CN112675063A

CN112675063A - Skin-tendering anti-wrinkle composition containing PDRN and application thereof

Info

Publication number: CN112675063A
Application number: CN202011608185.6A
Authority: CN
Inventors: 王超云; 高原
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2021-04-20

Abstract

The invention discloses a skin-tendering and anti-wrinkle composition containing PDRN (PDRN) and application thereof, wherein the skin-tendering and anti-wrinkle composition comprises PDRN (poly-deoxyribose nucleotide), dipeptide diaminobutyrylbenzyl amide diacetate and acetyl tetrapeptide-5. The mass ratio of PDRN, dipeptide diaminobutyrylbenzylamide diacetate to acetyl tetrapeptide-5 is (0.1-3): (1-3): (1-5). The skin-tendering and anti-wrinkle composition is applied to an anti-wrinkle cosmetic. The composition provided by the invention has a synergistic effect, can reduce oxidative stress damage, improve cell activity, retain water, stimulate the generation of collagen and elastin, and achieve the effect of resisting wrinkles. The formula disclosed by the invention has no conventional side effect, is safe and non-irritant, is suitable for people with various skin types, and can be applied to anti-wrinkle medical skin care products.

Description

Skin-tendering anti-wrinkle composition containing PDRN and application thereof

Technical Field

The invention belongs to the technical field of cosmetics, and particularly relates to a skin-tendering and anti-wrinkle composition containing PDRN and application thereof.

Background

The skin is an important tissue structure of the human body and is mainly composed of 3 parts of epidermis, dermis and subcutaneous tissue. The epidermis is divided into a basal layer, a spinous layer, a granular layer and a cuticle from inside to outside in sequence, the cuticle is positioned on the outermost layer of the epidermis and consists of a plurality of layers of cuticle cells and cuticle lipid, the cuticle cells are arranged tightly, can resist external friction, defend the invasion of pathogenic microorganisms, prevent the passage of water and electrolyte, have certain tolerance to some physicochemical factors such as acid, alkali and ultraviolet rays, and form an important natural protective layer of a human body. The dermis is composed of papillary layer, reticular layer and dermal connective tissue, wherein the connective tissue includes collagen fibers, elastic fibers and reticular fibers. They maintain the structural integrity and stability of skin, improve the living environment of skin cells and the metabolism of skin, promote wound healing, repair scars, retain moisture, and serve as dermis to maintain the strength, extensibility and elasticity of skin.

Skin is the tissue of the human body that is the earliest to develop aging, which includes physiological aging and extrinsic aging. With age, keratinocytes lose water retention, intercellular junctions are loose, the skin is dull, dry, elastic, collagen-reduced and abnormal cross-linking occurs, leading to laxity of the tissue and increased wrinkles. In addition, external factors can cause the weakening of the defense function of skin cells and the reduction of the activity of fibroblasts. The proliferation and differentiation of keratinocytes are inhibited under the combined influence of physiological (aging, disease, etc.) and exogenous factors (climate, sunlight, occupation, alcoholism, smoking, living conditions, etc.), the synthesis of collagen and elastin is reduced, the content of collagen and elastin is reduced, and skin aging phenomena such as skin thinning, loss of luster and elasticity, pigmentation, skin relaxation, and wrinkles are formed.

With age, facial wrinkles develop, which is intolerable to beauty-conscious women. In medicine, estrogen and superoxide dismutase are commonly used for removing wrinkles, but the effect is greatly reduced due to the lack of long-acting property, the skin cannot be reached because the skin cannot be damaged by gastric juice easily and the like. In recent years, with the rapid development of the medical and beauty industry, tretinoin, alpha-hydroxy acid, castor oil, coconut oil or cocoa butter are used for removing wrinkles, but the effect is general. In order to improve the wrinkle-removing effect, some compounds and peptides are widely applied in the medical and cosmetic industry, such as polypeptide Botulinum Toxin (BTX) has a remarkable effect on wrinkle removal, but the application of BTX is limited due to the high toxicity of BTX and the short-term effect of the wrinkle-removing effect of BTX.

As described above, we carefully analyzed the mechanism of skin wrinkles, and the dynamic wrinkles and the expression muscles were distributed in length, and wrinkles were formed only when the expression muscles contracted, and wrinkles were formed when the contraction power of the expression muscles was insufficient. Static wrinkles are associated with intracellular and extracellular dehydration, atrophy of the epidermis, the elastic components of the dermis, the blood vessels, the glands, the muscles and the bones, and fibrosis of the connective tissue.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a skin-tendering anti-wrinkle composition containing PDRN according to the generation mechanism of wrinkles, and the composition is applied to an anti-wrinkle cosmetic to provide short-term and long-term skin wrinkle-removing care for consumers; the composition has remarkable effect, is mild and non-irritant, and is suitable for medical cosmetics.

The specific technical scheme is as follows:

one of the objects of the present invention is to provide a skin rejuvenation and anti-wrinkle composition containing PDRN, which comprises PDRN (polydeoxyribonucleotide), dipeptide diaminobutyrylbenzylamide diacetate and acetyl tetrapeptide-5.

The PDRN (Poly Deoxy Ribo nucleotide) is a polydeoxyribonucleotide which is extracted from salmon testis, ovary, viscera or muscle tissue.

Dipeptide diaminobutyrylbenzylamide diacetate, a snake venom-like serum protein peptide, has CAS accession number of 823202-99-9, is an acetylcholine receptor blocker, can inhibit muscle contraction to reduce wrinkles, and has effects of smoothing skin and rapidly removing wrinkles.

The CAS registry number of the acetyltetrapeptide-5 is 820959-17-9, and can inhibit glycation, thereby reducing cross-linking of collagen, achieving the effects of reducing and resisting wrinkles, and improving skin elasticity and smoothness.

The components of the composition have obvious synergistic effect, not only have good wrinkle removing effect, but also can obviously improve the skin fineness, and the effect is obviously superior to that of single components and other combinations.

Further, the mass ratio of PDRN, dipeptide diaminobutyrylbenzylamide diacetate to acetyltetrapeptide-5 is (0.1-3): (1-3): (1-5).

Further, the mass ratio of PDRN, dipeptide diaminobutyrylbenzylamide diacetate to acetyltetrapeptide-5 is (0.5-1): (1-3): (1-3).

Furthermore, the molecular weight of the PDRN is 50 bp-2000 bp.

Further, the PDRN is obtained from the testis of male salmon.

Further, the preparation method of the PDRN comprises the following steps:

(1) mixing a male salmon testis and a trichloroacetic acid solution with the concentration of 0.8 wt% -1.5 wt%, homogenizing, centrifuging, and removing a supernatant; the mass ratio of the salmon testis to the trichloroacetic acid solution is 1: (3-10);

(2) adding a lysis solution into the precipitate obtained in the step (1), fully and uniformly mixing, carrying out heat treatment at 70-95 ℃ for 10-20 min, cooling to below 30 ℃, centrifuging, and taking a supernatant; the lysis solution contains 75-150 mM Tris-HCl, 100-250 mM NaCl, 2-6 mM EDTA and 0.15-0.25 wt% SDS, and the pH value is 7.0-8.5;

(3) and (3) adding isopropanol or absolute ethyl alcohol with the same volume to the supernatant obtained in the step (2), uniformly mixing, standing at the temperature of-18 to-25 ℃ for 25 to 40min, centrifuging at 7000 to 10000rpm for 8 to 15min, removing the supernatant, collecting the precipitate, and drying the precipitate to obtain PDRN.

And (3) further, centrifuging for 5-10 min at 4000-8000 rpm in the step (2).

Further, in the step (1), the pyrolysis heat treatment temperature is preferably 80-95 ℃.

The invention also aims to provide the application of the skin-tendering and anti-wrinkle composition in cosmetics.

The skin tendering and anti-wrinkle composition can be applied to various cosmetics with anti-wrinkle effects, has no conventional side effects, is safe and non-irritant, is suitable for people with various skin types, and is suitable for being applied to medical cosmetics. The cosmetic is preferably in the form of cream, emulsion, gel or aqueous preparation.

Furthermore, the skin-tendering anti-wrinkle composition is 0.001 wt% -40 wt%, preferably 0.1 wt% -30 wt%, and more preferably 5 wt% -20 wt% in the cosmetic.

Furthermore, the cosmetic also comprises one or more of an antioxidant, a humectant, an emulsifier, vegetable oil, a preservative and a thickening agent.

Still further, the antioxidant may be at least one of ascorbic acid and salts, vitamin E, coenzyme Q10, glutathione, thioredoxin, taurine, thiotaurine, hypotaurine, vitamin E acetate, and the like. Among them, at least one of ascorbic acid, glutathione, vitamin E, and taurine is preferable. The proportion of the antioxidant to be added is not particularly limited, but is usually 0.001 to 6 wt%, preferably 0.01 to 3 wt%, and more preferably 0.1 to 1.5 wt% with respect to the total amount of the composition for external application to the skin (e.g., anti-wrinkle nutritional liquid).

Still further, the humectant is at least one of sodium hyaluronate, sodium acetyl hyaluronate, sodium chondroitin sulfate, amino acids (amino acids such as alanine, serine, leucine, isoleucine, threonine, glycine, proline, hydroxyproline, aspartic acid, arginine, theanine, and the like, and derivatives thereof), ethylene glycol, 1, 3-propanediol, 1, 3-butanediol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, di-1, 3-propanediol, polyethylene glycol, glycerol, and sodium lactate. Among them, at least one of sodium hyaluronate, propylene glycol, glycerin and glycine is preferable. The proportion of the humectant is not particularly limited, but is usually 0.001 to 40 wt%, preferably 0.1 to 30 wt%, and more preferably 5 to 20 wt% based on the total weight of the composition for external application to the skin (e.g., an anti-wrinkle nutritional liquid).

Still further, the emulsifier is at least one selected from cetyl alcohol, C20-22 alcohol phosphate, glyceryl monostearate, tween, span, poloxamer 188, PEG-20 hydrogenated castor oil, glyceryl stearate/PEG-100, polyvinyl alcohol and polylactic acid-glycolic acid. The proportion of the emulsifier added is not particularly limited, but is usually 0.001 to 10 wt%, preferably 0.01 to 5 wt%, and more preferably 0.1 to 3 wt% with respect to the total amount of the composition for external application to the skin (e.g., anti-wrinkle nutritional liquid).

Still further, the vegetable oil and fat is at least one selected from the group consisting of rose hip oil, sweet almond oil, rose hip oil, jojoba oil, avocado oil, castor oil, avocado oil, coconut oil, palm oil, cocoa butter, palm kernel oil, aloe vera oil, sal fat, shea butter, macadamia nut oil, Chinese chestnut oil, seabuckthorn oil, passion flower oil, calendula, mango kernel oil and rose hip oil. Wherein the preferred oil is at least one of rosehip oil, jojoba oil and sweet almond oil. The ratio of the vegetable oil or fat to be added is not particularly limited, but is usually 0.001 to 10 wt%, preferably 0.01 to 8 wt%, and more preferably 0.1 to 5.0 wt% with respect to the total amount of the composition for external application to the skin (e.g., anti-wrinkle nutrient solution).

Still further, the preservative is at least one selected from benzyl alcohol, phenethyl alcohol, phenoxyethanol and lauroyl arginine ethyl ester, and is preferably lauroyl arginine ethyl ester. The proportion of the preservative added is not particularly limited, but is usually 0.001 to 10 wt%, preferably 0.01 to 5 wt%, and more preferably 0.1 to 2 wt% with respect to the total amount of the composition for external application to the skin (e.g., anti-wrinkle nutrient solution).

Still further, the thickening agent is at least one of carbomer, Arabic gum, sodium alginate, hydroxymethyl cellulose and cellulose acetate phthalate. The proportion of the thickener to be added is not particularly limited, but is usually 0.001 to 5 wt%, preferably 0.01 to 3 wt%, and more preferably 0.05 to 1 wt% with respect to the total amount of the composition for external application to the skin (e.g., anti-wrinkle nutrient solution).

The invention has the following beneficial effects:

the composition provided by the invention has a synergistic effect, can reduce oxidative stress damage, improve cell activity, retain water, stimulate the generation of collagen and elastin, and achieve the effect of resisting wrinkles. The formula of the invention has no conventional side effect, is safe and non-irritant, is suitable for people with various skin types, and can be applied to anti-wrinkle skin care products, in particular to medical skin care products.

Drawings

FIG. 1 is a gel electrophoresis image of PDRN obtained in examples 1, 2 and 3;

FIG. 2 shows the effect of the compositions of comparative examples 1 to 6 and examples 1 and 4 on cell viability in experiment 1;

FIG. 3 is a western blot electrophoresis of collagen III and elastin in experiment 1;

FIG. 4 shows the effect of different formulations on collagen expression III in experiment 1;

FIG. 5 shows the effect of different formulations on elastin expression in experiment 1;

FIG. 6 shows the improvement effect of example 7 on crow's feet in experiment 2.

In fig. 1: in FIG. 1, from left to right, lane 1 is PDRN prepared in example 1, lane 2 is PDRN prepared in example 2, lane 3 is PDRN prepared in example 3, lane 4 is blank, and lane 5 is marker.

Detailed Description

The principles and features of this invention are described below in conjunction with examples, which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

Example 1

1. Preparation of PDRN (polydeoxyribonucleotides), comprising the following steps:

(1) mixing a male salmon testis and a trichloroacetic acid solution with the concentration of 1 wt% according to the mass ratio of 1: 4, mixing, homogenizing, centrifuging at 4000rpm for 5min, and removing the supernatant;

(2) adding lysis solution into the precipitate obtained in the step (1), fully and uniformly mixing, heating in water bath at 90 ℃ for 15min, cooling to room temperature, centrifuging at 6000rpm for 10min, and taking supernatant; the lysis solution contains 100mM Tris-HCl, 200mM NaCl, 5mM EDTA and 0.2 wt% SDS, and the pH value is 8.0;

(3) and (3) adding equal volume of 4 ℃ precooled isopropanol into the supernatant obtained in the step (2), uniformly mixing, standing at-20 ℃ for 30min, centrifuging at 8000rpm for 10min, removing the supernatant, collecting the precipitate, and drying the precipitate until no ethanol smell exists, thereby obtaining PDRN.

2. The PDRN obtained from the above preparation was subjected to gel electrophoresis:

the method comprises the steps of accurately weighing PDRN, adding a TE solution, blowing and beating by using a gun head, placing in a constant-temperature water bath kettle at 50 ℃ for 2 hours for dissolving to obtain a sample with the concentration of more than 600ng/ul, adding a loading buffer of a sample loading solution according to the volume ratio of 1:6, and measuring the molecular weight of DNA in the sample by using gel electrophoresis, wherein the result is shown in figure 1 (in figure 1, from left to right, a lane 1 is the PDRN prepared and obtained in example 1, a lane 4 is a blank sample, and a lane 5 is a marker). As can be seen from FIG. 1, the molecular weight of the PDRN obtained as described above ranges from 50bp to 2000 bp.

3. The PDRN prepared by the method is used for obtaining a skin rejuvenation and anti-wrinkle composition containing the PDRN, which consists of the PDRN, dipeptide diaminobutyrylbenzylamide diacetate and acetyl tetrapeptide-5, wherein the mass ratio of the PDRN, the dipeptide diaminobutyrylbenzylamide diacetate to the acetyl tetrapeptide-5 is 0.1: 2: 2.

example 2

1. Preparation of PDRN (polydeoxyribonucleotides)

(1) Mixing a male salmon testis and a trichloroacetic acid solution with the concentration of 1 wt% according to the mass ratio of 1:10 mixing, homogenizing, centrifuging at 4000rpm for 5min, and discarding the supernatant;

(2) adding lysis solution into the precipitate obtained in the step (1), fully and uniformly mixing, heating in water bath at 95 ℃ for 10min, cooling to room temperature, centrifuging at 7000rpm for 10min, and taking supernatant; the lysis solution contains 150mM Tris-HCl, 200mM NaCl, 4mM EDTA and 0.25 wt% SDS, and the pH value is 8.2;

(3) and (3) adding equal volume of 4 ℃ precooled absolute ethyl alcohol into the supernatant obtained in the step (2), uniformly mixing, standing at-20 ℃ for 30min, centrifuging at 8000rpm for 10min, discarding the supernatant, collecting the precipitate, and drying the precipitate until no ethanol smell exists, thereby obtaining PDRN.

2. The PDRN obtained from the above preparation was subjected to gel electrophoresis: the molecular weight of DNA in the sample was measured by gel electrophoresis, and the results are shown in FIG. 1, from left to right, lane 2 shows the PDRN prepared in example 2. As can be seen from FIG. 1, the molecular weight of the PDRN obtained as described above ranges from 50bp to 2000 bp.

Example 3

1. Preparation of PDRN (polydeoxyribonucleotides)

(1) Mixing male salmon ovary and trichloroacetic acid solution with the concentration of 1 wt% according to the mass ratio of 1: 4, mixing, homogenizing, centrifuging at 4000rpm for 5min, and removing the supernatant;

(2) adding lysis solution into the precipitate obtained in the step (1), fully and uniformly mixing, heating in water bath at 70 ℃ for 20min, cooling to room temperature, centrifuging at 7000rpm for 10min, and taking supernatant; the lysis solution contains 100mM Tris-HCl, 150mM NaCl, 2mM EDTA and 0.15 wt% SDS, and the pH value is 8.0;

the molecular weight of DNA in the sample was measured by gel electrophoresis, and the results are shown in FIG. 1, from left to right, lane 3 shows the PDRN prepared in example 3. As can be seen from the electropherograms, the molecular weight of the PDRN obtained in example 3 is greater than 100bp, and a certain amount of components having a molecular weight greater than 2000bp are present, which is related to a lower cleavage temperature thereof.

Example 4

A skin rejuvenating anti-wrinkle composition containing PDRN was obtained using the same PDRN as in example 1, which differs from example 1 in that: the mass ratio of PDRN, dipeptide diaminobutyrylbenzylamide diacetate to acetyl tetrapeptide-5 is 1: 2: 2.

comparative example 1

A composition consisting of PDRN and dipeptide diaminobutyrylbenzylamide diacetate was obtained using the same PDRN as in example 1, in a mass ratio of PDRN to dipeptide diaminobutyrylbenzylamide diacetate of 0.1: 2.

Comparative example 2

A composition consisting of PDRN and dipeptide diaminobutyrylbenzylamide diacetate was obtained using the same PDRN as in example 1, in a mass ratio of PDRN to dipeptide diaminobutyrylbenzylamide diacetate of 1: 2.

Comparative example 3

A composition consisting of PDRN and acetyl tetrapeptide-5 was obtained using the same PDRN as in example 1, and the mass ratio of PDRN to acetyl tetrapeptide-5 was 0.1: 2.

Comparative example 4

A composition consisting of PDRN and acetyl tetrapeptide-5 was obtained using the same PDRN as in example 1, with a mass ratio of PDRN to acetyl tetrapeptide-5 of 1: 2.

Comparative example 5

Obtaining a composition consisting of dipeptide diaminobutyrylbenzylamide diacetate and acetyl tetrapeptide-5 in a mass ratio of dipeptide diaminobutyrylbenzylamide diacetate to acetyl tetrapeptide-5 of 1: 1.

Comparative example 6

Obtaining a composition consisting of dipeptide diaminobutyrylbenzylamide diacetate and acetyl tetrapeptide-5 in a mass ratio of dipeptide diaminobutyrylbenzylamide diacetate to acetyl tetrapeptide-5 of 1: 10.

Example 5

Based on the formula research of the composition, the skin-tendering and anti-wrinkle composition obtained in example 4 is used for preparing the anti-wrinkle cream, and the components and the mass percentage content of the components forming the anti-wrinkle cream are shown in table 1:

TABLE 1 anti-wrinkle cream ingredient table in example 5

Heating the oil phase components to 78 ℃, stirring and melting the components into liquid, and preserving heat for later use; slowly scattering powder raw materials in the water phase component into water, heating and uniformly dispersing while stirring, and heating to 82 ℃ to completely dissolve uniformly; slowly adding the water phase component into the oil phase component, homogenizing at 3000r/min for 5min until completely emulsifying uniformly, defoaming, and cooling; cooling to 70 deg.C, adding phase C component, stirring, cooling to 45 deg.C, adding skin caring and anti-wrinkle composition (PDRN, dipeptide diaminobutyrylbenzyl amide diacetate, acetyl tetrapeptide-5), sodium hyaluronate, antiseptic, and essence, stirring, and standing at 35 deg.C to obtain white fine uniform ointment.

Example 6

Based on formulation research on the composition, the skin-tendering anti-wrinkle composition obtained in example 4 was used to prepare an anti-wrinkle emulsion, and the components and mass percentage content thereof constituting the anti-wrinkle emulsion are shown in table 2:

TABLE 2 ingredient Table of wrinkle-resistant emulsion in example 6

Heating the component of the group A to 75 ℃, melting and uniformly stirring, sequentially dissolving the component B in sterile water according to the formula proportion, stirring and dissolving, adding the phenoxyethanol of the group C and the propylene glycol into the component B after mixing, cooling to 45 ℃, uniformly stirring and mixing the group A and the group B, and standing to form the PDRN anti-wrinkle emulsion.

Example 7

Based on formulation study on the composition, the skin-tendering and anti-wrinkle composition obtained in example 4 was used to prepare an anti-wrinkle essence, and the components and mass percentages of the components constituting the anti-wrinkle essence are shown in table 3:

TABLE 3 ingredient Table of anti-wrinkle essence in example 7

Components	Content in the Total System (wt%)
		Deionized water	Balance of
Hyaluronic acid	10.00
		Ethylene glycol	1.00
Skin-tendering and anti-wrinkle composition	5.00
		Preservative	Proper amount of

Mixing the above components in sequence, stirring to dissolve completely, inspecting, and packaging to obtain the final product.

Experiment 1

The effects of the compositions obtained in examples 1 and 4 and comparative examples 1 to 6 on the expression of collagen and elastin in human epidermal cells were investigated.

The compositions are prepared into 10mg/mL solutions respectively for later use. Comparative examples 1 to 6 and examples 1 and 4 are labeled in the order of groups a, b, c, d, e, f, g, and h.

Human epidermal cells (HaCaT, purchased from ATCC cell bank) were cultured and divided into 10 groups of normal group, model group, a (comparative example 1), b (comparative example 2), c (comparative example 3), d (comparative example 4), e (comparative example 5), f (comparative example 6), g (example 1) and H (example 4), and 150nM H was added to each of the groups except the normal group₂O₂Causing oxidative stress damage, adding the prepared sample solutions to a final concentration of 100 μ g/mL, and standing at 37 deg.C with 5% CO₂The cells were cultured for 48 hours in the environment, and the activity of each group of cells was measured by the MTT method, and the expression of collagen III (antibody derived from Abcam (ab184993)) and elastin (antibody derived from Abcam (ab269450)) in each group was measured by the western blot method.

The effect of different formulations on cell viability is shown in figure 2. (note:indicatesthe number of active sites in comparison to the normal group,^**p<0.01 was very significantly different from the normal group; # denotes comparison with a model set,^#p<0.05 was significantly different from the model group,^##p<0.01 was very significantly different from the model group,^###p<0.001 was very significantly different from the model group; delta indicates that compared to the PDRN formulation containing 0.1 ratio,^ΔΔp<0.01 is very different from the PDRN formula containing 0.1 proportion. )

The western blot of collagen III and elastin is shown in FIG. 3. In FIG. 3,

lanes

1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 represent the normal control group and H₂O₂Model group, a, b, c, d, e, f, g, h group.

The effect of different formulations on collagen expression III is shown in FIG. 4. (note:indicatesthe number of active sites in comparison to the normal group,^**p<0.01 was very significantly different from the normal group; # denotes comparison with a model set,^#p<0.05 was significantly different from the model group,^##p<0.001 was very significantly different from the model group; delta indicates that compared to the PDRN formulation containing 0.1 ratio,^Δp<0.05 was significantly different from the PDRN formulation containing 0.1 ratio. )

The effect of the different formulations on elastin expression is shown in figure 5. (Note: comparison with Normal group, p<0.01 was very significantly different from the normal group; # denotes comparison with model group, # p<0.05 significant difference, # p, compared to the model group<0.001 was very significantly different from the model group; delta indicates that compared to the PDRN formulation containing 0.1 ratio,^Δp<0.05 has obvious difference compared with the PDRN formula containing 0.1 proportion,^ΔΔp<0.01 is very different from the PDRN formula containing 0.1 proportion. )

As shown in figures 2, 3, 4 and 5, oxidative stress can obviously reduce the activity of HaCaT cells and inhibit the expression of collagen and elastin, the cell activity is obviously improved after different compositions are given, and the map shows that the proportion of PDRN can be increased to obviously improve the cell activity, increase the expression of collagen and elastin and reduce the damage of oxidative stress. A large number of researches show that PDRN has the effects of increasing cell growth factors, improving collagen/non-collagen synthesis, inducing cell regeneration and promoting wound healing; acetyl tetrapeptide-5 can inhibit saccharification, reduce mutual crosslinking of collagen, and dipeptide diaminobutyrylbenzylamide diacetate has obvious effect on promoting collagen/non-collagen synthesis by inhibiting sodium channels. The research shows that the formula in example 4 has the best effects on reducing oxidative stress injury, improving cell activity and increasing the expression of collagen and elastin, and the experimental result shows that the three components generate obvious synergistic effects on improving cell activity and increasing the expression of collagen and elastin.

Experiment 2

The wrinkle-removing cream, the emulsion and the essence obtained in examples 5 to 7 were tested and compared with the corresponding commercially available wrinkle-removing cream (comparative example 7), emulsion (comparative example 8) and essence (comparative example 9).

Firstly, testing physical and chemical indexes:

60 healthy skin volunteers were randomly selected and tested, and one group of examples or comparative examples was tested per group of 10 persons, and the test results were averaged.

The wrinkle-removing cream, lotion and essence of examples 5 to 7 and the cream, lotion and essence of comparative examples 7 to 9 were used for 3 times a day for 30 consecutive days.

1. The moisture content before and after use was measured using a skin moisture tester, and the% moisture content increase before and after use was calculated by the following method: skin moisture lifting rate (skin moisture content measured after use-skin moisture content measured before use)/skin moisture content before use × 100%.

2. The skin fineness and the wrinkle change are measured by adopting a Visia skin detector, and the fineness improvement rate is obtained by calculating according to the following method: the fineness increase rate is (skin fineness measured after use-skin fineness measured before use)/skin fineness measured before use × 100%.

3. The total score of the experience feeling score is 10 points, 8-10 points are good, 6-8 points are general, and 6 points or less are poor experience feeling.

4. After the use, the use of the test product is stopped and other moisturizing products are not used, and the water retention rate of the product is tested after 2 weeks. Moisture retention rate is skin moisture content measured after 2 weeks/skin moisture content measured immediately after use × 100%.

The effect on the change in the number of wrinkles is shown in table 4.

TABLE 4 Effect on the number of wrinkles

Test items	The number of people with remarkably reduced wrinkles	Number of people with slight wrinkles reduction	Number of people who did not have a change in wrinkles
				Example 5	10	0	0
Example 6	8	2	0
				Example 7	7	3	0
Comparative example 7	5	2	3
				Comparative example 8	3	1	6
Comparative example 9	3	2	5

As can be seen from Table 4, examples 5 to 7 are superior to comparative examples in the effect of reducing wrinkles as a whole. This shows that the formula of the invention has reasonable component matching.

The effect on skin moisture content and fineness is shown in table 5.

TABLE 5 influence on skin moisture content, fineness and feeling of use experience

Test items	Skin moisture lifting ratio (%)	Fineness raising ratio (%)	Experience score
				Example 5	58.6±4.2**	77.3±5.9**	8.9±1.2**
Example 6	51.5±3.7##	72.6±4.3##	8.3±1.4##
				Example 7	50.0±5.8++	73.7±6.8++	8.6±1.2++
Comparative example 7	36.6±7.3	49.1±8.5	6.2±1.2
				Comparative example 8	31.8±6.6	43.1±6.9	4.75±2.4
Comparative example 9	26.8±6.4	39.7±6.9	5±2.4

Note: indicates a very significant difference from comparative example 7, P < 0.01; # denotes a very significant difference, # P <0.01, compared to comparative example 8; + indicates a very significant difference of + P <0.01 compared to comparative example 9.

As can be seen from Table 5, the effects of examples 5 to 7 are superior to those of the comparative examples as a whole. The formula of the invention has reasonable component matching.

The effect on skin moisture retention is shown in table 6.

TABLE 6 Effect on skin moisture Retention

Test items	Water retention after 2 weeks (%)
		Example 5	89.7±5.3**
Example 6	84.6±5.3##
		Example 7	82.6±4.2++
Comparative example 7	65.4±5.0
		Comparative example 8	56.3±5.6
Comparative example 9	59.4±4.1

As can be seen from Table 6, the effects of examples 5 to 7 are superior to those of the comparative examples as a whole. This shows that the formula of the invention is reasonable in collocation.

Secondly, skin elasticity test:

30 healthy skin allergy-free volunteers were selected and randomized into three groups of 10 individuals each, namely, example 5, example 6 and example 7. Before applying the sample, each group of subjects washed their faces with the same mild cleanser, and the face was symmetrically positioned at about 5X 5cm on the left and right sides of the face²The area of size is regarded as the experimental area, scribbles about 0.5 g's embodiment sample on face left side, scribbles corresponding proportion of equivalent on face right side, and the frequency of use is once respectively morning and evening, uses continuously every day, avoids outdoor insolate. Before using the anti-wrinkle composition of the present invention, the elasticity n of each test site was measured in a test area using a skin comprehensive index meter₀A value; after 1, 2, 3, 4, 5 and 6 weeks of application, each test area was examined and the n-value of elasticity was recorded each time. For better evaluation of the data obtained, the average value of n for each test was divided by n for the unused samples₀The average value is used to obtain the elasticity rating of the part, and when the elasticity rating is larger, the test sample has better characteristic of increasing the skin elasticity. The results are shown in Table 7.

TABLE 7 Effect on skin elasticity

Test items	0 week	1 week	2 weeks	3 weeks	4 weeks	5 weeks	6 weeks
								Example 5	1	1.15	1.26	1.36	1.45	1.50	1.52
Example 6	1	1.13	1.20	1.32	1.42	1.46	1.49
								Example 7	1	1.14	1.21	1.34	1.46	1.50	1.52
Comparative example 7	1	1.07	1.10	1.13	1.16	1.19	1.22
								Comparative example 8	1	1.06	1.07	1.10	1.12	1.16	1.17
Comparative example 9	9	1.04	1.05	1.08	1.11	1.15	1.16

As shown in table 7, the elasticity evaluation values of the subjects increased from 1 to 1.52, 1.49, and 1.52 after six weeks using the anti-wrinkle composition of the present invention, and the elasticity evaluation values increased from 1 to 1.22, 1.17, and 1.16 after six weeks using the comparative example, respectively, and it was found that the anti-wrinkle composition of the present invention had a significant effect of enhancing skin elasticity.

And thirdly, testing improvement of the fishtail line:

the anti-wrinkle essence obtained in example 7 was applied to the canthus at a frequency of once every morning and once every night, and after 1 week of continuous daily application, 88% of the skin wrinkles became shallow in depth and reduced in wrinkles, as shown in fig. 6 (in fig. 6, the left side is before practical use and the right side is after one week of use), and as can be seen from fig. 6, the skin wrinkles became shallow in depth and the deep wrinkles were significantly reduced after 4 weeks of application of the cosmetic of the present invention. Meanwhile, the visual observation of the facial skin of the subject shows that the skin becomes ruddy and full and fine wrinkles are obviously improved.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A skin-tendering and anti-wrinkle composition containing PDRN is characterized by comprising PDRN, dipeptide diaminobutyrylbenzylamide diacetate and acetyl tetrapeptide-5.

2. The skin-tendering and anti-wrinkle composition as claimed in claim 1, wherein the mass ratio of PDRN, dipeptide diaminobutyrylbenzylamide diacetate and acetyl tetrapeptide-5 is (0.1-3): (1-3): (1-5).

3. The skin-tendering and anti-wrinkle composition as claimed in claim 2, wherein the mass ratio of PDRN, dipeptide diaminobutyrylbenzylamide diacetate and acetyl tetrapeptide-5 is (0.5-1): (1-3): (1-3).

4. The skin rejuvenation and anti-wrinkle composition as claimed in any one of claims 1 to 3 wherein the molecular weight of the PDRN is from 50bp to 2000 bp.

5. The skin rejuvenating and anti-wrinkle composition as claimed in any one of claims 1 to 3 wherein the PDRN is obtained from the testis of male salmon.

6. The skin rejuvenating and anti-wrinkle composition as claimed in any one of claims 1 to 3, wherein the PDRN is prepared by a method comprising the steps of:

7. Use of a skin rejuvenating anti-wrinkle composition as defined in any one of claims 1 to 6 in cosmetics.

8. The use according to claim 7, wherein the skin rejuvenating and anti-wrinkle composition is present in a cosmetic composition in an amount of from 0.001% to 40% by weight.

9. Use according to claim 7 or 8, wherein the cosmetic further comprises one or more of antioxidants, humectants, emulsifiers, vegetable fats and oils, preservatives and thickeners.