavelength range of 190-260 nm was measured by a circular dichroism spectrometer.

[0100] 2 Research results

[0101] As shown in Figure 3 , the structural changes of the non-denatured type II collagen in each sample during the gastrointestinal digestion process were analyzed. The intensity of the positive absorption peak in the circular dichroism spectrum reflects the integrity of the triple helix structure of type II collagen. If the triple helix structure is completely destroyed, the positive absorption peak will disappear. For the collagen binary complex, the ellipticity at 222 nm in the circular dichroism spectrum decreased rapidly after 1 h of gastric digestion, and after entering the intestinal digestion stage, the triple helix structure of the non-denatured type II collagen was rapidly degraded under the action of trypsin and bile salts, resulting in the ellipticity at 222 nm decreasing to near zero.

[0102] In contrast, the protein complex prepared in Example 1 of the present application had a slower digestion rate during gastric digestion, and the ellipticity was significantly higher than that of the collagen binary complex at 0.5 h and 1 h of intestinal digestion (P<0.05). When the intestinal digestion time increased from 0.5 h to 6 h, the triple helix structure of the non-denatured type II collagen in the protein complex prepared in Example 1 of the present application was slowly degraded, and about 24.23% was still retained after 6 h, and was significantly higher than that of the collagen binary complex at each time point (P<0.05). Therefore, the protein complex prepared in Example 1 of the present application can better maintain the activity of non-denatured type II collagen and play a better biological role in the intestinal tract.

[0103] Test Example 2 Effect of the composition of Example 2 on joint function of people with knee discomfort

[0104] 1 Purpose of the study

[0105] The purpose of this experiment is to evaluate the improvement effect of the composition of Example 2 on the knee function of people with knee discomfort in the running population, to quantify the changes of the subjects in the five scoring dimensions of knee symptoms, stiffness, pain, daily function, sports and entertainment function, and quality of life through the Knee Injury and Osteoarthritis Outcome Score (KOOS) scale, and to provide a basis for the functional efficacy of the product.

[0106] 2 Research plan

[0107] 2.1 Study subjects

[0108] This study recruited subjects and screened them through questionnaires from August 8, 2025 to August 12, and finally included 24 subjects who met the criteria. The subjects met the following criteria: 1. Having a long-term exercise habit for more than 1 year; 2. Currently having clear knee discomfort caused by exercise; 3. No intervention or related treatment of bone and joint health related products; 4. No other underlying diseases; 5. Voluntarily participate in this experiment and sign the informed consent form.

[0109] 2.2 Experimental design

[0110] This experiment used a self-control design, and all subjects were supplemented with nutrients according to a unified supplementation method. The effect was evaluated by changes in indicators before and after supplementation.

[0111] Supplementation method: This experiment used unified nutrient supplementation. The nutrient was the composition for treating exercise-induced osteoarthritis of Example 2. Method of consumption: The subjects consumed 2 tablets before going to bed every day for three consecutive weeks.

[0112] Quality control: The 24 subjects were divided into 6 groups (4 people per group), and each group was headed by an experimental staff member. The group was managed through a WeChat group; the subjects were supervised and confirmed to take the medication every day, and the group leader recorded the daily consumption compliance.

[0113] Observation index and evaluation tool: KOOS scale was used as the main evaluation tool, which included the following 5 dimensions:

[0114] Symptoms: Evaluate the swelling, friction / abnormal noise, jamming, flexion and extension ability of the knee joint in the past 1 week (S1-

[0115] S7);

[0116] Pain: Evaluate the frequency of knee pain in the past 1 week and the degree of pain in specific activities (such as twisting, going up and down stairs, walking, etc.) (P1-P9);

[0117] Daily activity function: Evaluate the difficulty of daily activities (such as going up and down stairs, getting up, shopping, dressing and undressing, etc.) caused by knee problems in the past 1 week (A1-A17);

[0118] Sports function: Evaluate the difficulty of high-intensity activities (such as squatting, running, jumping, kneeling, etc.) caused by knee problems in the past 1 week (SP1-SP5);

[0119] Quality of life: Evaluate the degree of concern about knee problems, lifestyle changes, confidence impact, and overall inconvenience in the past 1 week (Q1-Q4).

[0120] Scoring criteria: Each item is scored using a 5-point Likert scale (0 points = no problem, 1 point = mild problem, 2 points = moderate problem, 3 points = severe problem, 4 points = extreme problem), and the total score of each dimension is calculated after reversing the corresponding logical conversion of the reverse scoring items (such as S4, S5).

[0121] According to the benchmark value and threshold value of KOOS score established by Larsen et al. [2], the improvement of each dimension score after the supplement of the nutritional product is evaluated. The improvement of a certain dimension score of KOOS by ≥10 points indicates that the product significantly improves the dimension, and the score of a certain dimension of KOOS ≥ the benchmark value indicates that the dimension reaches the level of normal people.

[0122] Table 1 Benchmark value and improvement threshold of five dimensions of KOOS score

[0123]

[0124] Data collection: During the experiment, the scale follow-up was conducted once every 7 days, a total of 3 times. The subjects were reminded by the staff through WeChat group chat to complete the scale filling independently, and no language guidance was given to the subjects during the process. The follow-up scale was designed by questionnaire star for data collection.

[0125] 2.3 Data processing: The scoring method of the scale was referred to the research of Wang Ying et al. [1]. Statistical software was used for data analysis to calculate the baseline value of the subjects before the experiment and the KOOS dimension scores at each time point after the experiment. The differences in scores before and after the experiment were compared by paired sample T test, and P<0.05 was considered to have statistical significance.

[0126] 3 Research results and analysis:

[0127] 3.1 Compliance and adverse reactions of subjects

[0128] In this study, the 24 subjects strictly followed the supplement requirement of "eating 2 tablets before going to bed every day for three consecutive weeks", there was no case of withdrawal due to poor compliance, and the scale follow-up was completed as planned, once every 7 days for a total of 3 times. The overall compliance was good, which provided a reliable guarantee for the accuracy and effectiveness of the research data.

[0129] During the experiment, none of the 24 subjects had gastrointestinal discomfort, allergy, symptom aggravation and other adverse reactions, indicating that the product had low irritation and good safety.

[0130] 3.2 Effect of composition of Example 2 on KOOS symptom score

[0131] As shown in Table 2, for the symptom score, after 1 week of consuming the composition of Example 2, 79.17% of the subjects showed an increase compared to the baseline value, with an increase of 14.2%, and the difference was significant (P<0.05). At weeks 2 and 3, 83.33% and 87.50% of the subjects, respectively, showed an increase compared to the baseline value, and the amplitude further increased, by 17.7% and 21.1%, respectively, and the difference was significant (P<0.05); however, there was no significant difference between weeks 1, 2, and 3 (P>0.05).

[0132] In comparison with the KOOS score improvement threshold established by Larsen et al., the symptom score at week 3 increased by 15.37 points, reaching the clinical improvement threshold, indicating that the product was effective after consumption. At the same time, the score had exceeded the benchmark value (85.1 points), indicating that it had reached the level of normal people.

[0133] The composition of Example 2 used in this study added non-denatured type II collagen, hydrolyzed type II collagen, and other ingredients, activated the transforming growth factor-β / Smad signaling pathway related to cartilage synthesis, and promoted cartilage repair. The nutritional product used in this study may improve the stiffness, clicking, and other symptom scores of the subjects by repairing joint cartilage, maintaining the smoothness of the joint surface, and improving joint flexibility.

[0134] Table 2 Effect of the composition of Example 2 on the KOOS symptom score

[0135]

[0136] 3.3 Effect of the composition tablets of Example 2 on the KOOS pain score

[0137] As shown in Table 3, for the pain score, after 1 week of consuming the composition of Example 2, 75% of the subjects showed an increase compared to the baseline value, with an increase of 6.5%, and the difference was significant (P<0.05).

[0138] At week 2, 79.17% of the subjects showed an increase compared to the baseline value, with an increase of 8.6%, and the difference was significant (P<0.05); however, there was no significant difference between weeks 1 and 2 (P>0.05).

[0139] At week 3, 87.50% of the subjects showed an increase compared to the baseline value, and the amplitude further increased to 13.2%; and the difference between week 3 and week 1 was significant (P<0.05); however, there was no significant difference between weeks 2 and 3 (P>0.05).

[0140] Compared with the KOOS score improvement threshold established by Larsen et al., the pain score of the third week was improved by 10.98 points, reaching the clinical improvement threshold, indicating that the product was effective after being taken; at the same time, the score had exceeded the benchmark value (85.3 points), indicating that it had reached the level of normal people.

[0141] The composition of Example 2 used in the present study added non-denatured type II collagen, turmeric and other ingredients. Non-denatured type II collagen binds to the Peyer's lymph nodes in the intestinal tract, induces the production of specific regulatory T cells, activates the immune tolerance mechanism, and reduces the occurrence of inflammation [3] ; turmeric prevents NF-κB from entering the nucleus by inhibiting IKK, thereby reducing the production of inflammatory factors and alleviating joint inflammation [4] , and improving the pain score of the subjects.

[0142] Table 3 Effect of the composition of Example 2 on KOOS pain score

[0143]

[0144] 3.4 Effect of the composition of Example 2 on KOOS daily activity score

[0145] As can be seen from Table 4, compared with the baseline value, there was a certain improvement in the first, second and third weeks, but the differences were not significant (P>0.05), and the differences between the first, second and third weeks were also not significant (P>0.05). This may be due to the fact that the overall score at the baseline was already high, and the subsequent improvement space was limited, so no significant difference was shown.

[0146] Table 4 Effect of the composition of Example 2 on KOOS daily activity score

[0147]

[0148] 3.5 Effect of the composition of Example 2 on KOOS sports score

[0149] As can be seen from Table 5, for the sports score, after 1 week of use of the composition of Example 2, 62.5% of the subjects showed an improvement, with an improvement of 6.8%, and the difference was significant (P<0.05).

[0150] At the second week, compared with the baseline value, 66.67% of the subjects showed an improvement, with an improvement of 10.8%, and the difference was significant (P<0.05); however, the difference between the first and second weeks was not significant (P>0.05).

[0151] At the 3rd week, compared with the baseline value, 79.17% of the subjects had an increase, and the amplitude was further increased to 17.2%; and the difference between the 3rd week and the 1st week was significant (P<0.05); however, the difference between the 2nd week and the 3rd week was not significant (P>0.05).

[0152] Compared with the KOOS score improvement threshold established by Larsen et al., the sports score at the 3rd week was increased by 13.11 points, reaching the clinical improvement threshold, indicating that the product is effective after being taken; at the same time, the score has exceeded the benchmark value (70.9 points), indicating that it has reached the level of normal people.

[0153] As mentioned earlier, the composition of Example 2 used in this study improves the stiffness, clicking, pain, etc. of the knee joint of the subjects, improves the overall function of the knee joint of the subjects, and thus improves the sports ability of the knee joint of the subjects.

[0154] Table 5 Effect of the composition of Example 2 on the KOOS sports score

[0155]

[0156] 3.6 Effect of the composition of Example 2 on the KOOS life quality score

[0157] As can be seen from Table 6, for the life quality score, after 1 week of use of the composition of Example 2, compared with the baseline value, 66.67% of the subjects had an increase, with an increase of 14.2%, and the difference was significant (P<0.05).

[0158] At the 2nd week, compared with the baseline value, 70.83% of the subjects had an increase, with an increase of 24.2%, and the difference was significant (P<0.05); however, the difference between the 1st week and the 2nd week was not significant (P>0.05).

[0159] At the 3rd week, compared with the baseline value, 79.17% of the subjects had an increase, and the amplitude was further increased to 30.9%; and the difference between the 3rd week and the 1st week was significant (P<0.05); however, the difference between the 2nd week and the 3rd week was not significant (P>0.05).

[0160] Compared with the KOOS score improvement threshold established by Larsen et al., the life quality score at the 3rd week was increased by 13.11 points, reaching the clinical improvement threshold, indicating that the product is effective after being taken; at the same time, the score has exceeded the benchmark value (74.9 points), indicating that it has reached the level of normal people.

[0161] As mentioned above, the composition of Example 2 used in the present study improved the knee stiffness, clicking, pain, etc. of the subjects, improved the overall function of the knee of the subjects, and thus improved the life quality score of the subjects.

[0162] Table 6 Effect of the composition of Example 2 on KOOS life quality score

[0163]

[0164] 3.7 Effect of the composition of Example 2 on KOOS comprehensive score

[0165] As shown in Table 7, for the comprehensive score, after 1 week of using the composition of Example 2, 87.5% of the subjects showed an improvement compared to the baseline value, with an improvement of 7.1%, and the difference was significant (P<0.05).

[0166] At the 2nd week, 87.5% of the subjects still showed an improvement compared to the baseline value, and the improvement was further increased to 9.6%, and the difference was significant (P<0.05); however, there was no significant difference between the 1st week and the 2nd week (P>0.05).

[0167] At the 3rd week, all subjects showed an improvement compared to the baseline value, and the improvement was further increased to 12.7%; and the difference between the 3rd week and the baseline value and the 1st week was significant (P<0.05); however, there was no significant difference between the 2nd week and the 3rd week (P>0.05).

[0168] As mentioned above, the composition of Example 2 used in the present study improved the knee symptoms, pain, sports function, life quality, and comprehensive function of the subjects, and thus improved the comprehensive score of the subjects.

[0169] Table 7 Effect of the composition of Example 2 on KOOS comprehensive score

[0170]

[0171] 4 Research Conclusion

[0172] Based on the above research results, it is shown that the product can significantly improve the knee symptoms, pain, sports function, life quality, and comprehensive score after 1 week of consumption, among which the improvement rate of the comprehensive score is the highest, reaching 87.5%, and the improvement rate varies from 6.5% to 14.2%; with the extension of the consumption time of the product (1-3 weeks), the improvement rate and the improvement rate of each dimension (except for the daily activity function) gradually increase, especially at the 3rd week, the comprehensive score is improved by 100%, the pain score is improved by 13.2%, and the comprehensive score is improved by 12.7%.

[0173] The daily activity function showed no statistically significant improvement due to a higher baseline score, while the remaining dimensions (symptoms, pain, sports function, quality of life) and the overall score showed significant improvement. Therefore, it can be considered that the product has a certain improvement effect on the function of the knee joint in people with knee joint movement discomfort.

[0174] References

[0175] [1] Larsen P, Rathleff M S, Roos E M, et al. Knee injury and osteoarthritis outcome score (KOOS) – National record-based reference values [J]. Knee, The, 2023, 43(000):9. DOI:10.1016 / j.knee.2023.06.004.

[0176] [2] Wang Y, Wang G, Xu J L, et al. Reliability and validity evaluation of Chinese version of knee injury and osteoarthritis outcome score [J]. Guangdong Medicine, 2015, 36(16):4. DOI:CNKI:SUN:GAYX.0.2015-16-050.

[0177] [3] Gupta A, Maffulli N. Undenatured type II collagen for knee osteoarthritis. Ann Med. 2025;57(1):2493306. doi:10.1080 / 07853890.2025.2493306

[0178] [4] Swallow J, Seidler K, Barrow M. The mechanistic role of curcumin on matrix metalloproteinases in Osteoarthritis [J]. Fitoterapia, 2024, 174:105870.

[0179] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. Use of a composition in the preparation of a product for improving and / or treating sports osteoarthritis, the composition comprising a protein complex prepared from non-denatured type II collagen and a previously prepared CP complex prepared from curcumin and black pepper extract; the preparation method of the composition comprising the following steps: forming a CP complex; forming a non-denatured type II collagen solution with a pH value of 4-6; adding the CP complex to the non-denatured type II collagen solution at 35-39°C, slowly mixing to form a mixed solution; and freeze-drying the mixed solution at -10°C to -80°C to obtain the protein complex. wherein 2. The use according to claim 1, wherein the composition is prepared from 10.5-32 parts by weight of the CP complex and 2-64 parts by weight of the non-denatured type II collagen; and the CP complex is prepared from 10-30 parts by weight of curcumin and 0.5-2 parts by weight of black pepper extract.

3. The use according to claim 1, wherein the composition is prepared from 11-31 parts by weight of the CP complex and 3-63 parts by weight of the non-denatured type II collagen, and the CP complex is prepared from 11-29 parts by weight of curcumin and 0.6-1.8 parts by weight of black pepper extract.

4. The use according to claim 1, wherein the preparation method of the CP complex comprises the following steps: adding curcumin to a 4-10% ethanol solution to form a curcumin ethanol solution; adding black pepper extract to the curcumin ethanol solution and uniformly mixing for 3-5 min to form a CP mixture solution; and drying the CP mixture solution at 30-40°C to obtain the CP complex.

5. The use according to claim 1, wherein the CP complex is added to the non-denatured type II collagen solution and incubated for 30-40 min to form the mixed solution.

6. The use according to claim 5, wherein the pH value of the non-denatured type II collagen solution is 4.5-5.

5.

7. The use according to claim 5, wherein the concentration of the non-denatured type II collagen solution is 1.0-1.5 mg / mL.

8. A product containing the composition of any one of claims 1-7, wherein the product further comprises one or more of hydrolyzed type II collagen, vitamin B1, vitamin B2, vitamin D, milk mineral salt, maltitol powder, microcrystalline cellulose, magnesium stearate, silicon dioxide, a compound colorant, and a compound food additive.

9. The product according to claim 8, wherein the product is a tablet.

10. Use of the product of claim 8 or 9 in any one of a)-c): a. improving the gastrointestinal tract retention rate of non-denatured type II collagen; b. improving immunity; and c. improving osteoarthritis. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The product comprises the following components in mass parts: hydrolyzed type II collagen 18-36 parts, vitamin B1 0.4-1.2 parts, vitamin B2 0.2-0.8 parts, and vitamin D 2x10 -3 -4x10 -3 parts. ​ ​ ​ ​

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