A composition containing recombinant collagen and its preparation method and application

Abstract

The present invention discloses a composition containing recombinant collagen, its preparation method, and application, relating to the field of cosmetics technology. The composition comprises the following components: type I recombinant collagen, type III recombinant collagen, type XVII recombinant collagen, ergothioneine, hydrangea extract, and Lactobacillus plantarum / cristoma fennel fermentation product filtrate. The combination of these components achieves significant synergistic effects in promoting cellular COL-1 gene expression, cell migration and repair, and anti-oxidation. The composition has effective and safe anti-wrinkle and repair effects.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a composition containing recombinant collagen, and a preparation method and application thereof. Background Art

[0002] As people age and are affected by the environment, their skin gradually develops some unhealthy symptoms, the most obvious of which are the formation of wrinkles and skin sensitivity. Wrinkles not only affect people's appearance, but are also a direct reflection of skin health and youthfulness. The core principle of anti-wrinkle repair lies in improving the structure and elasticity of the skin, reducing the formation and depth of wrinkles, and enhancing the skin's barrier function. This is mainly achieved by repairing and promoting the production of collagen and elastic fibers in the skin and promoting skin cell regeneration. However, the effectiveness of anti-wrinkle repair products varies greatly. Many products cannot effectively achieve anti-wrinkle repair effects, or use a single ingredient with limited effect, or the amount of a certain active ingredient added is large. While achieving anti-wrinkle effects, it is also irritating and not very friendly to sensitive skin. Therefore, it is very necessary to provide a product that has anti-wrinkle repair effects without irritation. Summary of the Invention

[0003] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a composition containing recombinant collagen, which has excellent anti-wrinkle repair effect.

[0004] The present invention also provides a method for preparing the composition.

[0005] The present invention also provides use of the composition in preparing cosmetics.

[0006] The present invention also provides cosmetics comprising the above composition.

[0007] A composition containing recombinant collagen according to the first embodiment of the present invention comprises the following components:

[0008] Type I recombinant collagen, type III recombinant collagen, type XVII recombinant collagen, ergothioneine, hydrangea extract, Lactobacillus plantarum / cristoma foenum-flavor ferment filtrate.

[0009] The composition containing recombinant collagen according to the embodiment of the present invention has at least the following beneficial effects:

[0010] By compounding type I recombinant human collagen, type III recombinant human collagen, type XVII recombinant human collagen, ergothioneine, Sparassis Crispa extract, and Lactobacillus plantarum / crithmum maritimum fermentation product filtrate, significant synergistic effects are achieved in promoting cell COL-1 gene expression, promoting cell migration and repair, and anti-oxidation. The composition of the embodiment has effective and safe anti-wrinkle repair effects.

[0011] According to some embodiments of the present invention, the invention comprises, in parts by weight:

[0012]

[0013] According to some embodiments of the present invention, the invention comprises, in parts by weight:

[0014]

[0015] According to some embodiments of the present invention, the type I recombinant collagen is type I recombinant human collagen.

[0016] According to some embodiments of the present invention, the type III recombinant collagen is type III recombinant human collagen.

[0017] According to some embodiments of the present invention, the type XVII recombinant collagen is type XVII recombinant human collagen.

[0018] According to some embodiments of the present invention, the composition further comprises a solvent acceptable in the cosmetic field.

[0019] According to some embodiments of the present invention, the composition further comprises 30 to 40 parts by weight of a solvent, for example, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 parts by weight.

[0020] According to some embodiments of the present invention, the cosmetically acceptable solvent includes a polyol.

[0021] According to some embodiments of the present invention, the polyol includes at least one of glycerol, butanediol, 1,3-propanediol, propylene glycol, 1,2-pentanediol, and 1,2-hexanediol.

[0022] According to some embodiments of the present invention, the composition consists of the following components in percentage by mass:

[0023]

[0024] According to some embodiments of the present invention, the composition consists of the following components in percentage by mass:

[0025] Type I recombinant collagen 0.001% to 0.5%;

[0026] Type III recombinant collagen 0.01% to 5%;

[0027] Type XVII recombinant collagen 0.00001% to 0.1%;

[0028] Ergothioneine 0.001% ~ 1%;

[0029] Hydrangea extract 1% to 20%;

[0030] Lactobacillus plantarum / marine fennel fermentation product filtrate 40%-60%;

[0031] Solvent residue.

[0032] According to some embodiments of the present invention, based on the total mass of the composition as 100%, the content of the type I recombinant collagen may be 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%.

[0033] According to some embodiments of the present invention, based on the total mass of the composition as 100%, the content of the type III recombinant collagen can be 0.01%, 0.05%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%.

[0034] According to some embodiments of the present invention, based on the total mass of the composition as 100%, the content of the type XVII recombinant collagen may be 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05% or 0.1%.

[0035] According to some embodiments of the present invention, based on the total mass of the composition as 100%, the content of the ergothioneine can be 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%.

[0036] According to some embodiments of the present invention, based on the total mass of the composition as 100%, the content of the hydrangea extract can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%.

[0037] According to some embodiments of the present invention, based on the total mass of the composition as 100%, the content of the Lactobacillus plantarum / sea fennel fermentation product filtrate can be 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%.

[0038] The method for preparing the above-mentioned composition according to the second embodiment of the present invention comprises the following steps:

[0039] The composition is obtained by mixing the components.

[0040] According to the third aspect of the present invention, the composition is used in the preparation of cosmetics.

[0041] According to some embodiments of the present invention, the cosmetic is a lotion, an essence, an emulsion, a cream or a facial mask.

[0042] A cosmetic according to a fourth embodiment of the present invention comprises the above composition and cosmetically acceptable excipients. Since the cosmetic adopts all the technical solutions of the composition of the above embodiment, it has at least all the beneficial effects brought about by the technical solutions of the above embodiment.

[0043] According to some embodiments of the present invention, the mass percentage of the composition is 0.01% to 10% based on the total mass of the cosmetic, for example, 0.01%, 0.05%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.

[0044] According to some embodiments of the present invention, the auxiliary material includes at least one of an emollient, an emulsifier, a thickener, a moisturizer, a skin protectant, and a preservative.

[0045] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the present invention. DETAILED DESCRIPTION

[0046] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0047] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0048] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0049] As used herein, the terms "comprises," "including," "having," "containing" or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, or article that comprises a listed element is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, or article.

[0050] The conjunction "consisting of" excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed, excluding materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim, rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0051] "Parts by mass" refers to the basic unit of measurement used to express the mass ratio of multiple components. One part can represent any unit of mass, such as 1g or 2.689g. If we say that the mass of component A is a parts and the mass of component B is b parts, this means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, we could say that the mass of component A is aK and the mass of component B is bK (K is an arbitrary number representing a multiplication factor). It's important to note that, unlike parts by mass, the sum of the mass of all components is not limited to 100 parts.

[0052] Unless otherwise specified, "room temperature" in the present invention means (25±5)°C.

[0053] Recombinant human collagen type I, recombinant human collagen type II, recombinant human collagen type III, and recombinant human collagen type XVII were purchased from Jiangsu Chuangshi Medical Technology Co., Ltd.

[0054] Ergothioneine was purchased from Hubei Ruisentic Biotechnology Co., Ltd.

[0055] Sparassis Crispa extract (INCI name / English name: Sparassis Crispa EXTRACT) and Lactobacillus plantarum / crithmum maritimum fermentation product filtrate were purchased from Guangzhou Nova Biotechnology Co., Ltd.

[0056] Example 1

[0057] This example provides a composition containing recombinant collagen, which is composed of the following components in parts by weight:

[0058]

[0059] This example also provides a method for preparing the composition, the steps are as follows:

[0060] (1) adding type I recombinant human collagen, type III recombinant human collagen, type XVII recombinant human collagen, and ergothioneine to the filtrate of Lactobacillus plantarum / cristatum fructus fermentation product and stirring to completely dissolve to obtain a mixed solution;

[0061] (2) Adding the hydrangea extract, glycerol, and 1,2-hexanediol to the mixed solution obtained in step (1), stirring and dissolving the mixture completely, thereby obtaining a composition.

[0062] Example 2

[0063] This example provides a composition containing recombinant collagen, which is composed of the following components in parts by weight:

[0064]

[0065]

[0066] This example also provides a method for preparing the composition, the steps are as follows:

[0067] (1) adding type I recombinant human collagen, type III recombinant human collagen, type XVII recombinant human collagen, and ergothioneine to the filtrate of Lactobacillus plantarum / cristatum fructus fermentation product and stirring to completely dissolve to obtain a mixed solution;

[0068] (2) Adding the hydrangea extract, glycerol, butylene glycol, and 1,2-pentanediol to the mixed solution obtained in step (1), stirring and dissolving the mixture completely to obtain a composition.

[0069] Example 3

[0070] This example provides a composition containing recombinant collagen, which is composed of the following components in parts by weight:

[0071]

[0072] This example also provides a method for preparing the composition, the steps are as follows:

[0073] (1) adding type I recombinant human collagen, type III recombinant human collagen, type XVII recombinant human collagen, and ergothioneine to the filtrate of Lactobacillus plantarum / cristatum fructus fermentation product and stirring to completely dissolve to obtain a mixed solution;

[0074] (2) Adding the hydrangea extract, butanediol, 1,3-propylene glycol, 1,2-pentanediol, and 1,2-hexanediol to the mixed solution obtained in step (1), stirring and dissolving the mixture completely, thereby obtaining a composition.

[0075] Example 4

[0076] This example provides a composition containing recombinant collagen, which is composed of the following components in parts by weight:

[0077]

[0078]

[0079] This example also provides a method for preparing the composition, the steps are as follows:

[0080] (1) adding type I recombinant human collagen, type III recombinant human collagen, type XVII recombinant human collagen, and ergothioneine to the filtrate of Lactobacillus plantarum / cristatum fructus fermentation product and stirring to completely dissolve to obtain a mixed solution;

[0081] (2) Adding the hydrangea extract, 1,3-propanediol, propylene glycol, 1,2-pentanediol, and 1,2-hexanediol to the mixed solution obtained in step (1), stirring and dissolving the mixture completely to obtain a composition.

[0082] Example 5

[0083] This example provides a composition containing recombinant collagen, which is composed of the following components in parts by weight:

[0084]

[0085] This example also provides a method for preparing the composition, the steps are as follows:

[0086] (1) adding type I recombinant human collagen, type III recombinant human collagen, type XVII recombinant human collagen, and ergothioneine to the filtrate of Lactobacillus plantarum / cristatum fructus fermentation product and stirring to completely dissolve to obtain a mixed solution;

[0087] (2) Adding the hydrangea extract, butanediol, 1,3-propylene glycol, 1,2-pentanediol, and 1,2-hexanediol to the mixed solution obtained in step (1), stirring and dissolving the mixture completely, thereby obtaining a composition.

[0088] Example 6

[0089] This example provides a composition containing recombinant collagen, which is composed of the following components in parts by weight:

[0090]

[0091] This example also provides a method for preparing the composition, the steps are as follows:

[0092] (1) adding type I recombinant human collagen, type III recombinant human collagen, type XVII recombinant human collagen, and ergothioneine to the filtrate of Lactobacillus plantarum / cristatum fructus fermentation product and stirring to completely dissolve to obtain a mixed solution;

[0093] (2) Adding the hydrangea extract, butanediol, 1,3-propylene glycol, and 1,2-hexanediol to the mixed solution obtained in step (1), stirring and dissolving the mixture completely to obtain a composition.

[0094] Application Example 1

[0095] This application example provides a firming and anti-wrinkle repairing essence water, the formula of which is shown in Table 1.

[0096] Table 1

[0097]

[0098]

[0099] This application example also provides a method for preparing the firming, anti-wrinkle and repairing essence water, comprising the following steps:

[0100] (1) Dissolve the raw material of phase A at 60-65°C until transparent, then cool to below 40°C to obtain phase A;

[0101] (2) Pre-dispersing the raw materials of phase B evenly and completely to obtain phase B;

[0102] (3) After the phase C raw materials are mixed and dispersed evenly, continue to add phase A and phase B and stir until they are evenly dispersed;

[0103] (4) After passing the inspection, filter the material through 300 mesh.

[0104] Application Example 2

[0105] This application example provides a firming and anti-wrinkle repair essence lotion, the formula of which is shown in Table 2.

[0106] Table 2

[0107]

[0108] This application example also provides a method for preparing a firming and anti-wrinkle repairing essence lotion, comprising the following steps:

[0109] (1) Put the raw materials of phase A into an emulsifier, stir and disperse them uniformly, and heat to 70-80°C to obtain phase A;

[0110] (2) Put the raw materials of phase B into the oil phase pot, heat it to 70-80°C to dissolve and disperse it evenly, and obtain phase B; then slowly pump phase B into the emulsification pot, homogenize and emulsify for 10-15 minutes to obtain a mixture;

[0111] (3) After the emulsification pot is cooled to 40-45°C, add the raw materials of phase C into the emulsification pot and stir to disperse them evenly;

[0112] (4) After passing the inspection, filter the material through 300 mesh.

[0113] Application Example 3

[0114] This application example provides a firming, anti-wrinkle repairing facial mask, the formula of which is shown in Table 3.

[0115] Table 3

[0116]

[0117] This application example also provides a method for preparing a firming and anti-wrinkle repairing facial mask, comprising the following steps:

[0118] (1) Put the raw materials of phase A into an emulsifier, stir and disperse them uniformly, and heat to 70-80°C to obtain phase A;

[0119] (2) Preheat the raw materials of phase B to 50-60°C to dissolve and obtain phase B;

[0120] (3) After the emulsification pot is cooled to 40-45°C, add the raw materials of phase B and phase C into the emulsification pot and stir to disperse them evenly;

[0121] (4) After passing the inspection, filter the material through 300 mesh.

[0122] Application Example 4

[0123] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of application example 1, except that the composition of example 1 is replaced by the composition of example 2.

[0124] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0125] Application Example 5

[0126] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of application example 1, except that the composition of example 1 is replaced by the composition of example 3.

[0127] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0128] Application Example 6

[0129] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of application example 1, except that the composition of example 1 is replaced by the composition of example 4.

[0130] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0131] Application Example 7

[0132] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of application example 1, except that the composition of example 1 is replaced by the composition of example 5.

[0133] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0134] Application Example 8

[0135] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of application example 1, except that the composition of example 1 is replaced by the composition of example 6.

[0136] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0137] Comparative Example

[0138] This example provides 63 compositions containing recombinant collagen, and the component compositions by weight are shown in Table 4.

[0139] Table 4

[0140]

[0141]

[0142]

[0143] The preparation methods of the compositions of Comparative Examples 1 to 66 are the same as those of Example 1.

[0144] Comparative Application Example 1

[0145] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of Application Example 1, except that the composition of Example 1 is replaced by the composition of Comparative Example 1.

[0146] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0147] Application Comparative Example 2

[0148] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of Application Example 1, except that the composition of Example 1 is replaced by the composition of Comparative Example 2.

[0149] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0150] Application Comparative Example 3

[0151] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of Application Example 1, except that the composition of Example 1 is replaced by the composition of Comparative Example 3.

[0152] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0153] Comparative Application Example 4

[0154] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of Application Example 1, except that the composition of Example 1 is replaced by the composition of Comparative Example 4.

[0155] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0156] Application Comparative Example 5

[0157] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of Application Example 1, except that the composition of Example 1 is replaced by the composition of Comparative Example 5.

[0158] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0159] Application Comparative Example 6

[0160] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of Application Example 1, except that the composition of Example 1 is replaced by the composition of Comparative Example 6.

[0161] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0162] Application Comparative Example 7

[0163] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of Application Example 1, with the only difference being that the composition of Example 1 is replaced by the composition of Comparative Example 63.

[0164] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0165] Comparative Application Example 8

[0166] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of Application Example 1, with the only difference being that the composition of Example 1 is replaced by the composition of Comparative Example 64.

[0167] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0168] Comparative Application Example 9

[0169] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of Application Example 1, with the only difference being that the composition of Example 1 is replaced by the composition of Comparative Example 65.

[0170] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0171] Comparative Application Example 10

[0172] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of Application Example 1, with the only difference being that the composition of Example 1 is replaced by the composition of Comparative Example 66.

[0173] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0174] Comparative Application Example 11

[0175] This application example provides a firming, anti-wrinkle and repairing essence water, the formula of which is basically the same as that of application example 1, except that the composition of example 1 is replaced by a composition of deionized water.

[0176] The preparation method of the firming, anti-wrinkle and repairing essence water is the same as that of Application Example 1.

[0177] Test Example 1

[0178] Type I collagen (COL-1) is primarily found in skin, bones, muscles, and other connective tissues, playing a key role in maintaining tissue structural integrity and elasticity. When COL-1 is specifically degraded, the structure of collagen and elastic fibers in the body is disrupted, leading to aging phenomena such as wrinkles on the skin. This example examines the effects of the compositions of Examples 1 to 6 and Comparative Examples 1 to 63 (test samples) on the expression of COL-1 genes in fibroblasts. The experimental steps are as follows:

[0179] Human skin fibroblasts cultured to passage 3 were cultured at 5×10 5 Cells were seeded / well in a 96-well plate, mixed, and incubated at 37°C, 5% CO2 for 24 hours. For the blank group (BC), the culture medium was replaced with serum-free cell culture medium; for the control group (NC), the culture medium was replaced with serum-free cell culture medium containing 200 μmol / L H2O2; and for the experimental group, the culture medium was replaced with serum-free cell culture medium containing 200 μmol / L H2O2 and 0.01 wt% of the test sample. After 3 days of incubation at 37°C, 5% CO2, the COL-1 gene expression levels in each treatment group were measured by qPCR. The effect (X) on COL-1 expression in HSF cells was calculated as follows:

[0180] X=Q x / Q0;

[0181] Among them, Q x represents the gene expression level of each group determined by qPCR, and Q0 represents the gene expression level of the blank group BC of each group determined by qPCR.

[0182] The results are shown in Table 5.

[0183] Table 5

[0184]

[0185]

[0186] In the control group, the expression level of COL-1 in HSF cells was significantly reduced under the induction of H2O2. However, treatment with the compositions of Examples 1 to 6 significantly increased the expression level of COL-1 in HSF cells. The effect was significantly reduced when any one, two, three, four, or five of the compositions (excluding the solvent) were omitted. Compared to the composition of Comparative Example 63, the compositions of Comparative Examples 57 to 62, each containing only Lactobacillus plantarum / marine fennel fermentation product filtrate, Hydrangea extract, ergothioneine, type XVII recombinant human collagen, type III recombinant human collagen, and type I recombinant human collagen, had minimal effect on intracellular COL-1 expression. However, Example 1, through the combination of Lactobacillus plantarum / marine fennel fermentation product filtrate, Hydrangea extract, ergothioneine, type XVII recombinant human collagen, type III recombinant human collagen, and type I recombinant human collagen, significantly increased COL-1 expression in HSF cells, surpassing the combined promoting effects of each component alone. The difference in effect between Example 1 and Comparative Examples 64 to 66 indicates that replacing type XVII recombinant human collagen, type III recombinant human collagen, or type I recombinant human collagen with type II recombinant human collagen significantly reduced the effectiveness of the composition in increasing COL-1 expression in HSF cells. This demonstrates the irreplaceability of each recombinant human collagen in the composition of the present invention.

[0187] This shows that in terms of anti-wrinkle, there is a good synergistic effect between plant lactobacillus / sea fennel fermentation product filtrate, hydrangea extract, ergothioneine, type XVII recombinant human collagen, type III recombinant human collagen, and type I recombinant human collagen.

[0188] Test Example 2

[0189] The cell scratch test is a method for measuring cell migration and repair ability. It is similar to the in vitro wound healing process. In the in vitro culture system, a monolayer of adherent cells is used. The cells in the central part are physically removed, and then the cells are cultured for the time set in the experiment. The proliferation and migration ability of the surrounding cells is observed. The difference in the repair ability of cells in different groups for the scratch area can be used to judge the migration and repair ability of each group of cells. This example tests the cell migration and repair ability of the compositions (test samples) of Examples 1 to 6 and Comparative Examples 1 to 66. The experimental steps are as follows:

[0190] Use a marker to evenly mark horizontal lines across the cell wells on the back of the 6-well plate. The interval between the lines is about 0.5cm to 1cm, and each well should have at least 5 lines. 4Cells / well: Human immortalized keratinocytes HaCaT were seeded into the cell plate. The inoculation principle was that the fusion rate reached 100% after overnight. After 24 hours of cell attachment, a 100 μL pipette tip was used to scratch the cell layer perpendicular to the cell plane and perpendicular to the line on the back of the plate against a ruler. After the scratching was completed, the cells were washed 3 times with sterile DPBS to wash away the non-adherent cells (i.e., the cells removed during the streaking process) so that the gap left after the streaking was clearly visible, and then fresh serum-free culture medium was replaced. The sample to be tested was added to the cells in each experimental group to a final concentration of 0.01wt%; the blank group (BC) did not add any sample to be tested compared to the other experimental groups. The cells in each group were cultured at 37°C and 5% CO2 for 0, 12, 24, and 48 hours, and the cells were removed, observed, and photographed. The data were analyzed and processed using Image-J software.

[0191] The average migration rate is the average of the cell migration rates of three repeated experiments. The calculation formula of cell migration rate is:

[0192] Cell migration rate = [(initial scratch width - scratch width after culture) / initial scratch width] × 100%;

[0193] The calculation formula for the promotion effect is:

[0194] Promotion effect = [(average migration rate of cells in the experimental group - average migration rate of cells in the blank group) / average migration rate of cells in the blank group] × 100%.

[0195] The results are shown in Table 6.

[0196] Table 6

[0197]

[0198]

[0199] The composition treatment of embodiment 1~6 can significantly improve the migration and repair ability of cell.When any one, any two, any three, any four or any five kinds (except solvent) in the default composition, effect can be greatly deteriorated.Relative to the composition of comparative example 63, the composition of comparative example 57~62 only contains plant lactobacillus / sea fennel fermentation product filtrate, hydrangea extract, thioneine, XVII type recombinant human collagen, III type recombinant human collagen, I type recombinant human collagen respectively, and the promotion effect to the migration and repair ability of cell is very little, and embodiment 1 significantly improves the migration and repair ability of cell by plant lactobacillus / sea fennel fermentation product filtrate, hydrangea extract, thioneine, XVII type recombinant human collagen, III type recombinant human collagen, I type recombinant human collagen, is better than the superposition of the simple promotion effect of each component. The difference in efficacy between Example 1 and Comparative Examples 64-66 indicates that replacing recombinant human collagen type XVII, recombinant human collagen type III, or recombinant human collagen type I with recombinant human collagen type II significantly impairs the composition's ability to enhance cell migration and repair. This demonstrates the irreplaceable nature of each recombinant human collagen in the composition of the present invention.

[0200] This shows that in terms of promoting cell migration and repair ability, there is a good synergistic effect between Lactobacillus plantarum / marine fennel fermentation product filtrate, hydrangea extract, ergothioneine, type XVII recombinant human collagen, type III recombinant human collagen, and type I recombinant human collagen.

[0201] Test Example 3

[0202] Superoxide anion radical scavenging rate, hydroxyl radical scavenging rate, and ABTS radical scavenging rate are commonly used in vitro evaluation indicators of antioxidant capacity. The higher the scavenging rate, the better the antioxidant effect and the better the anti-aging and repair effect. This example tests the antioxidant capacity of the compositions (test samples) of Examples 1 to 6 and Comparative Examples 1 to 66. The experimental steps are as follows:

[0203] (1) Superoxide anion free radical scavenging ability evaluation test:

[0204] Preheat 4.5 mL of 0.05 mol / L Tris-HCl buffer solution (pH 8.2) in a 25°C water bath for 30 minutes. Add 1 mL of the sample to be tested and 0.4 mL of a 25 mol / L aqueous solution of pyrogallol. Mix thoroughly, and incubate in a 25°C water bath for 5 minutes. Terminate the reaction by adding 1 mL of an 8 mol / L aqueous hydrochloric acid solution. Measure the absorbance at 299 nm using the Tris-HCl buffer solution as a reference. For the blank control group, replace the sample to be tested with 1 mL of the corresponding solvent from the sample solution (using Example 1 as an example, the corresponding solvent for the blank control group is a mixture of 31 parts glycerol, 1 part 1,2-hexanediol, and 68 parts water). A 120 μM aqueous solution of Trolox is used as a positive control.

[0205] The calculation formula of superoxide anion radical scavenging rate is as follows:

[0206] Superoxide anion radical scavenging rate (%) = [1-(A2 / A1)] × 100%.

[0207] Where A1 is the absorbance value of the blank control group, and A2 is the absorbance value of the sample group.

[0208] The results are shown in Table 7.

[0209] Table 7

[0210]

[0211]

[0212] (2) Hydroxyl radical scavenging ability evaluation test:

[0213] Add 3 mL of 2 mmol / L FeSO4 aqueous solution and 3 mL of 1 mmol / L H2O2 aqueous solution to a 25 mL cuvette, shake well; then add 3 mL of 6 mmol / L salicylic acid solution (solvent is ethanol), shake well, heat in a 37°C water bath for 15 minutes, remove, and measure the absorbance A0 at 510 nm. Continue to add X mL of the sample with a mass fraction of 0.01% to the cuvette, shake well, continue to heat in a 37°C water bath for 15 minutes, remove, and measure the absorbance A0 at 510 nm. x A 120 μM Trolox aqueous solution was used as a positive control.

[0214] Replace the sample with the solvent of the corresponding sample to be tested and perform the test to obtain the absorbance A x0 .

[0215] The calculation formula of hydroxyl radical scavenging rate is as follows:

[0216] Hydroxyl radical scavenging rate (%) = [A0-Ax -(A0-A x0 )] / A0×100%.

[0217] The results are shown in Table 8.

[0218] Table 8

[0219]

[0220]

[0221] (3) ABTS method to detect the antioxidant activity of active components:

[0222] Dilute the ABTS solution with deionized water to an absorbance of 0.7 ± 0.02 at 734 nm to obtain the ABTS working solution. 10 μL of the sample to be tested and 190 μL of the ABTS working solution were added to a 96-well plate. After reacting at room temperature for 15 minutes, the absorbance at 734 nm was measured using a microplate reader. 样品 A 120 μM Trolox aqueous solution was used as a positive control. Each sample was repeated three times.

[0223] The calculation formula of ABTS free radical scavenging rate is as follows:

[0224] ABTS free radical scavenging rate = (1-A 样品 / 0.7)×100%.

[0225] The results are shown in Table 9.

[0226] Table 9

[0227]

[0228]

[0229] The composition of embodiment 1~6 has very high superoxide anion free radical, hydroxyl free radical, ABTS free radical scavenging ability.When any one, any two, any three, any four or any five kinds (except solvent) in the default composition, effect all can greatly deteriorate.Relative to the composition of comparative example 63, the composition of comparative example 57~62 only contains plant lactobacillus / sea fennel fermentation product filtrate, hydrazide extract, thioneine, XVII type recombinant human collagen, III type recombinant human collagen, I type recombinant human collagen respectively, its superoxide anion free radical, hydroxyl free radical, ABTS free radical scavenging effect are very poor, and embodiment 1 significantly improves superoxide anion free radical, hydroxyl free radical, ABTS free radical scavenging effect by the compound of plant lactobacillus / sea fennel fermentation product filtrate, hydrazide extract, thioneine, XVII type recombinant human collagen, III type recombinant human collagen, I type recombinant human collagen, is better than the superposition of the simple promotion effect of each component.

[0230] This shows that in terms of antioxidant properties, there is a good synergistic effect between the plant lactobacillus / sea fennel fermentation product filtrate, hydrangea extract, ergothioneine, type XVII recombinant human collagen, type III recombinant human collagen, and type I recombinant human collagen.

[0231] Test Example 4

[0232] In this example, the products of Examples 1-8 and Comparative Examples 1-11 were used as test substances, and the test was conducted in accordance with the human skin patch test method in the "Safety Technical Specifications for Cosmetics" (2015 edition). Thirty volunteers aged 18 to 60 years who met the test requirements were selected as test subjects (half male and half female). The test substance was added to the patch tester at a dosage of 0.020-0.025g. The control well served as a blank control (no substance was placed). The patch tester containing the test substance was applied to the flexor side of the subject's forearm using non-irritating tape. Using the palm of your hand, gently press the patch to evenly apply it to the skin. Mark the test site for easy observation. Apply the patch every 24 hours, remove the patch, and observe the skin reaction 30 minutes after the indentation disappears. If a suspicious reaction or a positive reaction occurs, the patch test is discontinued for the subject. If a negative reaction occurs, the patch test is repeated as described above at the same test site for a total of six times. The test subject's reaction results are recorded according to the skin adverse reaction grading criteria in Table 10. The test results are shown in Table 11.

[0233] Table 10

[0234] grade symbol Identification standards 0 - Negative reaction: no irritation, no erythema 1 ± Suspected reaction: mild erythema 2 + Weak positive reaction: erythema 3 ++ Strong positive reaction: erythema, papules, blisters 4 +++ Very strong positive reaction: severe edema, bullae

[0235] Table 11

[0236]

[0237]

[0238] The results in Table 11 show that there were no adverse reactions after six patch tests for Application Examples 1 to 8 and Comparative Examples 1 to 11, indicating that the products of Application Examples 1 to 8 and Comparative Examples 1 to 11 have good mildness and safety.

[0239] Test Example 5

[0240] Skin water loss TEWL is a very important parameter for evaluating the function of the skin's moisture protection layer and has been widely recognized internationally. The more intact the skin's protective layer, the higher the moisture content will be, and the lower the value of skin water loss TEWL will be. The skin moisture test can promptly detect whether the skin's barrier protection function has been damaged. This example tests the moisturizing ability of the firming and anti-wrinkle repairing essence water of Example 1, 4 to 8, and Comparative Examples 1 to 7, the firming and anti-wrinkle repairing essence milk of Example 2, and the firming and anti-wrinkle repairing facial mask of Example 3 (test samples). The steps are as follows:

[0241] The test instruments used were: Corneometer test probe for skin moisture content manufactured by Courage + Khazaka (CK) of Germany; Tewameter TM300 test probe for skin moisture loss (YEWL) manufactured by Courage + Khazaka (CK) of Germany.

[0242] A total of 570 volunteers aged 30 to 45, half male and half female, were randomly divided into 19 groups, with 15 males and 15 females in each group.

[0243] How to use: Apply on face for 15 minutes, then remove and wash off. Use once a day for 28 consecutive days.

[0244] Before the test, the subjects were asked to wash their faces and enter a climate-controlled room (22±1°C, relative humidity 50%) 2 hours after washing to sit quietly for more than 20 minutes and remain in a relaxed state.

[0245] During the experiment, half of the face was tested according to a random table, with the sample on one side and a blank on the other. The left and right nasolabial folds of the volunteers were selected for skin moisture content and water loss (TEWL) testing. The average of the three values was taken as the left and right nasolabial fold moisture content data (d0) and the nasolabial fold skin water loss (TEWL) data (d0). After the test, the volunteers were required to use the product according to the instructions, applying the sample product on one side of the face and leaving the other side blank. During the test, they should avoid using other products with the same efficacy. Use continued for 28 days, and the subjects were required to return for a follow-up visit on the 14th day (d14) and the 28th day (d28) of the test.

[0246] The results are shown in Table 12.

[0247] The calculation formula for the TEWL change rate is:

[0248] TEWL change rate (%) = (T1-T0) / T0×100%;

[0249] T0 is the TEWL value at d0, and T1 is the TEWL value at d14 or d18.

[0250] Table 12

[0251]

[0252] After 28 days of use, the products of Examples 1-8 significantly improved skin moisture content and reduced TEWL compared to those of Comparative Examples 1-11. This demonstrates the excellent repairing capabilities of the compositions of the present invention. However, the omission of any of the following: Lactobacillus plantarum / crista-marine fermentation filtrate, hydrangea extract, ergothioneine, recombinant human collagen type XVII, recombinant human collagen type III, or recombinant human collagen type I significantly reduced the repairing effects.

[0253] Test Example 6

[0254] In this example, the anti-wrinkle ability of the firming and anti-wrinkle repairing essence water of Example 1, 4 to 8, and Comparative Examples 1 to 11, the firming and anti-wrinkle repairing essence lotion of Example 2, and the firming and anti-wrinkle repairing facial mask of Example 3 (test samples) was tested.

[0255] 570 healthy subjects (35-45 years old, with normal skin and no history of cosmetic allergies) were randomly divided into 19 groups, with half male and half female. The subjects used the test sample (the dosage form of Application Example 3 is special and the frequency of use is once every night, one tablet at a time) once a day, morning and evening, with a dosage of 0.5g each time, for 28 consecutive days. On the 28th day, the subjects sat quietly for 30 minutes in a test environment with a temperature of 20±2°C and a humidity of 50±10%. VISIA was used to measure the number of facial wrinkles (lines), total wrinkle area, and maximum wrinkle length, and comprehensively evaluate the anti-wrinkle efficacy of the test sample on human skin.

[0256] Wrinkle Count (Rate of Change): This refers to the rate of change in the number of wrinkles after using the test sample compared to before use. A positive value indicates an increase in the number of wrinkles, meaning more wrinkles; a negative value indicates a decrease in the number of wrinkles, meaning fewer wrinkles.

[0257] Total wrinkle area change rate: This refers to the rate of change in the total wrinkle area after use of the test sample compared to before use. A positive value indicates an increase in the total wrinkle area, indicating more wrinkles; a negative value indicates a decrease in the total wrinkle area, indicating fewer wrinkles.

[0258] Maximum Wrinkle Length Change Rate: This refers to the rate of change in maximum wrinkle length after use of the test sample compared to before use. A positive value indicates an increase in maximum wrinkle length, meaning the wrinkle is longer; a negative value indicates a decrease in maximum wrinkle length, meaning the wrinkle is shorter.

[0259] The results are shown in Table 13.

[0260] Table 13

[0261]

[0262]

[0263] After 28 days of testing, the subjects who used the products of application examples 1 to 8 showed better improvement in the number of facial wrinkles (lines), total wrinkle area, and maximum wrinkle length than those who used the products of application examples 1 to 8. Among them, the number of facial wrinkles (lines), total wrinkle area, and maximum wrinkle length of the subjects who used the products of application example 1 for 28 days decreased by 9.38%, 10.56%, and 12.08%, respectively, while the corresponding values of the subjects who used the products of application examples 1 to 7 were the highest, with a decrease of 7.64%, 8.88%, and 9.08%, respectively. The omission of any of the following: Lactobacillus plantarum / sea fennel fermentation product filtrate, hydrangea extract, ergothioneine, type XVII recombinant human collagen, type III recombinant human collagen, and type I recombinant human collagen will greatly reduce the anti-wrinkle effect.

[0264] The embodiments of the present invention are described in detail above in conjunction with the embodiments, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A composition containing recombinant collagen, characterized in that: Includes the following components: Type I recombinant collagen, type III recombinant collagen, type XVII recombinant collagen, ergothioneine, hydrangea extract, Lactobacillus plantarum / cristoma foenum-flavor fermentation filtrate; In parts by weight, it comprises: Type I recombinant collagen 0.0001~1 part; Type III recombinant collagen 0.001~7 parts; Type XVII recombinant collagen 0.00001~0.5 parts; 0.0001~2 parts of ergothioneine; 0.5-23 parts of hydrangea extract; 37-63 parts of Lactobacillus plantarum / marine fennel fermentation product filtrate.

2. The composition according to claim 1, characterized in that In parts by weight, it comprises: Type I recombinant collagen 0.001~0.5 parts; Type III recombinant collagen 0.01~5 parts; Type XVII recombinant collagen 0.00001~0.1 part; Ergothioneine 0.001~1 part; 1-20 parts of Hydrangea extract; 40-60 parts of Lactobacillus plantarum / marine fennel fermentation product filtrate.

3. The composition according to claim 1, characterized in that The composition further comprises a cosmetically acceptable solvent.

4. The composition according to claim 3, characterized in that The cosmetically acceptable solvents include polyols.

5. The composition according to claim 4, characterized in that The polyol includes at least one of glycerol, butanediol, 1,3-propanediol, propylene glycol, 1,2-pentanediol, and 1,2-hexanediol.

6. The method for preparing the composition according to any one of claims 1 to 5, characterized in that: The following steps are involved: The composition is obtained by mixing the components.

7. Use of the composition according to any one of claims 1 to 5 in the preparation of cosmetics.

8. The use according to claim 7, characterized in that The cosmetics are lotion, essence, emulsion, cream or facial mask.

9. A cosmetic, characterized in that: The cosmetic comprises the composition according to any one of claims 1 to 5 and cosmetically acceptable excipients.

10. The cosmetic according to claim 9, characterized in that Based on the total mass of the cosmetic, the mass percentage of the composition is 0.01% to 10%.

11. The cosmetic according to claim 9, characterized in that The auxiliary materials include at least one of a moisturizer, an emulsifier, a thickener, a humectant, a skin protectant and a preservative.

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