Recombinant collagen repair composition, essence and preparation method thereof

Abstract

The invention discloses a recombinant collagen repairing composition, essence and a preparation method thereof, and belongs to the field of cosmetics. The recombinant collagen repairing composition comprises recombinant collagen and oligopeptide, the amino acid sequence of the recombinant collagen is shown as SEQ ID NO: 1, and the mass ratio of the recombinant collagen to the oligopeptide is (1-200): (0.1-1); cytology experiments show that the synergism of the recombinant collagen and the oligopeptide has better cytology activity. The invention further provides repairing essence and a mask, and the dosage of collagen and oligopeptide can be reduced. A human body efficacy evaluation result shows that the red area (VISIA CR + IPP analysis) of the repair essence on the sensitive skin can be reduced by more than 30% after the repair essence is used for 28 days, which indicates that the repair essence has a very good sensitive skin repair effect; in addition, the repairing essence further has the effects of relieving, resisting wrinkles, tightening, removing acnes, preserving moisture and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetic preparations, and relates to a recombinant collagen repair composition, an essence and a preparation method thereof. Background Art

[0002] Sensitive skin is a type of skin on various parts of the body, especially the facial skin, which is affected by factors such as genetics, environment, cosmetics, psychological stress, hormones, etc., and may undergo irritation reactions or overactivation of the immune system, resulting in problems such as itching, redness, burning, stinging, tightness, and rash.

[0003] Although existing peptide and plant extract combinations have achieved some success in the market, they still face several key challenges. The main problem is that many skin repair products are not sufficient in providing comprehensive effects, which are mainly caused by insufficient stability of active ingredients or lack of effective synergy between ingredients. In addition, some formulas may contain potentially irritating chemical ingredients, which may have a negative impact on the skin after long-term use, especially for consumers with sensitive skin.

[0004] More and more studies have shown that recombinant collagen has good anti-wrinkle and repair effects, but the price of recombinant collagen is high. For example, the price of high-quality recombinant collagen is generally 150,000-200,000 yuan / kg. In addition, the existing recombinant collagen generally has the problem of low skin absorption rate, so its addition amount is relatively large, resulting in high cost of skin care products. Scientifically and rationally adding recombinant collagen to skin care products can reasonably control costs while ensuring product efficacy. Therefore, it is necessary to develop a cost-effective recombinant collagen repair essence suitable for sensitive skin. Summary of the invention

[0005] The object of the present invention is to provide a recombinant collagen repair composition, an essence and a preparation method thereof.

[0006] The technical solution for achieving the purpose of the present invention is as follows:

[0007] In the first aspect, the present invention provides a recombinant collagen repair composition, comprising recombinant collagen and oligopeptides, wherein the recombinant collagen used in the present invention is human type XVII recombinant collagen, and its amino acid sequence is shown in SEQ ID NO: 1. Hereinafter, it is referred to as recombinant collagen or collagen.

[0008] SEQ ID NO:1

[0009] GSPGPKGDMGSPGPKGD RGDPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGEKGERGLQGLRGEVGLPGVKGDKGPMGPPPGPKGDQGEKGPPGQKGEMGTPPGKGD RGD AGPPGHPGPPGPRGHKGEKGDKGDQGPQGIAGQRGVVGLP

[0010] The oligopeptide used in the present invention is selected from one or more of oligopeptide-1, tripeptide-1 copper, and palmitoyl pentapeptide-4; preferably tripeptide-1 copper.

[0011] The present invention first screens the amount of collagen and oligopeptide required to promote cell proliferation through a cell proliferation experiment; secondly, the amount of collagen required to significantly promote cell proliferation is screened under a determined oligopeptide addition concentration. After completing the amount of each component in the combination of collagen and oligopeptide, the cost, efficacy, and the influence of cosmetic formula and other ingredients are taken into consideration, and finally the formula and preparation method of the recombinant collagen repair essence are determined.

[0012] Experiments have confirmed that in the recombinant collagen repair composition, the mass ratio of recombinant collagen to oligopeptide is 1-200:0.1-1; the preferred mass ratio is 10-100:1, and the recombinant collagen and oligopeptide synergistically improve cell proliferation activity.

[0013] The present invention screens out a combination of oligopeptides (oligopeptide-1 or tripeptide-1 copper) and recombinant collagen through cytological experiments, wherein the oligopeptide (oligopeptide-1 or tripeptide-1 copper) is added at a concentration of 0.0005%-0.001%, and the recombinant collagen is added at an amount of 0.005%-0.1%; wherein the optimal amounts of recombinant collagen and oligopeptide are 0.005% and 0.0005%, respectively.

[0014] In a second aspect, the present invention provides the use of the above-mentioned recombinant collagen repair composition in cosmetics, and the cosmetics include lotions, emulsions, essences, creams, facial masks, etc.

[0015] Specifically, the present invention provides a recombinant collagen repair essence, comprising the above-mentioned recombinant collagen repair composition and a cosmetic auxiliary material matrix; calculated by mass percentage, it comprises 0.005% to 0.1% recombinant collagen, 0.0005% to 0.001% oligopeptide, and the remainder is the cosmetic auxiliary material matrix.

[0016] The cosmetic auxiliary material matrix of the present invention includes moisturizers, pH regulators, preservative systems, other functional additives, thickeners, metal chelating agents, solvents, etc. commonly used in the cosmetics field.

[0017] Among them, the moisturizing agent includes but is not limited to one or more of glycerin, propylene glycol, butylene glycol, 1,2-hexanediol, tremella polysaccharide, sodium hyaluronate, and allantoin.

[0018] pH adjusters include sodium dihydrogen phosphate, triethanolamine, sodium hydroxide, etc. The appropriate pH adjuster and addition amount are selected according to the target pH of the cosmetics.

[0019] The preservative system can achieve the effect of preserving cosmetics, and can be a preservative, such as one or more of sodium methylparaben and phenoxyethanol; it can also be a composition with moisturizing and preservative effects, such as a composition comprising one or both of moisturizing agents 1,2-hexanediol (0.5-1.5%) and caprylyl glycol (0.5-1.5%) and an antioxidant p-hydroxyacetophenone (0.5-1%); or a preservative and a composition with moisturizing and preservative effects are used in combination, such as one or both of sodium methylparaben and phenoxyethanol are used in combination with one or both of moisturizing agents 1,2-hexanediol (0.5-1.5%) and caprylyl glycol (0.5-1.5%) and an antioxidant p-hydroxyacetophenone (0.5-1%).

[0020] Among them, other functional additives can be added according to the efficacy of the cosmetics, including plant extracts, such as one or more of rose water, Centella asiatica extract, ginseng extract, and aloe extract; or skin conditioners, such as bisabolol, etc. Thickeners such as carbomer, etc., and metal chelating agents such as EDTA, etc.

[0021] Preferably, the present invention provides a recombinant collagen repair essence, which comprises, by mass percentage: 0.005% to 0.1% recombinant collagen, 0.0005% to 0.001% oligopeptide, 5% to 10% moisturizer, 0.2% to 0.4% pH adjuster, 0.2% to 0.5% preservative, and the balance is water.

[0022] Furthermore, a recombinant collagen repair essence comprises the following components by mass percentage: 0.005% to 0.1% recombinant collagen, 0.0005% to 0.001% oligopeptide, 5% to 10% glycerol, 0.01% to 0.02% tremella polysaccharide, 0.01% to 0.05% sodium hyaluronate, 0.2% to 0.4% sodium dihydrogen phosphate, 0.1% to 0.2% sodium methylparaben, 0.2% to 0.5% phenoxyethanol, and the balance is water.

[0023] Furthermore, in the above-mentioned recombinant collagen repair essence, 0.1% sodium methylparaben and 0.2% phenoxyethanol are preferably added to achieve the characteristics of mild formula while achieving the preservation effect.

[0024] In a specific embodiment of the present invention, a collagen essence with soothing and repairing effects is provided, which includes the following components by mass fraction: 0.05% recombinant collagen, 0.005% tripeptide-1 copper, 5% glycerol, 0.01% tremella polysaccharide, 0.05% sodium hyaluronate, 0.3% sodium dihydrogen phosphate, 0.2% sodium methylparaben, 0.2% phenoxyethanol, and the balance is water.

[0025] In a specific embodiment of the present invention, a collagen essence with soothing and repairing effects is provided, which includes the following components by mass fraction: 0.005% recombinant collagen, 0.0005% oligopeptide-1, 6% glycerol, 0.02% tremella polysaccharide, 0.02% sodium hyaluronate, 0.25% sodium dihydrogen phosphate, 0.2% sodium methylparaben, 0.2% phenoxyethanol, 0.08% sodium chloride, and the balance is water.

[0026] Specifically, the present invention also provides a collagen essence facial mask, which is obtained by soaking a facial mask cloth in the facial mask essence and fully absorbing it.

[0027] The facial mask essence comprises the following components by mass percentage: 0.05%-0.1% recombinant collagen, 0.0005%-0.001% oligopeptide, 6% glycerol, 5% propylene glycol, 0.05% sodium hyaluronate, 0.08% carbomer, 0.2% sodium methylparaben, 0.02% sodium hydroxide, and the balance is water.

[0028] In a third aspect, the present invention provides a method for preparing a recombinant collagen repair essence, comprising the following steps:

[0029] According to the proportion, the cosmetic auxiliary material matrix is ​​evenly mixed and the pH is adjusted to the target value; collagen and oligopeptide-1 are dissolved in a small amount of water, filtered and sterilized through a 0.22um filter membrane, and then added to obtain recombinant collagen repair essence.

[0030] The human efficacy evaluation test confirms that the recombinant collagen repair essence provided by the present invention has the effects of repairing, soothing, being suitable for sensitive skin, being mild and non-irritating, as well as moisturizing and anti-wrinkle.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] The present invention uses recombinant collagen as the core active ingredient and combines it with oligopeptides (oligopeptide-1 or tripeptide-1 copper) in proportion. On the basis of achieving good efficacy, the amount of recombinant collagen added can be significantly reduced, which can effectively reduce costs and make the product more competitive in the market. Collagen and oligopeptides (oligopeptide-1 or tripeptide-1 copper) are compounded and continuously used for 28 days to significantly repair sensitive skin. At the same time, the product also has soothing, anti-wrinkle and acne-removing effects. The repair essence of the present invention has simple formula ingredients and a simple preparation process, which is conducive to the industrial production of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the result of MTT cell proliferation experiment with recombinant type XVII collagen.

[0034] Figure 2 This is the result of the tripeptide-1 copper MTT cell proliferation experiment.

[0035] Figure 3 This is the result of oligopeptide-1 MTT cell proliferation experiment.

[0036] Figure 4 This is the result of MTT cell proliferation experiment with tripeptide-1 copper and recombinant type XVII collagen.

[0037] Figure 5 This is the result of MTT cell proliferation experiment with oligopeptide-1 and recombinant type XVII collagen.

[0038] Figure 6 These are the VISIA CR red zone pictures before and 28 days after using the samples.

[0039] Figure 7 This is a graph showing the changes in the red zone area before using the sample and 28 days after using the sample.

[0040] Figure 8 This is a graph showing the changes in skin keratin moisture content before and 28 days after using the sample.

[0041] Fig. 9 This is a graph showing the changes in TEWL values ​​before and 28 days after using the sample.

[0042] Fig.10 These are the pictures of VISIA CR wrinkles before using the sample and 28 days after using the sample.

[0043] Fig.11 This is a graph showing the changes in the percentage of skin wrinkle area at the crow's feet and nasolabial folds before and 28 days after using the sample. DETAILED DESCRIPTION

[0044] The present invention is further described in detail with reference to the embodiments and the accompanying drawings. The examples are only used to explain the present invention but not to limit the scope of the present invention.

[0045] The collagen used in the following specific embodiments of the present invention is a recombinant human type XVII collagen, and its amino acid sequence is shown in SEQ ID NO: 1, which is a recombinant human type XVII collagen containing two RGD domains (underlined in SEQ ID NO: 1) by selecting 138 amino acids at positions 567-620 (54aa), 641-646 (6aa), 683-715 (33aa) and 1438-1482 (45aa) from the functional domain of type XVII collagen, and recombining them with amino acids 772-786 (GPQGIAGQRGVVGLP) in type I collagen, and then mutating the phenylalanine at position 20 and the proline at position 113 to aspartic acid to obtain a recombinant human type XVII collagen.

[0046] SEQ ID NO:1

[0047] GSPGPKGDMGSPGPKGD RGD PGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGEKGERGLQGLRGEVGLPGVKGDKGPMGPPPGPKGDQGEKGPPGQKGEMGTPPGKGD RGD AGPPGHPGPPGPRGHKGEKGDKGDQGPQGIAGQRGVVGLP

[0048] Those skilled in the art can obtain the recombinant collagen of the present invention by molecular biology, molecular cloning and other technical means. For example, the gene sequence of collagen is designed and synthesized, and after the sequence design optimization is completed, it is handed over to Tianjin Zhonghe Gene Technology Co., Ltd. for synthesis. After the sequence is synthesized, the 6xHis-SUMO expression gene can be fused to the 5' end of the type XVII collagen gene by overlap extension PCR to form the expression gene of 6xHis-SUMO-XVII. This DNA fragment is cloned and amplified by the PCR method, and Nde I and Xho I restriction sites are added by primers during amplification, and the target fragment is inserted into the pET28a vector by restriction digestion and connection. The recombinant vector is cloned and screened and sequenced to obtain the pET28a-6xHis-SUMO-XVII recombinant expression vector. The pET28a-6xHis-SUMO-XVII plasmid was transformed into the Escherichia coli expression strain BL21 (DE3), and a single clone was obtained by kanamycin resistance screening. The single clone was picked and cultured overnight in 5 mL of 2xYT medium, inoculated at a ratio of 1:100, and cultured at 37°C with shaking until OD600 was 0.6-0.8. The culture temperature was lowered to 20°C and IPTG was added to a final concentration of 0.2 mM. The culture was continued for 16 hours to induce protein expression, and the bacteria were collected by centrifugation. The desired collagen was isolated and purified.

[0049] Example 1 Cell proliferation experiment to screen the concentration of collagen added

[0050] Normally culture and passage BALB / c 3T3 cells. When the cell viability reaches more than 98%, the cells in good condition are inoculated into a 96-well culture plate, and a blank control without cells is set up. After culturing for 16 hours at 37°C and 5% CO2, the culture medium is replaced with DMEM medium, and the culture is continued for 16 hours until the cells are well attached to the wall. The original culture medium is aspirated, and 0.005%, 0.01%, 0.02% and 0.05% recombinant type XVII collagen are added respectively. The negative control group is DMEM medium, and the culture is continued for 48 hours. The positive control group is 200pg / ml EGF (epidermal growth factor) diluted in DMEM medium. 4 hours before the end of the culture, 5g / L MTT solution is added, and the culture is terminated after culturing for 4 hours; the liquid in the well is aspirated and discarded, washed once with PBS, DMSO is added to dissolve the crystals, and the absorbance value of each well is measured at a wavelength of 570nm.

[0051] Cell proliferation rate = (absorbance value of the assay well - absorbance value of the blank control group) / (absorbance value of the cell control group - absorbance value of the blank control group) × 100% (Formula 1).

[0052] In this example, the samples are 0.005%, 0.01%, 0.02% and 0.05% recombinant type XVII collagen, with 3 parallel samples in each group. Figure 1As shown, the experimental results show that under the concentration conditions of 0.005%, 0.01%, 0.02% and 0.05%, the proliferation promotion rates of recombinant type XVII collagen on BALB / c 3T3 cells are 109.9%, 115.4%, 129.6% and 129.8% respectively; when the addition amount of recombinant collagen is greater than 0.02%, it can significantly promote the proliferation of fibroblasts and has the biological activity of promoting cell proliferation.

[0053] Example 2 Cell proliferation experiment to screen the concentration of oligopeptides added

[0054] BALB / c 3T3 cells (mouse embryonic fibroblasts) in good logarithmic growth phase were selected, digested with 0.25% trypsin, resuspended in DMEM medium containing 10% serum after centrifugation and counted; BALB / c 3T3 cells were inoculated in 96-well culture plates at 6000 cells per well, and blank controls without cells were set up at the same time. After culturing for 16 hours at 37°C and 5% CO2, DMEM medium was replaced, and cultured for 16 hours until the cells adhered well. The original culture medium was aspirated, and oligopeptide-1 and tripeptide-1 copper were added respectively, and diluted to 0.1-10 μg / ml with DMEM medium, the negative control group was DMEM medium, and the positive control group was 200 pg / ml EGF (epidermal growth factor) diluted with DMEM medium, and cultured for 48 hours. 4 h before the end of culture, add 5 g / L MTT solution, continue to culture for 4 h and then terminate the culture; discard the liquid in the wells, wash once with PBS, add DMSO to dissolve the crystals, measure the absorbance value of each well at a wavelength of 570 nm, and calculate the cell proliferation rate according to formula 1.

[0055] In this example, the samples are oligopeptide-1 and tripeptide-1 copper, which are added to the cell culture medium at 0.1-10 μg / ml, with 3 parallel samples in each group. Figure 2 As shown in Figure 3, the experimental results showed that when the addition amount of oligopeptide-1 or tripeptide-1 copper was greater than 1 μg / ml, it could significantly promote the proliferation of fibroblasts, and when the addition amount of oligopeptide increased, it failed to further promote cell proliferation.

[0056] Example 3 Cell proliferation experiment to screen the added concentration of collagen and oligopeptide combination

[0057] Normally culture and passage BALB / c 3T3 cells. When the cell viability reaches more than 98%, the cells in good condition are inoculated into a 96-well culture plate, and a blank control without cells is set up. After culturing for 16 hours at 37°C and 5% CO2, the medium is replaced with DMEM medium and cultured for 16 hours until the cells are well attached. The original culture medium is aspirated, and different amounts of recombinant type XVII collagen and 1μg / ml oligopeptide-1 or tripeptide-1 copper are added. The negative control group is DMEM medium and cultured for 48 hours. 4 hours before the end of the culture, 5g / L MTT solution is added, and the culture is terminated after 4 hours of continuous culture; the liquid in the well is aspirated, washed once with PBS, DMSO is added to dissolve the crystals, and the absorbance value of each well is measured at a wavelength of 570nm. The cell proliferation rate is calculated according to formula 1.

[0058] In this embodiment, when the oligopeptide-1 or tripeptide-1 copper is 1 μg / ml, the amount of recombinant collagen added is 0.005%-0.05%, respectively, and the cell proliferation rate is higher than that of using oligopeptide or recombinant collagen alone, and this trend does not increase significantly with the increase of collagen addition amount; therefore, in vitro experiments show that the combination of 1 μg / ml oligopeptide and 0.005% collagen can significantly promote cell proliferation ( Figure 4 ,5).

[0059] Comprehensive examples 1-3, in vitro experiments show that when tripeptide-1 copper or oligopeptide-1 is used alone, the addition amount is 0.0001% to 0.001%, which can play a good role in cell proliferation; when recombinant collagen is used alone, the addition amount is greater than 0.02%, which can effectively stimulate cell proliferation. When oligopeptides (oligopeptide-1 and tripeptide-1 copper) and recombinant collagen are used in combination, the addition amount of oligopeptide-1 and tripeptide-1 copper is 0.0001%, and the addition amount of recombinant collagen is 0.005%, which can significantly stimulate cell proliferation (Table 1). Taking into account the effects of other cosmetic ingredients (preservatives, etc.) on the activity of recombinant collagen and oligopeptides, as well as factors such as absorption rate and cost, it is recommended that the addition amount of recombinant collagen should not be less than 0.005% and the addition amount of oligopeptides should not be less than 0.0005% when formulating skin care products.

[0060] Table 1 Cell proliferation rate after adding collagen alone, oligopeptide, and combination of collagen and oligopeptide

[0061]

[0062] Example 4 Preparation of collagen essence containing oligopeptide-1

[0063] The present embodiment provides a collagen essence with soothing effect, which includes the following components by mass fraction: 0.005% recombinant collagen, 0.0005% oligopeptide-1, 6% glycerol, 0.02% tremella polysaccharide, 0.02% sodium hyaluronate, 0.25% sodium dihydrogen phosphate, 0.2% sodium methylparaben, 0.2% phenoxyethanol, 0.08% sodium chloride, and the balance is water.

[0064] This embodiment also discloses a method for preparing collagen essence, comprising the following steps:

[0065] (1) Weigh the materials according to the formula table;

[0066] (2) adding sodium hyaluronate to glycerol, stirring and mixing, adding water, Tremella polysaccharide, and sodium dihydrogen phosphate, heating to 75° C., and stirring and dissolving for 25 minutes;

[0067] (3) Cooling to 35-40° C., adding phenoxyethanol and sodium methylparaben, and stirring to dissolve;

[0068] (4) Dissolve collagen and oligopeptide-1 in 2% water, filter through a 0.22 μm filter membrane, and then add; stir for 20 minutes to mix;

[0069] (5) Sampling and testing are carried out before packaging.

[0070] Example 5 Preparation of Collagen Essence Containing Tripeptide-1 Copper

[0071] This embodiment provides a collagen essence with soothing effect, which includes the following components by mass fraction: 0.05% recombinant collagen, 0.005% tripeptide-1 copper, 5% glycerol, 0.01% tremella polysaccharide, 0.05% sodium hyaluronate, 0.3% sodium dihydrogen phosphate, 0.2% sodium methylparaben, 0.2% phenoxyethanol, and the balance is water. The preparation method of the collagen essence containing tripeptide-1 copper is the same as that of Example 4.

[0072] Example 6 Preparation of collagen essence facial mask

[0073] The present embodiment provides a collagen essence mask with soothing effect, which includes the following components by mass fraction: 0.05% recombinant collagen, 0.0005% palmitoyl pentapeptide-4, 0.0005% oligopeptide-1, 6% glycerin, 5% propylene glycol, 0.05% sodium hyaluronate, 0.08% carbomer, 0.2% sodium methylparaben, 0.02% sodium hydroxide, and the balance is water.

[0074] This embodiment also discloses a method for preparing a collagen essence facial mask, comprising using the collagen essence described in the above claims, and comprising the following steps:

[0075] (1) Weigh the materials according to the formula table;

[0076] (2) Add sodium hyaluronate to glycerin, stir and mix, then add water and carbomer, heat to 75° C., and stir to dissolve for 25 minutes;

[0077] (3) Cooling to 35-40° C., adding phenoxyethanol and sodium methylparaben, and stirring to dissolve;

[0078] (4) Dissolve sodium hydroxide in 1% water and add;

[0079] (5) Dissolve collagen, oligopeptide-1 and tripeptide-1 copper in 2% water, filter through a 0.22 um filter membrane, and then add; stir for 20 minutes to mix;

[0080] (6) Fold the mask cloth and put it into the mask bag;

[0081] (7) Dispensing 30 g of the facial mask essence into the facial mask bag using a facial mask filling machine;

[0082] (8) Sampling and testing are carried out before packaging.

[0083] Example 7: Repair, soothing, suitable for sensitive skin, mild and non-irritating human efficacy evaluation test

[0084] This example verifies the efficacy of this product in repairing, soothing, being suitable for sensitive skin, and being mild and non-irritating by measuring the red area of ​​the sensitive skin of 33 sensitive skin test subjects before and after using this product (VISIA CR+IPP analysis).

[0085] The sample used in this example is the collagen essence described in Example 5.

[0086] 1. How to use: After cleaning your face, apply collagen essence evenly on your face and massage to help absorption.

[0087] Frequency and cycle of use: Use once a day in the morning and evening for 28 consecutive days.

[0088] 2. Subject requirements:

[0089] 2.1 Inclusion Criteria

[0090] (1) Healthy female subjects, aged 18-60 years;

[0091] (2) Facial redness;

[0092] (3) Sensitive skin (screening by West China Questionnaire);

[0093] (4) Able to cooperate well with the tester and maintain a regular life during the test period;

[0094] (5) Be able to read and understand all the contents of the informed consent form and voluntarily sign the informed consent form;

[0095] (6) Agree not to use any cosmetics, drugs or health products that may affect the results during the test;

[0096] (7) During the test period, similar products with the same efficacy as the test sample shall not be used;

[0097] (8) Not currently participating in and will not participate in any other research involving the test area during the study period;

[0098] (9) No medical aesthetic procedures may be performed during the test period.

[0099] 2.2 Exclusion criteria (anyone with any of the following conditions must be excluded from this study)

[0100] (1) Those with facial skin diseases that may affect the judgment of the test results;

[0101] (2) Those with highly allergic constitution;

[0102] (3) Women who are pregnant, breastfeeding, or planning to become pregnant during the trial;

[0103] (4) Those with severe heart, liver, or kidney damage and severe immunodeficiency;

[0104] (5) People with mental illness, severe endocrine diseases, or those taking oral contraceptives;

[0105] (6) Those who have participated in drug clinical trials or other trials within 30 days, or those who have systematically used drugs that may affect test results within the past 2 weeks;

[0106] (7) Those who have taken oral or external beauty products that may affect the test results within 2 weeks;

[0107] (8) Unable to cooperate with the test;

[0108] (9) Those who are deemed by the researcher to be unsuitable for participation in this study.

[0109] 2.3 Exit Criteria

[0110] Patients may withdraw from clinical research due to complications, adverse events or other reasons during the study, but the reasons for withdrawal should be clearly stated.

[0111] 2.4 Environmental conditions

[0112] The test environment conditions during the entire test process were controlled as follows: temperature 21±1°C, humidity: 50±10%RH.

[0113] 3. Test equipment

[0114] Facial image analysis system VISIA CR: Image acquisition + IPP analysis: Detect the skin wrinkle area ratio and red zone area. The smaller the measured value, the smaller the skin wrinkle area ratio / red zone area.

[0115] 4. Experimental process:

[0116] Before using the sample (D0):

[0117] (1) Volunteer subjects were recruited as required and signed a written informed consent. Before enrollment, subjects were asked a series of questions about their medical history, health status, etc. according to the inclusion and exclusion criteria;

[0118] (2) Cleaning the face and waiting: The subjects cleaned their faces with facial cleanser and dried their skin with dry facial tissues, and sat in a laboratory at 21±1°C and 50±10% RH for 30 minutes;

[0119] (3) Test baseline: laboratory technicians use VISIA to collect images of the subject's face;

[0120] (4) Sample distribution and use: Laboratory technicians instruct subjects on how to use samples, distribute test samples, and provide written sample instructions and usage logs.

[0121] After using the sample for 28 days (D28):

[0122] (5) Cleaning the face and waiting: The subjects cleaned their faces with facial cleanser and wiped them dry with dry facial tissue.

[0123] Skin, sit still in a laboratory at 21±1℃, 50±10%RH for 30 minutes;

[0124] (6) Testing: Laboratory technicians use VISIA to capture images of the subject's face;

[0125] (7) Sample recovery.

[0126] 5. Data Analysis

[0127] 5.1 Descriptive Statistics

[0128] The statistical analysis software SPSS was used to perform statistical analysis of the data and calculate the mean, standard deviation and change rate of each parameter at each time point. Calculation formula: Change rate = [(value after use - value before use) / value before use] × 100%

[0129] 5.2 Normal distribution test

[0130] A normal distribution test was performed on the parameter values ​​at each time point. If the asymptotic significance (two-sided) value of the normality test was > 0.050, the data series obeyed the normal distribution.

[0131] 5.3 Difference Analysis

[0132] Comparison before and after using samples: Perform difference analysis on the above parameter values. When the two groups of data are normally distributed at the same time, use paired T test to analyze the difference between the two groups of data; when the two groups of data are not normally distributed at the same time, use rank sum test to analyze the difference between the two groups of data.

[0133] 5.4 Evaluation Criteria

[0134] (1) When P < 0.010, it indicates a very significant difference;

[0135] (2) When 0.010≤P<0.050, it indicates a significant difference;

[0136] (3) When P ≥ 0.050, it indicates no significant difference.

[0137] 6. Basis for determination of results

[0138] (1) Soothing effect determination index: If the skin red area shows a significant decrease before and after using the sample (P<0.050), the test sample is deemed to have a soothing effect.

[0139] (2) Efficacy evaluation index for sensitive skin, mild and non-irritating: If no adverse reactions occur to the subjects during the use of the sample, the test sample is considered mild and non-irritating and suitable for sensitive skin.

[0140] 7. Analysis of experimental results

[0141] Table 2 Red zone area (VISIA CR+IPP analysis) test results

[0142]

[0143] Parameter Description: Figure 6 ,7, the smaller the measurement value, the smaller the red zone area;

[0144] Result interpretation: Compared with before using the samples, the red zone area of ​​33 subjects showed extremely significant difference after using the samples for 28 days.

[0145] It shows that the test sample has a soothing effect after using the sample for 28 days.

[0146] 8. Test conclusion

[0147] Efficacy conclusion: The test sample has a soothing effect after 28 days of use.

[0148] By observing 33 subjects with sensitive skin aged 27-55 years old, the test samples were used twice a day for 28 consecutive days under normal circumstances. The results showed that the 33 subjects had no adverse reactions after using the samples for 28 days. According to the experimental results, this product has the effects of "suitable for sensitive skin" and "mild and non-irritating".

[0149] 9. Adverse reaction assessment

[0150] During the entire testing process, none of the 33 subjects had any adverse reactions.

[0151] Example 8 Moisturizing and anti-wrinkle human efficacy evaluation test

[0152] This example verifies the moisturizing and anti-wrinkle effects of this product by measuring the water content of the stratum corneum and the transepidermal water loss (TEWL) value of 33 sensitive skin subjects before and after using this product, and by analyzing the percentage of skin wrinkle area.

[0153] The sample used in this example is the collagen essence described in Example 5.

[0154] 1. How to use: After cleaning your face, apply collagen essence evenly on your face and massage to help absorption.

[0155] Frequency and cycle of use: Use once a day in the morning and evening for 28 consecutive days.

[0156] 2. Subject requirements:

[0157] 2.1 Inclusion Criteria

[0158] (1) Healthy female subjects, aged 18-60 years;

[0159] (2) Skin sagging;

[0160] (3) crow's feet and nasolabial folds (wrinkle level 1-4);

[0161] (4) Corneometer stratum corneum moisture content is less than or equal to 60 c.u.;

[0162] (5) Tewameter test skin water loss value is greater than or equal to 15g / h / m 2 ;

[0163] (6) Able to cooperate well with the tester and maintain a regular life during the test period;

[0164] (7) Be able to read and understand all the contents of the informed consent form and voluntarily sign the informed consent form;

[0165] (8) Agree not to use any cosmetics, drugs or health products that may affect the results during the test;

[0166] (9) During the test period, similar products with the same efficacy as the test sample shall not be used;

[0167] (10) Not currently participating in and will not participate in any other research involving the test area during the study period;

[0168] (11) No medical aesthetic procedures may be performed during the test period.

[0169] 2.2 Exclusion criteria

[0170] Same as Example 7.

[0171] 2.3 Exit Criteria

[0172] Same as Example 7.

[0173] 2.4 Environmental conditions

[0174] The test environment conditions during the entire test process were controlled as follows: temperature 21±1°C, humidity: 50±10%RH.

[0175] 3. Test equipment

[0176] (1) Tewameter TM Hex: detects the transepidermal water loss (TEWL) value of the skin. The smaller the measured value, the less the transepidermal water loss per unit time and per unit cross-sectional area.

[0177] (3) Skin moisture tester Corneometer CM 825: detects the moisture content of the skin stratum corneum. The larger the measured value, the higher the moisture content of the skin stratum corneum.

[0178] 4. Experimental process:

[0179] Before using the sample (D0):

[0180] (1) Volunteer subjects were recruited as required and signed a written informed consent. Before enrollment, subjects were asked a series of questions about their medical history, health status, etc. according to the inclusion and exclusion criteria;

[0181] (2) Cleaning the face and waiting: The subjects cleaned their faces with facial cleanser and dried their skin with dry facial tissues, and sat in a laboratory at 21±1°C and 50±10% RH for 30 minutes;

[0182] (3) Test basic value: testing the moisture content of the stratum corneum and the transepidermal water loss (TEWL) value of the subjects’ skin;

[0183] (4) Sample distribution and use: Laboratory technicians instruct subjects on how to use samples, distribute test samples, and provide written sample instructions and usage logs.

[0184] After using the sample for 28 days (D28):

[0185] (5) Cleaning the face and waiting: The subjects cleaned their faces with facial cleanser and wiped them dry with dry facial tissue.

[0186] Skin, sit still in a laboratory at 21±1℃, 50±10%RH for 30 minutes;

[0187] (6) Testing: Testing the moisture content of the stratum corneum and the transepidermal water loss (TEWL) value of the subjects' skin;

[0188] (7) Sample recovery.

[0189] 5. Data Analysis

[0190] Same as Example 7.

[0191] 6. Basis for determination of results

[0192] (1) Repair efficacy determination index: If the transepidermal water loss (TEWL) value of the skin is significantly reduced before and after using the sample (P<0.050), the test sample is deemed to have a repair efficacy.

[0193] (2) Moisturizing efficacy determination index: before and after using the sample, if the moisture content of the stratum corneum of the skin is significantly increased (P<0.050) or the transepidermal water loss of the skin, TEWL value is significantly reduced (P<0.050), then the test sample is deemed to have moisturizing efficacy.

[0194] (3) Anti-wrinkle efficacy evaluation index: If the skin wrinkle area ratio shows a significant decrease before and after using the sample (P<0.050), the test sample is deemed to have anti-wrinkle efficacy.

[0195] 7. Analysis of experimental results

[0196] Table 3 Test results

[0197]

[0198]

[0199] Result analysis:

[0200] (1) Water content of the stratum corneum: The larger the measured value, the higher the water content of the stratum corneum; Figure 8 As shown, compared with before using the samples, the moisture content of the stratum corneum of the skin of 33 subjects after using the samples for 28 days was significantly different, indicating that the test samples have a moisturizing effect after using the samples for 28 days.

[0201] (2) Transepidermal water loss (TEWL): The smaller the value, the less transepidermal water loss per unit time and per unit cross-sectional area. Fig. 9 As shown, compared with before using the sample, the TEWL value of the skin of 33 subjects after using the sample for 28 days has a very significant difference. This shows that the test sample has a moisturizing effect after using the sample for 28 days. This shows that the test sample has a repairing effect after using the sample for 28 days.

[0202] (3) Skin wrinkle area ratio: The smaller the measured value, the smaller the skin wrinkle area ratio; Fig.10 , as shown in Figure 11, crow's feet: compared with before using the sample, the wrinkle area ratio of crow's feet in 33 subjects after using the sample for 28 days was significantly different. Nasal folds: compared with before using the sample, the wrinkle area ratio of nasolabial folds in 33 subjects after using the sample for 28 days was extremely significantly different. This shows that the test sample has anti-wrinkle effect after using the sample for 28 days.

[0203] Test Conclusion

[0204] Efficacy conclusion: After 28 days of use, the test sample has a moisturizing effect. After 28 days of use, the test sample has a repairing effect. After 28 days of use, the test sample has an anti-wrinkle effect.

[0205] Adverse reaction assessment

[0206] During the entire testing process, none of the 33 subjects had any adverse reactions.

[0207] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant collagen repair composition, characterized in that: It comprises recombinant collagen and oligopeptides, wherein the amino acid sequence of the recombinant collagen is shown as SEQ ID NO:

1.

2. The recombinant collagen repair composition according to claim 1, characterized in that: The mass ratio of the recombinant collagen to the oligopeptide is 1-200:0.1-1.

3. The recombinant collagen repair composition according to claim 1, characterized in that: The oligopeptide is one or more of oligopeptide-1, tripeptide-1 copper, and palmitoyl pentapeptide-4.

4. Use of the recombinant collagen repair composition according to claim 1 in cosmetics.

5. A recombinant collagen repair essence, characterized in that: It comprises the recombinant collagen repair composition according to claim 1 and a cosmetic auxiliary material matrix.

6. The recombinant collagen repair essence according to claim 5, characterized in that: Calculated by mass percentage, it includes 0.005% to 0.1% recombinant collagen, 0.0005% to 0.001% oligopeptide, and the remainder is a cosmetic auxiliary material matrix; the cosmetic auxiliary material matrix includes one or more of a moisturizer, a pH adjuster, a preservative, other functional additives, a thickener, a metal chelator, and a solvent.

7. The recombinant collagen repair essence according to claim 6, characterized in that: Calculated by mass percentage, it includes: recombinant collagen 0.005% ~ 0.1%, oligopeptide 0.0005% ~ 0.001%, moisturizer 5% ~ 10%, pH adjuster 0.2% ~ 0.4%, preservative 0.2% ~ 0.5%, and the balance is water.

8. The recombinant collagen repair essence according to claim 7, characterized in that: The moisturizer includes glycerin, tremella polysaccharide and sodium hyaluronate, the pH adjuster is sodium dihydrogen phosphate, and the preservatives include sodium methylparaben and phenoxyethanol; the recombinant collagen repair essence includes, by mass percentage: 0.005% ~ 0.1% recombinant collagen, 0.0005% ~ 0.001% oligopeptide, 5% ~ 10% glycerin, 0.01% ~ 0.02% tremella polysaccharide, 0.01% ~ 0.05% sodium hyaluronate, 0.2% ~ 0.4% sodium dihydrogen phosphate, 0.1% ~ 0.2% sodium methylparaben, and 0.2% ~ 0.5% phenoxyethanol.

9. A recombinant collagen repair essence facial mask, characterized in that: The mask cloth is soaked in the recombinant collagen repair essence liquid described in any one of claims 5 to 8 and then fully absorbed to obtain the essence liquid.

10. The method for preparing the recombinant collagen repair essence according to any one of claims 5 to 8, characterized in that: The cosmetic auxiliary material matrix is ​​mixed evenly and the pH is adjusted to the target value; collagen and oligopeptide-1 are dissolved in water, filtered and sterilized through a 0.22um filter membrane, and then added to obtain a recombinant collagen repair essence.

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