Recombinant collagen composition with skin repairing effect and skin care product

Abstract

The invention discloses a recombinant collagen composition with a skin repairing effect and a skin care product. In the recombinant collagen composition, the recombinant III-type collagen and the collagen peptide GSQ in a certain proportion have a synergistic effect in the aspect of oxidation resistance, and the recombinant I-type collagen and the collagen peptide GER in a certain proportion have a synergistic effect in the aspect of promoting fibroblast proliferation. Therefore, the recombinant collagen composition has a better repairing effect and can be applied to a skin care product with a skin barrier repairing effect.

Description

Technical Field

[0001] This patent application belongs to the field of skin care cosmetics. Specifically, it relates to a recombinant collagen composition with a synergistic skin repair effect and skin care cosmetics using the recombinant collagen composition as a raw material. Background Art

[0002] Collagen is a biological macromolecular fibrin, the most abundant and widely distributed functional protein in mammals. It is closely related to tissue formation, intercellular information transmission, and skin damage repair, and has now been widely used in fields such as daily cosmetics, medical plastic surgery, and medical devices.

[0003] In recent years, with the booming development of the medical beauty industry, more and more functional recombinant collagens have been developed and applied to various cosmetics.

[0004] However, the repair effect of a single recombinant collagen on barrier-damaged skin is insufficient, and the use effects of related products are often unsatisfactory. Regarding how to cooperate recombinant collagen with other ingredients to better exert its skin repair efficacy has not yet attracted the attention of researchers. Summary of the Invention

[0005] In view of the problem that the repair effect of a single recombinant collagen on barrier-damaged skin is insufficient in the prior art, the purpose of the present invention is to provide a recombinant collagen composition with a synergistic skin repair effect and skin care products using the recombinant collagen composition as a raw material.

[0006] The inventors conducted in-depth research on the synergistic effects of various ingredients with skin repair-related effects and found that: 1. If the skin lacks type III collagen, the tenderness, fineness, and elasticity of the skin will decline, and the recombinant type III collagen described in the specification of Chinese Patent Application No. 201310701942.8 can help the body repair aging and damaged skin; and a certain proportion of this recombinant type III collagen and GSQ tripeptide have a synergistic effect in antioxidant, and can quickly repair damaged skin after oxidative damage to the skin. 2. The recombinant type I collagen described in the specification of Chinese Patent Application No. 201310701767.2 can provide structural support and elastic tension for the dermis, promote epidermal cell proliferation, and repair damaged skin barriers; and a certain proportion of this recombinant type I collagen and GER tripeptide have a synergistic effect in promoting fibroblast proliferation, and can effectively promote the formation of elastic support for the skin structure. Based on the above scientific discoveries, the inventors completed the present invention.

[0007] That is, the present invention includes:

[0008] 1. A recombinant collagen composition, which comprises recombinant type III collagen and GSQ tripeptide as active ingredients, and a cosmetically acceptable carrier. In this composition, the weight ratio of the recombinant type III collagen to the GSQ tripeptide is 500:1 to 100:1;

[0009] The amino acid sequence of the recombinant type III collagen is as shown in SEQ ID No:2.

[0010] 2. The recombinant collagen composition according to item 1, wherein the weight ratio of the recombinant type III collagen to the GSQ tripeptide is 200:1 to 100:1.

[0011] 3. A recombinant collagen composition, which comprises recombinant type I collagen and GER tripeptide as active ingredients, and a cosmetically acceptable carrier. In this composition, the weight ratio of the recombinant type I collagen to the GER tripeptide is 200:1 to 50:1;

[0012] The amino acid sequence of the recombinant type I collagen is as shown in SEQ ID No:1.

[0013] 4. The recombinant collagen composition according to item 3, wherein the weight ratio of the recombinant type I collagen to the GER tripeptide is 100:1 to 50:1.

[0014] 5. A recombinant collagen composition, which comprises recombinant type III collagen, recombinant type I collagen, GSQ tripeptide and GER tripeptide as active ingredients, and a cosmetically acceptable carrier. In this composition, the weight ratio of the recombinant type III collagen to the GSQ tripeptide is 500:1 to 100:1, and / or the weight ratio of the recombinant type I collagen to the GER tripeptide is 200:1 to 50:1;

[0015] The amino acid sequence of the recombinant type III collagen is as shown in SEQ ID No:2;

[0016] The amino acid sequence of the recombinant type I collagen is as shown in SEQ ID No:1.

[0017] 6. The recombinant collagen composition according to item 5, wherein the weight ratio of the recombinant type III collagen to the GSQ tripeptide is 200:1 to 100:1, and / or the weight ratio of the recombinant type I collagen to the GER tripeptide is 100:1 to 50:1.

[0018] 7. A skin care product, which comprises the recombinant collagen composition according to any one of items 1 to 6.

[0019] 8. Use of the recombinant collagen composition according to any one of items 1 to 6 in the preparation of a cosmetic or drug for skin repair, wherein the cosmetic or drug is in a form suitable for application to the skin.

[0020] 9. The use according to item 8, wherein the cosmetic is made into essence water, essence milk or essence cream.

[0021] Advantages of the Invention

[0022] In the above recombinant collagen composition, recombinant type III collagen and GSQ tripeptide have a synergistic effect in antioxidant, and recombinant type I collagen and GER tripeptide have a synergistic effect in promoting fibroblast proliferation, which makes the recombinant collagen composition have better skin repair efficacy.

[0023] Detailed Embodiments of the Invention Examples

[0024] The main materials used in the following examples include:

[0025] Recombinant type III collagen: Prepared and purified according to the method disclosed in the specification of Chinese Patent Application No. 201310701942.8, and its amino acid sequence is as shown in SEQ ID No: 2.

[0026] Recombinant type I collagen: Prepared and purified according to the method disclosed in the specification of Chinese Patent Application No. 201310701767.2, and its amino acid sequence is as shown in SEQ ID No: 1.

[0027] GSQ tripeptide: A commercially available product.

[0028] GER tripeptide: A commercially available product.

[0029] Antioxidant Efficacy Test of Components and Compositions in Example 1

[0030] Prepare an aqueous solution of recombinant type III collagen (200 mg / mL), aqueous solutions of GSQ tripeptide I-V (the concentrations of GSQ tripeptide are 0.2 mg / mL, 0.4 mg / mL, 1 mg / mL, 2 mg / mL, 20 mg / mL), and compositions 1-5 (aqueous solutions, recombinant type III collagen 200 mg / mL + GSQ tripeptide 0.2 mg / mL, 0.4 mg / mL, 1 mg / mL, 2 mg / mL, 20 mg / mL).

[0031] Experimental method: The experiment was divided into 3 groups, namely the blank group, the AAPH control group and the sample group. The cells were seeded at 1×10 5Inoculate at a density of cells / mL in a Costa 96-well plate, add 200 μL of cell suspension to each well, and perform the experiment after adherent culture in an incubator at 37 °C and 5% CO2 for 24 h. Discard the culture medium. Add 120 μL of the above-prepared recombinant type III collagen aqueous solution, GSQ tripeptide aqueous solution (I-V), or composition (1-5) to the cells in each well of the sample group. Add an equal volume of FBS-free medium to the AAPH control group and the blank group. Incubate in the cell incubator for 2 h, and then wash the cells twice with PBS. To induce oxidative stress, add 120 μL of 200 μmol / L AAPH solution to each well of the sample group and the AAPH control group, and add an equal volume of FBS-free medium to the blank group. After incubating in the cell culture incubator for 3 h, wash the cells 3 times with PBS. Lyse the cells with RIPA lysis buffer containing 1 mmol / L phenylmethanesulfonyl fluoride at 4 °C for 10 min, collect the lysed cells, and centrifuge at 10,000 × g for 5 min to obtain the supernatant. Measure the GSH-Px enzyme activity in the supernatant.

[0032] At the same time, calculate the combination index CI = (A × B) / AB. When CI < 1, it indicates a synergistic effect in combination; when CI ≤ 0.9, it indicates a significant synergistic effect. Among them,

[0033] C = GSH-Px value of the blank group - GSH-Px value of the AAPH control group, representing the upper limit of the recovery of the GSH-Px value after drug administration;

[0034] T = GSH-Px value of each sample group - GSH-Px value of the AAPH control group, representing the actual recovery level of the GSH-Px value after drug administration;

[0035] A and B respectively represent the (1 + T / C) values when administering recombinant type III collagen or GSQ tripeptide alone;

[0036] AB represents the (1 + T / C) value when administering compositions 1-5.

[0037] The effects of the above recombinant type III collagen, GSQ tripeptide, and compositions 1-5 on the GSH-Px enzyme activity of cells, as well as the combination index of compositions 1-5, are shown in Table 1.

[0038] Table 1

[0039] Group GSH-Px (U / mg) CI Effect Blank group 217.53±13.28 / / AAPH control group 87.96±6.05 / / Recombinant type III collagen 100.35±5.78 / / GSQ tripeptide-I 98.59±7.32 / / GSQ tripeptide-II 105.87±4.51 / / GSQ tripeptide-III 116.54±5.69 / / GSQ tripeptide-IV 130.02±7.73 / / GSQ tripeptide-V 165.66±6.34 / / Composition 1 104.84±4.38 1.05 Not synergistic Composition 2 128.29±5.67 0.95 Synergistic Composition 3 168.31±7.02 0.83 Significantly synergistic Composition 4 175.31±6.33 0.87 Significantly synergistic Composition 5 181.98±8.41 1.02 Not synergistic

[0040] As can be seen from the results in Table 1 above, Compositions 1, 2, 3, 4, and 5 of the present invention can significantly increase the activity of GSH-Px in cells. Among them, the combination index of Compositions 2, 3, and 4 is less than 1, indicating that when the weight ratio of recombinant type III collagen to GSQ tripeptide in the composition is 500:1 to 100:1, there is a synergistic effect in improving the activity of GSH-Px enzyme in cells. Moreover, the combination index of Compositions 3 and 4 is less than 0.9, indicating that when the recombinant type III collagen:GSQ tripeptide is 200:1 to 100:1, the synergistic effect on improving the activity of GSH-Px enzyme in cells is significant. GSH-Px plays the role of an antioxidant in metabolism and can protect biological membranes from oxidative damage by catalyzing glutathione to reduce hydrogen peroxide. GSH-Px can bind harmful substances in the body, such as free radicals and heavy metal ions, and then excrete them from the body to prevent the body from suffering oxidative damage. Therefore, GSH-P X activity is one of the important indicators for antioxidant and anti-aging.

[0041] Test on the fibroblast proliferation-promoting efficacy of each component and composition in Example 2

[0042] Prepare an aqueous solution of recombinant type I collagen (42 mg / mL), aqueous solutions of GER tripeptide I-V (the concentrations of GER tripeptide are 0.042 mg / mL, 0.21 mg / mL, 0.42 mg / mL, 0.84 mg / mL, and 4.2 mg / mL), and Compositions 1-5 (aqueous solutions, 20 mg / mL of recombinant type I collagen + 0.042 mg / mL, 0.21 mg / mL, 0.42 mg / mL, 0.84 mg / mL, and 4.2 mg / mL of GER tripeptide).

[0043] Place the revived human skin fibroblasts in a cell culture incubator with saturated humidity. Use a low-glucose DMEM medium containing 10% FBS by volume ratio to culture the cells at 37 °C and 5% CO2 until the cell confluence reaches 85%. Then, digest and passage the cells with 0.25% trypsin, observe under an inverted microscope, and take the cells in the logarithmic growth phase for cell experiments.

[0044] Take the revived human skin fibroblasts, centrifuge and count them at 1000 r / min, and then seed them in a 96-well plate at a density of 1×10 4 cells / mL. Add 100 μL of cell suspension to each well. After adherent culture for 24 h, add 100 μL of the above-prepared sample solution. Place it in the incubator and culture for 24 h. After 24 h, add 10 μL of CCK-8 working solution to each well, incubate it in a CO2 incubator for 1 h, measure the absorbance at 450 nm, and calculate the cell proliferation rate.

[0045] Cell proliferation rate = OD of sample group × 100% / OD of blank group.

[0046] At the same time, the combined drug index CI = (A × B) / AB was calculated. When CI < 1, it indicates that the combined use has a synergistic effect; when CI ≤ 0.9, it indicates that the synergistic effect is significant. Among them, A and B are the cell proliferation rates of the recombinant type I human collagen group and the GER tripeptide-I to V group, respectively, and AB is the cell proliferation rate of the combination group 1 to 5.

[0047] Table 2 shows the effects of the above-mentioned recombinant type I collagen, GER tripeptide and compositions 1-5 on promoting fibroblast proliferation, as well as the combined drug index of compositions 1-5.

[0048] Table 2

[0049] Group Cell proliferation rate CI value Synergistic effect Recombinant human type I collagen 106.35% ± 2.43 / / GER tripeptide-I 107.57％+4.14 / / GER tripeptide-II 113.92％+4.86 / / GER tripeptide-III 125.94% ± 3.27 / / GER tripeptide-IV 135.19％+3.52 / / GER tripeptide-V 177.34% ± 2.32 / / Composition 1 111.07％+3.91 1.03 Not synergistic Composition 2 127.53％+5.23 0.95 Synergistic Composition 3 153.34％+3.63 0.87 Significantly synergistic Composition 4 166.61％+3.65 0.86 Significantly synergistic Composition 5 I78.80% ± 2.97 1.05 Not synergistic

[0050] As can be seen from the results of Table 2 above, Composition 1, Composition 2, Composition 3, Composition 4, and Composition 5 of the present invention can significantly promote fibroblast proliferation. Among them, the combined medication index of Composition 2, Composition 3, and Composition 4 is less than 1, indicating that the weight ratio of recombinant type I collagen: GER tripeptide has a synergistic effect in promoting fibroblast proliferation when it is 200:1 to 50:1. Moreover, the synergistic index of Composition 3 and Composition 4 is less than 0.9, indicating that the synergistic effect of recombinant type I collagen: GER tripeptide in promoting fibroblast proliferation is significant when it is 100:1 to 50:1. In the early stage of the repair process, fibroblasts divide and proliferate in large numbers and gather at damaged skin barriers. In the later stage of the repair process, fibroblasts secrete collagenase to participate in skin tissue reconstruction. Therefore, in the repair process, fibroblasts play a very important role.

[0051] Example 3 Product efficacy test

[0052] As an example of raw materials, a recombinant collagen composition is prepared, which includes: recombinant type I collagen, recombinant type III collagen and Mini small molecule recombinant collagen peptide, wherein the weight ratio of recombinant type III collagen to Mini small molecule recombinant collagen peptide GSQ is in the range of 100:1 to 500:1, and the weight ratio of recombinant type I collagen to Mini small molecule recombinant collagen peptide GER is in the range of 200:1 to 50:1.

[0053] The same amount of the above recombinant collagen composition raw material and commercially available collagen were used as the only effective ingredient, and the same production process was used to produce essence water for the following product efficacy test. The test results are shown in Table 3 below, which shows that this product is significantly better than the commercially available collagen product.

[0054] Test item: Visia-CR skin red area ratio

[0055] Test instrument: Facial image analyzer Visia-CR

[0056] Test subjects: 32 Chinese healthy male / female subjects aged 18 - 35 years old, with an average age of 27.81 ± 0.85 years old, were selected. Their facial skin was rough with obvious visible pores; their facial skin had certain redness / thread-like red blood streaks; their skin barrier function was poor and the TEWL screening value ≥ 15; they self-identified as sensitive skin and the lactic acid stimulation score ≥ 3 points.

[0057] Test method: The before-and-after comparison method was adopted. The proportion of the red area of the subjects' skin was measured before using the test sample and 2 weeks after continuous use of the sample.

[0058] Measurement area: Cheeks. The essence water was coated on the left and right cheeks respectively, and a single coating was carried out according to (20 ± 0.1) mg / cm 2 for each.

[0059] Test principle: Visia-CR uses multiple light sources, greatly improving the visibility of the skin to be analyzed and providing the conditions of melanin and blood vessels in the lower layer of the skin. It is a new method for detecting, displaying, and analyzing the lower layer of the skin.

[0060] Environmental conditions: The test environment temperature was 20.1℃ - 21.1℃, and the relative humidity was 50.6%RH - 59.7%RH.

[0061] Table 3: Test results of the proportion of the red area of the skin

[0062]

[0063] The present invention has been described through specific embodiments and examples above. However, those skilled in the art should understand that these are not intended to limit the scope of the present invention, and the scope of the present invention should be determined by the claims.

[0064] Industrial applicability

[0065] The present invention provides a recombinant collagen composition with a synergistic skin repair effect and skin care products using the recombinant collagen composition as a raw material.

[0066] Sequence information

[0067] SEQ ID NO: 1: The complete amino acid sequence of recombinant type I collagen

[0068] QLSYGYDEKS TGGISVPGPM GPSGPRGLPG PPGAPGPQGF QGPPGEPGEP

[0069] GASGPMGPRG PPGPPGKNGD DGEAGKPGRP GERGPPGPQG ARGLPGTAGL

[0070] PGMKGHRGFS GLDGAKGDAG PAGPKGEPGS PGENGAPGQM GPRGLPGERG

[0071] RPGAPGPAGA RGNDGATGAA GPPGPTGPAG PPGFPGAVGA KGEAGPQGPR

[0072] GSEGPQGVRG EPGPPGPAGA AGPAGNPGAD GQPGAKGANG APGIAGAPGF

[0073] PGARGPSGPQ GPGGPPGPKG NSGEPGAPGS KGDTGAKGEP GPVGVQGPPG

[0074] PAGEEGKRGA RGEPGPTGLP GPPGERGGPG SRGFPGADGV AGPKGPAGER

[0075] GSPGPAGPKG SPGEAGRPGE AGLPGAKGLT GSPGSPGPDG KTGPPGPAGQ

[0076] DGRPGPPGPP GARGQAGVMG FPGPKGAAGE PGKAGERGVP GPPGAVGPAG

[0077] KDGEAGAQGP PGPAGPAGER GEQGPAGSPG FQGLPGPAGP PGEAGKPGEQ

[0078] GVPGDLGAPGPSGARGERGF PGERGVQGPP GPAGPRGANG APGNDGAKGD

[0079] AGAPGAPGSQ GAPGLQGMPG ERGAAGLPGP KGDRGDAGPK GADGSPGKDG

[0080] VRGLTGPIGP PGPAGAPGDK GESGPSGPAG PTGARGAPGD RGEPGPPGPA

[0081] GFAGPPGADG QPGAKGEPGD AGAKGDAGPP GPAGPAGPPG PIGNVGAPGA

[0082] KGARGSAGPP GATGFPGAAG RVGPPGPSGN AGPPGPPGPA GKEGGKGPRG

[0083] ETGPAGRPGE VGPPGPPGPA GEKGSPGADG PAGAPGTPGP QGIAGQRGVV

[0084] GLPGQRGERG FPGLPGPSGE PGKQGPSGAS GERGPPGPMG PPGLAGPPGE

[0085] SGREGAPGAE GSPGRDGSPG AKGDRGETGP AGPPGAPGAP GAPGPVGPAG

[0086] KSGDRGETGP AGPAGPVGPVGARGPAGPQG PRGDKGETGE QGDRGIKGHR

[0087] GFSGLQGPPG PPGSPGEQGPSGASGPAGPR GPPGSAGAPG KDGLNGLPGP

[0088] IGPPGPRGRT GDAGPVGPPG PPGPPGPPGP PSAGFDFSFL PQPPQEKAHD

[0089] GGRYYRA

[0090] SEQ ID NO: 2: Full Amino Acid Sequence of Recombinant Type III Collagen

[0091]

Claims

1. A recombinant collagen composition having skin repairing effect, comprising recombinant type III collagen and GSQ tripeptide as active ingredients, and a cosmetically acceptable carrier, wherein: In the composition, the weight ratio of the recombinant type III collagen to the GSQ tripeptide is 500:1 to 100:1; The amino acid sequence of the recombinant type III collagen is shown in SEQ ID No:

2.

2. The recombinant collagen composition according to claim 1, wherein The weight ratio of the recombinant type III collagen to the GSQ tripeptide is 200:1 to 100:

1.

3. A recombinant collagen composition comprising recombinant type I collagen and GER tripeptide as active ingredients, and a cosmetically acceptable carrier, wherein: In the composition, the weight ratio of the recombinant type I collagen to the GER tripeptide is 200:1 to 50:1; The amino acid sequence of the recombinant type I collagen is shown in SEQ ID No:

1.

4. The recombinant collagen composition according to claim 3, wherein The weight ratio of the recombinant type I collagen to the GER tripeptide is 100:1 to 50:

1.

5. A recombinant collagen composition comprising as active ingredients recombinant type III collagen, recombinant type I collagen, GSQ tripeptide and GER tripeptide, and a cosmetically acceptable carrier, wherein: In the composition, the weight ratio of the recombinant type III collagen to the GSQ tripeptide is 500:1 to 100:1, and / or the weight ratio of the recombinant type I collagen to the GER tripeptide is 200:1 to 50:1; The amino acid sequence of the recombinant type III collagen is shown in SEQ ID No: 2; The amino acid sequence of the recombinant type I collagen is shown in SEQ ID No:

1.

6. The recombinant collagen composition according to claim 5, wherein The weight ratio of the recombinant type III collagen to the GSQ tripeptide is 200:1 to 100:1, and / or the weight ratio of the recombinant type I collagen to the GER tripeptide is 100:1 to 50:

1.

7. A skin care product comprising the recombinant collagen composition according to any one of claims 1 to 6.

8. Use of the recombinant collagen composition according to any one of claims 1 to 6 in the preparation of cosmetics or medicines for skin repair, wherein the cosmetics or medicines are prepared in a form suitable for application to the skin.

9. The use according to claim 8, wherein The cosmetic is made into essence water, essence milk or essence cream.

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