Compositions for improving and / or treating exercise-induced osteoarthritis and methods of making and using the same

Abstract

This invention relates to compositions for improving and / or treating exercise-induced osteoarthritis, methods of preparation, and applications thereof. The compositions comprise protein complexes made from CP complexes and undenatured type-II collagen (UC-II), wherein the CP complex is made from curcumin and black pepper extract. The invention first forms a CP composition from curcumin and black pepper extract. This CP composition is then combined with undenatured type-II collagen to form a protein complex. This protein complex formation enhances the gastrointestinal retention rate of undenatured type-II collagen (UC-II), thereby improving the effect of undenatured type-II collagen (UC-II) through an immune-mediated oral tolerance mechanism.

Description

Technical Field

[0001] This invention relates to compositions for improving and / or treating exercise-induced osteoarthritis, methods for their preparation, and applications. Background Technology

[0002] Osteoarthritis of the joint (OA) is a common degenerative joint disease characterized by articular cartilage degeneration, inflammation, and pain, severely impacting patients' quality of life. Although its exact etiology is not fully understood, numerous studies have shown that the occurrence of OA is closely related to multiple risk factors and complex pathophysiological mechanisms, involving biomechanics, inflammation, and abnormal cellular metabolism. One of the pathophysiological features of OA is the elevated levels of pro-inflammatory cytokines (such as IL-1β, TNF-α, IL-6, and IL-17) in the synovium and cartilage. Non-denatured type II collagen can inhibit the production of these pro-inflammatory cytokines, thereby alleviating joint inflammation.

[0003] While existing treatments and nutritional supplements can alleviate symptoms of osteoarthritis to some extent, several shortcomings remain. First, the bioavailability of the active ingredients in many products is low, leading to unsatisfactory therapeutic effects. Second, existing compositions often lack multi-target integration mechanisms, failing to comprehensively regulate immunity and bone metabolism. Furthermore, current research largely focuses on the effects of single components, lacking in-depth exploration of the synergistic effects of multiple components. Particularly when using non-denatured type II collagen (such as UC-II) as a dietary supplement to improve and / or alleviate osteoarthritis, its efficacy is diminished by pepsin degradation during administration, as it operates through an immune-mediated oral tolerance mechanism, necessitating further improvements.

[0004] Therefore, how to develop a novel method to maintain the stability of non-denatured type II collagen (such as UC-II) in gastric juice, improve its immune oral tolerance, and effectively integrate the advantages of multiple components to enhance the effect of improving or / and treating osteoarthritis has become an urgent technical problem to be solved. Summary of the Invention

[0005] This invention provides compositions for improving and / or treating exercise-induced osteoarthritis, methods for their preparation, and applications.

[0006] To address the aforementioned technical problems, the present invention 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 undenatured 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 includes the following steps:

[0008] Formation of CP complex;

[0009] A non-denatured type II collagen solution is formed, wherein the pH value of the non-denatured type II collagen solution is 4-6;

[0010] The CP complex was added to the non-denatured type II collagen solution at 35-39°C and slowly mixed to form a mixed solution.

[0011] The mixed solution was freeze-dried at -10°C to -80°C to obtain the protein complex.

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

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

[0014] In this invention, curcumin can be turmeric or turmeric extract with a curcumin content of 5% or more. As long as it conforms to the formula composition, it can be calculated as curcumin. If turmeric or turmeric extract is used, its curcumin content is 5%, 6%, 7%, 8%, 15%, 30%, 50%, 70%, 80% or more.

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

[0016] In one embodiment of the present invention, the method for preparing the CP complex includes the following steps:

[0017] Curcumin is added to a 4-10% ethanol solution to form a curcumin ethanol solution;

[0018] Add black pepper extract to curcumin ethanol solution and mix evenly for 3-5 minutes to form CP mixture solution;

[0019] The CP mixture solution was dried at 30-40℃ to obtain the CP complex.

[0020] In one embodiment of the present invention, the CP complex is added to the non-denatured type II collagen solution and incubated for 30-40 min to prepare a mixed solution.

[0021] In one embodiment of the present invention, 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-denatured 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. Piperine's structural features include benzodioxane, N-acylpiperidine, and tertiary carboxamide.

[0030] In this invention, turmeric or turmeric extract can be used in the preparation of the composition. The amount added must meet the curcumin content requirements of this product. Curcumin is a natural polyphenolic compound extracted from the rhizome of turmeric, a plant in the ginger family. Figure 1 The diagram shows the three-dimensional spatial structure of the curcumin molecule. A represents the three-dimensional spatial structure of the curcumin molecule, and B represents the two-dimensional spatial structure of the curcumin molecule. Its chemical formula is C. 21 H 20 O6, with a molecular weight of 368.38 g / mol. Curcumin is a diarylheptane compound, with two ortho-substituted aryloxy groups linked by a seven-carbon chain (heptadienedione). Its structure contains multiple functional groups, including phenolic hydroxyl groups and β-diketone structures, which are key to its diverse biological activities.

[0031] Curcumin and piperine can form a stable complex, known as the CP complex, through non-covalent interactions such as hydrogen bonding, hydrophobic interactions, van der Waals forces, and π-π stacking. A homogeneous CP complex matrix is ​​formed by uniformly mixing curcumin and black pepper extract at the molecular level, providing a foundation for further and more comprehensive binding with undenatured type II collagen. Specifically, hydrogen bonds are formed between the amino groups of piperine and the phenolic hydroxyl groups of curcumin, while the hydrophobic interaction between them further enhances the stability of the complex. The theoretical adsorption energy of this complex is -12.6 kcal / mol, indicating that its formation process is exothermic. Electronegativity analysis shows that the electronegativity of the CP complex (4.312 eV) is lower than that of the individual components, indicating that there is charge transfer between curcumin and piperine, thus affecting the overall electronic structure of the complex. Furthermore, piperine not only acts as a solubilizer for curcumin but also enhances the binding affinity between curcumin and proteins by binding to allosteric sites, stabilizing their interaction.

[0032] Undenatured type-II collagen (UC-II) can self-assemble into nanofiber structures with diameters ranging from 50 to 200 nm under conditions of 37 °C 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 and has limited kinetic stability. At the physiological temperature of 37 °C, UC-II exhibits the fastest self-assembly rate, which is conducive to the formation of a dense network structure.

[0033] The formation of the protein complex may involve curcumin and piperine first forming a stable CP complex, which then binds to undenatured type-II collagen (UC-II). The main driving forces of the complexation include hydrophobic interactions and hydrogen bonding. The hydrophobic groups in the CP complex can bind to the hydrophobic regions exposed after the unwinding of UC-II collagen, while the amide and ether bonds of piperine and the hydroxyproline, lysine, and other residues of UC-II collagen can form a hydrogen bond network.

[0034] Although undenatured type-II collagen (UC-II) is close to its isoelectric point (Zeta potential of -5 to +5 mV) in the pH range of 4.5-5.5 and is therefore prone to aggregation, the CP complex interacts electrostatically with amino acid residues (such as lysine and glutamic acid) on the surface of undenatured type-II collagen through its phenolic hydroxyl and carbonyl groups. This interaction regulates the surface charge distribution, increases the absolute value of the Zeta potential, and thus inhibits aggregation and stabilizes the system.

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

[0036] In this invention, curcumin and piperine, as small molecules, form a stable complex with undenatured type-II collagen (UC-II). This complex, through cross-linking with UC-II, imparts high thermal stability to the collagen. Simultaneously, the complex physically protects the triple helix structure of collagen from attack by gastric acid and pepsin. This protection reduces collagen dissociation and degradation in the stomach, thereby improving the integrity of UC-II and allowing it to be absorbed into the intestines in a more intact form.

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

[0038] Advantages of this invention:

[0039] This invention first forms a CP composition by combining curcumin and black pepper extract. The CP composition then forms a protein complex with undenatured type-II collagen (UC-II). This protein complex enhances the stability of undenatured type-II collagen, thereby increasing its gastrointestinal digestibility and retention rate. This ensures that more intact undenatured type-II collagen enters the intestine and is fully absorbed, thus improving the effectiveness of undenatured type-II collagen (UC-II) through its immune-mediated oral tolerance mechanism.

[0040] Meanwhile, the protein complex of the present invention also contains hydrolyzed type II collagen, non-denatured type II collagen, B vitamins and vitamin D, etc., to improve calcium ion absorption and significantly improve and / or treat the effects of exercise-induced osteoarthritis.

[0041] The anti-inflammatory effect of curcumin in the composition of this invention 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. The addition of B vitamins and vitamin D enhances bone health. Overall, it achieves a synergistic effect of multiple targets, improves bioavailability, and ultimately improves joint function and quality of life for patients with sports osteoarthritis. Attached Figure Description

[0042] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0043] Figure 1This is a three-dimensional spatial structure diagram of the curcumin molecule of the present invention. A is the three-dimensional spatial structure of the curcumin molecule, and B is the two-dimensional structure of the curcumin molecule.

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

[0045] Figure 3 The ellipticity curves at 222 nm in circular dichroism spectroscopy are shown at various time points, where T: protein complex group; B: collagen binary complex group; C: control group. Detailed Implementation

[0046] Preferred embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. Before description, it should be understood that the terminology used in the specification and appended claims should not be construed as limited to its general and dictionary meaning, but rather should be interpreted based on the principle of allowing the inventors to appropriately define the terminology for the best interpretation, and based on its meaning and concept corresponding to the technical level of the invention. Therefore, the description herein is merely a preferred example for illustrative purposes and is not intended to limit the scope of the invention; thus, it should be understood that other equivalent implementations and modifications can be made without departing from the spirit and scope of the invention.

[0047] Example 1: Preparation of protein complexes

[0048] The protein complex in this embodiment consists of a CP complex and non-denatured type II collagen, wherein: the mass of the CP complex is 11.0 g, which is made from 10.0 g of curcumin and 1.0 g of black pepper extract; and the mass of the non-denatured type II collagen is 16.0 g.

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

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

[0051] Black pepper extract was added to curcumin ethanol solution and mixed evenly for 3-5 min to form a CP mixture solution with a mass concentration of 5 g / L.

[0052] The CP complex was prepared by drying the CP mixture solution at 30°C.

[0053] A 1.0 mg / mL undenatured type II collagen solution was prepared with a pH of 4.5-5.5. The concentration of undenatured type II collagen had a significant impact on the homogeneity of the complex, in order to balance the integrity of the fiber network and the dispersion stability.

[0054] The CP complex was added to a 35°C non-denatured type II collagen solution and incubated for 30 min. After mixing, a protein mixture solution was formed.

[0055] The protein mixture was freeze-dried at -10°C to -80°C to obtain the protein complex.

[0056] Example 2: Composition for treating exercise-induced osteoarthritis

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

[0058] The preparation method of this protein complex includes the following steps:

[0059] Preparation of CP complex: Curcumin was added to a 4% ethanol solution to form a curcumin ethanol solution;

[0060] Black pepper extract was added to curcumin ethanol solution and mixed evenly for 3-5 min to form a CP mixture solution with a mass concentration of 5 g / L.

[0061] The CP complex was prepared by drying the CP mixture solution at 30°C.

[0062] A 1.0 mg / mL undenatured type II collagen solution was prepared with a pH of 4.5-5.5. The concentration of undenatured type II collagen had a significant impact on the homogeneity of the complex, in order to balance the integrity of the fiber network and the dispersion stability.

[0063] The CP complex was added to a 35°C non-denatured type II collagen solution and incubated for 30 min. After mixing, a protein mixture solution was formed.

[0064] The protein mixture was freeze-dried at -10°C to -80°C to obtain the protein complex.

[0065] The composition for treating sports-related osteoarthritis in this embodiment comprises 27.0 g of the aforementioned protein complex, 24 g of hydrolyzed type II collagen, 0.5 g of vitamin B1, 0.3 g of vitamin B2, and 2.5 × 10 g of vitamin D. -3 The tablets are made by adding appropriate amounts of milk mineral salts, maltitol powder, microcrystalline cellulose, magnesium stearate, silicon dioxide, compound coloring agents and compound food additives, with each tablet containing 0.5g.

[0066] Example 3: A composition for treating exercise-induced osteoarthritis

[0067] The protein complex in this embodiment consists of a CP complex and non-denatured type II collagen, wherein:

[0068] The CP complex weighed 32g and was made from 30g of curcumin and 2g of black pepper extract; the non-denatured type II collagen weighed 64g.

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

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

[0071] Black pepper extract was added to curcumin ethanol solution and mixed evenly for 3-5 min to form a CP mixture solution with a mass concentration of 8 g / L.

[0072] The CP complex was obtained by drying the CP mixture solution at a temperature of 38°C.

[0073] A 1.2 mg / mL undenatured type II collagen solution was prepared with a pH of 4.5-5.5. The concentration of undenatured type II collagen had a significant impact on the homogeneity of the complex, in order to balance the integrity of the fiber network and the dispersion stability.

[0074] The CP complex was added to a non-denatured type II collagen solution at 38°C and incubated for 35 min. After mixing, a protein mixture solution was formed.

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

[0076] The composition for treating sports-related osteoarthritis of this embodiment comprises 96g of the aforementioned protein complex, 36g of hydrolyzed type II collagen, 1.2g of vitamin B1, 0.8g of vitamin B2, and 4×10g of vitamin D. -3 The tablets are made by adding appropriate amounts of milk mineral salts, maltitol powder, microcrystalline cellulose, magnesium stearate, silicon dioxide, compound coloring agents and compound food additives, with each tablet containing 0.5g.

[0077] Example 4: Composition for treating exercise-induced osteoarthritis

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

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

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

[0081] Black pepper extract was added to curcumin ethanol solution and mixed evenly for 3-5 min to form a CP mixture solution with a mass concentration of 10 g / L.

[0082] The CP complex was obtained by drying the CP mixture solution at a temperature of 30-40℃.

[0083] A 1.5 mg / mL undenatured type II collagen solution was prepared with a pH of 4.5-5.5. The concentration of undenatured type II collagen had a significant impact on the homogeneity of the complex, in order to balance the integrity of the fiber network and the dispersion stability.

[0084] The CP complex was added to a 39°C non-denatured type II collagen solution and incubated for 30 min. After mixing, a protein mixture solution was formed.

[0085] The protein mixture was freeze-dried at -10°C to -80°C to obtain the protein complex.

[0086] The composition for treating sports-related osteoarthritis in this embodiment comprises 12.5 g of the aforementioned protein complex, 18 g of hydrolyzed type II collagen, 0.4 g of vitamin B1, 0.2 g of vitamin B2, and 2 × 10 g of vitamin D. -3 The tablets are made by adding appropriate amounts of maltitol powder, microcrystalline cellulose, magnesium stearate, silicon dioxide, compound coloring agents and compound food additives, with each tablet containing 0.5g.

[0087] As a possible implementation, the protein combination can be combined with other nutrients to prepare other forms of nutritional supplements such as powders, liquids, jellies, and capsules.

[0088] Experimental Example 1: Protein complex improves the gastrointestinal retention rate of undenatured type II collagen.

[0089] 1 Research Methods

[0090] 1.1 Preparation of experimental samples

[0091] Protein complex group (T group): The composition prepared in Example 1 was used as the experimental sample and dissolved in 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 was adjusted to 5.0. 28 g of curcumin powder was added and mixed. The mixture was stirred at 500 rpm for 5 h, centrifuged at 25℃ and 8000×g for 10 min, and the supernatant was collected as the collagen binary complex sample.

[0093] Control group (Group C): Non-denatured type II collagen powder was used as the control sample, and was dissolved in 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, a 1.25-fold concentration of simulated gastric juice was preheated to 37°C and then added to the experimental sample solution at a 1:1 (v / v) ratio. The pH of the resulting mixture was adjusted to 1.2 with 1 mol / L HCl, and after the pH stabilized for 5 min, calcium chloride dihydrate solution was added. Then, porcine pepsin (final system concentration 2000 U / mL) was added, and the 1.25-fold concentration of simulated gastric juice was diluted to 1-fold concentration with deionized water. The mixture was incubated in a 37°C shaker for 0.5 h or 1.0 h. After the simulated digestion was completed, the sample was removed and immediately placed in a refrigerator to terminate the pepsin activity.

[0096] 1.3 Simulated intestinal digestion

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

[0098] 1.4 Circular dichroism analysis

[0099] The simulated gastric and intestinal digestive fluids prepared in steps 1.2 and 1.3 were diluted 20-fold and 10-fold, respectively, with phosphate buffer (0.1 mol / L, pH 7.0). The diluted solutions were placed in 1 mm quartz cuvettes, and their absorbance in the wavelength range of 190–260 nm was measured using a circular dichroism chromatograph.

[0100] 2. Research Results

[0101] like Figure 3 As shown, the structural changes of non-denatured type II collagen in various samples during gastrointestinal digestion were analyzed. The intensity of the positive absorption peak in circular dichroism spectroscopy 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, after 1 hour of gastric digestion, the ellipticity at 222 nm in the circular dichroism spectroscopy decreased rapidly. Furthermore, after entering the intestinal digestion stage, the triple helix structure of non-denatured type II collagen was rapidly degraded by 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 this invention exhibits slower digestion during gastric digestion, and its ellipticity is significantly higher than that of the collagen binary complex at 0.5 and 1 hour of intestinal digestion (P < 0.05). When the intestinal digestion time increases from 0.5 h to 6 h, the triple helix structure of undenatured type II collagen in the protein complex prepared in Example 1 of this invention degrades slowly, retaining approximately 24.23% after 6 h, and this percentage is significantly higher than that of the collagen binary complex at all time points (P < 0.05). Therefore, the protein complex prepared in Example 1 of this invention can better maintain the activity of undenatured type II collagen and exert a better biological effect in the intestine.

[0103] Experimental Example 2: Effect of the composition of Example 2 on joint function in individuals with knee discomfort.

[0104] 1. Research Objective

[0105] This experiment aims to evaluate the effect of the composition of Example 2 on improving knee function in runners with knee discomfort. The changes in subjects across five dimensions of knee symptoms, stiffness, pain, daily function, sports and recreational function, and quality of life were quantitatively analyzed using the Knee Injury and Osteoarthritis Outcome Score (KOOS) to provide a basis for the efficacy of the product.

[0106] 2 Research Plan

[0107] 2.1 Research Subjects

[0108] This study recruited and screened participants through questionnaires from August 8th to August 12th, 2025, ultimately including 24 eligible participants. Participants met the following criteria: 1. They had a long-term exercise habit of more than one year; 2. They currently experienced clearly defined knee discomfort caused by exercise; 3. They had not received any interventions or treatments related to bone and joint health; 4. They had no other underlying diseases; 5. They voluntarily participated in this study and signed an informed consent form.

[0109] 2.2 Experimental Design

[0110] This experiment employed a self-controlled pre- and post-treatment design, with all participants receiving nutritional supplements according to a standardized method. The effects were assessed by evaluating changes in indicators before and after supplementation.

[0111] Supplementation Method: This experiment used a standardized nutritional supplement. The supplement was the composition from Example 2 for treating exercise-induced osteoarthritis. Dosage: Subjects took two tablets daily before bedtime for three consecutive weeks.

[0112] Quality control: The 24 subjects were divided into 6 groups (4 people in each group). Each group was led by a research staff member and managed through a WeChat group. The staff member supervised and confirmed the subjects' medication daily, and the group leader recorded the daily medication adherence.

[0113] Observation indicators and assessment tools: The KOOS scale is used as the main assessment tool, which includes the following 5 dimensions:

[0114] Symptoms: Assess knee swelling, grinding / clicking, locking sensation, and flexion / extension ability (S1-) over the past week.

[0115] S7);

[0116] Pain: Assess the frequency of knee pain over the past week and the degree of pain during specific activities (such as twisting, climbing stairs, walking, etc.) (P1-P9).

[0117] Daily activities function: Assess the degree of difficulty in daily activities (such as climbing stairs, getting up, shopping, dressing and undressing, etc.) caused by knee problems in the past week (A1-A17).

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

[0119] Quality of life: Assess the level of attention paid to knee problems, lifestyle changes, impact on confidence, and overall inconvenience in the past 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). Reverse-scored items (such as S4 and S5) are converted according to the corresponding logic to calculate the total score for each dimension.

[0121] Referring to the KOOS scoring baseline and threshold established by Larsen et al. [2], the improvement of scores in each dimension after nutritional supplementation was evaluated. An improvement of ≥10 points in a certain dimension of KOOS indicates that the product has a significant effect on the improvement of that dimension, and a score ≥ the baseline value in a certain dimension of KOOS indicates that the dimension has reached the level of normal people.

[0122] Table 1. Benchmark values ​​and improvement thresholds for the five dimensions of the KOOS score.

[0123]

[0124] Data Collection: The questionnaire was followed up every 7 days during the experiment, for a total of 3 follow-ups. Staff members reminded participants to complete the questionnaire independently via WeChat group chat, without providing verbal guidance. The follow-up questionnaires were entered and collected using a questionnaire platform.

[0125] 2.3 Data processing: The scale scoring method was based on the study of Wang Ying et al. [1]. Statistical software was used for data analysis to calculate the baseline values ​​of the subjects before the experiment and the scores of each dimension of KOOS 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 be statistically significant.

[0126] 3. Research Results and Analysis:

[0127] 3.1 Subject compliance and adverse reactions

[0128] In this study, all 24 participants strictly adhered to the supplementary requirement of "taking 2 tablets before bedtime daily for three consecutive weeks." There were no cases of withdrawal due to poor adherence, and the participants completed the scale follow-up three times every seven days as planned. Overall, the adherence was good, providing a reliable guarantee for the accuracy and validity of the research data.

[0129] During the experiment, none of the 24 subjects experienced adverse reactions such as gastrointestinal discomfort, allergies, or worsening of symptoms, indicating that the product has low irritation and good safety.

[0130] 3.2 Effect of the composition used in Example 2 on KOOS symptom scores

[0131] As shown in Table 2, regarding symptom scores, after one week of consumption of the composition in Example 2, 79.17% of the subjects showed improvement compared to baseline, an increase of 14.2%, and the difference was statistically significant (P < 0.05). In the second and third weeks, 83.33% and 87.50% of the subjects showed improvement compared to baseline, respectively, with further increases in the percentages of improvement (17.7% and 21.1%, respectively), and the differences were statistically significant (P < 0.05). However, there was no statistically significant difference between the first, second, and third weeks (P > 0.05).

[0132] Compared with the KOOS score improvement threshold established by Larsen et al., the symptom score improved by 15.37 points in week 3, reaching the clinical improvement threshold, indicating that the product was effective; at the same time, the score exceeded the baseline value (85.1 points), indicating that it had reached the level of a normal person.

[0133] The composition used in Example 2 of this study, fortified with non-denatured type II collagen and hydrolyzed type II collagen, activates the transforming growth factor-β / Smad signaling pathway related to cartilage synthesis, promoting cartilage repair. The nutrients used in this study may improve the subjects' stiffness and clicking symptoms by repairing articular cartilage, maintaining smooth joint surfaces, and enhancing joint flexibility.

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

[0135]

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

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

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

[0139] In week 3, 87.50% of the subjects showed improvement compared to baseline, with the increase further to 13.2%; and the differences between week 3 and week 1 were statistically significant (P < 0.05); however, there were no statistically significant differences between week 2 and week 3 (P > 0.05).

[0140] Compared with the KOOS score improvement threshold established by Larsen et al., the pain score improved by 10.98 points in week 3, reaching the clinical improvement threshold, indicating that the product was effective; at the same time, the score exceeded the baseline value (85.3 points), indicating that it had reached the level of a normal person.

[0141] The composition used in Example 2 of this study was supplemented with non-denatured type II collagen, turmeric, and other ingredients. Non-denatured type II collagen induces the production of specific regulatory T cells by binding to Pear lymph nodes in the intestine, thereby activating the immune tolerance mechanism and reducing the occurrence of inflammation. [3] Turmeric, on the other hand, reduces joint inflammation by inhibiting IKK, thereby preventing NF-κB from entering the cell nucleus and reducing the production of inflammatory factors. [4] It improved the pain scores of the subjects.

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

[0143]

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

[0145] As shown in Table 4, there were some improvements in weeks 1, 2, and 3 compared to the baseline values; however, the differences were not statistically significant (P > 0.05), and there were no significant differences among weeks 1, 2, and 3 (P > 0.05). This may be because the overall score of athletic ability was already high at baseline, leaving limited room for further improvement, hence the lack of significant differences.

[0146] Table 4. Effect of the composition used in Example 2 on KOOS daily activity scores.

[0147]

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

[0149] As shown in Table 5, for sports performance scores, after one week of use of the composition in Example 2, 62.5% of the subjects showed an improvement compared to the baseline value, with an improvement of 6.8%, and the difference was statistically significant (P < 0.05).

[0150] In week 2, 66.67% of the subjects showed improvement compared to baseline, an increase of 10.8%, and the difference was statistically significant (P < 0.05); however, there was no statistically significant difference between week 1 and week 2 (P > 0.05).

[0151] In week 3, 79.17% of the subjects showed an improvement compared to baseline, with the increase further to 17.2%; and the differences between week 3 and week 1 were statistically significant (P < 0.05); however, there were no statistically significant differences between week 2 and week 3 (P > 0.05).

[0152] Compared with the KOOS score improvement threshold established by Larsen et al., the physical activity score improved by 13.11 points in week 3, reaching the clinical improvement threshold, indicating that the product was effective; at the same time, the score exceeded the baseline value (70.9 points), indicating that it had reached the level of a normal person.

[0153] As previously stated, the composition of Example 2 used in this study improved knee stiffness, clicking, and pain in the subjects, improved the overall function of the subjects' knee joints, and thus enhanced the subjects' knee joint mobility.

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

[0155]

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

[0157] As shown in Table 6, for quality of life scores, after one week of use of the composition in Example 2, 66.67% of the subjects showed an improvement compared to the baseline value, with an improvement of 14.2%, and the difference was statistically significant (P < 0.05).

[0158] In week 2, 70.83% of the subjects showed an improvement compared to baseline, an increase of 24.2%, and the difference was statistically significant (P < 0.05); however, there was no statistically significant difference between week 1 and week 2 (P > 0.05).

[0159] In week 3, 79.17% of the subjects showed improvement compared to baseline, with the increase further to 30.9%; and the differences between week 3 and week 1 were statistically significant (P < 0.05); however, there were no statistically significant differences between week 2 and week 3 (P > 0.05).

[0160] Compared with the KOOS score improvement threshold established by Larsen et al., the quality of life score improved by 13.11 points in week 3, reaching the clinical improvement threshold, indicating that the product was effective; at the same time, the score exceeded the baseline value (74.9 points), indicating that it had reached the level of a normal person.

[0161] As previously stated, the composition of Example 2 used in this study improved subjects' knee stiffness, clicking, and pain, and improved the overall function of the subjects' knee joints, thereby improving the subjects' quality of life scores.

[0162] Table 6. Effect of the composition used in Example 2 on KOOS quality of life scores.

[0163]

[0164] 3.7 Effect of the composition used in Example 2 on the KOOS overall score

[0165] As shown in Table 7, for the overall score, after one week of use of the composition in 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 statistically significant (P < 0.05).

[0166] In week 2, 87.5% of the subjects showed an improvement compared to baseline, with the increase reaching 9.6%, and the difference was statistically significant (P < 0.05); however, there was no statistically significant difference between week 1 and week 2 (P > 0.05).

[0167] In week 3, all subjects showed an improvement compared to baseline, with the increase reaching 12.7%; and the differences between week 3 and week 1 were statistically significant (P < 0.05); however, there were no statistically significant differences between week 2 and week 3 (P > 0.05).

[0168] As previously stated, the composition of Example 2 used in this study improved the subjects' knee joint symptoms, pain, physical activity function, and quality of life across four dimensions, comprehensively improving the subjects' overall knee joint function and thus enhancing their overall score.

[0169] Table 7. Effect of the composition used in Example 2 on the KOOS overall score.

[0170]

[0171] 4. Research Conclusions

[0172] Based on the above research results, this product can significantly improve knee joint symptoms, pain, sports function, quality of life, and overall scores within one week of consumption. The overall score improvement rate is the highest, reaching 87.5%, with an improvement range of 6.5% to 14.2%. As the consumption period of this product is extended (1-3 weeks), the improvement rate and improvement range of each dimension (except for daily activity function) show a gradual upward trend. In particular, the overall score achieves 100% improvement at 3 weeks, with a 13.2% improvement in pain score and a 12.7% improvement in overall score.

[0173] Daily activity function did not show statistically significant improvement due to a high baseline score, while other dimensions (symptoms, pain, athletic ability, and quality of life) and the overall score all showed significant improvement. Therefore, it can be concluded that this product has a certain improving effect on knee function in individuals with knee joint discomfort.

[0174] References

[0175] [1] Larsen P, Rathleff MS, Roos EM, 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 Ying, Wang Gang, Xu Jingli, et al. Reliability and validity evaluation of Chinese version of the knee joint injury and osteoarthritis outcome score[J]. Guangdong Medical Journal, 2015, 36(16):4. DOI:CNKI:SUN:GAYX.0.2015-16-050.

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

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

[0179] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The use of the composition in the preparation of a product for improving the gastrointestinal digestibility retention of undenatured type II collagen, the composition comprising a protein complex prepared from undenatured type II collagen and a pre-prepared CP complex, the CP complex being prepared from curcumin and black pepper extract; in, The method for preparing the composition includes the following steps: Formation of CP complex; A non-denatured type II collagen solution is formed, wherein the pH value of the non-denatured type II collagen solution is 4-6; The CP complex was added to the non-denatured type II collagen solution at 35-39°C and slowly mixed to form a mixed solution. The mixed solution was freeze-dried at -10°C to -80°C to obtain the protein complex; The method for preparing the CP complex includes the following steps: Curcumin is added to a 4-10% ethanol solution to form a curcumin ethanol solution; Add black pepper extract to curcumin ethanol solution and mix evenly for 3-5 minutes to form CP mixture solution; The CP mixture solution was dried at 30-40℃ to obtain the CP complex.

2. The use as described in claim 1, characterized in that, The composition is made from the following raw materials in parts by weight: 10.5-32 parts of CP complex and 2-64 parts of non-denatured type II collagen; The CP complex is made from the following raw materials in parts by weight: 10-30 parts curcumin and 0.5-2 parts black pepper extract.

3. The use as described in claim 1, characterized in that, The composition is made from the following active ingredients in parts by weight: 11-31 parts of CP complex and 3-63 parts of non-denatured type II collagen, wherein the CP complex is made from the following raw materials in parts by weight: 11-29 parts of curcumin and 0.6-1.8 parts of black pepper extract.

4. The use as described in claim 1, characterized in that, The CP complex was added to the non-denatured type II collagen solution and incubated for 30-40 minutes to prepare a mixed solution.

5. The use as described in claim 1, characterized in that, The pH value of the non-denatured type II collagen solution is 4.5-5.

5.

6. The use as described in claim 1, characterized in that, The concentration of the non-denatured type II collagen solution is 1.0-1.5 mg / mL.

7. A product comprising the composition used in any of claims 1-6, characterized in that, The product also includes 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.

8. The product as described in claim 7, characterized in that, The product comprises the following components in parts by weight: 18-36 parts hydrolyzed type II collagen, 0.4-1.2 parts vitamin B1, 0.2-0.8 parts vitamin B2, and 2×10 parts vitamin D. -3 -4×10 -3 share.

9. The use of the product of claim 7 or 8 for non-disease treatment purposes in improving the gastrointestinal digestibility retention of non-denatured type II collagen.

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