A collagen conjugate, its preparation method and application

Abstract

This invention discloses a collagen conjugate, its preparation method, and its application, belonging to the field of cosmetic technology. The collagen conjugate is obtained by conjugating human recombinant collagen with glycosaminoglycans; the human recombinant collagen includes at least one of human recombinant type I, III, IV, V, VII, and XVII collagen; the glycosaminoglycans include at least one of hyaluronic acid and its sodium salt, chondroitin sulfate and its sodium salt, and dermatan sulfate and its sodium salt. This collagen conjugate not only forms a protective film on the skin surface, providing immediate repair and firming effects and reducing damage to the skin from external stimuli; it also decomposes to produce beneficial substances, promoting the growth of probiotics on the skin surface and maintaining the skin's microecological balance; furthermore, after degradation, the collagen conjugate promotes collagen production, enhancing its effectiveness in improving or preventing wrinkles. This collagen conjugate can be used in the preparation of cosmetics.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and more specifically, to a collagen conjugate, its preparation method, and its application. Background Technology

[0002] Collagen is an important structural protein widely found in skin, bones, and connective tissue. Due to its moisturizing, repairing, and skin-elasticity-enhancing properties, collagen has become a widely used active ingredient in the cosmetics industry. However, traditional collagen applications in cosmetics have at least one of the following limitations:

[0003] (1) Low absorption efficiency: Natural collagen has a large molecular weight, making it difficult to be effectively absorbed through the stratum corneum of the skin, thus making it difficult to fully exert its effects;

[0004] (2) Poor stability: Collagen is easily degraded in the formulation, especially in environments containing moisture and specific pH values, which limits the product's shelf life and stability.

[0005] (3) Single function: Traditional collagen mainly provides moisturizing and basic repair functions, but lacks more skin care functions.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a collagen conjugate, its preparation method, and its application, so as to solve or improve the above-mentioned technical problems.

[0008] This invention can be implemented as follows:

[0009] In a first aspect, the present invention provides a collagen conjugate obtained by conjugating human recombinant collagen with glycosaminoglycans;

[0010] Among them, recombinant human collagen includes at least one of recombinant human type I collagen, recombinant human type III collagen, recombinant human type IV collagen, recombinant human type V collagen, recombinant human type VII collagen and recombinant human type XVII collagen; glycosaminoglycans include at least one of hyaluronic acid and its sodium salt, chondroitin sulfate and its sodium salt, and dermatan sulfate and its sodium salt.

[0011] In an optional embodiment, the mass ratio of human recombinant collagen to glycosaminoglycan is (0.8–1.2):(0.8–1.2).

[0012] In an optional embodiment, the mass ratio of human recombinant collagen to glycosaminoglycan is 1:1.

[0013] Secondly, the present invention provides a method for preparing a collagen conjugate as described in the foregoing embodiments, comprising the following steps: dissolving human recombinant collagen and glycosaminoglycan to form a mixed solution; adjusting the pH of the mixed solution to 4.8-5.2; adding a catalyst to the pH-adjusted mixed solution and reacting the glycosaminoglycan in the mixed solution with the human recombinant collagen to modify the structure of the human recombinant collagen with glycosaminoglycan.

[0014] In an optional embodiment, the catalyst includes EDC.

[0015] In an optional embodiment, the amount of catalyst added to the mixed solution is 8 mmol / L to 12 mmol / L;

[0016] In an optional embodiment, the amount of catalyst added to the mixed solution is 10 mmol / L.

[0017] In an optional embodiment, the reaction temperature is 15°C to 30°C, and / or the reaction time is 2h to 3h.

[0018] In an optional implementation, the reaction is carried out under oscillating conditions.

[0019] Thirdly, the present invention provides an application of the collagen conjugate as described in the foregoing embodiments, which is used to promote the proliferation of beneficial bacteria on the epidermis.

[0020] In an optional implementation, the collagen conjugate is used to promote the proliferation of Staphylococcus epidermidis.

[0021] In an optional embodiment, the effective concentration of the collagen conjugate is 0.01 ppm to 100 ppm.

[0022] Fourthly, the present invention provides an application of the collagen conjugate as described above, which is used to promote the proliferation of human epidermal keratinocytes.

[0023] In an optional embodiment, the effective concentration of the collagen conjugate is 1 ppm to 100 ppm.

[0024] Fifthly, the present invention provides an application of the collagen conjugate as described above, which is used to inhibit the expression of inflammatory factors.

[0025] In an optional implementation, the collagen conjugate is used to inhibit TNF-α expression.

[0026] In an optional embodiment, the effective concentration of the collagen conjugate is 0.01 ppm to 100 ppm.

[0027] In an optional embodiment, the effective concentration of the collagen conjugate is 1 ppm to 100 ppm.

[0028] In a sixth aspect, the present invention provides an application of the collagen conjugate as described above, which is used to promote the expression of type I collagen COL1A1.

[0029] In an optional embodiment, the effective concentration of the collagen conjugate is 0.01 ppm to 100 ppm.

[0030] In an optional embodiment, the effective concentration of the collagen conjugate is 10 ppm to 100 ppm.

[0031] In a seventh aspect, the present invention provides a cosmetic product, wherein the raw materials for preparing the cosmetic product include the collagen conjugate described in the foregoing embodiments.

[0032] In optional embodiments, the cosmetic is a cosmetic for preventing or improving wrinkles; and / or, the cosmetic is a cosmetic for promoting the proliferation of beneficial bacteria on the skin; and / or, the cosmetic is a cosmetic for relieving or improving impaired skin barrier function; and / or, the cosmetic is a cosmetic for soothing and improving firmness.

[0033] The beneficial effects of this invention include:

[0034] The collagen conjugate provided by this invention exhibits good absorption efficiency and stability, and offers a variety of benefits. For example, it enhances film-forming repair: forming a protective film on the skin surface provides immediate repair and firming effects, reducing damage from external stimuli. It is suitable not only for normal skin but also for sensitive skin. Furthermore, it promotes probiotic growth: through a microecological degradation mechanism, the collagen conjugate decomposes to produce beneficial substances, promoting the growth of probiotics on the skin surface and maintaining the skin's microecological balance. Finally, it enhances anti-aging effects: after degradation, the collagen conjugate promotes collagen production, improving its effectiveness in improving or preventing wrinkles.

[0035] The aforementioned collagen conjugates overcome the limitations of traditional collagen and can provide more comprehensive skin care solutions for cosmetics. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 The graph shows the effect of collagen conjugate on promoting the proliferation of Staphylococcus epidermidis in Experiment Example 1.

[0038] Figure 2 This is a graph showing the effect of collagen conjugates on promoting keratinocyte proliferation in Experiment Example 2;

[0039] Figure 3 The graph shows the TNF-α inhibition results of collagen conjugate, collagen alone, and glycosaminoglycan alone in Experiment Example 3.

[0040] Figure 4 The graph shows the effects of collagen conjugate, collagen alone, and glycosaminoglycan alone on promoting COL1A1 expression in Experiment Example 4. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0042] The collagen conjugates provided by this invention, their preparation methods, and their applications are described in detail below.

[0043] The present invention provides a collagen conjugate obtained by conjugating human recombinant collagen with glycosaminoglycans.

[0044] Among them, recombinant human collagen includes at least one of recombinant human type I collagen, recombinant human type III collagen, recombinant human type IV collagen, recombinant human type V collagen, recombinant human type VII collagen and recombinant human type XVII collagen; glycosaminoglycans include at least one of hyaluronic acid and its sodium salt, chondroitin sulfate and its sodium salt, and dermatan sulfate and its sodium salt.

[0045] In some embodiments, a single type of recombinant human collagen may be coupled with a single type of glycosaminoglycan; in some other embodiments, multiple types of recombinant human collagen may be coupled with a single type of glycosaminoglycan; in some other embodiments, a single type of recombinant human collagen may be coupled with multiple types of glycosaminoglycans; in some other embodiments, multiple types of recombinant human collagen may be coupled with multiple types of glycosaminoglycans.

[0046] The collagen conjugate provided by this invention exhibits good absorption efficiency and stability, and offers a variety of benefits. For example, it enhances film-forming repair: forming a protective film on the skin surface provides immediate repair and firming effects, reducing damage from external stimuli. It is suitable not only for normal skin but also for sensitive skin. Furthermore, it promotes probiotic growth: through a microecological degradation mechanism, the collagen conjugate decomposes to produce beneficial substances, promoting the growth of probiotics on the skin surface and maintaining the skin's microecological balance. Finally, it enhances anti-aging effects: after degradation, the collagen conjugate promotes collagen production, improving its effectiveness in improving or preventing wrinkles.

[0047] Therefore, the collagen conjugate provided by this invention overcomes the limitations of traditional collagen and can provide a more comprehensive skin care solution.

[0048] In some optional embodiments, the mass ratio of the recombinant human collagen to glycosaminoglycan can be (0.8–1.2):(0.8–1.2), such as 1:1, 0.8:1.0, 0.8:1.2, 0.9:0.8, 0.9:1.0, 0.9:1.2, 1:0.8, 1:1.2, 1.2:0.8, or 1.2:1.0, or other values ​​within the range of (0.8–1.2):(0.8–1.2). In some preferred embodiments, the mass ratio of the recombinant human collagen to glycosaminoglycan is 1:1.

[0049] Accordingly, the present invention provides a method for preparing the above-mentioned collagen conjugate, which may include the following steps: dissolving human recombinant collagen and glycosaminoglycan to form a mixed solution; adjusting the pH value of the mixed solution to 4.8-5.2; adding a catalyst to the pH-adjusted mixed solution and reacting the glycosaminoglycan in the mixed solution with the human recombinant collagen to modify the structure of the human recombinant collagen with glycosaminoglycan.

[0050] In some optional embodiments, the solvent in the mixed solution can be water, and the amount of water used is sufficient to dissolve both the recombinant human collagen and the glycosaminoglycan. For example, the content of recombinant human collagen in the mixed solution can be 0.8 wt% to 1.2 wt%, and the content of glycosaminoglycan in the mixed solution can also be 0.8 wt% to 1.2 wt%. In some specific embodiments, the content of recombinant human collagen in the mixed solution is 1 wt%, and the content of glycosaminoglycan in the mixed solution is also 1 wt%.

[0051] In some alternative embodiments, hydrochloric acid can be used to adjust the pH of the mixed solution to 4.8–5.2, such as 4.8, 4.9, 5.0, 5.1, or 5.2. In some specific embodiments, hydrochloric acid can be used to adjust the pH of the mixed solution to 5.0.

[0052] In some alternative embodiments, the catalyst may, by way of example but not limitation, include EDC. The amount of catalyst added to the mixed solution may be from 8 mmol / L to 12 mmol / L, such as 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, or 12 mmol / L. In some specific embodiments, the amount of catalyst added to the mixed solution is 10 mmol / L.

[0053] In some optional embodiments, the reaction temperature between glycosaminoglycans and recombinant human collagen can be 15°C to 30°C (which can also be understood as room temperature). The reaction time between glycosaminoglycans and recombinant human collagen can be 2 hours to 3 hours, such as 2 hours, 2.5 hours, or 3 hours.

[0054] In some preferred embodiments, the reaction is carried out under oscillating conditions to make the modification and grafting more uniform and effective.

[0055] Furthermore, the reaction mixture obtained after the reaction can be post-processed. Post-processing may include, for example, ultrafiltration to remove small molecules not modified onto the recombinant collagen, followed by drying of the remaining material.

[0056] For example, the reaction mixture after the reaction can be transferred to a dialysis bag (the molecular weight cutoff can be, for example, 8000 to 14000), and dialyzed for 10 to 16 hours. The dialysate is obtained by mixing deionized water and the reaction solution at a mass ratio of 20:1 to 30:1, and the dialysate is changed every 4 hours.

[0057] Furthermore, the present invention also provides an application of the above-mentioned collagen conjugate, which can be used to promote the proliferation of beneficial bacteria on the epidermis.

[0058] In some embodiments, the collagen conjugate can be used to promote the proliferation of Staphylococcus epidermidis. Accordingly, the effective concentration of the collagen conjugate is 0.01ppm to 100ppm, such as 0.01ppm, 0.1ppm, 1ppm, 5ppm, 10ppm, 50ppm or 100ppm, or other values ​​in the range of 0.01ppm to 100ppm.

[0059] In addition, the present invention provides an application of the above-mentioned collagen conjugate, which can be used to promote the proliferation of human epidermal keratinocytes. Accordingly, the effective concentration of the collagen conjugate is 1ppm to 100ppm, such as 1ppm, 5ppm, 10ppm, 50ppm or 100ppm, or other values ​​in the range of 1ppm to 100ppm.

[0060] Furthermore, the present invention also provides an application of the above-mentioned collagen conjugate, which is used to inhibit the expression of inflammatory factors.

[0061] In some embodiments, the collagen conjugate is used to inhibit TNF-α expression. Accordingly, the effective concentration of the collagen conjugate is 0.01 ppm to 100 ppm, such as 0.01 ppm, 0.1 ppm, 1 ppm, 5 ppm, 10 ppm, 50 ppm, or 100 ppm, or other values ​​within the range of 0.01 ppm to 100 ppm. In some preferred embodiments, the effective concentration of the collagen conjugate is 1 ppm to 100 ppm. Within this preferred range, the inhibitory effect on inflammatory factors is superior to that of unconjugated collagen or glycosaminoglycans.

[0062] Furthermore, the present invention also provides an application of the above-mentioned collagen conjugate for promoting the expression of type I collagen COL1A1. Accordingly, the effective concentration of the collagen conjugate is 0.01 ppm to 100 ppm, such as 0.01 ppm, 0.1 ppm, 1 ppm, 5 ppm, 10 ppm, 50 ppm, or 100 ppm, or other values ​​within the range of 0.01 ppm to 100 ppm. In some preferred embodiments, the effective concentration of the collagen conjugate is 10 ppm to 100 ppm. Within this preferred range, the effect on promoting the expression of type I collagen COL1A1 is superior to that of unconjugated collagen or glycosaminoglycans.

[0063] In addition, the present invention also provides a cosmetic product in which the above-mentioned collagen conjugate is included in the raw materials for preparation.

[0064] In some alternative embodiments, the above-mentioned cosmetic has at least one of the following effects:

[0065] (1) Prevent or improve wrinkles; (2) Promote the proliferation of beneficial bacteria in the skin; (3) Relieve or improve impaired skin barrier function; (4) Repair; (5) Soothe; (6) Firm.

[0066] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0067] Example 1

[0068] This embodiment provides a method for preparing a collagen conjugate, comprising the following steps: dissolving human recombinant collagen and glycosaminoglycan in water to form a mixed solution, wherein the amount of human recombinant collagen and glycosaminoglycan in the mixed solution is 1 wt%. Adjusting the pH of the mixed solution to 5.0 using hydrochloric acid; adding 10 mmol / L EDC to the pH-adjusted mixed solution and reacting the glycosaminoglycan with the human recombinant collagen at room temperature with shaking for 3 hours to modify the structure of the human recombinant collagen with glycosaminoglycan. Transferring the reaction mixture to a dialysis bag (molecular weight cutoff, for example, 10,000), dialyzing for 14 hours, the dialysate being deionized water and the reaction solution at a mass ratio of 25:1, changing the dialysate every 4 hours. Drying the remaining material after dialysis to obtain the collagen conjugate.

[0069] Example 2

[0070] This embodiment provides a method for preparing a collagen conjugate, comprising the following steps: dissolving human recombinant collagen and glycosaminoglycan in water to form a mixed solution, wherein the amounts of human recombinant collagen and glycosaminoglycan in the mixed solution are 0.8 wt% and 1.2 wt%, respectively. Adjusting the pH of the mixed solution to 4.8 using hydrochloric acid; adding 8 mmol / L EDC to the pH-adjusted mixed solution and reacting the glycosaminoglycan with the human recombinant collagen at room temperature with shaking for 2.5 h to modify the structure of the human recombinant collagen with glycosaminoglycan. Transferring the reaction mixture to a dialysis bag (molecular weight cutoff, for example, 8000), dialyzing for 16 h, the dialysate being deionized water and the reaction solution at a mass ratio of 20:1, changing the dialysate every 4 h. Drying the remaining material after dialysis to obtain the collagen conjugate.

[0071] Example 3

[0072] This embodiment provides a method for preparing a collagen conjugate, comprising the following steps: dissolving human recombinant collagen and glycosaminoglycan in water to form a mixed solution, wherein the amounts of human recombinant collagen and glycosaminoglycan in the mixed solution are 1.2 wt% and 0.8 wt%, respectively. Adjusting the pH of the mixed solution to 5.2 using hydrochloric acid; adding 12 mmol / L EDC to the pH-adjusted mixed solution and reacting the glycosaminoglycan with the human recombinant collagen at room temperature with shaking for 2 hours to modify the structure of the human recombinant collagen with glycosaminoglycan. Transferring the reaction mixture to a dialysis bag (molecular weight cutoff, for example, 14000), dialyzing for 10 hours, the dialysate being deionized water and the reaction solution at a mass ratio of 30:1, changing the dialysate every 4 hours. Drying the remaining material after dialysis to obtain the collagen conjugate.

[0073] Experimental Example 1

[0074] The effect of collagen conjugates on the proliferation of beneficial bacteria on the skin.

[0075] Staphylococcus epidermidis (accession number CMCC26069, purchased from the China Center for Medical Bacteriological Culture Collection), stored at -80℃, was reactivated in LB broth at 37℃ and 200 rpm for approximately 24 hours. The activated strain was collected by centrifugation at 4000 rpm for 2 minutes, washed twice with sterile water, and the broth was removed. The bacterial cells were resuspended in sterile water, and the OD of the bacterial suspension was adjusted. 600 The value was approximately 0.6. Five 600 μl aliquots of bacterial suspension were added to 24-well plates with collagen conjugate at final concentrations of 0.01 ppm, 0.1 ppm, 1 ppm, 10 ppm, and 100 ppm, respectively. The plates were incubated continuously at 37°C and 100 rpm for 8 hours, with OD values ​​measured hourly using a microplate reader. 600 Values ​​were calculated, and a growth curve was plotted. Data from 8 hours were as follows: Figure 1 As shown.

[0076] The collagen conjugate used in this experiment was prepared according to the method provided in Example 1, wherein the human recombinant collagen was human recombinant type III collagen and the glycosaminoglycan was chondroitin sulfate sodium.

[0077] Depend on Figure 1 It can be seen that collagen conjugates have the effect of promoting the proliferation of beneficial bacteria in the epidermis within the concentration range of 0.01ppm to 100ppm, which helps to establish a healthy skin microecological barrier.

[0078] Experimental Example 2

[0079] Collagen conjugates can alleviate or improve impaired skin barrier function.

[0080] HaCaT cells in good growth condition were cultured in 96-well plates. After 24 hours, collagen conjugate was added to final concentrations of 0.01 ppm, 0.1 ppm, 1 ppm, 10 ppm, and 100 ppm, respectively. The cells were then further cultured at 37°C for 48 hours. Triple replicates were used, and cell viability was assessed using the CCK-8 assay. Results are shown below. Figure 2 As shown.

[0081] The collagen conjugate used in this experiment was prepared according to the method provided in Example 1, wherein the human recombinant collagen was human recombinant type III collagen and the glycosaminoglycan was chondroitin sulfate sodium.

[0082] Depend on Figure 2It can be seen that collagen conjugates have the effect of promoting the proliferation of human epidermal keratinocytes in the concentration range of 1ppm to 100ppm, which helps to alleviate or improve the damage to the skin barrier function.

[0083] Experimental Example 3

[0084] The skin-soothing effect of collagen conjugates.

[0085] HaCaT cells in good growth condition were cultured in 6-well plates. After 24 hours, collagen conjugate was added to final concentrations of 0.01 ppm, 0.1 ppm, 1 ppm, 10 ppm, and 100 ppm, respectively. The cells were then cultured at 37°C for 48 hours, with three replicates. Total RNA was extracted using the QIAGEN RNeasy Mini Kit and diluted to the same concentration. TNF-α expression was detected using quantitative real-time qPCR. Comparisons were also set up using collagen alone and glycosaminoglycan alone at the same concentrations and experimental methods. The results are shown below. Figure 3 As shown.

[0086] The collagen conjugate used in this experiment was prepared according to the method provided in Example 1, wherein the human recombinant collagen was human recombinant type III collagen and the glycosaminoglycan was chondroitin sulfate sodium.

[0087] Depend on Figure 3 It can be seen that collagen conjugates have the effect of inhibiting the inflammatory factor TNF-α in the concentration range of 0.01ppm to 100ppm, and the effect is better than that of collagen or glycosaminoglycans alone in the range of 1ppm to 100ppm.

[0088] Test Example 4

[0089] Collagen conjugates can alleviate or improve skin wrinkles.

[0090] HDF cells in good growth condition were cultured in 6-well plates. After 24 hours, collagen conjugate was added to final concentrations of 0.01 ppm, 0.1 ppm, 1 ppm, 10 ppm, and 100 ppm, respectively. The cells were then cultured at 37°C for 48 hours, with three replicates. Total RNA was extracted using the QIAGEN RNeasy Mini Kit and diluted to the same concentration. The expression level of COL1A1 was detected using quantitative real-time qPCR. Comparisons were also set up using collagen alone and glycosaminoglycan alone at the same concentrations and experimental methods. The results are shown below. Figure 4 As shown.

[0091] The collagen conjugate used in this experiment was prepared according to the method provided in Example 1, wherein the human recombinant collagen was human recombinant type III collagen and the glycosaminoglycan was chondroitin sulfate sodium.

[0092] Depend on Figure 4 It can be seen that collagen conjugates can promote the expression of type I collagen COL1A1 in the concentration range of 0.01ppm to 100ppm, and the effect is significantly better than collagen or glycosaminoglycans alone in the range of 10ppm to 100ppm.

[0093] Experimental Example 5

[0094] The influence of collagen type on the efficacy of collagen conjugates.

[0095] HDF cells in good growth condition were cultured in 6-well plates. After 24 hours, different collagen conjugates (the human recombinant collagen used is shown in Table 1) were added to a final concentration of 10 ppm for each conjugate. The cells were then cultured at 37°C for 48 hours, with three replicates. Total RNA was extracted using the QIAGEN RNeasy Mini Kit and diluted to the same concentration. The expression level of COL1A1 was detected using quantitative real-time qPCR, and the results are shown in Table 1.

[0096] Table 1. Effects of different types of collagen on the efficacy of collagen conjugates.

[0097] Collagen categories COL1A1 expression promotion rate (%) Human recombinant type I collagen 142.6 Human recombinant type III collagen 143.8 Human recombinant type IV collagen 138.8 Human recombinant type V collagen 137.2 Human recombinant type VII collagen 140.1 Human recombinant type XVII collagen 134.9

[0098] As shown in Table 1, the collagen conjugates prepared from different types of human recombinant collagen provided by this invention all have a high COL1A1 expression promotion rate.

[0099] Experimental Example 6

[0100] The effect of different types of glycosaminoglycans on the efficacy of collagen conjugates.

[0101] HaCaT cells in good growth condition were cultured in 6-well plates. After 24 hours, different collagen conjugates (the glycosaminoglycans used are shown in Table 2) were added to a final concentration of 10 ppm for each conjugate. The cells were then cultured at 37°C for 48 hours, with three replicates. Total RNA was extracted using the QIAGEN RNeasy Mini Kit and diluted to the same concentration. TNF-α expression was detected using quantitative real-time qPCR, and the results are shown in Table 2.

[0102] Table 2. Effects of different types of glycosaminoglycans on the efficacy of collagen conjugates.

[0103] Glycosaminoglycans TNF-α expression inhibition rate (%) Chondroitin sulfate 42.7 Sodium chondroitin sulfate 43.2 Sodium dermatin sulfate 39.8 Sodium hyaluronate 41.1

[0104] As shown in Table 2, the collagen conjugates prepared according to different glycosaminoglycan types provided by the present invention all have high TNF-α expression inhibition rates.

[0105] Experimental Example 7

[0106] The recombinant human collagen involved in this experiment was all recombinant human type III collagen, and the glycosaminoglycans were all chondroitin sulfate sodium.

[0107] This experimental example includes multiple comparative examples:

[0108] Among them, the test sample of Comparative Example 1 was a mixture obtained by directly mixing human recombinant type III collagen and glycosaminoglycan at a mass ratio of 1:1 at room temperature.

[0109] The test sample of Comparative Example 2 was a collagen conjugate prepared according to the preparation method of Example 1, wherein the mass ratio of human recombinant collagen to glycosaminoglycan was changed to 1:0.5.

[0110] The test sample of Comparative Example 3 was a collagen conjugate prepared according to the preparation method of Example 1, with the mass ratio of human recombinant collagen to glycosaminoglycan changed to 0.5:1.

[0111] The test sample of Comparative Example 4 was prepared according to the preparation method of Example 1 without the addition of a catalyst.

[0112] The test sample of Comparative Example 5 was a collagen conjugate prepared according to the preparation method of Example 1, wherein the amount of catalyst added was set to 5 mmol / L.

[0113] The test sample of Comparative Example 6 was a collagen conjugate prepared according to the preparation method of Example 1, wherein the amount of catalyst added was set to 15 mmol / L.

[0114] The test samples obtained from Comparative Examples 1 to 6 were tested at a concentration of 10 ppm according to the methods of Test Examples 1 to 4, and the results are shown in Tables 3 to 6.

[0115] Table 3. Results of Staphylococcus epidermidis proliferation rate.

[0116] Staphylococcus epidermidis proliferation rate (%) Example 1 24.6 Comparative Example 1 23.7 Comparative Example 2 23.1 Comparative Example 3 14.7 Comparative Example 4 22.7 Comparative Example 5 19.3 Comparative Example 6 20.0

[0117] Table 4. Results of Keratinocyte Proliferation Rate

[0118] Keratinocyte proliferation rate (%) Example 1 10.0 Comparative Example 1 3.1 Comparative Example 2 6.8 Comparative Example 3 2.6 Comparative Example 4 3.0 Comparative Example 5 4.2 Comparative Example 6 7.3

[0119] Table 5. Results of TNF-α Inhibition Rate

[0120] TNF-α expression inhibition rate (%) Example 1 43.6 Comparative Example 1 23.5 Comparative Example 2 32.0 Comparative Example 3 37.6 Comparative Example 4 30.1 Comparative Example 5 39.8 Comparative Example 6 34.7

[0121] Table 6 Results of the Promotion Rate of COL1A1 Expression Level

[0122] COL1A1 expression promotion rate (%) Example 1 143.8 Comparative Example 1 124.6 Comparative Example 2 135.7 Comparative Example 3 113.5 Comparative Example 4 122.8 Comparative Example 5 137.0 Comparative Example 6 130.3

[0123] In summary, the collagen conjugate provided by this invention not only forms a protective film on the skin surface, providing immediate repair and firming effects and reducing damage to the skin from external stimuli, but also decomposes to produce beneficial substances, promoting the growth of probiotics on the skin surface and maintaining the skin's microecological balance. Furthermore, the degradation of this collagen conjugate promotes collagen production, enhancing its effectiveness in improving or preventing wrinkles. This collagen conjugate can be used in the preparation of cosmetics.

[0124] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A collagen conjugate, characterized by, The preparation of the collagen conjugate comprises the following steps: dissolving human recombinant collagen and glycosaminoglycan to form a mixed solution; adjusting the pH value of the mixed solution to 4.8-5.2; adding a catalyst to the mixed solution after adjusting the pH value and allowing the glycosaminoglycan in the mixed solution to react with the human recombinant collagen to modify the glycosaminoglycan to the structure of the human recombinant collagen. The human recombinant collagen comprises at least one of human recombinant type I collagen, human recombinant type III collagen, human recombinant type IV collagen, human recombinant type V collagen, human recombinant type VII collagen and human recombinant type X VII collagen; and the glycosaminoglycan comprises at least one of hyaluronic acid and its sodium salt, chondroitin sulfate and its sodium salt, dermatan sulfate and its sodium salt. The mass ratio of the human recombinant collagen to the glycosaminoglycan is (0.8-1.2):(0.8-1.2). The catalyst comprises EDC; the amount of the catalyst added to the mixed solution is 8-12 mmol / L; the reaction temperature is 15-30°C; and the reaction time is 2-3 hours.

2. The collagen conjugate of claim 1, wherein, The mass ratio of the human recombinant collagen to the glycosaminoglycan is 1:

1.

3. A method for preparing the collagen conjugate according to claim 1 or 2, characterized by, The preparation of the collagen conjugate comprises the following steps: dissolving human recombinant collagen and glycosaminoglycan to form a mixed solution; Adjusting the pH value of the mixed solution to 4.8-5.2; Adding a catalyst to the mixed solution after adjusting the pH value and allowing the glycosaminoglycan in the mixed solution to react with the human recombinant collagen to modify the glycosaminoglycan to the structure of the human recombinant collagen. The mass ratio of the human recombinant collagen to the glycosaminoglycan is (0.8-1.2):(0.8-1.2). The catalyst comprises EDC; the amount of the catalyst added to the mixed solution is 8-12 mmol / L; the reaction temperature is 15-30°C; and the reaction time is 2-3 hours.

4. The production method according to claim 3, characterized by, The amount of the catalyst added to the mixed solution is 10 mmol / L.

5. The preparation method according to claim 3, characterized in that, The reaction is carried out under shaking conditions.

6. The use of the collagen conjugate as claimed in claim 1 or 2, characterized by The collagen conjugate is used for preparing a product for maintaining the skin microecological balance.

7. Use according to claim 6, characterized in that, The collagen conjugate is used for promoting the proliferation of Staphylococcus epidermidis.

8. Use according to claim 7, characterized in that, The action concentration of the collagen conjugate is 0.01-100 ppm.

9. The use of the collagen conjugate as claimed in claim 1 or 2, characterized by The collagen conjugate is used for preparing a product for relieving or improving the damage of skin barrier function.

10. Use according to claim 9, characterized in that, The action concentration of the collagen conjugate is 1-100 ppm.

11. The use of the collagen conjugate as claimed in claim 1 or 2, wherein The collagen conjugate is used for preparing a product with skin repair effect.

12. Use according to claim 11, characterized in that, The collagen conjugate is used for inhibiting the expression of TNF-α.

13. Use according to claim 12, characterized in that, The action concentration of the collagen conjugate is 0.01-100 ppm.

14. Use according to claim 13, characterized in that, The action concentration of the collagen conjugate is 1-100 ppm.

15. Use of the collagen conjugate as claimed in claim 1 or 2, wherein, The collagen conjugate is used for preparing a product for improving or preventing wrinkles.

16. Use according to claim 15, characterized in that, The action concentration of the collagen conjugate is 0.01-100 ppm.

17. Use according to claim 16, characterized in that, The action concentration of the collagen conjugate is 10-100 ppm.

18. A cosmetic product, characterized by, The collagen conjugate according to claim 1 or 2 is included in a raw material for preparing the cosmetic.

19. The cosmetic product of claim 18, wherein, The cosmetic is a cosmetic for preventing or improving wrinkles; and / or, the cosmetic is a cosmetic for promoting skin microflora balance; and / or, the cosmetic is a cosmetic having the effect of alleviating or improving impaired skin barrier function; and / or, the cosmetic is a cosmetic having the effect of soothing and improving firming.

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