Preparation method and application of anti-inflammatory and anti-aging composition

Abstract

The invention relates to the technical field of compositions, and discloses a preparation method and application of an anti-inflammatory and anti-aging composition, the composition comprises a COLIII-HA-M solution and additives, and the additives are at least three of chondroitin sulfate, fibronectin, elastin, human type 17 collagen and type I collagen. The preparation method comprises the following steps: 1, preparing an HA-M solution, namely dispersing low-molecular-weight hyaluronic acid and high-molecular-weight hyaluronic acid in deionized water in equal proportion, heating, polymerizing, and cooling to obtain a polymer HA-M solution; step 2, preparing a COLIII-HA-M solution: slowly adding type III collagen into the polymer HA-M solution, uniformly mixing, and purifying to obtain the COLIII-HA-M solution; and step 3, mixing with additives to obtain the anti-inflammatory and anti-aging composition. According to the scheme, the composition is obtained by mixing and reacting the polymer HA-M and COLIII and then mixing with the additive, so that the stability of COLIII-HA-M is effectively improved, and the antioxidant and anti-aging effects of the composition are remarkably improved due to the synergistic interaction among different raw materials.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-inflammatory and anti-aging compositions, and in particular to a preparation method and application of the anti-inflammatory and anti-aging compositions. Background Art

[0002] The extracellular matrix (ECM) is a broad class of products secreted by cells, including growth factors, cytokines, and cell adhesion factors. Typically, the ECM forms a complex 3D network on the cell surface. The ECM dynamically responds to changes in the intracellular microenvironment, influencing cell behavior. In addition to its physical protective effects on cells, the ECM participates in various cell signaling pathways, including those related to inflammatory responses and aging, and is crucial for maintaining homeostasis in tissues throughout the body.

[0003] Collagen is a major component of the extracellular matrix (ECM), accounting for over 30% of the body's total protein content. It is found throughout various organs and tissues, serving as the structural framework of the extracellular matrix (ECM). Collagen fibers form the ECM framework, providing a living space for cells and supporting the physiological morphology of tissues. They also regulate cell adhesion, proliferation, and differentiation, immune cell responses, and tissue regeneration through signal transduction. Type XVII collagen (COL17) is a transmembrane protein in the basal layer of the epidermis, maintaining the tight junction between the epidermis and dermis, promoting epidermal renewal, and stabilizing the basement membrane. Type III collagen accounts for approximately 10-15% of the skin's content and is primarily distributed in the superficial dermis. Type III collagen fibers are fine, forming a loose, reticular structure. A deficiency in type III collagen can lead to a decrease in skin firmness and elasticity. Fibronectin is composed of two similar subunits, cross-linked by two disulfide bonds near their carboxyl termini, forming a dimer. Each subunit has multiple functional domains that can bind to various ECM components.

[0004] In practice, single ECM components are often supplemented to achieve skin-filling, anti-inflammatory, and anti-aging benefits. However, single ECM supplementation is difficult to achieve overall ECM regulation, resulting in a short-term repair effect and compromised anti-inflammatory and anti-aging benefits. While simple physical mixing of multiple ECM components can somewhat mitigate the drawbacks of single component supplementation, the resulting mixture is unstable and typically lacks a tight molecular arrangement. The properties of the individual components cannot complement each other, thus failing to achieve the desired anti-aging and anti-inflammatory effects.

[0005] In view of this, the development of a complex intervention plan that can synergistically regulate multiple ECM components and rebuild the dynamic homeostasis of the skin has become a technical challenge that urgently needs to be overcome in this field. Summary of the Invention

[0006] The present invention aims to provide a preparation method and application of an anti-inflammatory and anti-aging composition to solve the technical problem that the mixture formed by the existing simple mixing of multiple components is unstable and cannot achieve the ideal anti-aging and anti-inflammatory effects.

[0007] To achieve the above-mentioned object, the present invention adopts the following technical solution: a method for preparing an anti-inflammatory and anti-aging composition, wherein the composition comprises a COLIII-HA-M solution, and the preparation method comprises the following steps:

[0008] Step 1: Preparation of HA-M solution: Disperse low molecular weight hyaluronic acid and high molecular weight hyaluronic acid in deionized water at equimolar mass, heat to polymerize, and then cool to obtain a polymer HA-M solution of high molecular weight hyaluronic acid and low molecular weight hyaluronic acid;

[0009] Step 2: Prepare COLⅢ-HA-M solution: slowly add type III collagen to the above polymer HA-M solution, mix well and purify to obtain the intermolecular complex COLⅢ-HA-M solution.

[0010] Preferably, as an improvement, the heating polymerization is carried out at 115-150° C. for 10-30 minutes.

[0011] Preferably, as an improvement, the cooling is performed to cool to 10-37° C. within 5-6 minutes.

[0012] Preferably, as an improvement, the purification adopts affinity chromatography, including the following contents: dissolving type III collagen with a HIS tag in a HA-M solution, incubating at room temperature for 1 to 1.5 hours, and then passing the sample through a nickel column; first washing the column three times with Tris-HCL, Nacl solution, and then eluting the sample with Tris-HCL, Nacl, and imidazole solution. After the sample is concentrated in a concentrator tube, it is dissolved in deionized water to obtain a COLⅢ-HA-M solution.

[0013] Preferably, as an improvement, the mass concentration of type III collagen in the COLⅢ-HA-M solution is 1-5%.

[0014] Preferably, as an improvement, the method further comprises step three, mixing with additives: adjusting the pH value of the COLⅢ-HA-M solution to 5-6, placing the solution at room temperature for 2-12 hours, and then sequentially adding additives at 4°C with stirring to obtain an anti-inflammatory and anti-aging composition.

[0015] Preferably, as an improvement, the additives are at least three of chondroitin sulfate, fibronectin, elastin, human type 17 collagen, and type I collagen, and the mass percentage of each additive is 0.01 to 0.5%.

[0016] Preferably, as an improvement, this solution also provides an anti-inflammatory and anti-aging composition, which is prepared by the above preparation method.

[0017] Preferably, as an improvement, the present solution also provides the use of the above composition in the preparation of anti-inflammatory preparations, antioxidant preparations or anti-aging preparations.

[0018] Preferably, as an improvement, the present invention also provides the use of the above composition in the preparation of any one of anti-inflammatory, antioxidant, and anti-aging cosmetic preparations, daily chemical preparations, medical beauty preparations, medical device preparations, and biopharmaceutical preparations.

[0019] The principles and advantages of this solution are:

[0020] 1. This solution disperses low-molecular-weight hyaluronic acid and high-molecular-weight hyaluronic acid in deionized water at equimolar mass. Under high temperature (125°C for 10 minutes), HA initiates intermolecular polymerization. Upon rapid cooling (to 25°C within 5 minutes), it preferentially forms intermolecular polymerization with low-molecular-weight HA, resulting in a highly stable polymer. When combined with COLⅢ, the resulting COLⅢ-HA-M contains more active sites, further enhancing the anti-inflammatory effect of COLⅢ-HA-M. This in turn fully utilizes the combined effects of the various raw materials, significantly enhancing the anti-inflammatory and anti-aging effects of the composition.

[0021] 2. This solution adds a variety of raw materials in combination, and the different raw materials synergize and enhance each other, which not only effectively increases the target of the composition, but also significantly enhances the antioxidant and anti-aging effects of the composition. Specifically, first, hyaluronic acid + chondroitin sulfate + collagen form a "water-locking-structure" network, which effectively moisturizes and then strengthens the skin barrier, reducing inflammation caused by external stimuli. Secondly, fibronectin guides cell repair, type XVII collagen maintains stem cell activity, and chondroitin sulfate inhibits inflammatory pathways, accelerating damage repair in multiple ways, and taking into account both anti-inflammatory and repair. Furthermore, type I / III collagen provides support, elastin maintains resilience, and type XVII collagen delays epidermal atrophy. When used in combination, it effectively reshapes the anti-aging structure and comprehensively combats sagging and wrinkles. Finally, chondroitin sulfate effectively scavenges free radicals, and collagen reduces ultraviolet damage, synergistically delaying photoaging.

[0022] 3. The functions of hyaluronic acid (such as moisturizing and viscoelasticity) are highly dependent on the integrity of the molecular weight. This solution helps inhibit thermal degradation of hyaluronic acid during dissolution by cooling the solution to 4°C before adding hyaluronic acid, thereby maintaining its molecular weight and ensuring its full effect. This significantly improves the effect of the composition at a smaller usage amount. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1This is the effect of the composition COLⅢ-HA-M in Example 2 of the present invention on the concentration of the inflammatory factor IL-6.

[0024] Figure 2 This is the effect of the compositions MIX1 to MIX7 in Experimental Example 3 of the present invention on the concentration of the inflammatory factor IL-6.

[0025] Figure 3 This is the effect of the compositions MIX1 to MIX7 in Experimental Example 3 of the present invention on the concentration of the inflammatory factor TNF-a.

[0026] Figure 4 This is the effect of the compositions MIX1 to MIX7 in Experimental Example 5 of the present invention on the number of DCFH-DA positive cells.

[0027] Figure 5 This is the effect of MIX1 and MIX7 on the IL-1 concentration in skin tissue in Experimental Example 6 of the present invention.

[0028] Figure 6 This is the effect of MIX1 and MIX7 on IL-6 concentration in skin tissue in Experimental Example 6 of the present invention.

[0029] Figure 7 This is the effect of MIX1 and MIX7 on the concentration of MMP3 in skin tissue in Experimental Example 6 of the present invention.

[0030] Figure 8 This is the effect of MIX1 and MIX7 on the concentration of MMP9 in skin tissue in Experimental Example 6 of the present invention.

[0031] Figure 9 This is the effect of MIX1 and MIX7 on the β-galactosidase content in skin tissue in Experimental Example 6 of the present invention.

[0032] Figure 10 This is the effect of MIX1 and MIX7 on the arrangement of collagen fibers in skin tissue in Experimental Example 6 of the present invention.

[0033] Figure 11 This is the effect of MIX1 and MIX7 on the integrated optical density of collagen fibers in Experimental Example 6 of the present invention. DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used are all commercially available.

[0035] Example 1

[0036] This solution provides an anti-inflammatory and anti-aging composition, which is a COL III-HA-M solution. The preparation method of the composition in this embodiment includes the following steps:

[0037] Step 1: Preparation of HA-M solution: low molecular weight hyaluronic acid and high molecular weight hyaluronic acid are dispersed in deionized water at equimolar mass, heated and polymerized at 125°C for 10 minutes (actually, treatment at 115-150°C for 10-30 minutes is optional), and then rapidly cooled to 25°C within 5 minutes (actually, cooling to 10-37°C within 5-6 minutes is optional) to obtain a polymer HA-M solution of high molecular weight hyaluronic acid and low molecular weight hyaluronic acid;

[0038] Step 2: Prepare COLⅢ-HA-M solution: Slowly add type III collagen to the polymer HA-M solution, mix thoroughly, and purify to obtain the intermolecular complex COLⅢ-HA-M solution. This purification protocol utilizes affinity chromatography, which includes the following steps: dissolving HIS-tagged type III collagen in the HA-M solution, incubating at room temperature for 1 to 1.5 hours, and then passing the sample through a nickel column. The column is first washed three times with 5ml of Tris-HCl, 10mM NaCl solution; then, the column is eluted with Tris-HCl, 10mM NaCl, and 200mM imidazole solution. The sample is concentrated in a concentrator tube and dissolved in deionized water to obtain COLⅢ-HA-M solution. The mass concentration of type III collagen in the COLⅢ-HA-M solution is 1 to 5%.

[0039] Example 2

[0040] The present invention provides an anti-inflammatory and anti-aging composition, which also includes additives, wherein the additives are at least three of chondroitin sulfate, fibronectin, elastin, human type 17 collagen (COLⅩⅦ), and type I collagen (COLⅠ), and the mass percentage of each additive in the composition is 0.01 to 0.5%.

[0041] The preparation of the composition in this embodiment also includes step three, mixing the COLⅢ-HA-M solution with additives: adjusting the pH value of the COLⅢ-HA-M solution to 5-6 with citric acid, leaving it at room temperature for 2-12 hours, and then adding the additives in sequence at 4°C with stirring to obtain an anti-inflammatory and anti-aging composition.

[0042] According to the difference in the types of additives, compositions MIX1 to MIX7 were prepared in this example. The compositions of the raw materials of MIX1 to MIX7 are shown in Table 1.

[0043] Table 1 Composition of raw materials in MIX1 to MIX7

[0044] COLⅢ-HA-M Chondroitin sulfate Fibronectin Elastin COLⅩⅦ COLⅠ MIX1 + + + + + + MIX2 + + + - + + MIX3 + + + + + - MIX4 + - + + + + MIX5 + + - + + + MIX6 + + + + - - MIX7 + + - - + +

[0045] This solution also provides the use of the above composition in the preparation of anti-inflammatory preparations, antioxidant preparations or anti-aging preparations.

[0046] This solution also provides the use of the above composition in the preparation of any one of anti-inflammatory, antioxidant, and anti-aging cosmetic preparations, daily chemical product preparations, medical beauty preparations, medical device preparations, and biopharmaceutical preparations.

[0047] Experimental Example 1: Total sugar concentration of intermolecular complexes prepared from COLⅢ and HA treated with different methods

[0048] Equal amounts of HIS-tagged type III collagen (1 mg) were dissolved in 100 ml of 0.1% HA solution (all treated by weight). The solutions were incubated at room temperature for 1 hour. The samples were then passed through a nickel column and washed three times with 5 ml of Tris-HCl, 10 mM NaCl solution. The column was then eluted with 100 mM Tris-HCl, 200 mM imidazole solution. The sample was concentrated in a concentrator tube and the buffer exchanged with deionized water to a final volume of 10 ml. A 100 μl sample was tested for total sugar concentration (as a reference, using the anthrone-sulfuric acid method). The results are shown in Table 2.

[0049] Table 2 Total sugar concentration of intermolecular complexes prepared from COLⅢ and HA treated with different methods

[0050] sample Bound sugar concentration COLⅢ-HA-L 0.0008mg COLⅢ-HA-H 0.0022mg COLⅢ-HA-M 0.0059mg COLⅢ 0.0001mg

[0051] Experimental data show that the intermolecular complex COLⅢ-HA-M prepared by mixing COLⅢ and HA-M has the highest concentration of bound sugars, effectively increasing the active sites and thereby enhancing the anti-inflammatory effect of COLⅢ-HA-M.

[0052] Experimental Example 2: Anti-inflammatory effect of the composition COLⅢ-HA-M obtained in Example 1

[0053] After culturing RAW246.7 cells on a plate for 24 hours, carefully aspirate the original culture medium, wash 3 times with PBS, and divide the cells into 96-well plates at a density of 10,000 cells per well. After culturing for 8 hours, remove the supernatant, prepare 100 μL of complete culture medium containing 1 μg / mL LPS, and add 0.1% concentration of HA-L polysaccharide, HA-H polysaccharide, HA-M polysaccharide, and HA-L, HA-H, HA-M and the conjugates prepared with COLⅢ (i.e. COLⅢ-HA-L, COLⅢ-HA-H, COLⅢ-HA-M) to the cell wells. Set up 6 replicates for each group, set up a control group with only LPS added, and incubate in the incubator for 8 hours. Carefully aspirate the supernatant for ELISA analysis of IL-6. For detailed results, see Figure 1 .

[0054] like Figure 1 As shown, the anti-inflammatory ability of type III collagen combined with HA-M (i.e., COLⅢ-HA-M) was significantly improved.

[0055] Experimental Example 3: Anti-inflammatory effects of compositions MIX1 to MIX7

[0056] After culturing RAW246.7 cells for 24 hours, carefully remove the original culture medium, wash 3 times with PBS, and split the cells into 96-well plates at a density of 10,000 cells per well. After culturing for 8 hours, remove the supernatant, prepare 100 μL of complete culture medium containing 1 μg / mL LPS and add 1% concentration of MIX1 / MIX2 / MIX3 / MIX4 / MIX5 / MIX6 / MIX7 to the cell wells, with 6 replicates per group. Set up a control group with only LPS added. After incubation in the incubator for 8 hours, carefully remove the supernatant and perform ELISA analysis of IL-6 and TNF-a. For detailed results, see Figure 2 and Figure 3 .

[0057] like Figure 2 and Figure 3 As shown, MIX1-MIX7 in all experimental groups significantly increased cell viability and promoted cell proliferation. The MIX1 group significantly outperformed the other test groups in promoting cell proliferation. The results suggest that the combined presence of fibronectin and elastin can produce a more synergistic effect, a synergistic effect that requires the participation of chondroitin sulfate or type XVII or type I collagen.

[0058] Experimental Example 4: Anti-aging effects of the compositions MIX1 to MIX7 on skin cells

[0059] HDFn cells were digested and plated at 1×10 6 Cells were plated at a density of 1 mL / well into 6-well plates. After 12 hours of culture, DMEM containing 1% MIX1 / MIX2 / MIX3 / MIX4 / MIX5 / MIX6 / MIX7 was added. After 48 hours of culture, the cell culture medium was aspirated, the cells were washed once with PBS, and 1 ml of staining fixative (4% paraformaldehyde) was added and fixed at room temperature for 15 minutes. The fixative was aspirated, and the cells were washed three times with PBS for 3 minutes each. The PBS was aspirated, and 1 ml of SA-β-gal staining solution was added to each well. The cells were incubated overnight at 37°C and counted under a standard light microscope. The results are shown in Table 3.

[0060] Table 3 Anti-aging effect of the composition on skin cells

[0061] Test object Percentage of positive cells SD control group 26.2 5.07 MIX1 6.75 3.8 MIX2 12.87 5.6 MIX3 18.7 5.81 MIX4 12.34 6.73 MIX5 17.97 5.28 MIX6 13.85 3.64 MIX7 9.09 3.15

[0062] The results showed that the number of SA-β-gal-stained cells in all experimental groups was significantly lower than that in the control group, indicating that all test substances had anti-aging capabilities. Among them, the MIX1 and MIX3 test groups had the fewest SA-β-gal-positive cells, suggesting that they had the best anti-aging capabilities.

[0063] Experimental Example 5: Analysis of the Antioxidant Effects of Compositions MIX1 to MIX7

[0064] HDFn cells were digested and plated at 1×10 6 The cells were divided into 6-well plates at a density of 100 μg / mL and cultured for 12 h. Then, DMEM culture medium containing 1% MIX1 / MIX2 / MIX3 / MIX4 / MIX5 / MIX6 / MIX7 was added respectively. After 48 h of culture, the cell culture medium was aspirated and the cells were washed three times with serum-free cell culture medium. The cells were digested with 0.25% trypsin, and after 5 min, the cells were observed to have fallen off under a microscope. The cells were collected into a 15 mL centrifuge tube and centrifuged at 2000 rpm at 4°C for 10 min. The positive control cells were suspended in 1 mL of serum-free culture medium. The reactive oxygen species positive control (Rosup, 50 mg / ml) was diluted with serum-free culture medium at a ratio of 1:1000 with DCFH-DA to a final concentration of 10 μM / L. The diluted reactive oxygen species control was added to the positive control cells and stimulated for 20 min. After the experimental group MIX1 / MIX2 / MIX3 / MIX4 / MIX5 / MIX6 / MIX7 and untreated cells were collected and suspended in diluted DCFH-DA at a concentration of 1 million per ml. They were incubated in a 37°C cell culture incubator for 20 minutes. The cells were inverted and mixed every 3 to 5 minutes to ensure full contact between the probe and the cells. The cells were washed three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells. The results were analyzed by flow cytometry, as shown in Figure 2. Figure 4 shown.

[0065] The results showed that MIX1 to MIX7 in the experimental group all had the ability to reduce cellular reactive oxygen species, as manifested by a decrease in the number of DCFH-DA-positive cells, among which MIX1 and MIX3 had the most significant ability to inhibit cellular reactive oxygen species.

[0066] Experimental Example 6: Study on the anti-aging effects of the composition MIX1 and the composition MIX7 on skin cells at the animal level

[0067] Thirty female nude mice (22 ± 1.5 g) were selected and fed for 5 days. After acclimation, the mice were numbered and randomly divided into four groups (blank, model, positive, MIX1, and MIX7). The blank group was not irradiated with UVB and applied with matrix. The model group was irradiated with UVB and subcutaneously injected with D-galactose (D-GAL) and applied with matrix. The positive group was irradiated with UVB and subcutaneously injected with D-GAL and applied with matrix mixed with vitamin E. The MIX1 and MIX7 groups were irradiated with UVB and subcutaneously injected with D-GAL and applied with matrix mixed with MIX1 (2%). The matrix formula was as follows: 8% glycerol (10%), ethylparaben (0.1%). Six hours after modeling, the matrix, positive drug, MIX1, and MIX7 were applied to the exposed skin of the mice's backs at a rate of 100 μL per mouse once daily for four weeks. The mice were then sacrificed and their back skin tissues were harvested for subsequent testing.

[0068] The subcutaneous tissue at the smear site was cut and weighed, rinsed in 4°C 0.9% NaCl solution, dried with absorbent paper, chopped, and added with 4°C protein lysis buffer mixed with protease inhibitor (PMSF). The tissue was homogenized using a tissue homogenizer (90 seconds each time, repeated 3 times). The homogenate was centrifuged at 3000r / min for 15 minutes, and the supernatant was removed. After repeating 3 times, the protein was determined according to the BCA protein quantification kit and the protein concentration of the supernatant was diluted to the same level. An appropriate amount of supernatant was taken to detect and calculate the content of IL-1, IL-6, MMP3, MMP9 and β-galactosidase in skin tissue according to the ELISA kit instructions. The results are detailed in [ 14 ]. Figures 5 to 11 .

[0069] like Figure 5 and Figure 6 As shown in the results, compared with the blank group, the expression levels of IL-1β and IL-6 in the model group were significantly increased. After treatment with vitamin E (1 mg / g) and the MIX1 and MIX7 groups, the levels of the three inflammatory factors were significantly decreased (P<0.01).

[0070] like Figures 7 to 9 As shown, compared with the blank group, the expression of MMP3 and MMP9 in the model group was significantly upregulated (P<0.01). Compared with the model group, the MIX1 and MIX7 groups significantly downregulated the expression of MMP3 in the mouse skin (P<0.01) and the expression of MMP9 (P<0.05). The results of the measurement of β-galactosidase content in mouse skin showed that the expression of β-galactosidase in the model group was significantly increased compared with the blank group. After treatment with VE or the MIX1 and MIX7 groups, the β-galactosidase content in the mouse skin was significantly downregulated (P<0.01), indicating that the MIX1 and MIX7 groups alleviated the aging-related upregulation of β-galactosidase expression.

[0071] like Figures 10 and 11 As shown, Masson staining analysis of mouse dorsal skin tissue revealed that collagen fibers in the blank group were regularly and tightly arranged, while those in the model group were loosely curled. Treatment with MIX1 and MIX7 significantly increased collagen fiber expression, surpassing that of the blank group, and collagen fiber arrangement was more regular and tightly arranged than in the model group. This suggests that the MIX1 composite matrix and MIX7 treatments effectively repair the loss of skin collagen caused by UVB combined with D-GAL subcutaneous administration.

[0072] The above is only an embodiment of the present invention, and the common knowledge such as the specific technical solutions and / or characteristics in the solution are not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. A method for preparing an anti-inflammatory and anti-aging composition, characterized in that: The composition includes a COLIII-HA-M solution, and the preparation method includes the following steps: Step 1: Preparation of HA-M solution: Disperse low molecular weight hyaluronic acid and high molecular weight hyaluronic acid in deionized water at equimolar mass, heat to polymerize, and then cool to obtain a polymer HA-M solution of high molecular weight hyaluronic acid and low molecular weight hyaluronic acid; Step 2: Prepare COLⅢ-HA-M solution: slowly add type III collagen to the above polymer HA-M solution, mix well and purify to obtain the intermolecular complex COLⅢ-HA-M solution.

2. The preparation method according to claim 1, wherein: The heating polymerization is carried out at 115-150° C. for 10-30 minutes.

3. The preparation method according to claim 1, wherein: The cooling is performed to cool the temperature to 10-37° C. within 5-6 minutes.

4. The preparation method according to claim 1, wherein: The purification adopts affinity chromatography, which includes the following steps: dissolving type III collagen with a HIS tag in a HA-M solution, incubating at room temperature for 1 to 1.5 hours, and then passing the sample through a nickel column; first washing the column three times with a Tris-HCl and NaCl solution, then eluting the sample with a Tris-HCl, NaCl, and imidazole solution, concentrating the sample in a concentrator tube, and dissolving it in deionized water to obtain a COLⅢ-HA-M solution.

5. The preparation method according to claim 1, wherein: The mass concentration of type III collagen in the COLⅢ-HA-M solution is 1-5%.

6. The preparation method according to claim 1, wherein: The method further includes step three, mixing with additives: adjusting the pH value of the COLⅢ-HA-M solution to 5-6, placing the solution at room temperature for 2-12 hours, and then sequentially adding additives at 4°C with stirring to obtain an anti-inflammatory and anti-aging composition.

7. The preparation method according to claim 6, characterized in that: The additives are at least three of chondroitin sulfate, fibronectin, elastin, human type 17 collagen, and type I collagen, and the mass percentage of each additive is 0.01-0.5%.

8. An anti-inflammatory and anti-aging composition, characterized in that: The composition is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the composition according to claim 8 in the preparation of anti-inflammatory preparations, antioxidant preparations or anti-aging preparations.

10. Use of the composition according to claim 8 in the preparation of any one of anti-inflammatory, antioxidant, and anti-aging cosmetic preparations, daily chemical product preparations, medical beauty preparations, medical device preparations, and biopharmaceutical preparations.

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