Use of non-denatured type i collagen in the manufacture of an oral product for improving joint cartilage health

Abstract

The application discloses application of non-denatured type I collagen in preparation of oral joint cartilage health improvement products and belongs to the technical field of biological health care. The non-denatured type I collagen with a complete natural triple helix structure is adopted, and through oral administration, the collagen can be used for multi-target point regulation of a cartilage microenvironment, down-regulation of expression of cartilage degeneration related factors, inhibition of cartilage matrix degradation and cell aging, and has good biocompatibility and safe and non-irritating taking. Animal experiments prove that the collagen can effectively thicken the cartilage layer, reduce loss of proteoglycans and improve cartilage degenerative damage. The application has the advantages of easy raw material acquisition, stable process, suitability for industrialized production, convenient and non-invasive taking, adaptation to maintenance needs of people with different degrees of cartilage degeneration, effective delay of cartilage degeneration and improvement of joint health.

Description

Technical Field

[0001] This invention belongs to the field of bio-health technology and provides an application of non-denatured type I collagen in the preparation of oral products to improve joint cartilage health. It is especially suitable for long-term dietary supplementation for people with mild, moderate and severe joint cartilage degeneration, and can simultaneously relieve joint discomfort and promote cartilage health. Background Technology

[0002] Osteoarthritis (OA) is a common chronic degenerative disease, and its incidence increases with age. Osteoarthritis damages the cartilage and surrounding tissues such as connective tissue in the joints. Articular cartilage and subchondral bone are frequently affected, and the lubricating and anti-wear properties of cartilage and synovial fluid are significantly reduced. This manifests as joint pain, swelling, stiffness, and difficulty walking due to joint dysfunction. Current treatments for osteoarthritis include cartilage repair for early-stage osteoarthritis. Commonly used clinical methods include autologous cartilage transplantation, microfractures, and stem cell transplantation. However, all of these techniques have significant drawbacks. Autologous cartilage transplantation involves extracting cartilage cells from non-weight-bearing areas, expanding them in vitro, and then implanting them back into the body, which can cause secondary damage. Furthermore, tissue loss may occur at the cell extraction site. Microfractures involve drilling holes in the joint bone tissue to allow cells from the bone marrow to migrate to the cartilage defect for repair. However, because the cells originate from the bone marrow, the newly formed cartilage is prone to fibrosis, thus affecting its physical properties. Stem cell transplantation faces challenges such as immune rejection and significant individual differences among donors; therefore, this technology is rarely used in clinical practice.

[0003] In recent years, injectable hydrogels have been widely proposed and researched in the field of cartilage repair in osteoarthritis. However, the application of injectable hydrogels still leaves wounds on the skin around the joints of osteoarthritis patients and can even cause pain in some patients. At the same time, oral collagen preparations are mostly traditional hydrolyzed collagen, which has problems such as low digestibility and absorption rate and lack of active ingredients, making it difficult to effectively reach the joint site to exert its effects.

[0004] In recent years, although some studies have proposed using biomaterials for localized invasive repair, such methods still cause trauma or discomfort to patients. Meanwhile, most commercially available oral collagen products are hydrolyzed collagen or gelatin, whose triple-helix structure has been destroyed. They can only provide amino acid nutrition and cannot effectively bind to cell membrane receptors to activate anabolic pathways. Although there is some research on non-denatured type II collagen (UC...) II) Reports indicate its use in immune regulation, but its production is low and its cost is high, making it unsuitable for long-term use by ordinary consumers.

[0005] Therefore, there is an urgent need to develop an oral non-denatured type I collagen product with complete structure and high yield to effectively protect and actively repair the health of articular cartilage, and to provide greater comfort during use. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides the application of non-denatured type I collagen in the preparation of oral products to improve the health of articular cartilage. This collagen, through a multi-target mechanism, can not only rapidly alleviate degeneration caused by damage to the joint microenvironment, but also promote cartilage matrix synthesis and inhibit cartilage degradation. Moreover, it has high oral safety, providing a new solution that addresses both the symptoms and the root cause of articular cartilage health.

[0007] To achieve the above objectives, the present invention provides the following technical solution: the application of non-denatured type I collagen in the preparation of oral products for improving joint cartilage health, wherein the non-denatured type I collagen has a complete natural triple helix structure.

[0008] Furthermore, the non-denatured type I collagen achieves the effects of relieving joint discomfort and delaying and reversing articular cartilage degeneration by regulating chondrocyte aging, inhibiting cartilage matrix degradation, and improving the cartilage microenvironment.

[0009] Furthermore, the non-denatured type I collagen can downregulate the expression of MMP13, COLX, and P21 degeneration-related factors in inflammatory chondrocyte stem cells, thereby inhibiting cartilage matrix degradation and chondrocyte aging.

[0010] Furthermore, the non-denatured type I collagen is telopeptide-free soluble collagen powder.

[0011] Furthermore, the effective in vitro concentration range of the non-denatured type I collagen is 0.4 mg / mL to 12.8 mg / mL.

[0012] Furthermore, the effective in vivo dosage range of the oral product for improving joint cartilage health is 8 mg / kg·d to 26.4 mg / kg·d, and the continuous oral intervention period is no less than 4 weeks.

[0013] Furthermore, the dosage varies depending on the sex; the optimal intervention dose for female individuals is 26.4 mg / kg·d, and the optimal intervention dose for male individuals is 8 mg / kg·d.

[0014] Furthermore, the oral product for improving joint cartilage health can be any one of dietary supplements, functional health foods, or oral health preparations.

[0015] The present invention also provides an oral cartilage maintenance preparation for improving the health of articular cartilage, wherein the active ingredient of the preparation is non-denatured type I collagen with a complete natural triple helix structure.

[0016] Furthermore, the cartilage maintenance preparation is suitable for people with mild, moderate and severe articular cartilage degeneration, and can help delay the progression of joint deformities in people with severe cartilage wear.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: This invention provides an application of non-denatured type I collagen in the preparation of oral products for improving joint cartilage health. It utilizes non-denatured type I collagen that retains its complete natural triple helix structure, distinguishing it from commercially available hydrolyzed collagen and gelatin products that have lost their structural integrity. This complete natural structure ensures that the collagen possesses genuine cartilage microenvironment regulation capabilities, effectively intervening in chondrocyte aging and matrix degradation processes, rather than simply providing basic amino acid nutrition. This core structural aspect guarantees the actual efficacy of joint cartilage care.

[0018] The non-denatured type I collagen of this invention can specifically downregulate the expression of degeneration-related factors such as MMP13, COLX, and P21 in inflammatory chondrocyte stem cells, inhibiting the degradation of cartilage matrix and blocking the aging process of chondrocytes at the molecular level, effectively improving the damaged cartilage microenvironment. It can not only relieve the discomfort symptoms caused by joint degeneration, but also delay and reverse the degenerative lesions of articular cartilage, achieving a dual effect of symptom relief and tissue structure maintenance.

[0019] This invention clarifies the effective range of collagen through cell experiments and in vivo animal experiments. It shows good biocompatibility and no cytotoxicity within a specific concentration range in vitro. The oral dosage can be adapted to the cartilage repair needs of different genders. The optimal intervention dose is matched according to physiological differences, avoiding the problem of uneven intervention effects with uniform dosage, making cartilage maintenance intervention more targeted and effective.

[0020] This invention provides an oral administration product that can be prepared into various oral health supplement formulations. It is convenient to take, non-invasive, and has no gastrointestinal irritation or side effects. Long-term use is well-tolerated. The product is suitable for individuals with mild, moderate, and severe articular cartilage degeneration, and is particularly effective in slowing the progression of joint deformities in individuals with severe cartilage wear. It has a wide range of applicable populations and boasts excellent safety and patient compliance. Attached Figure Description

[0021] Figure 1 Scanning electron microscope image of the non-denatured type I collagen of this invention.

[0022] Figure 2 The present invention provides an SDS-PAGE image of non-denatured type I collagen.

[0023] Figure 3 CD diagram of non-denatured type I collagen of this invention.

[0024] Figure 4 The immunoblotting results (a) and grayscale analysis (b) of COL X protein expression after treatment of inflammatory chondrocyte stem cells with gradient concentrations of non-denatured type I collagen in this invention.

[0025] Figure 5 The present invention presents statistical results on the cell viability of chondrocyte stem cells treated with different concentrations of non-denatured type I collagen.

[0026] Figure 6 The statistical results of MMP13 expression level (a) and P21 expression level (b) after treatment of inflammatory chondrocyte stem cells with gradient concentrations of COL I in this invention.

[0027] Figure 7 The results of H&E staining of mouse bone and joint sections after gavage administration of different concentrations of non-denatured type I collagen (a) and the statistical results of the average and maximum cartilage layer thickness of different groups of mice (bg).

[0028] Figure 8 The results of safranin-fast green staining of mouse bone and joint sections after gavage administration of different concentrations of non-denatured type I collagen (a) and the statistical results of the average and maximum cartilage layer thickness of different groups of mice (bg). Detailed Implementation

[0029] To better understand the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but the content of the present invention is not limited to the following embodiments.

[0030] Example 1: The non-denatured type I collagen is derived from Guangdong Shengchi's determinate peptide-soluble collagen powder. It uses bovine genuine leather as raw material and employs acidic pepsin-specific enzymatic hydrolysis technology to fully preserve the triple helix backbone structure of collagen. Through a series of processes including raw material refining and impurity removal, low-temperature enzymatic hydrolysis, salting-out enrichment, fractional ultrafiltration, deep endotoxin removal, and aseptic freeze-drying, a high-purity medical-grade determinate peptide-soluble collagen powder soluble in cold water and weak acids is obtained.

[0031] Scanning electron microscopy (SEM): Collagen is placed on a sample stage, fixed with conductive adhesive, sputter-coated with gold, and scanned images are obtained using an accelerating voltage of 30 kV for observation. Figure 1 The prepared collagen exhibits a sheet-like and membrane-like structure, with interwoven fibrous bundles arranged in an irregular three-dimensional stacked state, containing irregular pores tens of micrometers in size and curled edges. This indicates that the present invention successfully preserves the natural fibrous structure of collagen.

[0032] SDS-PAGE: The relative molecular mass of proteins in COLI was identified using polyacrylamide gel electrophoresis. COLI was dissolved in distilled water, and protein loading buffer was added at a specific ratio. The mixture was incubated at 95°C for 15 min to denature the proteins, causing conformational dissociation, charge neutralization, and the formation of SDS-bound molecules. After preparing the gel using an SDS-PAGE gel preparation kit, 10 μL of sample was loaded into each well. The protein sample was compressed through a stacking gel at 90 V, and electrophoresis was performed at 150 V until the bands migrated to the bottom of the separating gel. The gel was then removed, stained with Coomassie Brilliant Blue for 1 h, destained overnight, and then observed using a gel imaging system. Figure 2 There are two very distinct bands between 90 and 130 kDa, which are the α1 and α2 chains of COLI, with molecular weights of about 110 kDa and 120 kDa, respectively. There is a fainter band at about 200 kDa, confirming that it is type I collagen.

[0033] Circular dichroism (CD) chromatography: Dissolve COLI in distilled water. After complete dissolution, filter through a 0.22 μm filter membrane to remove insoluble matter, and adjust the protein concentration to approximately 0.1–0.2 mg / mL. Add an appropriate amount of sample to a 1 mm quartz cuvette. Set the test wavelength range to 190–260 nm, the scan rate to 50–100 nm / min, the data interval to 0.5–1 nm, the bandwidth to 1 nm, and the experimental temperature to 25 °C. Figure 3 As shown, a positive peak is observed at approximately 220 nm and a negative peak at approximately 198 nm, which is a typical triple helix conformation feature, proving that the extraction process successfully preserved the non-denatured natural structure of collagen.

[0034] Example 2: Cell-level effect verification experiment COL X protein expression detection (Western blot): Cartilage stem cells were seeded into 12-well plates. After the cells were stably adhered, inflammation was induced in the cartilage stem cells by interleukin IL-1β. After removing the complete culture medium containing IL-1β, the cells were washed twice with PBS buffer to completely remove IL-1β. Then, the cells were cultured in complete culture medium containing gradient concentrations of COL X for 24 h. When the cells proliferated to 80%~90% of the plate area, the cells were lysed and total cellular protein was extracted.

[0035] Prepare the separating and stacking gels according to the kit. Compress the bands in the electrophoresis buffer at a constant voltage of 80 V. After entering the separating gel, electrophoresis is performed at a constant voltage of 120 V until the bromophenol blue is near the bottom of the separating gel. Remove the gel and cut off a portion of the gel corresponding to the molecular weight of the target protein. Cut a polyvinylidene fluoride (PVDF) membrane of the appropriate size, activate it in methanol, place the membrane on the gel sheet, remove air bubbles, and prepare a transfer sandwich. Place the membrane in a transfer tank and transfer it in a constant current ice bath at 250 mA. After transfer, block the PVDF membrane with 5% BSA for 2 h, then incubate it overnight at 4°C with primary antibody. After incubation, recover the primary antibody and wash the PVDF membrane three times with TBST for 10 min each time. After washing, incubate with secondary antibody at room temperature for 1 h. Repeat the washing process, and then image using a chemiluminescence imaging system. Perform grayscale analysis on the protein immunoblot bands, measuring the grayscale values ​​of the target protein band and the internal control protein band. The ratio of the gray value of the target protein to the gray value of the corresponding internal reference protein in each sample was used as the result of the first normalization. The average value of the first normalization result of the control group was used as the benchmark to perform a second normalization on each group of samples. Finally, the expression level of the target protein was expressed as the relative expression level.

[0036] Figure 4 The expression levels of the three osteoarthritis-related factors shown indicate that the expression trends of P21, MMP13, and COLX in inflammatory chondrocyte stem cells after treatment with different concentrations of COLI are not entirely consistent, but they show a certain regulatory trend within certain concentration ranges. Moreover, they show significant differences compared with the IL-1β group at specific concentration ranges, indicating that COLI has a certain regulatory effect on chondrocyte stem cell degeneration-related factors under inflammatory conditions. This effect may not be a simple linear dose-response relationship.

[0037] Cell viability assay (CCK-8 assay): Chondrocyte stem cells passaged to passage 3-4 were selected and counted using a hemocytometer. The cells were seeded into 96-well plates at a density of 5000 cells per well. After stable cell attachment, a certain concentration of COLI was dissolved in complete culture medium containing 15% fetal bovine serum for cell culture. A control group was also included. After 48 hours, the culture medium was removed, and CCK-8 reagent was added in the dark. The cells were incubated at 37°C with 5% carbon dioxide for 2 hours. The OD value was then measured using a microplate reader at 450 nm. Figure 5 As shown, the cell viability of each COLI treatment group was not significantly different from that of the control group (p>0.05), indicating that the non-denatured type I collagen of the present invention has no obvious cytotoxicity to chondrocyte stem cells and no obvious inhibition of metabolic activity, indicating that COLI has good cell compatibility.

[0038] Detection of inflammation and catabolism-related factors: After chondrocyte stem cells were cultured and stabilized, they were subjected to inflammatory stimulation with a certain concentration of interleukin-1β for 12 hours when the cells reached approximately 50% confluence with the culture dish surface, thus constructing an inflammatory chondrocyte stem cell model. Cells were cultured in complete medium containing gradient concentrations of COLI for 24 hours. 1 mL of Trizol was added to each well, and cells were lysed on ice for 5 minutes. The resulting lysate was transferred to centrifuge tubes for total RNA extraction. The total RNA concentration was measured, and the RNA content was adjusted to a consistent 500 ng. Real-time quantitative PCR was performed using cDNA obtained from reverse transcription as a template. Ct values ​​were recorded for each group, and melting curves were analyzed. GAPDH was used as an internal reference gene, and the relative expression levels of the target gene were normalized using the 2^-ΔΔCt method. Figure 6 After treatment with different concentrations of COLI, the expression of P21 and MMP13 in chondrocyte stem cells showed a non-linear trend, but their response patterns were not entirely consistent. P21 was significantly decreased at concentrations of 0.4 mg / mL and 0.8 mg / mL (p<0.05), with the 0.4 mg / mL group lower than the 0.8 mg / mL group. It increased at 1.6 mg / mL and was higher than the IL-1β group, then decreased at 3.2 mg / mL, decreased again at 6.4 mg / mL with a significant difference from the IL-1β group, and increased again at 12.8 mg / mL, but was still lower than the IL-1β group with no significant difference.

[0039] The MMP13 expression results indicate that COLI's regulatory effect on chondrocyte stem cells under inflammatory stimulation exhibits a significant non-linear concentration-dependent effect. Since P21 and MMP13 reflect different biological processes such as cell senescence cycle arrest and matrix degradation, respectively, their response thresholds and patterns to COLI stimulation may differ. COLI only shows a good intervention effect on cell degeneration-related phenotypes within a specific concentration range, while its regulatory effect weakens at excessively high concentrations and may even induce additional stress responses. This suggests that COLI's regulation of inflammation-related genes may be a non-linear biological response. This may be because MMP13 and P21 genes are regulated by multiple signaling pathways, and various feedback regulation and compensatory expression changes may occur under different COLI stimulation intensities.

[0040] Example 3: Animal in vivo effect verification experiment (1) Animal model establishment: Forty 6-8 week old C57BL / 6 mice, weighing 20-25 g, were used. After acclimatization for 1 week, a knee osteoarthritis model was established by medial meniscus instability (DMM) surgery. In the sham surgery group, only the joint capsule was opened and then sutured.

[0041] (2) Grouping and administration: The model mice were randomly divided into 4 groups (n=10): model control group (administered with physiological saline), low-dose group (administered by gavage 2 mg / mL COLI, at 8 mL / kg, i.e. 26.4 mg / kg), medium-dose group (administered by gavage 6.6 mg / mL COLI, i.e. 80 mg / kg), and high-dose group (administered by gavage 20 mg / mL COLI, i.e. 200 mg / kg). A sham-operated group (administered with physiological saline) was also set up. The mice were administered by gavage once a day for 4 consecutive weeks.

[0042] (3) Histological evaluation: At the end of day 28 of the animal experiment, the mice were anesthetized and euthanized. The skin of the knee on one side of the model was dissected with a scalpel, the attached adipose tissue was removed, the entire knee joint was cut off, and the surrounding muscle and adipose tissue were removed. The joint was fixed in 4% paraformaldehyde for 48 h, and then transferred to 10% EDTA solution for decalcification. The joint was placed in a shaker at room temperature for decalcification, and the decalcification solution was changed daily to accelerate the decalcification process. After decalcification, the knee joint was rinsed under running water, and then dehydrated sequentially in 70% ethanol, 80% ethanol, 95% ethanol and anhydrous ethanol, with each dehydration step lasting 1 h. The joint was then cleared twice in xylene, 30 min each time. After clearing, the bone joint was immersed in molten paraffin at 60℃ three times, 1 hour each time, to ensure full penetration of the paraffin into the tissue. Finally, it was embedded. After the paraffin had completely solidified, the paraffin block was removed and cut into 4-5 μm sections using a paraffin microtome. These sections were then spread in 40℃ warm water and fixed onto glass slides to form slides. H&E staining and Safranin O-Fixed Green staining were then performed. Results are as follows... Figure 7 and Figure 8 As shown: H&E staining results showed significant wear on the cartilage surface in the medial meniscus instability surgery (DMM) group, with thinning of the cartilage layer and even exposure to subchondral bone. In some areas, cartilage tissue almost disappeared, accompanied by obvious fissures, indicating a successful osteoarthritis model. In contrast, in both sexes, the degree of cartilage surface damage in the 8 mg / kg / day and 26.4 mg / kg / day COLI groups was significantly lower than that in the DMM and 80 mg / kg / day groups, with relatively intact cartilage structure. Statistical results showed that female mice had the greatest cartilage thickness in the 26.4 mg / kg / day group, while male mice showed a more significant thickening trend in the 8 mg / kg / day group.

[0043] Safranin O-Fixed Green staining results showed that, compared with the control group and the 80 mg / kg·d concentration group, the 8 mg / kg·d and 26.4 mg / kg·d intervention groups exhibited milder cartilage damage in both female and male mice. Specifically, the cartilage surface wear and structure were relatively more intact, and the safranin staining intensity was stronger, indicating a lower degree of proteoglycan loss. Statistical results showed that, compared with the control group, the OARSI scores of all different dose groups decreased, with the 8 mg / kg·d and 26.4 mg / kg·d dose groups showing a greater decrease in scores. The moderate-dose intervention was more effective in improving cartilage degeneration, and the effect was not a simple linear dose-dependent relationship, but rather exhibited gender-related dose-response differences.

[0044] This invention provides an application of non-denatured type I collagen in the preparation of oral products for improving articular cartilage health. The non-denatured type I collagen raw material used is widely available, environmentally friendly, and inexpensive. Experimental results demonstrate that this non-denatured type I collagen has good activity and shows great promise for application in articular cartilage repair and tissue regeneration.

Claims

1. The application of a non-denatured type I collagen in the preparation of oral products for improving joint cartilage health, characterized in that, The undenatured type I collagen has a complete natural triple helix structure.

2. The application according to claim 1, characterized in that, The non-denatured type I collagen achieves the effects of relieving joint discomfort and delaying and reversing articular cartilage degeneration by regulating chondrocyte aging, inhibiting cartilage matrix degradation, and improving the cartilage microenvironment.

3. The application according to claim 1, characterized in that, The non-denatured type I collagen can downregulate the expression of MMP13, COLX, and P21 degeneration-related factors in inflammatory chondrocyte stem cells, thereby inhibiting cartilage matrix degradation and chondrocyte aging.

4. The application according to claim 1, characterized in that, The non-denatured type I collagen is telopeptide-free soluble collagen powder.

5. The application according to claim 1, characterized in that, The effective in vitro concentration range of the non-denatured type I collagen is 0.4 mg / mL to 12.8 mg / mL.

6. The application according to claim 1, characterized in that, The effective in vivo dosage range of the oral product for improving joint cartilage health is 8 mg / kg·d to 26.4 mg / kg·d, and the continuous oral intervention period is no less than 4 weeks.

7. The application according to claim 6, characterized in that, There are sex-appropriate differences in dosage; the optimal intervention dose for females is 26.4 mg / kg·d, and for males it is 8 mg / kg·d.

8. The application according to claim 1, characterized in that, The oral products for improving joint cartilage health are any one of the following: dietary supplements, functional health foods, and oral health preparations.

9. An oral cartilage maintenance preparation for improving the health of articular cartilage, characterized in that, The active ingredient in the formulation is non-denatured type I collagen with a complete natural triple helix structure.

10. The oral cartilage maintenance preparation for improving articular cartilage health according to claim 9, characterized in that, The cartilage maintenance preparation is suitable for people with mild, moderate and severe articular cartilage degeneration, and can help delay the progression of joint deformity in people with severe cartilage wear.

Images