Imitated type II collagen amino acid and plant extract composition for promoting joint health and application of imitated type II collagen amino acid and plant extract composition

Abstract

The present disclosure relates to amino acid and plant extract compositions and uses thereof, and also to joint care compositions comprising the amino acid and plant extract. The composition disclosed by the invention can increase bone hardness, prevent inflammatory response, relieve arthralgia and / or improve joint flexibility, so that systematic support is provided for bone and joint health.

Description

Technical Field

[0001] This disclosure relates to compositions of amino acids and plant extracts and their applications, and also to compositions for joint health care comprising the amino acids and plant extracts. Background Technology

[0002] Collagen is an important structural protein in human connective tissue, widely found in tissues such as skin, cartilage, bone, and joints. Type II collagen plays a crucial role in maintaining the structure and function of cartilage tissue, supporting joint elasticity and stability. With age, the body's natural collagen synthesis gradually declines. Reduced collagen in bone tissue weakens the bone matrix, increasing the risk of bone loss and fractures. Therefore, supplementing or promoting collagen production has become an important means of delaying skin aging and maintaining bone health.

[0003] Currently, most collagen supplements are derived from animal tissues (such as cow, pig, fish skin, or fish scales), and are hydrolyzed to obtain small molecule peptides to improve bioavailability. However, these products have several limitations, including potential allergic reactions, the risk of pathogen transmission, and the fact that some consumers cannot accept animal-derived ingredients due to subjective preferences, ethical considerations, or dietary habits. Furthermore, from an environmental and sustainable development perspective, over-reliance on animal sources also presents certain limitations.

[0004] To address these issues, the concept of biomimetic collagen has been proposed in recent years. Biomimetic collagen uses plant-based biomimetic technology to mimic the amino acid composition of human collagen, providing a purely plant-based, sustainable, and allergy-free collagen option.

[0005] Bone and joint health is fundamental to the normal functioning of the human musculoskeletal system, and its maintenance depends on the balance of bone tissue metabolism, the integrity of cartilage, and the lubrication of the joint cavity. However, with the increasing trend of national fitness and population aging, joint problems are no longer limited to the elderly but are gradually affecting younger people and those who exercise more. Young people, due to prolonged desk work, lack of sunlight, and insufficient calcium intake, experience decreased bone density and reduced bone hardness in the knee, hip, and spinal joints. Fitness enthusiasts are prone to micro-injuries to the cartilage in the knee, hip, and ankle joints due to strenuous exercise. Without sufficient repair, they are more likely to experience joint stiffness and pain prematurely. In the elderly, due to decreased osteoblast activity and increased osteoclast activity, bone remodeling imbalances are prone to occur, leading to bone loss. At the same time, reduced cartilage matrix synthesis and narrowing of the joint cavity result in joint stiffness, pain, and limited mobility.

[0006] Therefore, there is an urgent need to develop a joint health composition that can increase bone density, prevent inflammatory responses, relieve joint pain, and / or improve joint flexibility, thereby providing systemic support for bone and joint health. Summary of the Invention

[0007] In view of the aforementioned problems and needs in the art, this disclosure provides an amino acid composition that mimics type II human collagen. This amino acid composition, when combined with berry extracts (such as elderberry extract) or turmeric plant extracts (such as turmeric extract), can synergistically increase bone density, prevent inflammatory responses, relieve joint pain, and / or improve joint flexibility.

[0008] In a first aspect, this disclosure provides an amino acid composition, wherein, by weight percentage, the amino acid composition comprises the following components:

[0009] 20.0%-28.0% glycine or its salt, 13.0%-18.0% proline or its salt, 6.0%-10.0% alanine or its salt, 4.0%-7.0% aspartic acid or its salt, 4.0%-7.0% glutamine or its salt, 3.5%-6.0% glutamic acid or its salt, 3.5%-5.0% arginine or its salt, 3.0%-4.5% lysine or its salt, 3.0%-4.5% serine or its salt, 2. 5%-4.0% leucine or its salt, 2.0%-3.2% threonine or its salt, 1.8%-2.8% valine or its salt, 1.5%-2.5% methionine or its salt, 1.5%-2.5% isoleucine or its salt, 1.0%-2.0% phenylalanine or its salt, 0.4%-0.8% tyrosine or its salt, 0.3%-0.6% histidine or its salt, 0.3%-0.6% tryptophan or its salt, and optional additives.

[0010] In one embodiment, the additive comprises 1.5%-6.0%, preferably 3.0%-5.0% of maltodextrin and / or 2.0%-4.0% of vitamin C, relative to the total weight of the amino acid composition.

[0011] In one embodiment, the amino acid composition comprises, by weight percentage: 24.03% glycine or a salt thereof, 15.98% proline or a salt thereof, 7.93% alanine or a salt thereof, 5.56% aspartic acid or a salt thereof, 5.44% glutamine or a salt thereof, 4.68% glutamic acid or a salt thereof, 4.26% arginine or a salt thereof, 3.97% lysine or a salt thereof, 3.96% serine or a salt thereof, 3.31% leucine or a salt thereof, 2.60% threonine or a salt thereof, 2.25% valine or a salt thereof, 2.07% methionine or a salt thereof, 2.01% isoleucine or a salt thereof, 1.48% phenylalanine or a salt thereof, 0.59% tyrosine or a salt thereof, 0.47% histidine or a salt thereof, 0.41% tryptophan or a salt thereof, 2.25% maltodextrin, and 3.00% vitamin C.

[0012] In one embodiment, the amino acid composition comprises, by weight percentage: 24.03% glycine or a salt thereof, 15.98% proline or a salt thereof, 7.93% alanine or a salt thereof, 5.56% aspartic acid or a salt thereof, 5.44% glutamine or a salt thereof, 4.68% glutamic acid or a salt thereof, 4.26% arginine or a salt thereof, 3.97% lysine or a salt thereof, 3.96% serine or a salt thereof, 3.31% leucine or a salt thereof, 2.60% threonine or a salt thereof, 2.25% valine or a salt thereof, 2.07% methionine or a salt thereof, 2.01% isoleucine or a salt thereof, 1.48% phenylalanine or a salt thereof, 0.59% tyrosine or a salt thereof, 0.47% histidine or a salt thereof, 0.41% tryptophan or a salt thereof, 3.25% maltodextrin, and 3.00% vitamin C.

[0013] In one embodiment, the amino acid composition comprises, by weight percentage: 24.03% glycine or a salt thereof, 15.98% proline or a salt thereof, 7.93% alanine or a salt thereof, 5.56% aspartic acid or a salt thereof, 5.44% glutamine or a salt thereof, 4.68% glutamic acid or a salt thereof, 4.26% arginine or a salt thereof, 3.97% lysine or a salt thereof, 3.96% serine or a salt thereof, 3.31% leucine or a salt thereof, 2.60% threonine or a salt thereof, 2.25% valine or a salt thereof, 2.07% methionine or a salt thereof, 2.01% isoleucine or a salt thereof, 1.48% phenylalanine or a salt thereof, 0.59% tyrosine or a salt thereof, 0.47% histidine or a salt thereof, 0.41% tryptophan or a salt thereof, 5.00% maltodextrin, and 3.00% vitamin C.

[0014] In one embodiment, each amino acid component in the amino acid composition is of industrial origin, of renewable plant origin, or prepared from glucose by fermentation.

[0015] In a second aspect, this disclosure provides the use of a combination of the amino acid composition according to the first aspect with a berry extract in the preparation of a composition for increasing bone density.

[0016] In one embodiment, the berry extract is elderberry extract.

[0017] In one embodiment, the composition is used to increase bone mineral density.

[0018] In one embodiment, the composition is used to promote osteoblast differentiation.

[0019] In one embodiment, the composition is used to inhibit osteoclast differentiation.

[0020] In a third aspect, this disclosure provides the use of the combination of the amino acid composition according to the first aspect with berry extract in the preparation of a composition for preventing inflammatory responses.

[0021] In one embodiment, the berry extract is elderberry extract.

[0022] In one embodiment, the composition is used to reduce inflammatory factor levels. In another embodiment, the composition is used to reduce TNF-α, IL-1β, and iNOS levels.

[0023] In one embodiment, the composition is used to prevent inflammation of chondrocytes.

[0024] In a fourth aspect, this disclosure provides the use of the combination of the amino acid composition according to the first aspect with a turmeric plant extract in the preparation of a composition for relieving joint pain.

[0025] In one embodiment, the turmeric plant extract is a turmeric extract.

[0026] In one embodiment, the composition is used to prevent joint wear.

[0027] In one embodiment, the composition is used to promote the secretion of cartilage collagen.

[0028] In one embodiment, the composition is used to promote the secretion of glucosamine.

[0029] In a fifth aspect, this disclosure provides the use of the combination of the amino acid composition according to the first aspect with a turmeric plant extract in the preparation of a composition for improving joint flexibility.

[0030] In one embodiment, the turmeric plant extract is a turmeric extract.

[0031] In one embodiment, the composition is used to improve joint stiffness.

[0032] In one embodiment, the composition is used to promote the secretion of cartilage collagen.

[0033] In one embodiment, the composition is used to promote the secretion of glucosamine.

[0034] In one embodiment, the composition comprises a berry extract, wherein the berry extract comprises 2.0%-3.5% by weight, based on the total weight of the amino acid composition and the berry extract as 100%.

[0035] In one embodiment, the composition comprises a turmeric plant extract, wherein the turmeric plant extract comprises 0.5% to 3.0% by weight, based on the total weight of the amino acid composition and the turmeric plant extract as 100%.

[0036] In one embodiment, the composition comprises a berry extract and a turmeric plant extract, wherein the berry extract comprises 2.0%-3.5% by weight and the turmeric plant extract comprises 0.5%-3.0% by weight, based on the total weight of the amino acid composition, the berry extract, and the turmeric plant extract as 100%.

[0037] In one implementation, the berry extract is derived from the fruit of the black elderberry (Sambucus nigra).

[0038] In one embodiment, the turmeric plant extract is derived from the rhizome of turmeric (Curcuma longa).

[0039] In one embodiment, the composition comprises a food-grade excipient.

[0040] In one implementation, food-grade excipients include, but are not limited to: carriers / diluents such as maltodextrin, starch, lactose, glucose, fructose, mannitol, sorbitol, and dietary fiber; binders such as gum arabic, gelatin, pectin, hydroxypropyl methylcellulose (HPMC), and guar gum; disintegrants (for tablets / capsules) such as croscarmellose sodium, starch derivatives, and microcrystalline cellulose; lubricants / anti-caking agents such as magnesium stearate, silica, and talc; and coating agents (such as...). Tablets or granules): such as hydroxypropyl methylcellulose, polyvinyl alcohol, shellac; sweeteners / flavoring agents: such as xylitol, erythritol, steviol glycosides, monk fruit extract, honey powder; flavorings / fragrances: such as lemon powder, orange juice powder, vanilla powder; coloring agents (natural sources): such as beet red, chlorophyll, curcumin, carotene; anti-caking agents: such as tricalcium phosphate, calcium carbonate, silicon dioxide; beverage bases (such as instant powder, functional beverage type): such as whey protein, plant protein, fruit juice powder, coconut powder, oat powder.

[0041] In one implementation, food-grade excipients are food-grade carriers, binders, anti-caking agents, and / or natural sweeteners.

[0042] In one embodiment, the composition is a powder, tablet, capsule, soft capsule, granule, oral liquid, pill, or suspension, preferably a powder.

[0043] The amino acid compositions of the present invention, and compositions comprising them, have the following advantages:

[0044] The amino acid composition of the present invention can increase bone density, prevent inflammatory response, relieve joint pain, and improve joint flexibility.

[0045] The amino acid composition of the present invention can activate the expression of genes related to collagen synthesis in chondrocytes and promote the secretion of collagen, chondroitin and hyaluronic acid in cartilage.

[0046] The amino acid composition of the present invention can inhibit the production of nitric oxide (NO) by inflammatory macrophages, reduce the secretion of pro-inflammatory substances, and lower the level of inflammatory factors, thereby alleviating joint discomfort and protecting joint tissues.

[0047] The amino acid composition of the present invention can inhibit cartilage degeneration, significantly reduce the inflammatory response of chondrocytes, and inhibit the degenerative process of articular cartilage.

[0048] The amino acid composition of the present invention can promote osteoblast differentiation, enhance bone formation, and inhibit osteoclast differentiation and maturation, thus preventing osteoporosis.

[0049] The amino acid composition of the present invention can be used as a non-therapeutic health product, food product or food additive to increase bone density, prevent inflammatory response, relieve joint pain and improve joint flexibility. In other words, it can also treat and prevent osteoporosis, treat arthritis, relieve joint pain and improve limited mobility caused by joint problems. Attached Figure Description

[0050] Figure 1 The effect of sample 1 on the expression level of collagen-related genes in chondrocytes is shown.

[0051] Figure 2A The expression of glucosamine (containing chondroitin and hyaluronic acid) in chondrocytes was shown after Alcian blue staining.

[0052] Figure 2B It shows Figure 2A Quantitative analysis of glucosamine expression in chondrocytes.

[0053] Figure 3 The comparison of nitric oxide (NO) production in different treatment groups in an LPS-induced macrophage model is shown.

[0054] Figure 4 The results of qPCR experiments are shown, comparing the gene expression levels of multiple inflammatory factors in chondrocytes between different treatment groups.

[0055] Figure 5A The experimental results of the effect of Sample 1 on osteogenic differentiation of mesenchymal stem cells are shown, including the results of Alizarin Red S staining of cells in the control group and the experimental group (Sample 1).

[0056] Figure 5B It shows Figure 5A Quantitative analysis of osteoblast differentiation.

[0057] Figure 6 The inhibitory effect of sample 1 on osteoclast differentiation and maturation is shown, including fluorescence microscopy observations of the control group and the experimental group (sample 1).

[0058] Figure 7 The T-values ​​of the subject's spine are shown after taking sample 2 for 12 weeks.

[0059] Figure 8 The Z-score of the subject's spine is shown after taking sample 2 for 12 weeks.

[0060] Figure 9 The study showed the serum TNF-α levels in subjects after taking sample 2 for 12 weeks.

[0061] Figure 10 The WOMAC scores of the subjects were shown after taking sample 3 for 12 weeks. Detailed Implementation

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0063] In this document, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”).

[0064] The present disclosure will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the disclosure. Experimental methods in the embodiments that do not specify specific conditions are performed under conventional conditions known in the art or under conditions recommended by the manufacturer.

[0065] Example 1: Preparation of a composition of type II collagen-like amino acids and plant extracts

[0066] The following ingredients, by weight percentage: 24.03% glycine, 15.98% L-proline, 7.93% L-alanine, 5.56% L-aspartic acid, 5.44% L-glutamine, 4.68% L-glutamic acid, 4.26% L-arginine, 3.97% L-lysine hydrochloride, 3.96% L-serine, 3.31% L-leucine, 2.60% L-threonine, 2.25% L-valine, 2.07% L-methionine, 2.01% L-isoleucine, 1.48% L-phenylalanine, 0.59% L-tyrosine, 0.47% L-histidine, 0.41% L-tryptophan, and 2.75% elderberry extract (from black elderberry (Sambucus)). The extract was obtained by fruit extraction of turmeric (available from Hunan Huacheng Biotechnology Co., Ltd.), 1.00% turmeric extract (obtained from the rhizome extraction of turmeric (Curcuma longa) available from ARJUNA NATURAL PRIVATE LTD), 2.25% maltodextrin, and 3.00% vitamin C were added to a mixer and mixed to obtain a preliminary mixture. The preliminary mixture was sieved through a sieve (40 mesh) to obtain an intermediate mixture with uniform particle size. The intermediate mixture was homogenized (homogenizer: Boang, G560 (2-434437)) to obtain a homogeneous mixture, namely the type II collagen amino acid and plant extract composition of Example 1 (sample 1).

[0067] Example 2: Promoting collagen secretion by chondrocytes

[0068] Sample 1 obtained in Example 1 was used in chondrocyte cell line experiments to evaluate its effect on collagen gene expression.

[0069] Specifically, the mouse cartilage precursor cell line ATDC5 (Sigma, 99072806) was used at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 100 cells / well in 24-well culture plates and cultured in DMEM / F12 medium (Gibco, 3086139) supplemented with 10% FBS (Gibco, M3009125RP) and 1% antibiotic AA (Simply, CC501-0100) for 24 hours at 37°C in a 5% CO2 incubator.

[0070] Subsequently, the test compositions were added to the culture medium separately, and the groups were as follows:

[0071] Control group: blank culture medium

[0072] Experimental group: Sample 1 with a concentration of 0.0625 mg / mL

[0073] After culturing cells for another 48 hours, the culture medium was discarded, and the cells were washed with 1×DPBS. 600 μL of LRB Buffer (Geneaid, RB300) was added to each well to lyse the cells and extract total RNA. The obtained RNA was used for subsequent qPCR detection to evaluate the expression levels of collagen-related genes. Experimental data were statistically analyzed using Student's-test in Excel (** indicates P < 0.01 relative to the control group, *** indicates P < 0.001 relative to the control group).

[0074] The primer sequences for each gene are as follows:

[0075]

[0076] Collagen, as an important structural component of articular cartilage, can enhance the toughness and elasticity of cartilage, reduce friction on the cartilage surface during exercise, slow down the vibration and impact caused by exercise, thereby further improving joint function, significantly enhancing joint flexibility, and preventing joint wear and tear, thus relieving joint pain.

[0077] like Figure 1 As shown, the experimental results indicate that Sample 1 can upregulate the expression levels of type II collagen (Collagen II) and other collagen-related genes (CollagenIXA2, CollagenIXA3, and Collagen XIA2). Specifically, compared to the control group (set as 100%), the relative expression levels of type II collagen were significantly upregulated by 49.8%, Collagen IXA2 by 22.6%, CollagenIXA3 by 33.8%, and Collagen XIA2 by 79.7%. These results suggest that Sample 1 can promote collagen secretion from chondrocytes, significantly improving cartilage matrix synthesis, promoting joint repair, and maintaining joint health.

[0078] Example 3: Promoting the secretion of glucosamine by chondrocytes

[0079] Sample 1 obtained in Example 1 was used in chondrocyte experiments to evaluate its effect on glucosaminoglycan (GAG) secretion.

[0080] Specifically, the human immortalized chondrocyte cell line C28 / I2 (Merck, SC043) was used at a concentration of 1×10⁻⁶. 5Cells were seeded at a density of 100 cells / well in 24-well culture plates and cultured in DMEM / F12 medium (Gibco, 3086139) supplemented with 10% FBS (Gibco, M3009125RP) and 1% antibiotic AA (Simply, CC501-0100) for 24 hours at 37°C in a 5% CO2 incubator.

[0081] Subsequently, the samples to be tested were added to the culture medium, and the groups were as follows:

[0082] Control group: blank culture medium

[0083] Experimental group: Sample 1 with a concentration of 0.0625%

[0084] Continue culturing for 48 hours, remove the culture medium and wash three times with 1×PBS, then add 0.5 mL of 10% paraformaldehyde to each well and fix at room temperature for 30 minutes. Discard the fixative and wash three more times with 1×PBS. Subsequently, stain with 0.5 mL of 1% Alcian Blue solution (pH = 2.5; Sigma, A5268-10G) prepared with 0.1 M HCl, and react overnight at room temperature.

[0085] After staining, the sample was washed three times with 1×PBS, and the blue signal of Alcian Blue bound to glucosamine was observed under a microscope (ZEISS Axiovert, ATCID00055). Further, 1 mL of 6M Guanidine-HCl (Sigma, G4505-25G) was added to each well and incubated at room temperature for 10 minutes to dissolve the dye. Then, 200 μL was transferred to a 96-well plate, and the absorbance at 620 nm was measured using a microplate reader (BIOTECH, Epoch 1703289). Experimental data were statistically analyzed using Student's-test in Excel (* indicates P < 0.05 relative to the control group).

[0086] Glucosamine is an important component of synovial fluid and cartilage matrix. The increase of glucosamine not only helps to strengthen the structure of cartilage matrix, but also enhances the lubrication function of synovial fluid in the joint cavity, thereby effectively reducing friction during joint movement, improving joint smoothness, and thus improving joint flexibility and preventing joint wear.

[0087] like Figure 2A Experimental results showed that after co-culturing chondrocytes with sample 1, the GAG ​​staining signal was significantly enhanced, indicating that sample 1 can promote the secretion of glucosamine by chondrocytes. Figure 2BAs shown, compared with the control group, the content of glucosamine secreted by chondrocytes increased by 5.3%, indicating that sample 1 can promote the secretion of glucosamine by chondrocytes, thereby effectively preventing joint wear and tear, relieving joint pain, and improving joint flexibility by increasing joint lubrication.

[0088] Example 4: Inhibiting nitric oxide (NO) production by inflammatory macrophages

[0089] Sample 1 obtained in Example 1 was used in macrophage experiments to evaluate its regulatory effect on inflammation-related nitric oxide (NO) production.

[0090] Specifically, the mouse macrophage cell line RAW264.7 (ATCC, TIB-71) was selected and cultured in DMEM medium (Gibco, 3124477) supplemented with 10% FBS (Gibco, M3009125RP) and 1% antibiotic AA (Simply, CC501-0100). Cells were cultured at a density of 1 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 100 cells / well in 96-well culture plates, with 200 μL of culture medium added to each well, and incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0091] After the culture was completed, the culture medium was discarded and replaced with FBS-free medium, and the media were divided into the following groups:

[0092] Control group: 500 ng / mL lipopolysaccharide (LPS; Sigma, SI-L2880-25 mg) was added.

[0093] Experimental group: 500 ng / mL LPS and 2 mg / mL sample 1

[0094] After 24 hours of incubation, 150 μL of culture supernatant was removed from each well and transferred to a new 96-well plate, each well pre-contained with 130 μL of sterile distilled water. Then, 20 μL of Griess reagent (a 1:1 mixture of reagent A and reagent B; Invitrogen, G7921) was added to each well and incubated in the dark for 30 minutes. Afterward, the absorbance (OD) was measured at 548 nm using a microplate reader (BIOTECH, Epoch 1703289). A higher OD value indicates a higher NO concentration. Experimental data were statistically analyzed using Student's t-test in Excel (* indicates P < 0.05 relative to the control group).

[0095] In an inflammatory state, macrophages produce nitric oxide, which stimulates other immune cells to further participate in the inflammatory response, thereby exacerbating the inflammatory process and leading to joint damage and degeneration.

[0096] like Figure 3As shown, the experimental results indicate that, compared with the control group containing only LPS, the treatment group with added sample 1 can effectively reduce the nitric oxide produced by macrophages by 30%, proving that the composition can effectively inhibit the release of nitric oxide by macrophages under inflammatory conditions, thereby preventing inflammatory responses.

[0097] Example 5: Reducing inflammatory response in chondrocytes

[0098] Sample 1 obtained in Example 1 was used in a chondrocyte inflammation model experiment to evaluate its regulatory effect on the expression of inflammation-related factors.

[0099] Specifically, the mouse chondrocyte cell line ATDC5 (Sigma, 99072806) was selected, and the cells were cultured at a concentration of 1×10⁶ cells / year. 5 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured at 37°C and 5% CO2 for 24 hours. After cell adhesion, 200 μg / mL monosodium urate (MSU; Sigma, U2875) was added to each well to induce an inflammatory response. Then, experimental samples were added, and the plates were incubated at 37°C and 5% CO2 for 48 hours, with the following groups:

[0100] Control group: MSU concentration of 200 μg / mL

[0101] Experimental group: Sample 1 with a concentration of 200 μg / mL MSU and a concentration of 0.125 mg / mL

[0102] After culture, the culture medium was discarded, and the cells were washed with 1×DPBS. 600 μL of RB Bμffer was added to each well to lyse the cells. Total RNA was then extracted and analyzed by reverse transcription and qPCR gene quantification. Experimental data were statistically analyzed using Student's t-test in Excel (*** indicates P < 0.001 relative to the control group).

[0103] The target genes for detection include the inflammatory cytokines IL-1β and TNF-α, as well as inducible nitric oxide synthase (iNOS). The primer sequences are as follows:

[0104]

[0105] When chondrocytes are damaged due to various reasons (such as age and mechanical wear), the damaged cells release a large number of pro-inflammatory factors, such as IL-1β, TNF-α, and nitric oxide (NO). These signaling molecules trigger an inflammatory response, and the persistent inflammatory environment further inhibits cartilage repair, accelerates cell apoptosis, and leads to more extensive joint and cartilage degeneration, ultimately resulting in limited joint function.

[0106] like Figure 4As shown, the experimental results indicate that Sample 1 significantly reduced the expression levels of multiple inflammatory factors in chondrocytes. Compared with the control group (set as 100%), the relative expression levels of IL-1β gene were significantly reduced by 80%, TNF-α gene by 84%, and iNOS gene by 83%. Therefore, Sample 1 can effectively inhibit the inflammatory response of chondrocytes, reduce the expression of pro-inflammatory factors, thereby exerting an anti-inflammatory protective effect and effectively delaying the degenerative process of articular cartilage.

[0107] Example 6: Promoting osteoblast differentiation

[0108] Sample 1 obtained in Example 1 was used in an osteoblast differentiation experiment to evaluate its role in bone health.

[0109] First, human adipose-derived mesenchymal stem cells (AdMSCs) (ATCC, CRL-2749) were seeded in 24-well culture plates at a seeding density of 8 × 10⁻⁶ cells / well. 4 Cells per well, culture medium volume 500 μL / well, αMEM medium (Gibco, 12000-022) supplemented with 5% fetal bovine serum FBS (Gibco, M3009125RP) and 1% antibiotic AA (Simply, CC501-0100), and cultured in a CO2 incubator for 1 day. Subsequently, the culture medium was replaced with MSCgo. TM Osteogenic differentiation medium (05-440-1B) was used for continuous differentiation for 7 days, with the differentiation medium being changed every 2-3 days.

[0110] Seven days later, the samples to be tested were added, and the groups were as follows:

[0111] Control group: blank culture medium

[0112] Experimental group: Sample 1 with a concentration of 0.5%

[0113] After adding the sample, the cells were cultured for 14 days, with the culture medium changed every 2-3 days. After culture, the culture medium was removed, and the cells were washed twice with PBS, followed by fixation with 4% paraformaldehyde at 37°C for 10 minutes. The fixed cells were then washed twice with ddH2O and stained with Alizarin Red S staining solution (2g Alizarin Red S / 100mL ddH2O; Sigma, A5533) for 1 minute. After removing the staining solution, the cells were thoroughly washed with ddH2O to obtain the staining results. Cellular calcification nodules were observed using a microscope (ZEISS Axiovert, ATCID00055).

[0114] Further, the stained samples were added to CPC solution (350 mg CPC / 10 mL ddH2O; Sigma, C9002-25 G) and incubated at 37°C for 2 hours to dissolve Alizarin Red S dye. Subsequently, 50 μL of solution was transferred from each well to a 96-well plate, and OD was measured using a microplate reader (BIOTECH, Epoch 1703289). 405 The t-test was used to quantitatively evaluate the degree of cell calcification. Experimental data were statistically analyzed using Student's t-test in Excel (* indicates P < 0.05 relative to the control group).

[0115] Calcium in bones can be stained red by Alizarin Red S, such as Figure 5A As shown, the increased red color in the experimental group indicates that sample 1 can promote osteoblast differentiation. The ability of sample 1 to promote osteoblast differentiation was further demonstrated by quantitative detection of the degree of cell calcification, and the results are as follows. Figure 5B As shown, compared to the control group (set as 100%), Sample 1 significantly increased the relative number of differentiated osteoblasts by 41.9%. Osteoblasts are the main functional cells in bone formation, responsible for the synthesis, secretion, and mineralization of the bone matrix. Human bone tissue is constantly being rebuilt; the bone rebuilding process includes bone breakdown and resorption, as well as new bone formation, with osteoblasts responsible for new bone formation. Through new bone formation, bone density and bone mass can be increased, thereby increasing bone hardness and promoting bone health.

[0116] Example 7: Inhibition of osteoclast differentiation and maturation

[0117] Sample 1 obtained in Example 1 was used in an in vitro experiment to evaluate its effect on the differentiation of peripheral blood mononuclear cells (PBMCs) into osteoclasts.

[0118] First, peripheral blood was collected from healthy volunteers, and 20 mL of blood was diluted with 15 mL of PBS. 15 mL of Ficoll (GE Healthcare) was added to a 50 mL centrifuge tube, and then 35 mL of diluted blood was slowly added on top of the Ficoll layer. The tube was centrifuged at 400 g for 40 minutes. After centrifugation, a thin layer consisting of peripheral blood mononuclear cells (PBMCs) was observed between the Ficoll layer and the plasma layer. The PBMCs were carefully transferred and washed twice with PBS (400 g, 15 minutes).

[0119] The obtained PBMCs were 1×10 6 10 cells / mL to 2×10 6Cells were seeded at a density of [number] cells / mL and cultured in osteoclast differentiation medium along with sample 1. The osteoclast differentiation medium consisted of α-MEM (Gibco, 12000-022) supplemented with 10% FBS (Gibco, M3009125RP), 1×P / S, human RANKL (40 ng / mL; Peprotech, 310-01-10UG), and human M-CSF (25 ng / mL; Peprotech, 300-25-10UG). Cells were cultured continuously at 37°C in a 5% CO2 incubator for 14 days, with the culture medium changed every 3 days.

[0120] The groups are as follows:

[0121] Control group: blank culture medium

[0122] Experimental group: Sample 1 with a concentration of 5 mg / mL

[0123] After 14 days of continuous culture, a large number of multinucleated osteoclasts were observed to form in the control group. Subsequently, one drop of Actin Green was added. T 488 Reagent (Invitrogen, A11008) and Hoechst 33342 (Thermo, 62249; 1:20000 dilution) were used to treat cells at 37°C for 15 minutes. Cells were then fluorescently stained, and the expression level of osteoclasts in each group was observed using a fluorescence microscope (ZEISS Axiovert, ATCID00055).

[0124] In young bones, the activity of osteoblasts and osteoclasts is in dynamic equilibrium, maintaining bone health and density. However, with age, changes in cytokine levels lead to excessive osteoclast proliferation and abnormally high activity. This imbalance between osteogenic and osteoclast processes causes bone breakdown to far outpace synthesis, resulting in significant calcium loss from bone tissue, reduced bone mass, and weakened, more brittle bones.

[0125] like Figure 6 As shown, compared with the control group (mature multinucleated osteoclasts), the number of osteoclasts in the experimental group (undifferentiated monocytes) was significantly reduced (by 70%), indicating that sample 1 can significantly inhibit the differentiation and maturation of osteoclasts, effectively slow down bone loss, thereby increasing bone density, increasing bone hardness, and promoting bone health.

[0126] Example 8: Preparation of a composition of type II collagen-like amino acids and plant extracts

[0127] The following ingredients, by weight percentage: 24.03% glycine, 15.98% L-proline, 7.93% L-alanine, 5.56% L-aspartic acid, 5.44% L-glutamine, 4.68% L-glutamic acid, 4.26% L-arginine, 3.97% L-lysine hydrochloride, 3.96% L-serine, 3.31% L-leucine, 2.60% L-threonine, 2.25% L-valine, 2.07% L-methionine, 2.01% L-isoleucine, 1.48% L-phenylalanine, 0.59% L-tyrosine, 0.47% L-histidine, 0.41% L-tryptophan, 3.25% maltodextrin, and 2.75% elderberry extract (for black elderberry (Sambucus)). The extract was obtained by fruit extraction of *C. nigra* (available from Hunan Huacheng Biotechnology Co., Ltd.) and 3.00% vitamin C. These were added to a mixer and mixed to obtain a preliminary mixture. The preliminary mixture was then sieved through a 40-mesh sieve to obtain a uniform intermediate mixture. This intermediate mixture was homogenized (using a homogenizer: Boang, G560(2-434437)) to obtain a homogeneous mixture, namely the type II collagen amino acid and plant extract composition of Example 8 (Sample 2).

[0128] Example 9: Preparation of a composition of type II collagen-like amino acids and plant extracts

[0129] By weight percentage, it comprises 24.03% glycine, 15.98% L-proline, 7.93% L-alanine, 5.56% L-aspartic acid, 5.44% L-glutamine, 4.68% L-glutamic acid, 4.26% L-arginine, 3.97% L-lysine hydrochloride, 3.96% L-serine, 3.31% L-leucine, and 2.60%... 2.25% L-threonine, 2.07% L-valine, 2.01% L-methionine, 1.48% L-phenylalanine, 0.59% L-tyrosine, 0.47% L-histidine, 0.41% L-tryptophan, 5.00% maltodextrin, 3.00% vitamin C, and 1.00% turmeric extract (obtained from the rhizome of turmeric (Curcuma longa), available from ARJUNA NATURAL PRIVATE LTD) were added to a mixer and mixed to obtain a preliminary mixture. The preliminary mixture was sieved through a 40-mesh sieve to obtain an intermediate mixture with uniform particle size. The intermediate mixture was homogenized (homogenizer: Boang, G560 (2-434437)) to obtain a homogeneous mixture, namely the type II collagen amino acid and plant extract composition of Example 9 (sample 3).

[0130] Example 10: Human Efficacy Trial

[0131] This embodiment provides the efficacy of Sample 2 obtained in Example 8 on bone and joint health.

[0132] Sample: Sample 2 of Example 8 (5 g / day)

[0133] Subjects: 10 adults

[0134] Inclusion criteria: Adults at high risk of fracture (low bone mineral density, postmenopausal women, age > 65 years).

[0135] Methods: A self-controlled trial was conducted. Subjects took 5 grams of the sample daily before meals for 3 months. Bone mineral density values ​​were collected at month 0 and month 3. Experimental data were statistically analyzed using Student's t-test in Excel (** indicates P < 0.01 relative to week 0, *** indicates P < 0.001 relative to week 0).

[0136] Test items: Spinal bone mineral density scan (dual-energy X-ray absorptiometry, DEXA): T-score, Z-score

[0137] As shown in Table 2 below, the T-score reflects the comparison of bone mineral density (BMD) with the mean BMD of young, healthy adults of the same sex. The T-score can be used to diagnose osteopenia and osteoporosis.

[0138] Table 2.

[0139] T-value illustrate -1 or more Normal bone density Less than -1 to greater than -2.5 Osteopenia (below normal BMD) -2.5 or less Osteoporosis (low BMD)

[0140] As shown in Table 3 below, the Z-score compares the subject's BMD with the average BMD of their age group, taking into account gender and body mass index (BMI).

[0141] Table 3.

[0142] Z-value illustrate -1.5 or more Typical BMD of peers Below -1.5 Atypical BMD, requires investigation.

[0143] Test results

[0144] like Figure 7 As shown, after taking sample 2 for 12 weeks, the subjects' spinal T-scores significantly improved from -0.92 to -0.72, with a change rate of 21.7%, and 80% of the subjects showed effective improvement.

[0145] like Figure 8 As shown, after taking sample 2 for 12 weeks, the subjects' spinal Z-score significantly increased from -0.140 to 0.270, with a change rate of 293%, and 90% of the subjects showed effective improvement.

[0146] Decreased bone mineral density is a major cause of reduced bone hardness and increased fracture risk. Sample 2 can significantly increase bone mineral density, thereby reducing the fracture risk in postmenopausal women or older adults, and can slow down bone loss, improve bone strength, increase bone hardness, and increase bone support.

[0147] Example 11: Human Efficacy Trial

[0148] This embodiment provides the efficacy of Sample 2 obtained in Example 8 on bone and joint health.

[0149] Sample: Sample 2 of Example 8 (5 g / day)

[0150] Subjects: 10 adults

[0151] Inclusion criteria: 20 years and older.

[0152] Methods: A self-controlled trial was conducted. Subjects took 5 grams of the sample daily before meals for 3 months. Blood samples were collected at month 0 and month 3. Experimental data were statistically analyzed using Student's t-test in Excel (* indicates P < 0.05 relative to week 0).

[0153] Test item: Serum TNF-α level (inflammatory marker).

[0154] Testing method: After collecting fasting blood, the samples were sent to Liren Laboratory for testing. TNF-α was tested using ELISA KIT (Elabscience, E-EL-H0 1 09).

[0155] Test results

[0156] It is well known in the art that TNF-α is one of the core inflammatory factors in the occurrence and development of arthritis. Studies have shown that TNF-α can not only directly damage chondrocytes, but also stimulate synovial fibroblasts and chondrocytes to produce inflammatory mediators, including matrix metalloproteinases (MMPs), accelerating the degradation of the extracellular matrix, leading to articular cartilage destruction and persistent inflammation.

[0157] like Figure 9 As shown, after 12 weeks of continuous administration of sample 2, TNF-α levels decreased by 59.1%, and 90% of subjects experienced effective improvement. This indicates that sample 2 can reduce inflammatory cytokine levels, thereby helping to prevent inflammatory responses.

[0158] Example 12: Human Efficacy Trial

[0159] This embodiment provides the efficacy of sample 3 obtained in Example 9 on bone and joint health.

[0160] Sample: Sample 3 of Example 9 (5 g / day)

[0161] Subjects: 10 adults

[0162] Inclusion criteria: Adults aged 20 and older with joint discomfort.

[0163] Methods: A self-controlled trial was conducted. Participants took 5 grams of the sample daily before meals for 3 months. Self-assessment questionnaires were collected at weeks 0, 4, and 12. The self-assessment questionnaire assessed the participants' total WOMAC (The Western Ontario and McMaster Universities Osteoarthritis Index) score, joint pain score, stiffness score, and range of motion score. Statistical analysis of the experimental data was performed using Student's t-test in Excel (* indicates P < 0.05 relative to week 0, ** indicates P < 0.01 relative to week 0).

[0164] Test results

[0165] like Figure 10 As shown, after taking Sample 3 for 12 weeks, the WOMAC score decreased significantly by 58%, the joint pain score decreased by 44%, the joint stiffness score decreased by 66.7%, and the range of motion score (physical function) decreased by 60.5%. This indicates that the composition of Sample 3 of this application can effectively improve joint stiffness in people with joint discomfort, thereby improving joint flexibility and effectively relieving joint pain, so as to improve joint function and physical performance.

[0166] All publications, patent applications, patents, nucleic acid and amino acid sequences, and other references mentioned in this disclosure are incorporated herein by reference in their entirety.

[0167] While this disclosure has been illustrated and described with reference to certain preferred embodiments, those skilled in the art should understand that the above description is a further detailed explanation of the disclosure in conjunction with specific embodiments, and should not be construed as limiting the specific implementation of this disclosure to these descriptions. Various changes in form and detail can be made by those skilled in the art, including some simple deductions or substitutions, without departing from the spirit and scope of this disclosure.

Claims

1. An amino acid composition, wherein, The amino acid composition comprises the following components by weight percentage: 20.0%-28.0% glycine or its salt, 13.0%-18.0% proline or its salt, 6.0%-10.0% alanine or its salt, 4.0%-7.0% aspartic acid or its salt, 4.0%-7.0% glutamine or its salt, 3.5%-6.0% glutamic acid or its salt, 3.5%-5.0% arginine or its salt, 3.0%-4.5% lysine or its salt, 3.0%-4.5% serine or its salt. Salt, 2.5%-4.0% leucine or its salt, 2.0%-3.2% threonine or its salt, 1.8%-2.8% valine or its salt, 1.5%-2.5% methionine or its salt, 1.5%-2.5% isoleucine or its salt, 1.0%-2.0% phenylalanine or its salt, 0.4%-0.8% tyrosine or its salt, 0.3%-0.6% histidine or its salt, 0.3%-0.6% tryptophan or its salt, and Optional additives.

2. The amino acid composition according to claim 1, wherein, The additive comprises, relative to the total weight of the amino acid composition, 1.5%-6.0%, preferably 3.0%-5.0% of maltodextrin and / or 2.0%-4.0% of vitamin C.

3. The use of the combination of the amino acid composition of claim 1 or 2 with the berry extract in the preparation of a composition for increasing bone density.

4. The application according to claim 3, wherein, The berry extract is elderberry extract.

5. The application according to claim 3 or 4, wherein, The composition is used to increase bone mineral density.

6. The application according to claim 3 or 4, wherein, The composition is used to promote osteoblast differentiation.

7. The application according to claim 3 or 4, wherein, The composition is used to inhibit osteoclast differentiation.

8. Use of the combination of the amino acid composition of claim 1 or 2 with the berry extract in the preparation of a composition for preventing inflammatory responses.

9. The application according to claim 8, wherein, The berry extract is elderberry extract.

10. The application according to claim 8 or 9, wherein, The composition is used to reduce the level of inflammatory factors.

11. The application according to claim 10, wherein, The composition is used to reduce TNF-α, IL-1β, and iNOS levels.

12. The application according to claim 8 or 9, wherein, The composition is used to prevent inflammation of chondrocytes.

13. The application according to any one of claims 3 to 12, wherein, The berry extract has a weight percentage content of 2.0%-3.5% based on the total weight of the amino acid composition and the berry extract, which is 100%.

14. The application according to any one of claims 3 to 13, wherein, The berry extract is derived from the fruit of the black elderberry (Sambucus nigra).

15. The use of the combination of the amino acid composition of claim 1 or 2 with a turmeric plant extract in the preparation of a composition for relieving joint pain.

16. The application according to claim 15, wherein, The turmeric plant extract is turmeric extract.

17. The application according to claim 15 or 16, wherein, The composition is used to prevent joint wear.

18. The application according to claim 17, wherein, The composition is used to promote the secretion of cartilage collagen.

19. The application according to claim 17, wherein, The composition is used to promote the secretion of glucosamine.

20. The use of the combination of the amino acid composition of claim 1 or 2 with the extract of a turmeric plant in the preparation of a composition for improving joint flexibility.

21. The application according to claim 20, wherein, The turmeric plant extract is turmeric extract.

22. The application according to claim 20 or 21, wherein, The composition is used to improve joint stiffness.

23. The application according to claim 20 or 21, wherein, The composition is used to promote the secretion of cartilage collagen.

24. The application according to claim 20 or 21, wherein, The composition is used to promote the secretion of glucosamine.

25. The application according to any one of claims 15 to 24, wherein, The total weight percentage of the amino acid composition and the turmeric plant extract is 0.5%-3.0%, based on 100% of the total weight of the turmeric plant composition and the turmeric plant extract.

26. The application according to any one of claims 15 to 25, wherein, The turmeric plant extract is derived from the rhizome of turmeric (Curcuma longa).

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