A composition containing a polypeptide and collagen and uses thereof

Abstract

The application discloses a kind of polypeptide and collagen-containing composition and its application, the composition includes polypeptide and collagen;The polypeptide includes palmitoyl pentapeptide-4, decarboxyl carnosine, acetyl octapeptide-3 and hexapeptide-11;The collagen is recombinant type XVII collagen;The mass ratio of polypeptide and collagen is 12:10 or 12:20.The application adds recombinant type XVII collagen in polypeptide composition, can significantly improve the permeability of polypeptide composition on the one hand, and can improve the generation of collagen, the compounding of polypeptide composition and recombinant type XVII collagen can achieve synergistic effect, improve COL7A1 and COL17A1 gene relative expression, to enhance the overall anti-aging effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cosmetics, in particular to a composition containing polypeptides and collagen and application thereof. BACKGROUND

[0002] In recent years, with the continuous growth of the color value economy and self-pleasure consumption, the public's anti-aging awareness is becoming younger, and anti-aging has become a must-have for beauty across multiple age groups, and is also the most concerned skin care demand of consumers. According to reports, among the Chinese anti-aging population, 80.2% have started using anti-aging products before the age of 35, and consumers aged 23-28 have begun to focus on concepts such as anti-oxidation. This has led to anti-aging skin care products leading the consumer boom in functional skin care, and the market demand for related technologies has increased.

[0003] Patent document CN119454551A discloses that by compounding plant extracts (high mountain fireweed, peach resin, gentian root, marigold) with composite polypeptides (palmitoyl tripeptide-1, tetrapeptide-7, etc.) and recombinant collagen proteins (I / IV / VII type), the skin elasticity and anti-wrinkle effect are improved. Patent document CN119280095A discloses the combination of okra hydrolyzed glycoprotein, bidens pilosa extract, recombinant XVII / III type collagen protein and cordyceps sinensis extract, emphasizing the anti-wrinkle effect by supplementing collagen protein and activating endogenous production. Although these compositions introduce recombinant collagen protein, they hardly verify the correlation with the expression of collagen protein-related genes (such as COL1A1, COL4A1, COL17A1, etc.), and lack targeted repair of collagen protein. Secondly, the stability of some natural ingredients in the composition is poor, and they are easily affected by heat, light and other factors, resulting in a decrease in their activity.

[0004] Patent document CN114010526A discloses an anti-aging polypeptide composition containing a complex of hexapeptide-11 and trifluoroacetyl tripeptide-2. It slows down the aging process by reducing the level of oxidative stress (ROS). However, the target of this patent composition is relatively single, limited to the reduction of oxidative stress, and cannot comprehensively solve the multiple problems of skin aging, such as collagen synthesis and improvement of skin elasticity.

[0005] In addition, most of the anti-aging ingredients in the prior art, although having certain activity, are difficult to penetrate into the deep layer of the skin due to their large molecular structure or the limitation of physical and chemical properties, resulting in a significant reduction in effect. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a composition containing polypeptides and collagen and application thereof.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] In a first aspect, the present application provides a polypeptide and collagen-containing composition, which comprises polypeptide and collagen;

[0009] The polypeptide comprises palmitoyl pentapeptide-4, decarboxy carnosine, acetyl octapeptide-3 and hexapeptide-11.

[0010] The collagen is recombinant collagen type XVII.

[0011] The mass ratio of the polypeptide to the collagen is 12:5-12:25.

[0012] As a preferred solution, the mass ratio of the polypeptide to the collagen is 12:10-12:20.

[0013] As a preferred solution, the mass ratio of the polypeptide to the collagen is 12:18-22, and most preferably, the mass ratio is 12:20.

[0014] As a preferred solution, the mass ratio of the polypeptide to the collagen is 12:10 or 12:20.

[0015] As a preferred solution, the polypeptide comprises palmitoyl pentapeptide-4, decarboxy carnosine, acetyl octapeptide-3 and hexapeptide-11 with a mass ratio of 0.0001-5:0.0001-5:0.0001-5:0.0001-5.

[0016] As a preferred solution, the polypeptide comprises palmitoyl pentapeptide-4, decarboxy carnosine, acetyl octapeptide-3 and hexapeptide-11 with a mass ratio of 1:1:1:1.

[0017] In a second aspect, the present application provides use of the aforementioned polypeptide and collagen-containing composition in the preparation of an anti-aging product.

[0018] As a preferred solution, the mass percentage of the composition in the anti-aging product is 0.0001-5%.

[0019] As a preferred solution, the anti-aging product is an anti-aging skin care product, and the dosage form of the skin care product comprises any one of water, emulsion, cream, gel, oil, powder, tablet, mud, aerosol, patch, film or nano preparation.

[0020] In a third aspect, the present application provides an anti-aging skin care product, which comprises the aforementioned polypeptide and collagen-containing composition; and the mass percentage of the composition in the anti-aging skin care product is 0.0001-5%.

[0021] As a preferred solution, the skin care product further comprises at least one of a solvent, an oil, a chelating agent, an emulsifier, a humectant, a thickening agent, a solubilizer, an emollient, a rheology modifier, an antioxidant, a whitening agent, a conditioning agent, a soothing agent, a fragrance, a pigment.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] 1) The present application can significantly improve the permeability of the polypeptide composition by adding recombinant collagen XVII to the polypeptide composition. The transdermal permeation test results show that after adding recombinant collagen XVII, the relative permeability of the polypeptide composition at 0.5 h, 2 h, 4 h and 6 h is increased by 815.6%, 70.6%, 70.4% and 47.7% respectively.

[0024] 2) The present application can improve the generation of collagen by adding recombinant collagen XVII to the polypeptide composition. And through the compounding of the polypeptide composition and the recombinant collagen XVII, the relative expression of COL7A1 and COL17A1 genes can be improved, so as to enhance the overall anti-aging effect. BRIEF DESCRIPTION OF DRAWINGS

[0025] Other features, objects and advantages of the present application will become more apparent from the following detailed description of non-limiting embodiments with reference to the attached drawings:

[0026] Figure 1 The test results of cell viability using the polypeptide composition, recombinant collagen XVII or a combination of the two;

[0027] Figure 2 The distribution diagram of the test sample using the polypeptide composition at different depths of human in vivo skin;

[0028] Figure 3 The distribution diagram of the test sample using the polypeptide composition compounded with recombinant collagen XVII at different depths of human in vivo skin;

[0029] Figure 4 The relative permeability change curve of the polypeptide composition when using the test sample of the polypeptide composition and the test sample of the polypeptide composition compounded with recombinant collagen XVII. DETAILED DESCRIPTION

[0030] The present application will be described in detail below with specific examples. The following examples will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made. These all belong to the protection scope of the present application.

[0031] In the early research results of the applicant's research and development team, a kind of polypeptide composition with synergistic effect (CN118141702A) is developed, which can play the roles of antioxidant, promoting collagen production, inhibiting elastase and the like through reasonable combination of different polypeptides, so as to delay skin aging. However, the skin permeability of the polypeptide composition is still insufficient, and the effect of promoting collagen production still needs to be improved. Based on this, the present application tries to add collagen type XVII to the polypeptide composition scheme to promote the skin permeability of other polypeptides and further improve the production of collagen, and enhance the overall anti-aging effect.

[0032] The technical solutions of the present application will be described in detail below through examples.

[0033] Example 1

[0034] (1) In vitro cell safety evaluation

[0035] 1.1 Experimental materials

[0036] Test sample: recombinant collagen type XVII and polypeptide composition

[0037] Blank control group (BC): serum-free DMEM medium

[0038] The test sample group was treated as follows: 1) recombinant collagen type XVII group: take 1 mL of recombinant collagen type XVII solution (the mass concentration of recombinant collagen type XVII is 100 ppm), add 4 mL of DEME medium containing 10% serum (FBS) to prepare 20% recombinant collagen type XVII solution, and then dilute to 10%, 5%, 2.5%, 1.25%, 0.625%, and 0.3125% recombinant collagen type XVII solution for standby; 2) polypeptide composition group: weigh palmitoyl pentapeptide-4, decarboxylated carnosine, acetyl octapeptide-3, and hexapeptide-11 and add them to solvent (pure water) to prepare a polypeptide composition solution (the mass concentration of palmitoyl pentapeptide-4, decarboxylated carnosine, acetyl octapeptide-3, and hexapeptide-11 is 5 ppm each); weigh 1 mL of the polypeptide composition solution, add 4 mL of DEME medium containing 10% serum (FBS) to prepare 20% polypeptide composition solution, and then dilute to 10%, 5%, 2.5%, 1.25%, 0.625%, and 0.3125% polypeptide composition solution for standby; 3) polypeptide composition: recombinant collagen type XVII = 6:1 group (the mass ratio of palmitoyl pentapeptide-4, decarboxylated carnosine, acetyl octapeptide-3, hexapeptide-11, and recombinant collagen type XVII is 3:3:3:3:10): take 1200 μL of the aforementioned prepared polypeptide composition solution, add 200 μL of recombinant collagen type XVII solution to prepare 100% 6:1 finished product solution, take 1 mL of 100% 6:1 finished product solution, add 4 mL of DEME medium containing 10% serum (FBS) to prepare 20% 6:1 finished product solution, and then dilute to 10%, 5%, 2.5%, 1.25%, 0.625%, and 0.3125% 6:1 finished product solution for standby; 4) polypeptide composition: recombinant collagen type XVII = 3:1 group (the mass ratio of palmitoyl pentapeptide-4, decarboxylated carnosine, acetyl octapeptide-3, hexapeptide-11, and recombinant collagen type XVII is 3:3:3:3:20): take 1200 μL of the aforementioned prepared polypeptide composition solution, add 400 μL of recombinant collagen type XVII solution to prepare 100% 3:1 finished product solution, take 1 mL of 100% 3:1 finished product solution, add 4 mL of DEME medium containing 10% serum (FBS) to prepare 20% 3:1 finished product solution, and then dilute to 10%, 5%, 2.5%, 1.25%, 0.625%, and 0.3125% 3:1 finished product solution for standby. The samples of different concentrations were incubated for 24 h.

[0039] Cell source: human immortalized keratinocytes HaCaT from the Chinese Academy of Sciences Cell Bank.

[0040] 1.2 Main reagents and instruments (as shown in Table 1 and Table 2)

[0041] Table 1 Raw materials and reagents

[0042]

[0043] Table 2 Instruments and equipment

[0044]

[0045] 1.3 Experimental method

[0046] The sample group and the blank control group were set up respectively for comparison experiment, and the cell experiment was set up with 6 repeats (n=6). The logarithmic growth phase cells were inoculated in 96-well plates at 1 10 4 cells / well, and incubated in a 37°C, 5% CO2 incubator for 24 h. Then the supernatant was discarded, and the cells were washed twice with PBS. Each concentration gradient solution prepared by each sample group was added to the well plate at 100 μL per well, and incubated for 24 h. Then 10 μL of CCK-8 reagent was added and incubated for 1 h, and the OD value at 450 nm was measured. The cell viability (i.e. cell survival rate) was calculated according to the following formula:

[0047]

[0048] In the formula: V (%) - cell viability, %; OD sample - absorbance of the reaction system containing the sample to be tested; OD blank control - absorbance of the empty plate without any substance; OD cell control - absorbance without the sample to be tested.

[0049] 1.4 Data processing

[0050] The data was expressed as mean ± standard error using GraphPad Prism 8 software.

[0051] 1.5 Experimental results and analysis

[0052] Cell survival rate is usually used as an indicator to evaluate the cytotoxicity of components, and CCK-8 method can determine the number of living cells in the cells, which has the advantages of high sensitivity and no radioactivity. For example, Figure 1As shown, the cell survival rate was greater than 90% at a concentration of 0.3125% to 5% of the recombinant collagen XVII, the cell survival rate was greater than 90% at a concentration of 0.3125% to 10% of the polypeptide composition, and the cell survival rate was greater than 90% at a concentration of 0.3125% to 2.5% of the polypeptide composition:recombinant collagen XVII = 6:1 or 3:1. Based on the above results, 2.5% of the polypeptide composition, the recombinant collagen XVII, and the polypeptide composition:recombinant collagen XVII = 6:1 or 3:1 were selected for subsequent experiments.

[0053] (2) In vitro efficacy test

[0054] 2.1 Experimental materials

[0055] Test sample: 2.5% sample solution prepared from each sample group in step 1.1

[0056] Blank control group (BC): serum-free DMEM medium starvation for 2 h, 10% serum (FBS) DMEM medium culture for 24 h

[0057] Model (UVB) group: serum-free medium starvation for 2 h, 60 mJ / cm 2 10% serum (FBS) DMEM medium culture for 24 h after UVB irradiation

[0058] Test sample group treatment: serum-free medium starvation for 2 h, 60 mJ / cm 2 Add sample solution of corresponding concentration and culture for 24 h after UVB irradiation

[0059] Positive control (PC) group: serum-free medium starvation for 2 h, 60 mJ / cm 2 Add 10% serum (FBS) DMEM medium containing VC+VE and culture for 24 h after UVB irradiation

[0060] Cell source: human immortalized keratinocytes HaCaT, P14

[0061] 2.2 Main reagents and instruments (as shown in Tables 3 and 4)

[0062] Table 3 Reagents

[0063]

[0064] Table 4 Instruments and equipment

[0065]

[0066] 2.3 Experimental grouping (as shown in Table 5)

[0067] Table 5 Experimental grouping

[0068]

[0069] 2.4 Experimental method

[0070] 1) Logarithmic growth phase HaCaT cells were inoculated in 6-well plates at 1 10 6 cells per well, and cultured in a 37°C 5% CO2 incubator for 24 h;

[0071] 2) Discard the culture medium, add 2 mL serum-free DMEM medium to each well, and starve for 2 h;

[0072] 3) After discarding the serum-free medium, UVB (60 mJ / cm 2 ) treated cells (blank control group without UVB treatment)

[0073] 4) After washing with PBS twice, sample solution was added to the sample group, and 10% serum DMEM medium was added to the blank control group and model group, and cultured in a 37°C 5% CO2 incubator for 24 h;

[0074] 5) Discard the supernatant, wash with PBS twice, and collect the cells;

[0075] 6) Extract RNA and reverse transcribe into cDNA for RT-qPCR.

[0076] 7) The relative expression of target genes was calculated by 2 -△△Ct method using GAPDH as an internal reference.

[0077] 8) The values obtained by calculation were imported into Graphpad Prism 8 for statistical analysis and graphing.

[0078] 2.5 Data processing

[0079] Graphpad Prism 8 software was used, and the data were expressed as mean ± standard error. The difference between the blank control group and the model group was analyzed using T-test; the difference between the experimental group and the model group was analyzed using one-way ANOVA, P<0.05 was considered to be significantly different, P<0.01 was considered to be extremely significantly different, and P<0.001 was considered to be extremely significantly different.

[0080] 2.6 Experimental results and analysis

[0081] Table 6 Analysis of COL4A1 gene expression results

[0082]

[0083] Table 7 Summary of COL7A1 gene expression result analysis

[0084]

[0085] Table 8 Summary of COL17A1 gene expression result analysis

[0086]

[0087] Table 9 Summary of COL1A1 gene expression result analysis

[0088]

[0089] COL1A1 is a gene encoding collagen type I alpha 1 chain, involved in maintaining the integrity and elasticity of the skin, COL17A1 plays an important role in keeping the skin young. It maintains tissue health by promoting stem cell competition, while promoting stronger cells to proliferate; ECM is mainly composed of collagen IV (COL4), which is essential for maintaining tissue structure and function, and collagen VII (COL7) encoded by COL7A1 gene forms anchoring fibers to connect the epidermis and dermis.

[0090] The relative expression of COL4A1, COL1A1, COL17A1 and COL1A1 genes was determined by RT-qPCR using HaCaT cells, and the results are shown in Tables 6, 7, 8 and 9. Compared with the model group, the use of 2.5% polypeptide composition, recombinant collagen type XVII, and the complex of polypeptide composition: recombinant collagen type XVII = 6:1 and 3:1 can significantly increase the relative expression of COL4A1, COL7A1, COL17A1 and COL1A1 genes.

[0091] Further, as can be seen from the results of Table 6 and Table 9, when the polypeptide composition: recombinant type XVII collagen = 6:1 or 3:1 is compounded at 2.5%, the relative expression of COL4A1 and COL1A1 genes is increased at a rate between the increase rate of the polypeptide composition alone and the increase rate of the recombinant type XVII collagen alone. As can be seen from the results of Table 7, when the polypeptide composition: recombinant type XVII collagen = 6:1 is compounded at 2.5%, the relative expression of COL4A1 and COL1A1 genes is increased at a rate between the increase rate of the polypeptide composition alone and the increase rate of the recombinant type XVII collagen alone; and when the polypeptide composition: recombinant type XVII collagen = 3:1 is compounded at 2.5%, the relative expression of COL7A1 gene is increased at a rate higher than the increase rate of the polypeptide composition alone or the recombinant type XVII collagen alone, indicating that the combination of the polypeptide composition and the recombinant type XVII collagen at a ratio of 3:1 can exert synergistic effect to increase the relative expression of COL7A1 gene. As can be seen from the results of Table 8, when the polypeptide composition: recombinant type XVII collagen = 6:1 or 3:1 is compounded at 2.5%, the relative expression of COL17A1 gene is increased at a rate higher than the increase rate of the polypeptide composition alone or the recombinant type XVII collagen alone, indicating that the combination of the polypeptide composition and the recombinant type XVII collagen can exert synergistic effect to increase the relative expression of COL17A1 gene.

[0092] The combination of the polypeptide composition and the recombinant type XVII collagen can significantly increase the relative expression of COL7A1 and COL17A1 genes, so that the expression of the two genes can be promoted by the combination of the polypeptide composition and the recombinant type XVII collagen, thereby significantly improving the anti-aging effect.

[0093] (3) Transdermal penetration test

[0094] 3.1 Purpose and method of the experiment

[0095] The human forearm region was selected for human Raman test, wherein the samples for testing were the polypeptide composition (sample group 1 in Table 5), the polypeptide composition: recombinant type XVII collagen = 3:1 (sample group 4 in Table 5), and the test skin area was selected to be 1×1 cm 2 , and the tests were performed at 0.5 h, 2 h, 4 h, and 6 h time points; the number of test persons was 1, and the test area was 3 pieces of 1×1 cm 2 test skin area, and 5 points in each test area were selected for parallel test 3 times.

[0096] Specific method: T / SHRH 064-2024《Cosmetic ingredients transdermal penetration test Human in vivo Raman spectroscopy》

[0097] 3.2 Test sample

[0098] Test sample: Polypeptide composition, Polypeptide composition: Recombinant collagen type XVII = 3:1

[0099] Method of use: Apply, use amount 2 mg / cm 2

[0100] 3.3 Test instrument

[0101] LabRAM Soleil ultra-high resolution microscopic confocal Raman spectrometer

[0102] 3.4 Test environment

[0103] Temperature: 22℃ ± 2℃; Humidity: 50% ± 10% RH

[0104] 3.5 Test process

[0105] Test instructions were given to the subjects, and after the subjects visited, the test area skin was cleaned with clean water, and after cleaning, the subjects sat in the constant temperature and humidity room for 30 min. Then the product was applied to the test area for Raman testing of the light peptide sample.

[0106] 3.6 Result statistical method

[0107] Raman spectrum imaging data processing includes spectrum preprocessing and data analysis. The spectrum preprocessing process includes cosmic ray removal, spectrum smoothing, background noise removal, baseline calibration and spectrum normalization. Univariate data analysis is mainly aimed at a specific peak position corresponding to a biochemical substance, and Raman spectrum data analysis is performed to show the distribution of the substance in human skin.

[0108] Data analysis uses Labspec software to calibrate the baseline and confirm the characteristic peak position of the Raman spectrum, calculates the obtained Raman spectrum, including peak intensity, peak shift, peak area, half-peak width, etc.; at the same time, Labspec software is used for numerical analysis of peak intensity corresponding to different depths and drawing of spatial distribution.

[0109] The penetration behavior of the sample is confirmed by the characteristic Raman signal of the product that is different from the intrinsic signal of the skin, and its distribution in different skin depth spaces is confirmed.

[0110] 3.7 Test results

[0111] Through depth analysis of the Raman image, the distribution of the polypeptide composition in the human in-vivo skin at different depths in the polypeptide composition test sample is obtained, and the distribution graph is as shown in the following figure Figure 2 As shown, through analysis, it is concluded that the relative penetration rate of the polypeptide composition within 0.5 h, 2 h, 4 h and 6 h after the polypeptide composition sample is used on the human in-vivo skin is 0.45%, 7.08%, 12.52% and 19.73% respectively.

[0112] Through depth analysis of the Raman image, the distribution of the polypeptide composition in the human in-vivo skin at different depths in the polypeptide composition test sample is obtained, and the distribution graph is as shown in the following figure Figure 3 As shown, through analysis, it is concluded that the relative penetration rate of the polypeptide composition within 0.5 h, 2 h, 4 h and 6 h after the polypeptide composition sample is used on the human in-vivo skin is 0.45%, 7.08%, 12.52% and 19.73% respectively.

[0113] Through depth analysis of the Raman image, the distribution of the polypeptide composition in the human in-vivo skin at different depths in the polypeptide composition test sample is obtained, and the distribution graph is as shown in the following figure Figure 4 Through analysis of the penetration rate change curve, it can be known that the recombinant type XVII collagen promotes the penetration of the polypeptide composition in the skin, and after the polypeptide composition is compounded with the recombinant type XVII collagen, the relative penetration rate of the polypeptide composition at 0.5 h, 2 h, 4 h and 6 h is increased by 815.6%, 70.6%, 70.4% and 47.7% respectively, compared with the polypeptide composition used alone.

[0114] The present application has many specific application approaches, and the above description is only the preferred embodiment of the present application. It should be noted that the above examples are only used to illustrate the present application, and are not used to limit the protection scope of the present application. For ordinary skilled persons in the art, several improvements can be made without departing from the principles of the present application, and these improvements should also be considered as the protection scope of the present application.

Claims

1. A composition containing polypeptides and collagen, characterized in that, The composition comprises polypeptides and collagen; The polypeptides comprise palmitoyl pentapeptide-4, decarboxy carnosine, acetyl octapeptide-3 and hexapeptide-11; The collagen is recombinant collagen XVII, which is purchased from Zhejiang Weiming Biological Technology Co., Ltd. The mass ratio of the polypeptides to the collagen is 12:10 or 12:

20.

2. The polypeptide and collagen-containing composition of claim 1, wherein, The polypeptides comprise palmitoyl pentapeptide-4, decarboxy carnosine, acetyl octapeptide-3 and hexapeptide-11 at a mass ratio of 0.0001-5:0.0001-5:0.0001-5:0.0001-5.

3. The polypeptide and collagen-containing composition of claim 1, wherein, The polypeptides comprise palmitoyl pentapeptide-4, decarboxy carnosine, acetyl octapeptide-3 and hexapeptide-11 at a mass ratio of 1:1:1:

1.

4. Use of the polypeptide and collagen-containing composition according to any one of claims 1-3 in the preparation of an anti-aging product.

5. Use according to claim 4, characterized in that, The mass percentage of the composition in the anti-aging product is 0.0001-5%.

6. Use according to claim 4 or 5, characterized in that, The anti-aging product is an anti-aging skin care product, and the dosage form of the skin care product comprises any one of water, emulsion, cream, gel, oil, powder, tablet, mud, aerosol, patch, film or nano preparation.

7. An anti-aging skin care product, characterized by, The composition comprises polypeptides and collagen according to any one of claims 1-3, and the mass percentage of the composition in the anti-aging skin care product is 0.0001-5%.

8. The anti-aging skin care product according to claim 7, characterized in that, The skin care product further comprises at least one of a solvent, oil, chelating agent, emulsifier, humectant, thickening agent, solubilizing agent, emollient, rheology modifier, antioxidant, whitening agent, conditioning agent, soothing agent, fragrance and pigment.

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