Use of an anti-aging protein or a recombinant protein thereof in the preparation of a cosmetic product

Abstract

The present application belongs to the field of genetic engineering technology and cosmetics, and particularly relates to use of an anti-aging protein or a recombinant protein thereof in preparation of cosmetics. The present application provides use of an anti-aging protein or a recombinant protein thereof in preparation of at least one product or composition selected from the following: a product or composition for improving gene expression level of a cell anti-wrinkle factor; a product or composition for improving gene expression level of a cell inflammation factor; a product or composition for improving skin tightness of a subject; a product or composition for improving skin elasticity value of a subject; a product or composition for improving wrinkle-related parameters of a subject; a product or composition for delaying natural skin aging; a product or composition for reducing cell aging caused by photoaging; and the amino acid sequence of the anti-aging protein is shown as SEQ ID NO:1. The cosmetic prepared from the anti-aging protein can realize the effects of tightening skin, improving skin elasticity and reducing eye wrinkles.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering technology and cosmetics, and specifically relates to a use of an anti-aging protein or a recombinant protein thereof in the preparation of a cosmetic. BACKGROUND

[0002] With the extension of the average life span of human beings and the aggravation of population aging, the health problems of aging have been concerned. Aging is a necessary process of living beings, and "anti-aging" means that people hope to "healthily age" while extending the life span. For the skin, the factors leading to aging are mainly divided into two categories-natural aging and photoaging. Natural aging mainly refers to aging caused by uncontrollable factors such as heredity, endocrine and immune function existing in the body with age increasing; photoaging mainly refers to the process of natural aging accelerated due to the action of ultraviolet rays; climate change and environmental pollution also have a direct effect on the photoaging of the skin. Although aging is an inevitable natural phenomenon, people can usually slow down the pace of aging through some means, and "anti-aging" cosmetics are among them. "Anti-aging" cosmetics are roughly divided into moisturizing cosmetics, antioxidant cosmetics and biologically active cosmetics according to different functions or effects (Li Xiang, Hu Junjiao, Li Qiong, et al. Perfume and Cosmetics [J]. 2013, 5.).

[0003] Chinese patent application 202210715721.5 discloses an anti-aging skin care product and its preparation method and application. The anti-aging skin care product comprises 7-45% active functional ingredients by mass percentage; the active functional ingredients comprise secretin, fermentate of schizosaccharomyces pombe, hexapeptide and pentapeptide; the anti-aging skin care product can promote epidermal cell proliferation and renewal, promote collagen generation, improve skin elasticity, and has a strong skin anti-aging effect.

[0004] Chinese patent application 201911286742.4 discloses an anti-aging composition comprising the following components by mass fraction: Pinus pinaster bark extract 20-25 parts; Bletilla striata root extract 10-15 parts; Radix Polygoni Multiflori extract 8-12 parts; solvent 17-25 parts; the composition can promote the absorption of effective components in the anti-aging composition by human body, scavenge free radicals, thereby delaying the speed of skin aging, and has more remarkable anti-aging effect.

[0005] In recent years, foreign studies have found the importance of inflammatory aging, and consider that the proinflammatory cytokine network as one of the phenomena in the aging process, and other aging mechanisms such as oxidative stress damage can form a vicious cycle, accelerate cell aging (Jylhava J, Hurme M. Gene variants as determinants of longevity: focus on the inflammatory factors [J]. Pflugers Arch, 2010; 459: 239-46; Giunta S. Exploring the complex relations between inflammation and aging (inflamm-aging): anti-inflamm-aging remodeling of inflamm-aging, from robustness to frailty [J]. Inflamm Res, 2008; 57: 558-63). Recently, the integrated oxidation-inflammation theory of aging (oxi-inflamm-aging) caused by oxidative stress and inflammatory stress is proposed as one of the main reasons for aging. Chronic oxidative stress affects all cells, especially the regulatory system such as the nervous, endocrine and immune systems, and the interconnection of the above systems, leading to the prevention of sufficient homeostasis, and thus affecting the maintenance of health (De la Fuente M, Miquel J. An update of the oxidation-inflammation theory of aging: the involvement of the immune system in oxi-inflamm-aging [J]. Curr Pharm Des, 2009; 15: 3003-26).

[0006] Anti-wrinkle factors mainly include Collagen I, Collagen III, Collagen IV, Collagen VII, Decorin, VCAN, BGN, MMP-1, MMP-3, Smad3, Smad7, c-fos, and c-jun. Each anti-wrinkle factor participates in the aging process through different mechanisms.

[0007] At present, there is a lack of anti-aging cosmetics that can simultaneously regulate inflammatory factors and anti-wrinkle factors in the field. SUMMARY

[0008] Epicoccum nigrum is widely present in air, soil, and the surface of senescent, just dead or young plant tissues. Epicoccum nigrum belongs to the fourth type of pathogenic microorganisms, most of which are not pathogenic and do not cause human or animal diseases under normal circumstances, and the source is safe.

[0009] The inventors unexpectedly isolated a novel protein (named "Folded Peptide-162", code: HJ-P2) from Epicoccum nigrum, i.e. the anti-aging protein of the present application, which was found to have excellent anti-aging effect through various efficacy experiments, and could significantly regulate the expression amount of inflammatory factors and anti-aging factors, thereby achieving the effects of tightening skin, improving skin elasticity, and reducing eye wrinkles, and having important application value in the field of cosmetics.

[0010] In order to achieve the above technical purposes, the technical scheme of the present application is as follows:

[0011] In one aspect, the present application provides a use of an anti-aging protein or a recombinant protein thereof in the preparation of at least one of the following products or compositions:

[0012] (1) a product or composition for improving the expression level of a cell anti-wrinkle factor gene;

[0013] (2) a product or composition for improving the expression level of a cell inflammatory factor gene;

[0014] (3) a product or composition for improving the skin tightening degree of a subject;

[0015] (4) a product or composition for improving the skin elasticity value of a subject;

[0016] (5) a product or composition for improving the wrinkle-related parameters of a subject;

[0017] (6) a product or composition for delaying the natural aging of skin;

[0018] (7) a product or composition for reducing cell senescence caused by photoaging;

[0019] The amino acid sequence of the anti-aging protein is shown as SEQ ID NO: 1,

[0020] The nucleotide sequence encoding the anti-aging protein is shown as SEQ ID NO: 2.

[0021] SEQ ID NO: 1:

[0022] MQFFTVASALFAAALAAPAPQTSDCPNPAHCGGSPPDPSTYENIDISDFYVRKNNGIQN AGFKLTGKNATDLSCTIGATDLPSNVVTCGDSDYRFGLTKGDTTEFGLAIYHQTSPFAGLW AIGDAPTYCHAGGNGPDDFVCQQTGPTTIVIVGQNYPGGADN.

[0023] SEQ ID NO:2:

[0024] ATGCAATTTTTCACAGTAGCTTCAGCACTATTCGCTGCTGCCTTGGCAGCGCCGGCACCGCAAACTTCGGACTGCCCGAATCCGGCGCACTGCGGAGGTAGCCCGCCGGACCCGTCCACCTACGAGAACATTGATATTAGCGACTTCTATGTTCGTAAAAACAACGGCATCCAAAACGCCGGTTTTAAACTGACCGGTAAGAACGCGACCGATCTGTCTTGTACCATTGGTGCGACCGATCTTCCGAGCAATGTTGTCACCTGCGGCGACTCCGACTACCGCTTCGGCCTGACGAAGGGTGATACCACCGAATTTGGCTTGGCGATTTATCACCAGACCAGCCCATTTGCGGGCCTGTGGGCAATCGGCGACGCTCCGACGTACTGCCATGCGGGTGGTAATGGTCCGGACGATTTCGTTTGTCAGCAGACCGGCCCTACTACGATCGTGATCGTGGGTCAGAATTATCCGGGTGGCGCGGATAAC.

[0025] In some examples, the recombinant protein further comprises a tag protein which assists its expression and / or purification, or an active protein which enhances its activity and / or prolongs its half-life.

[0026] In some examples, the tag protein can be a His tag, preferably a 6xHis tag.

[0027] In some preferred examples, the amino acid sequence of the recombinant protein is as shown in SEQ ID NO:3.

[0028] In some preferred examples, the nucleotide encoding the recombinant protein is as shown in SEQ ID NO:4.

[0029] SEQ ID NO: 3:

[0030] MQFFTVASALFAAALAAPAPQTSDCPNPAHCGGSPPDPSTYENIDISDFYVRKNNGIQN AGFKLTGKNATDLSCTIGATDLPSNVVTCGDSDYRFGLTKGDTTEFGLAIYHQTSPFAGLW AIGDAPTYCHAGGNGPDDFVCQQTGPTTIVIVGQNYPGGADNLEHHHHHH.

[0031] SEQ ID NO: 4:

[0032] ATGCAATTTTTCACAGTAGCTTCAGCACTATTCGCTGCTGCCTTGGCAGCGCCGGCACCGCAAACTTCGGACTGCCCGAATCCGGCGCACTGCGGAGGTAGCCCGCCGGACCCGTCCACCTACGAGAACATTGATATTAGCGACTTCTATGTTCGTAAAAACAACGGCATCCAAAACGCCGGTTTTAAACTGACCGGTAAGAACGCGACCGATCTGTCTTGTACCATTGGTGCGACCGATCTTCCGAGCAATGTTGTCACCTGCGGCGACTCCGACTACCGCTTCGGCCTGACGAAGGGTGATACCACCGAATTTGGCTTGGCGATTTATCACCAGACCAGCCCATTTGCGGGCCTGTGGGCAATCGGCGACGCTCCGACGTACTGCCATGCGGGTGGTAATGGTCCGGACGATTTCGTTTGTCAGCAGACCGGCCCTACTACGATCGTGATCGTGGGTCAGAATTATCCGGGTGGCGCGGATAACCTCGAGCACCACCACCACCACCACTGA.

[0033] In some examples, the subject is a human or an animal, preferably a human.

[0034] In some examples, the product or composition comprises at least 1.5 mg / L of an anti-aging protein or a recombinant protein thereof.

[0035] In some preferred examples, the product or composition can comprise 1.5-30 mg / L of the anti-aging protein or the recombinant protein thereof, for example: 1.5 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, 20 mg / L, 21 mg / L, 22 mg / L, 23 mg / L, 24 mg / L, 25 mg / L, 26 mg / L, 27 mg / L, 28 mg / L, 29 mg / L, 30 mg / L.

[0036] In some examples, the anti-aging factor includes, but is not limited to, Collagen I, Collagen III, Collagen IV, Collagen VII, Decorin, VCAN, BGN, MMP-1, MMP-3, Smad3, Smad7, c-fos or c-jun, preferably Collagen I, Collagen VII, Decorin, VCAN, BGN, MMP-1, Smad7 or c-fos.

[0037] In some examples, the inflammation factor includes, but is not limited to, TNF-a, IL-1b, IL-6, IL-1a or IL-8, preferably TNF-a and / or IL-1b.

[0038] In some examples, the wrinkle includes, but is not limited to, crow's feet, forehead lines, glabellar lines, under-eye lines, marionette lines, perioral lines, chevron lines or neck lines, preferably under-eye lines.

[0039] In some examples, the wrinkle-related parameter includes, but is not limited to, the number of wrinkles, the perimeter of wrinkles, the area of wrinkles, the roughness of wrinkles, the average depth of wrinkles or the proportion of wrinkle area, preferably the number of wrinkles, the perimeter of wrinkles, the area of wrinkles and the roughness of wrinkles.

[0040] In another aspect, the present application provides use of an anti-aging protein or a recombinant protein thereof in the preparation of a cosmetic product, wherein the amino acid sequence of the anti-aging protein is shown as SEQ ID NO: 1,

[0041] The nucleotide sequence encoding the anti-aging protein is shown as SEQ ID NO: 2.

[0042] In some examples, the recombinant protein further comprises a tag protein that assists in its expression and / or purification, or an active protein that enhances its activity and / or prolongs its half-life.

[0043] In some examples, the tag protein can be a His tag, preferably a 6xHis tag.

[0044] In some preferred examples, the amino acid sequence of the recombinant protein is set forth in SEQ ID NO: 3.

[0045] In some preferred examples, the nucleotide encoding the recombinant protein is set forth in SEQ ID NO: 4.

[0046] In some examples, the cosmetic is used for:

[0047] 1) improving skin tightness of a subject; and / or

[0048] 2) improving skin elasticity value of a subject; and / or

[0049] 3) improving wrinkle-related parameters of a subject.

[0050] In some examples, the wrinkle includes, but is not limited to, crow’s feet, forehead lines, glabellar lines, under-eye lines, marionette lines, perioral lines, chevron lines, or neck lines, preferably under-eye lines.

[0051] In some examples, the wrinkle-related parameters include, but are not limited to, wrinkle number, wrinkle perimeter, wrinkle area, or wrinkle roughness, wrinkle average depth, or wrinkle area ratio, preferably wrinkle number, wrinkle perimeter, wrinkle area, and wrinkle roughness.

[0052] In some examples, the 3) includes:

[0053] (I) reducing wrinkle number; and / or

[0054] (II) reducing wrinkle perimeter; and / or

[0055] (III) reducing wrinkle area; and / or

[0056] (IV) reducing wrinkle roughness.

[0057] In some examples, the subject is a human.

[0058] In some examples, the cosmetic comprises at least 1.5 mg / L of the anti-aging protein or the recombinant protein thereof.

[0059] In some preferred examples, the cosmetic can comprise 1.5-30 mg / L of the anti-aging protein, for example: 1.5 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, 20 mg / L, 21 mg / L, 22 mg / L, 23 mg / L, 24 mg / L, 25 mg / L, 26 mg / L, 27 mg / L, 28 mg / L, 29 mg / L, 30 mg / L.

[0060] In yet another aspect, the present application provides a cosmetic comprising an effective amount of an anti-aging protein or a recombinant protein thereof, wherein the amino acid sequence of the anti-aging protein is set forth in SEQ ID NO: 1.

[0061] In some examples, the nucleotide sequence encoding the anti-aging protein is set forth in SEQ ID NO: 2.

[0062] In some examples, the recombinant protein further comprises a tag protein that facilitates its expression and / or purification, or an active protein that enhances its activity and / or prolongs its half-life.

[0063] In some examples, the tag protein can be a His tag, preferably a 6xHis tag.

[0064] In some preferred examples, the amino acid sequence of the recombinant protein is set forth in SEQ ID NO: 3.

[0065] In some preferred examples, the nucleotide encoding the recombinant protein is set forth in SEQ ID NO: 4.

[0066] In some examples, the cosmetic further comprises a cosmetically acceptable carrier or excipient, which includes, but is not limited to, at least one of a surfactant, an oil, an emulsifier, a high-fat ingredient, a consistency agent, a thickening agent, a polymer, a silicon-containing compound, a fat, a wax, a stabilizer, a biologically active ingredient, a deodorant, a film-forming agent, an expanding agent, an antioxidant, an inorganic coloring pigment, a water-miscible solvent, a preservative, a solubilizer, a right-precise and a colorant.

[0067] In some embodiments, the cosmetically acceptable carrier or excipient is selected from, but not limited to, one or more of deionized water, trehalose, niacinamide, betaine, glycerin, disodium EDTA, 1,2-hexanediol, xanthan gum, pentaerythrityl tetraethylhexanoate, dimethicone, glyceryl stearate PEG-100, stearate PEG-100, diisopropyl sebacate, myristyl myristate, cetyl alcohol ethylhexanoate, phytosteryl, behenyl alcohol, octyldodecyl lauramide, tocopheryl acetate, jojoba esters, tocopherol (vitamin E), sodium stearoyl glutamate, sodium polyacrylate, butylene glycol, p-hydroxyacetophenone, denatonium benzoate, hyaluronic acid, glucosyl hesperidin, and alginyl stearoxyl polyether-20.

[0068] In some examples, the dosage form of the cosmetic includes, but is not limited to, an aqueous or oily solution, an aqueous cream, an aqueous or oily gel, a mask, or a water-free product, etc.

[0069] In some preferred examples, the dosage form of the cosmetic includes, but is not limited to, a body wash, a shampoo, an emulsion, a cream, a microemulsion, a nanoemulsion, a mask, a serum, a lotion, an ointment, a foam, a patch, a cosmetic powder, a cosmetic cotton, an eye serum, an eye mask, an eye shadow, a sunscreen, an eye gel, or an eye cream.

[0070] In some examples, the cosmetic comprises at least 1.5 mg / L of the anti-aging protein or the recombinant protein thereof.

[0071] In some preferred examples, the cosmetic can comprise 1.5-30 mg / L of the anti-aging protein, for example: 1.5 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, 8 mg / L, 9 mg / L, 10 mg / L, 11 mg / L, 12 mg / L, 13 mg / L, 14 mg / L, 15 mg / L, 16 mg / L, 17 mg / L, 18 mg / L, 19 mg / L, 20 mg / L, 21 mg / L, 22 mg / L, 23 mg / L, 24 mg / L, 25 mg / L, 26 mg / L, 27 mg / L, 28 mg / L, 29 mg / L, 30 mg / L.

[0072] In yet another aspect, the present application provides a method of care for non-therapeutic purposes, the method of care comprising:

[0073] topically, preferably ocularly, administering to an external surface of a skin tissue of a subject an effective amount of any of the aforementioned products or compositions, or any of the aforementioned cosmetics.

[0074] In yet another aspect, the present application provides a method of preparing any of the aforementioned cosmetics, the method of preparation comprising the steps of:

[0075] mixing the cosmetic base with the anti-aging protein or the recombinant protein to obtain a cosmetic.

[0076] In some examples, the preparation method comprises the following steps:

[0077] I) Put the water phase materials into an emulsifying pot, mix and stir to heat to a first temperature to obtain a water phase;

[0078] II) Put the oil phase materials into an oil phase pot, mix and stir to heat to a second temperature, denoted as an oil phase;

[0079] III) Homogeneously emulsify the oil phase into the stirring water phase, stir to cool to a third temperature, add other materials, and mix uniformly to obtain a cosmetic base;

[0080] IV) Mix the cosmetic base with the anti-aging protein to obtain a cosmetic.

[0081] In some examples, the first temperature and the second temperature are 70-80°C.

[0082] In some examples, the homogenization emulsification in step III) is performed for 3-5 min.

[0083] In some examples, the third temperature is 40-45°C.

[0084] Compared with the prior art, the use of the anti-aging protein or the recombinant protein provided by the present application has at least the following beneficial effects:

[0085] (1) Better safety, without showing obvious cytotoxicity;

[0086] (2) Significantly reducing cell aging caused by photoaging, with a decrease of about 74% in the activity of β-galactosidase relative to the model group;

[0087] (3) Significantly prolonging the average lifespan of Caenorhabditis elegans, with a maximum increase of about 7 days in the average lifespan relative to the control group;

[0088] (4) Significantly down-regulating the expression of two inflammatory factor genes TNF-α and IL-1β, with a decrease of about 33% in the relative expression of TNF-α and a decrease of about 14% in the relative expression of IL-1β relative to the model group;

[0089] (5) Significantly improved the expression of 8 anti-wrinkle factors, including Collagen I, Collagen VII, Decorin, VCAN, BGN, MMP-1, Smad7 and c-fos; among them, Collagen I was up-regulated by 19.51%, Collagen VII was up-regulated by 31.88%, Decorin was up-regulated by 65.79%, VCAN was up-regulated by 21.05%, BGN was up-regulated by 64.29%, MMP-1 was down-regulated by 31.44%, Smad7 was down-regulated by 13.74%, and c-fos was down-regulated by 26.85%.

[0090] (6) Significantly improved skin tightness, skin elasticity, and under-eye wrinkles (number, perimeter, area, roughness); among them, after using the eye serum test sample containing 3mg / L HJ-P2 protein for 4 weeks, the skin tightness was improved by about 13%, the skin elasticity value was increased by about 8%, the number of wrinkles was reduced by about 11%, the perimeter of wrinkles was reduced by about 19%, the area of wrinkles was reduced by about 15%, and the roughness of wrinkles was reduced by about 5%. BRIEF DESCRIPTION OF DRAWINGS

[0091] Figure 1 It is the protein SDS-PAGE electrophoretogram in the purification process of HJ-P2 protein.

[0092] Figure 2 It is the cell morphology chart of HJ-P2 protein cytotoxicity test.

[0093] Figure 3 It is the cell viability curve chart of HJ-P2 protein cytotoxicity test.

[0094] Figure 4 The effect of HJ-P2 protein on the β-galactosidase activity of cells is shown.

[0095] Figure 5 It is the survival analysis chart of Caenorhabditis elegans administered with different doses of HJ-P2 protein.

[0096] Figure 6 It is the survival cycle chart of Caenorhabditis elegans administered with different doses of HJ-P2 protein.

[0097] Figure 7 The effect of HJ-P2 protein on the relative expression of inflammatory factors is shown.

[0098] Figure 8 The experimental results of the in vitro anti-wrinkle efficacy test of HJ-P2 protein are shown.

[0099] Figure 9 The improvement of HJ-P2 protein on different anti-wrinkle factors is shown.

[0100] Figure 10 The change in the mean value of skin tightness F4 of each group (test group and control group) at different time points is shown (N=14).

[0101] Figure 11 The improvement in the mean value of skin tightness F4 of different groups (test group and control group) at different time points is shown (N=14).

[0102] Figure 12 The change in the mean value of skin elasticity Q1 of each group (test group and control group) at different time points is shown (N=14).

[0103] Figure 13 The change in the mean value of skin elasticity Q1 of different groups (test group and control group) at different time points is shown (N=14).

[0104] Figure 14 The change in the mean value of the number of under-eye wrinkles of each group (test group and control group) at different time points is shown (N=14).

[0105] Figure 15 The change in the mean value of the perimeter of under-eye wrinkles of each group (test group and control group) at different time points is shown (N=14).

[0106] Figure 16 The change in the mean value of the area of under-eye wrinkles of each group (test group and control group) at different time points is shown (N=14).

[0107] Figure 17 The change in the mean value of the roughness of under-eye wrinkles of each group (test group and control group) at different time points is shown (N=14). DETAILED DESCRIPTION

[0108] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied, for the purposes of interpreting this description, and apply uniformly throughout this description, unless an otherwise expressly set out definition applies. As used in interpreting this disclosure, the following terms shall have the following directives, except to the extent otherwise indicated herein.

[0109] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0110] As used herein, the term "about" means a range of ±20% of the value that follows. In some embodiments, the term "about" means a range of ±10% of the value that follows. In some embodiments, the term "about" means a range of ±5% of the value that follows.

[0111] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. The specific conditions not mentioned in the embodiments are carried out according to the conventional conditions or the conditions suggested by the manufacturers. All reagents or instruments not mentioned by the manufacturers are the conventional products that can be purchased in the market. In order to better illustrate the present application, numerous specific details are given in the following specific embodiments. The specific embodiments described herein are only used to explain the present application and do not constitute any limitation on the present application. In addition, in the following description, the description of the well-known structures and techniques is omitted to avoid unnecessary confusion of the concept of the present application. Such structures and techniques are also described in many publications, such as Molecular Cloning: A Laboratory Manual (Fourth Edition) (Cold Spring Harbor Laboratory Press), Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience.

[0112] Example 1 Preparation of HJ-P2 protein

[0113] The inventors creatively isolated an HJ-P2 protein from Epicoccum nigrum, and the amino acid sequence of the HJ-P2 protein is shown as SEQ ID NO: 1. In order to study the biological function thereof in the subsequent research, the inventors linked the nucleotide sequence of HJ-P2 to the pET-21a expression vector (enzyme cutting sites are Nde I and Xho I), obtained a recombinant plasmid HJ-P2-pET-21a, and the C-terminal of which is fused with the 6xHis tag in the vector. The plasmid was artificially synthesized.

[0114] 1.1 Induced expression of HJ-P2 protein

[0115] The synthesized HJ-P2 plasmid was dissolved with sterile water, respectively, and then transformed into E. coli OverExpress C43 (DE3). The recombinant E. coli was cultured in solid LB medium in a constant temperature incubator at 37°C overnight. 12 mL of sterile shaking tubes were added with 5 mL of sterile LB medium (containing 100 μg / mL of ampicillin), and the grown C43 single colony was inoculated into the medium with a sterile pipette gun head, and placed in a constant temperature shaker overnight. The culture conditions were 37°C, 200 rpm, and 15 h. The bacterial solution after culture could be preserved with 17.5% glycerol and placed in a -80°C refrigerator, or subjected to the next step of large-scale culture.

[0116] Add 75 mL of liquid LB medium (pre-sterilized by autoclaving and cooled to room temperature) to a 250 mL Erlenmeyer flask, add ampicillin to a final concentration of 100 μg / mL, inoculate 1.5 mL of the above-mentioned proliferating bacterial culture, and incubate in a constant temperature shaker at 37°C and 220 rpm for approximately 2.5 h until the OD value is reached. 600 After reaching approximately 0.7, add the inducer IPTG to a final concentration of 0.2 mmol / L, and incubate overnight at 25°C for 15 h.

[0117] 1.2 Isolation and purification of HJ-P2 protein

[0118] The buffer solution used for protein separation and purification in this embodiment is shown below:

[0119] Disruption buffer: 50 mmol / L Tris-HCl, pH 7.8;

[0120] Binding buffer: 50 mmol / L Tris-HCl, 300 mmol / L NaCl, 10 mmol / L Imidazole, pH 7.8;

[0121] Elution buffer: 50 mmol / L Tris-HCl, 300 mmol / L NaCl, 500 mmol / L Imidazole, pH 7.8;

[0122] Desalting buffer: 50 mmol / L Tris-HCl, pH 7.8.

[0123] Add 1 mL of disruption buffer to every 2 mL of fermentation broth, then add disruption buffer to the collected bacterial cells and resuspend the cells. Disrupt the cells using an ultrasonic cell disruptor until the liquid is homogeneous and transparent. Centrifuge the disrupted bacterial solution at 4°C and 7000 rpm for 30 min, collect the supernatant (i.e., the "crude" sample), filter it through a 0.45 μm aqueous filter membrane, and store it on ice for later use.

[0124] Affinity chromatography: Proteins were purified using an AKTA protein purifier and a nickel ion affinity chromatography column. The procedure is as follows:

[0125] • Place both pump A and pump B in pure water for pump rinsing; after rinsing, set 50% B and flow rate to 1 mL / min, and install the nickel column; set the flow rate to 5 mL / min and rinse the system until the baseline is stable.

[0126] Replace pump A with binding buffer and pump B with elution buffer, and perform pump flushing; after flushing, set to 100% B and flush the system until the baseline is stable.

[0127] • Set to 100% A, flush the system until the baseline is stable, then zero it.

[0128] • Put A pump into sample, set flow rate 2 mL / min, start loading, until about 0.5 mL sample left at the bottom, collect the flow-through, which is the "through" sample.

[0129] • Rinse A pump head with pure water, put A pump back into binding buffer, set flow rate 3 mL / min, rinse the impurities to baseline stable.

[0130] • Set flow rate 5 mL / min, set linear elution condition: 0% to 50% B, 60 mL, and collect the elution peak sample (i.e. "Ni pure" sample) for subsequent desalting purification.

[0131] • Set 100% B, rinse the system to baseline stable.

[0132] • Put both A and B pumps into pure water, perform pump rinse, after rinse, set 50% B, rinse the system to baseline stable.

[0133] • Put both A and B pumps into 20% ethanol, rinse for at least 2 column volumes, remove the nickel column and shut down the system.

[0134] Desalting purification: use AKTA purifier and desalting column for desalting, the operation procedure is as follows:

[0135] • Put A pump into pure water, perform pump rinse; after rinse, set 100% A, flow rate 1 mL / min, install the desalting column, set flow rate 2 mL / min, rinse the system for more than 1 column volume.

[0136] • Change A pump into desalting buffer, perform pump rinse, after rinse, set flow rate 3 mL / min, rinse the system to baseline stable, then zero.

[0137] • Put A pump into sample, start loading, until about 0.5 mL sample left at the bottom.

[0138] • Rinse A pump head with pure water, put A pump back into desalting buffer, collect the elution peak protein (i.e. "desalted" sample) according to UV-280. The loading volume should not exceed 1 / 3 of the column volume, otherwise the protein elution peak (UV-280) might overlap with the salt ion elution peak (Conductivity);

[0139] • Put A pump into pure water, perform pump rinse, rinse the system to baseline stable.

[0140] • Put both A and B pumps into 20% ethanol, perform pump rinse; rinse the system for more than 1 column volume, remove the desalting column and shut down the system.

[0141] Ultrafiltration concentration: the desalted protein solution was ultrafiltrated and concentrated by 10 kDa ultrafiltration tube, the ultrafiltration condition was set as: 4℃, 3000xg, and the protein concentration was above 1 g / L after ultrafiltration (i.e. "concentrated" sample). The samples of each step of the above purification were taken for SDS-PAGE protein electrophoresis analysis, and the electrophoresis results are shown in Figure 1 Figure 2, the molecular weight of HJ-P2 protein is consistent with the expected value of 17.9 kDa.

[0142] Protein concentration determination: the protein concentration standard curve was established according to the instruction of the BCA protein quantitative kit, and the concentration of the target protein after ultrafiltration and concentration was determined. According to the BCA protein concentration standard curve, the measured OD 562 of the sample was substituted into the standard curve equation, and the concentration of the HJ-P2 pure protein was calculated to be 2.45 g / L. Figure 1

[0143] Example 2: Cytotoxicity test

[0144] • Cell inoculation: fibroblasts were inoculated into a 96-well plate at a seeding density of 8x10 3 cells / well, and incubated in an incubator (37℃, 5% CO2) overnight.

[0145] • Test grouping: the test was set up with a zero setting group, a solvent control group, a positive control group and a sample group. In the sample group, 8 concentration gradients were set for each sample, and 3 repeated wells were set for each concentration gradient.

[0146] • Liquid preparation: different concentrations of sample working solution were prepared according to the following gradient: 0.0781%, 0.1563%, 0.3125%, 0.625%, 1.25%, 2.5%, 5%, 10% (v / v). The concentration of 100% HJ-P2 protein stock solution in this test was 300 mg / L.

[0147] • Drug administration: when the plating rate of cells in the 96-well plate reached 40%-60%, drug administration was performed. The solvent control group was added with 200 μL of culture medium per well; the positive control group was added with 200 μL of culture medium containing 10% DMSO per well; the sample group was added with 200 μL of culture medium containing the corresponding concentration of sample per well; the zero setting group was not inoculated with cells, and only 200 μL of cell culture medium was added. After drug administration, the 96-well plate was placed in an incubator (37℃, 5% CO2) for 24 h.

[0148] • Detection: after 24 h of rich culture of cells, the supernatant was discarded, MTT working solution (0.5 mg / mL) was added, and incubation was performed at 37℃ in the dark for 4 h. After incubation, the supernatant was discarded, 150 μL of DMSO was added per well, and the OD value was read at 490 nm.

[0149] ​• Cell relative viability calculation: According to the formula, cell relative viability (%) = (sample well OD - zero well OD) / (solvent control well OD - zero well OD) x 100%.

[0150] MTT and morphological results showed that sample HJ-P2 protein did not exhibit significant cytotoxicity to fibroblasts at a concentration range of 10% (v / v), and was safe Figures 2-3 and Table 1).

[0151] Table 1 MTT detection results of sample HJ-P2 protein

[0152]

[0153] Example 3 Effect of HJ-P2 protein on β-galactosidase activity of cells

[0154] Studies have shown that when cells age, β-galactosidase (β-GAL) in cells is overexpressed, so the activity and expression of β-galactosidase can be used as an indicator for detecting cell aging. β-galactosidase can decompose p-nitrophenyl-β-D-galactopyranoside to generate p-nitrophenol, which has a maximum absorption peak at 400 nm, so the absorbance value generated by detecting p-nitrophenol can reflect the activity of β-galactosidase, thereby characterizing the aging of cells.

[0155] • Preparation of cells: NHDF cells (normal human fibroblasts) in good growth state were prepared into a cell suspension, 500 μL of the diluted cell suspension was added to each well of a 24-well plate, so that the concentration was 5.0 x 10 4 cells / well.

[0156] • Put into a CO2 incubator for 16 h.

[0157] • Add drugs and incubate for 24 h.

[0158] • Select UVA ultraviolet intensity (5 J / cm 2 ) to irradiate the cells.

[0159] • Continue to culture for 24 h.

[0160] • Cell collection: collect the cells in the 24-well plate into two groups of centrifuge tubes and centrifuge at 10,000 rpm for 5 min.

[0161] • Protein quantification detection: discard the excess culture medium in one group of centrifuge tubes, add 100 μL of cell lysis solution, mix thoroughly, and perform protein quantification detection according to the BCA protein quantification kit.

[0162] Cell disruption: Take the remaining centrifuge tube group and discard the excess culture medium, add 125 μL extraction solution to each tube, low-temperature ultrasonic cell disruption, and centrifuge the disrupted cells at 15000 x g, 4°C, 10 min, and take the supernatant for testing.

[0163] Dilution of standard solution: Dilute the standard solution (5 μnmol / mL p-nitrophenol solution) to 0, 6.25, 12.5, 25, 50, 100, and 200 nmol / mL according to the β-galactosidase activity kit instructions.

[0164] Determination of samples (add reagents in sequence according to Table 2 in a 96-well plate):

[0165] Table 2 Amount of each reagent added in this example

[0166]

[0167] After preparation according to Table 2, mix thoroughly, and measure the absorbance value A at 400 nm, and record A determination tube, A control tube, A standard tube, and A blank tube, respectively. Calculate ΔA determination = A determination tube - A control tube, and ΔA standard = A standard tube - A blank tube.

[0168] Calculation of β-galactosidase activity:

[0169] 1) Establishment of standard curve:

[0170] Establish a standard curve according to the absorbance value (x, ΔA standard) and concentration (y, nmol / ml) of the standard tube, and substitute ΔA (x, ΔA determination) into the standard curve to calculate the amount of product generated by the sample (y, nmol / ml).

[0171] 2) Calculation of β-GAL activity:

[0172] β-GAL activity (U / mg prot) = (y x V total) ÷ (V sample x Cpr) ÷ T

[0173] Wherein: Cpr: sample protein concentration (mg / ml); V total: total volume of the reaction system; V sample: sample volume added to the reaction system; T: reaction time. (1 nmol of p-nitrophenol produced per mg of protein per hour is defined as one enzyme activity unit.)

[0174] The results of repeated verification are as follows: Figure 4As shown, the β-galactosidase activity in the NHDF cells irradiated by UVA was significantly higher than that of the blank group without UVA irradiation, and it was considered that the experimental model was established. After treatment with 5 mg / L of the HJ-P2 sample, the β-galactosidase activity of the NHDF cells was significantly reduced, and the β-galactosidase activity was reduced by about 74% relative to the model group. Therefore, the HJ-P2 sample at the detection concentration can reduce cell senescence caused by photoaging.

[0175] Example 4 Effect of HJ-P2 protein on the lifespan length of Caenorhabditis elegans

[0176] 4.1 Synchronization of nematodes (hypochlorite lysis method)

[0177] · Collect nematodes:

[0178] Take the nematode plate, observe the nematodes under a microscope, and most of the nematodes are in the egg-laying stage, so the synchronization experiment can be performed. Suck SM buffer into the nematode plate, suck and wash the nematodes on the plate, collect about nematode liquid into a centrifuge tube, centrifuge at 3500 rpm for 1 min, discard the supernatant, and resuspend with SM buffer. This step is repeated 3 times.

[0179] · Lysis of nematodes:

[0180] Prepare a nematode lysis solution with 5M NaOH and 5% NaClO, with a ratio of 5M NaOH:5% NaClO=1:2, and use it immediately. Mix the nematode suspension and the nematode lysis solution in a volume ratio of 2:1, shake continuously, and observe the lysis state of the nematodes in the tube until no complete nematodes are visible to the naked eye. The process does not exceed 5 min. Immediately centrifuge at 3500 rpm for 2 min, and carefully discard the supernatant. Gently resuspend the eggs with SM buffer and wash repeatedly for more than 3 times.

[0181] · Starvation culture:

[0182] Resuspend the nematode eggs washed with the lysis solution with SM buffer, then transfer them to a centrifuge tube, add an appropriate amount of SM buffer, and place them in a 20°C shaking incubator for 24 h.

[0183] · Transfer:

[0184] Centrifuge at 3500 rpm for 2 min to collect the synchronized nematodes, and transfer the collected nematodes to a 90mm NGM culture plate, and incubate at 20°C for two days to the L4 stage for subsequent experiments.

[0185] 4.2 Nematode lifespan experiment

[0186] · Preparation of NGM culture medium plate for lifespan experiment

[0187] NGM solid medium was prepared, sterilized and cooled to 50℃, 5-Fu was added and mixed evenly, 4 mL medium was taken with a pipette gun and placed in a plate with a diameter of 35 mm, the medium was evenly distributed in the whole plate, about 50 plates were poured, the excess plates were sealed and stored at 4℃ for standby.

[0188] HJ-P2 protein aqueous solution with concentrations of 3 mg / L, 1.5 mg / L and 0.75 mg / L was prepared respectively, 400 μL was added to the prepared NGM plate, the positive control was chlorogenic acid, and the blank control was sterile water. After the sample working solution was immersed in the NGM plate and dried, 80 μL of inactivated OP50 bacterial suspension was added to the NGM plate and dried.

[0189] · picking worms

[0190] Two repeated plates were set up, and 20 nematodes in the L4 late stage after synchronization were picked from each working plate and placed in a 20℃ incubator.

[0191] · Nematode plate changing and life span observation

[0192] Observation and recording were started from the second day of plating, and the head or tail of the nematode was gently touched with an inoculation needle every other day. If it did not respond, it was recorded as dead. The dead nematodes were picked out and recorded, and the data was counted until all the nematodes died. The life span curve graph was made. During the life span experiment, the plates were changed according to the actual situation, and the plates were replaced in time if contamination occurred.

[0193] · Test results

[0194] In this experiment, chlorogenic acid was used as the positive control, and a blank control was added. The test concentrations of HJ-P2 protein samples were 3 mg / L, 1.5 mg / L and 0.75 mg / L, respectively, with a total of 5 treatment groups.

[0195] The experimental results showed that 3 mg / L HJ-P2 could extend the average life span of nematodes by about 7 days, 1.5 mg / L HJ-P2 could extend the average life span of nematodes by about 3 days, and 0.75 mg / L HJ-P2 had no significant extension ability on the life span of nematodes. This indicates that 3 mg / L and 1.5 mg / L HJ-P2 can significantly extend the life span of nematodes, and have significant anti-aging effect Figure 5 and Figure 6 ).

[0196] Example 5 Influence of HJ-P2 protein on the relative expression amount of inflammatory factors

[0197] · Plating: THP-1 was inoculated in a 24-well plate, 5 × 10 5 cells were inoculated in each well.

[0198] • Sample preparation: Determine the final test concentration of each sample based on the maximum safe concentration of the sample measured in the preliminary experiment; pre-dilute the sample with an appropriate solvent so that the concentration of the sample dilution is 100 times the final test concentration.

[0199] • Sample addition: Add 10 μL of solvent to the negative control group (NC) and the model group (M); add 10 μL of the corresponding sample diluent to the corresponding well of each sample group (Sn), and add 10 μL of 1 mM dexamethasone stock solution (final concentration 10 μM) to the corresponding well of the positive control group (PC); incubate in an incubator for 24 h.

[0200] • LPS treatment: Add 10 μL of 100 μg / mL LPS (final concentration 1 μg / mL) to the wells of the model group (M), positive control group (PC), and each sample group (Sn), respectively, and add 10 μL of culture medium to the wells of the negative control group (NC); incubate in an incubator for 24 h.

[0201] • RNA extraction and reverse transcription: Cells from each group were collected, and total RNA was extracted from each group using the RaPure Total RNA Kit. The total RNA from each group was reverse transcribed into cDNA using the HiScript IIQ RT SuperMix for qPCR (+gDNA wiper).

[0202] qPCR testing

[0203] The relative expression levels of TNF-α and IL-1β were detected by qPCR using cDNA templates from each group (with the GAPDH gene as an internal reference gene).

[0204] In this embodiment, three independent replicate experiments were conducted, and the mean (Mean) and standard deviation (SD) of the relative expression levels of each group were calculated. The results are expressed as Mean ± SD. The comparisons between groups were performed using t-test statistical analysis (two-tailed). P < 0.05 was considered statistically significant, and P < 0.01 was considered extremely significant.

[0205] The test results showed that the relative expression levels of all inflammatory factors in the negative control group were significantly lower than those in the model group (LPS treatment), indicating that the inflammatory cell model was established. The relative expression levels of all inflammatory factors in the positive control group (dexamethasone treatment) were significantly lower than those in the model group, indicating that the positive control group was established. In summary, the quality control indicators of this experiment met the requirements, and the detection system was established.

[0206] Repeated validation showed that the relative expression levels of TNF-α and IL-1β, two inflammatory factors, in the 5 mg / L HJ-P2 sample group were lower than those in the model group. This indicates that 5 mg / L HJ-P2 can inhibit the LPS-induced upregulation of TNF-α and IL-1β expression.Figure 7 ). Wherein the relative expression of TNF-a was down-regulated by about 33% relative to the model group after administration of 5 mg / L HJ-P2. The relative expression of IL-1 β was down-regulated by about 14% relative to the model group.

[0207] Example 6 Effect of HJ-P2 protein on anti-wrinkle factors

[0208] In this example, after UVA irradiation of fibroblasts, the anti-wrinkle efficacy of the test sample was evaluated by detecting the changes in the expression of 13 anti-wrinkle factor genes. The mechanism of action of the 13 anti-wrinkle factors is shown in Table 3.

[0209] Table 3 Mechanism of action of anti-wrinkle factors

[0210]

[0211]

[0212] Test method

[0213] • Inoculation: inoculate cells into a 6-well plate at an inoculation density of 2 x 10 5 cells / well, and incubate in an incubator (37°C, 5% CO2) overnight.

[0214] • Solution preparation: prepare the test sample working solution according to the test grouping of Table 4.

[0215] • Dosing: according to the test grouping of Table 3, when the plating rate of cells in the 6-well plate reaches 40-60%, dosing is performed according to the grouping, with a dosing amount of 2 mL per well, and 3 replicate wells per group. Incubate in an incubator (37°C, 5% CO2) for 24 h.

[0216] • UVA irradiation: according to the experimental grouping, perform UVA irradiation at 30 J / cm 2 for the groups with UVA irradiation, and continue to incubate in an incubator (37°C, 5% CO2) for 24 h.

[0217] • Cell collection: after the end of incubation, discard the old liquid, wash twice with PBS, add 1 mL of RNAiso Plus per well, blow and lyse the cells, and then collect the sample.

[0218] • Gene expression detection: after extraction of RNA and reverse transcription to cDNA, perform fluorescence quantitative PCR detection, and calculate the results using the 2-△△CT method.

[0219] • Statistical analysis of results: use GraphPad Prism for plotting, and the results are expressed as Mean ± SD. Use t-test for statistical analysis between groups. Statistical analysis is two-tailed. P < 0.05 is considered to be significantly different, and P < 0.01 is considered to be extremely significantly different.

[0220] Table 4 Test Grouping for this Example

[0221]

[0222] The results show ( Figure 8 and Figure 9 Based on fibroblasts, compared with the control group, the HJ-P2 protein sample at a concentration of 3 mg / L showed significantly upregulated expression levels of Collagen I, Collagen VII, Decorin, VCAN, and BGN genes, and significantly downregulated expression levels of MMP-1, Smad7, and c-fos genes. Specifically, Collagen I was upregulated by 19.51%, Collagen VII by 31.88%, Decorin by 65.79%, VCAN by 21.05%, and BGN by 64.29%, while MMP-1 was downregulated by 31.44%, Smad7 by 13.74%, and c-fos by 26.85%. Based on the above, the sample at a concentration of 3 mg / L can achieve anti-wrinkle effects by upregulating the expression levels of Collagen I, Collagen VII, Decorin, VCAN, and BGN genes and downregulating the expression levels of MMP-1, Smad7, and c-fos genes.

[0223] Example 7: Application of HJ-P2 protein in the preparation of eye serum

[0224] Materials used in this embodiment:

[0225] Aqueous phase materials: deionized water, trehalose, nicotinamide, betaine, glycerol, disodium EDTA, 1,2-hexanediol, xanthan gum;

[0226] Oil phase materials: Pentaerythritol tetra(ethylhexanoate), polydimethylsiloxane, glyceryl stearate PEG-100, stearate PEG-100, diisopropyl sebacate, myristyl myristate, cetyl ethylhexanoate, phytosterols, behenyl alcohol, octyl dodecyl lauroyl glutamate, tocopheryl acetate, jojoba esters, tocopherol (vitamin E), sodium stearoyl glutamate, sodium polyacrylate;

[0227] Other materials: Butylene glycol, p-hydroxyacetophenone, benzyl denatum, hyaluronic acid, glucosyl hesperidin, and fucus stearyl alcohol polyether-20.

[0228] Preparation method

[0229] (1) Weigh out the aqueous phase material according to the ratio and add it to the emulsification pot. Mix and heat to 70-80℃, and record it as the aqueous phase.

[0230] (2) According to the proportion, the oil phase materials are weighed and added into the oil phase pot for stirring and heating to 70-80℃, which is recorded as the oil phase;

[0231] (3) The oil phase is added into the stirring water phase for homogenization and emulsification for 3-5 minutes, and the stirring is cooled to 40-45℃, and other materials weighed according to the proportion are added, and stirred uniformly, which is the "eye essence base";

[0232] (4) Half of the "eye essence base" is taken and added into HJ-P2 protein with a final concentration of 3 mg / L, and stirred uniformly, which is the "eye essence test sample".

[0233] Example 8 Safety evaluation of eye essence

[0234] ·Experimental technical basis information

[0235] Test product: "eye essence base" and "eye essence test sample" prepared in Example 6

[0236] Number of subjects: 30 people

[0237] Age: 23-45 years old

[0238] Gender: 28 women and 2 men

[0239] Health status: The subjects have healthy skin, no history of skin allergy, and meet the voluntary selection criteria for subjects.

[0240] ·Experimental method

[0241] A qualified patch tester is selected, and the test substance about 0.020 mL-0.025 mL is dropped into the patch tester in a closed patch test, and a special adhesive tape is applied to the inner side of the arm of the subject, and the skin reaction at 0.5, 24 and 48 h is observed, and the results are recorded according to the skin reaction grading standard in the "Cosmetic Hygiene Standard".

[0242] ·Experimental results

[0243] The results of human skin patch test show that 30 subjects pass the patch test of "eye essence base" and "eye essence test sample" described in the application, and the skin reaction is observed at 0.5, 24 and 48 h, among which 0 cases have skin adverse reactions, which indicates that the "eye essence base" and "eye essence test sample" described in the application are safe for use.

[0244] Example 9 Anti-aging effect test of eye essence

[0245] Select the periocular region of Asian adult subjects, and randomly allocate the left and right periocular regions to use two different test samples. By measuring the changes in the values of tightness (F4), elasticity (Q1), and under-eye wrinkle parameters (number, perimeter, area, roughness Rz) of the periocular region of the subjects before using the sample (W0), 1 week after using the sample (W1), 2 weeks after using the sample (W2), and 4 weeks after using the sample (W4), the efficacy of the sample in tightening, enhancing elasticity, and anti-wrinkling is evaluated.

[0246] 9.1 Test basic information

[0247] · Study subjects: 14 healthy adult women

[0248] · Age: 18-60 years old

[0249] · Inclusion criteria:

[0250] Periorbital wrinkles, under-eye wrinkles grade 2-6 (wrinkle grade evaluation reference: Skin Aging Atlas (Asian people));

[0251] Periorbital skin elasticity Q1 baseline value 0.35-0.55;

[0252] Periorbital skin tightness F4 baseline value >5

[0253] · Sample information:

[0254] Test group: "Eye Serum Test Sample"

[0255] Control group: "Eye Serum Base"

[0256] · Usage method: After cleansing in the morning and evening, take an appropriate amount of sample on one side of the eye, evenly apply it to the periocular skin, and gently massage until absorbed

[0257] · Main instruments: Skin elasticity tester (MPA580, Germany), skin rapid optical imaging system (VISIO 4D, France)

[0258] 9.2 Test procedure

[0259] ① W0 first visit: The technician explains and signs the informed consent form to the subject, and completes the preliminary screening;

[0260] ② On the day of the test, the subject does not apply any material to the face, and after cleansing with water, sits and rests in a constant temperature and humidity room for 30 minutes; After 30 minutes, the technician measures the subject: skin rapid optical imaging system takes pictures, and skin elasticity tester measures;

[0261] ③ After the W0 measurement of the included subjects, the technician distributes the sample to the subject and explains the sample usage method, and the subject tries it on site once;

[0262] ④W1, W2, W4 follow-up tests: The testing process is the same as steps ②-③;

[0263] ⑤ Technicians at each follow-up testing site must confirm the sample collection, weighing, and usage.

[0264] ⑥ After the test is completed, organize the data and issue a report.

[0265] 9.3 Test Items

[0266] • Firmness F4: The F4 value is measured three times in parallel on the test area using a skin elasticity tester, and the average value is taken. The smaller the value, the firmer the skin.

[0267] • Elasticity value Q1: The Q1 value is measured three times in parallel on the test area using a skin elasticity tester, and the average value is taken. A value closer to 1 indicates better skin elasticity.

[0268] • Wrinkle parameters:

[0269] Number of wrinkles: The smaller this value, the fewer wrinkles there are.

[0270] Wrinkle circumference: The smaller this value, the fewer wrinkles there are.

[0271] Wrinkle area: The smaller this value, the fewer wrinkles there are.

[0272] Wrinkle roughness Rz: The smaller this value, the fewer wrinkles there are.

[0273] The results showed that compared with before the use of the sample, the experimental group showed significant improvements in skin firmness (F4), skin elasticity (Q1), and under-eye wrinkles (number, circumference, area, and roughness); compared with the control group, the experimental group also showed significant improvements in skin firmness (F4), skin elasticity (Q1), and under-eye wrinkles (number, circumference, area, and roughness). Compared with before use, after 4 weeks of using the eye serum test sample, the subjects showed approximately 13% improvement in skin firmness, approximately 8% increase in skin elasticity, approximately 11% reduction in the number of wrinkles, approximately 19% reduction in wrinkle circumference, approximately 15% reduction in wrinkle area, and approximately 5% reduction in wrinkle roughness. This indicates that the "eye serum test sample" containing HJ-P2 protein has the effect of firming the skin, improving skin elasticity, and reducing under-eye wrinkles. Figures 10-17 ).

[0274] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Use of an anti-aging protein or a recombinant protein thereof in the preparation of at least one of the following products or compositions: (1) Products or compositions that improve the expression level of anti-wrinkle factor genes in cells; (2) Products or compositions that improve the expression levels of cellular inflammatory factor genes; (3) Products or compositions that improve the skin firmness of the subjects; (4) Products or compositions that improve the skin elasticity of the subject; (5) Products or compositions that improve wrinkle-related parameters in subjects; (6) Products or compositions that delay the natural aging of the skin; (7) Products or compositions that reduce cell aging caused by photoaging; The amino acid sequence of the anti-aging protein is shown in SEQ ID NO: 1, and the nucleotide sequence encoding the anti-aging protein is shown in SEQ ID NO: 2; The amino acid sequence of the recombinant protein is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the recombinant protein is shown in SEQ ID NO:

4.

2. The use according to claim 1, characterized in that, The product or composition contains at least 1.5 mg / L of an anti-aging protein or its recombinant protein.

3. The use according to claim 1, characterized in that, The anti-wrinkle factor includes Collagen I, Collagen Ⅲ, Collagen Ⅳ, Collagen VII, Decorin, VCAN, BGN, MMP-1, MMP-3, Smad3, Smad7, c-fos or c-jun ; and / or The inflammatory factors include TNF-α, IL-1β, IL-6, IL-1α or IL-8 ; and / or The wrinkles include crow's feet, forehead wrinkles, frown lines, under-eye wrinkles, nasolabial folds, perioral wrinkles, frown lines, or neck wrinkles; and / or The wrinkle-related parameters include the number of wrinkles, wrinkle perimeter, wrinkle area, wrinkle roughness, average wrinkle depth, or wrinkle area percentage.

4. The use of an anti-aging protein or its recombinant protein in the preparation of cosmetics, characterized in that, The amino acid sequence of the anti-aging protein is shown in SEQ ID NO: 1, and the nucleotide sequence encoding the anti-aging protein is shown in SEQ ID NO: 2; the amino acid sequence of the recombinant protein is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the recombinant protein is shown in SEQ ID NO:

4.

5. The use according to claim 4, characterized in that, The cosmetics are used for: 1) Improves skin firmness in subjects; and / or 2) Improves the subject's skin elasticity value; and / or 3) Improves wrinkle-related parameters in subjects; The subjects were humans.

6. The use according to claim 5, characterized in that, The wrinkle-related parameters include the number of wrinkles, wrinkle perimeter, wrinkle area, wrinkle roughness, average wrinkle depth, or wrinkle area percentage.

7. A cosmetic product, characterized in that, The cosmetic contains an anti-aging protein or a recombinant protein thereof, wherein the amino acid sequence of the anti-aging protein is shown in SEQ ID NO: 1, and the nucleotide sequence encoding the anti-aging protein is shown in SEQ ID NO: 2; the amino acid sequence of the recombinant protein is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the recombinant protein is shown in SEQ ID NO:

4.

8. The cosmetic product according to claim 7, characterized in that, The cosmetic contains at least 1.5 mg / L of an anti-aging protein or its recombinant protein.

9. The cosmetic product according to claim 7, characterized in that, The cosmetic also includes cosmetically acceptable carriers or excipients selected from at least one of surfactants, oils, high-fat ingredients, thickeners, polymers, silicone compounds, fats, waxes, stabilizers, bioactive ingredients, deodorants, film-forming agents, swelling agents, antioxidants, water-based cosolvents, preservatives, solubilizers, and colorants.

10. The cosmetic product according to claim 7, characterized in that, The dosage form of the cosmetic is selected from at least one of the following: shower gel, shampoo, lotion, facial mask, serum, wash, ointment, foaming agent, patch, cosmetic powder, cotton pad, eye serum, eye mask, eyeshadow, sunscreen, eye gel, and eye cream.

11. A method for preparing a cosmetic according to any one of claims 7-10, characterized in that, The preparation method includes the following steps: The cosmetic base is mixed and stirred evenly with anti-aging protein or its recombinant protein to obtain the cosmetic.

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