CN114042009A

CN114042009A - Polypeptide composition with antioxidation and repair effects and application thereof

Info

Publication number: CN114042009A
Application number: CN202111458580.5A
Authority: CN
Inventors: 丁文锋; 彭晏; 陈雪; 赵文豪
Original assignee: Yutai Biology Dongguan Co ltd
Current assignee: Yutai Biology Dongguan Co ltd
Priority date: 2021-12-01
Filing date: 2021-12-01
Publication date: 2022-02-15

Abstract

The invention discloses a polypeptide composition with oxidation resistance and repair functions and application thereof, belonging to the technical field of beauty polypeptide, wherein the composition comprises a polypeptide for promoting endogenous synthesis of coenzyme Q10, improving skin energy and a polypeptide for inhibiting lipid peroxidation damage, and the concentration ratio of the polypeptide to the polypeptide is 1: 4. The polypeptide for promoting endogenous synthesis of coenzyme Q10 and improving skin energy is selected from pentapeptide-34 trifluoroacetate. The polypeptide for inhibiting lipid peroxidation damage is selected from carnosine, decarboxylated carnosine or acetyl carnosine. The polypeptide composition disclosed by the invention is high in safety, can effectively resist oxidation by virtue of a synergistic effect of polypeptide combinations of different targets, and can be used for reducing oxidative damage of proteins and lipids; remarkably promotes the generation of collagen, and can be used for improving skin, slowing down skin aging and repairing aged skin.

Description

Polypeptide composition with antioxidation and repair effects and application thereof

Technical Field

The invention belongs to the technical field of beauty polypeptide, and provides a polypeptide composition with antioxidant and repairing functions and application thereof.

Background

Human skin is exposed to a variety of harmful substances, of which ultraviolet rays play an important role in skin damage. Damage caused by ultraviolet light includes direct DNA damage and oxidative damage of DNA, proteins and lipids caused by Reactive Oxygen Species (ROS). Mitochondria are particularly susceptible to light stress as the major site of ROS production in cells. The resulting mitochondrial dysfunction may adversely affect many essential cellular processes.

If the DNA damage is not repaired, mutations will occur. Mitochondrial DNA (mtdna) is particularly sensitive in this case, since this type of DNA not only encodes the essential protein of the respiratory chain, but also is in close proximity to the respiratory chain. Mutations in the gene encoding the respiratory chain protein result in increased mtROS production. mtROS accelerate mtDNA damage and mutations, thereby creating a vicious cycle. Mitochondrial dysfunction can negatively impact many of the fundamental processes in the cell, including reduced ATP production and susceptibility to apoptosis and necrosis. Therefore, an increase in mtROS is considered to be a major cause of aging of cells and organisms.

To break the vicious cycle of ROS and DNA damage, the body will try to counteract ROS with natural substances such as coenzyme Q10 and the like. Coenzyme Q10(CoQ10) has long been recognized as an effective ingredient that helps people to remain young. This fat-soluble component, which is naturally present in the body and located in the mitochondrial cell membrane, is essential for energy production and action, and acts as a powerful antioxidant against premature aging. In skin cells, coenzyme Q10 protects against damage and oxidative stress caused by uv light. Daniel et al^[1]It was found that after UV irradiation, mitochondrial membrane potential was maintained in CoQ 10-treated skin fibroblasts, mitochondrial dysfunction was reduced, and cellular ATP levels were accelerated in regeneration. CoQ10 can also reduce UVA irradiation induced ROS production and DNA in human keratinocytesAnd (4) damaging. In addition, CoQ10 was shown to reduce UVA-induced MMPs in cultured human dermal fibroblasts^[2]。

With age, the level of coenzyme Q10 decreases, impeding the cells' ability to resist oxidative defenses and produce energy. The general cosmetic measures against this problem are to directly apply coenzyme Q10 externally, but studies have shown that the external supply of coenzyme Q10 may increase mainly the level of coenzyme Q10 in sebum, failing to restore the level of coenzyme Q10 in the epidermis.

On the other hand, UV causes peroxidation damage to proteins and lipids, which generates various radicals, thereby abstracting hydrogen from the proteins to generate protein radicals, polymerizing and crosslinking the proteins. Lipid-peroxidized carbonyl compounds (aldehydes, ketones) react with the amino and thiol groups of proteins, causing cross-linking of the protein molecules. Lipid peroxidation destroys the normal structure of biological membranes. The biological membrane is a flowing mosaic structure composed of lipid and protein, membrane protein can move on the membrane to complete a certain function, and the cross-linking of the membrane protein caused by lipid peroxidation limits the movement of the protein and seriously damages the function of the biological membrane. Unsaturated lipids in the skin are unstable and relatively easily oxidized, and the number, configuration and relative position of double bonds are three important factors related to the depth of oxidation. The double bond is considered to be directly oxidized, and more experiments now prove that the alpha-methylene adjacent to the double bond is mainly subjected to oxidative dehydrogenation, and the alpha-methylene is subjected to dehydrogenation to form a free radical and form p-pi conjugation with the double bond, so that the double bond is easier to oxidize. The components for isolating double bonds such as linoleic acid and linoleic acid are more easily dehydrogenated due to the activation of the alpha-methylene group in the middle of the double bond by the double bonds on both sides, so that the oil containing linoleic acid and linoleic acid is more easily oxidized than other oils. Linoleic acid is essential fatty acid for human body and is key component for tight connection of human skin cuticle. In addition, various biological membranes are phospholipid bilayers and contain a large amount of unsaturated fatty acid to ensure the mobility of the biological membranes. Lipid peroxidation will have serious consequences. Lipid peroxidation directly affects the aging and longevity of humans. In appearance, lipid peroxidation leads to the appearance of signs of aging such as wrinkles, dullness, etc. on the skin.

Based on the mechanism of aging, the invention provides a polypeptide composition for preventing or resisting pathological symptoms related to oxidation process by adopting the combination of a polypeptide for promoting the endogenous synthesis of coenzyme Q10 and a polypeptide for inhibiting lipid peroxidation damage.

Disclosure of Invention

Through a large number of experimental researches, the inventor unexpectedly discovers that the composition containing the specific polypeptide is beneficial to the skin to promote the endogenous synthesis of CoQ10, inhibit lipid oxidation caused by non-metallic ions, promote the generation of collagen, protect the skin from oxidative damage and repair aged skin, and has excellent use safety, thereby opening new treatment and beauty prospects.

Therefore, the invention aims to provide a polypeptide composition with antioxidant and repair effects. The composition is obtained by compounding different polypeptides in a specific ratio, can exert a synergistic effect, and can be used for resisting oxidation and treating, preventing or repairing skin aging or photoaging signs.

The invention relates to a polypeptide composition with antioxidant and repairing effects, which comprises the following components:

(1) a polypeptide for promoting endogenous synthesis of coenzyme Q10 and increasing skin energy, selected from the group consisting of: pentapeptide-34 trifluoroacetate salt;

(2) a polypeptide that inhibits lipid peroxidation damage selected from the group consisting of: carnosine, decarboxylated carnosine, or acetyl carnosine;

in the polypeptide composition, the concentration ratio of the polypeptide for promoting endogenous synthesis of coenzyme Q10 and improving skin energy to the polypeptide for inhibiting lipid peroxidation damage is 1: 4.

Pentapeptide-34 trifluoroacetate promotes endogenous synthesis of coenzyme Q10(CoQ10) by up-regulating PDSS1 (propyl diphosphate synthase subunit 1)^[3]The product has effects in increasing coenzyme Q10 content in skin, reducing oxidative stress injury caused by ultraviolet, increasing ATP synthesis level, improving skin, and resisting aging.

Carnosine (β -alkyl-L-histidine), desmosine (β -alkyl histidine), N-acetyl carnosine (N-acetyl- β -alkyl histidine) are important components with a variety of biological and pharmacological chaperone properties in human tissues. The imidazole groups contained in the compounds have strong capability of complexing metal ions, so that the phenomenon that free metal ions catalyze and form free radicals to further cause lipid peroxidation can be avoided. Meanwhile, due to the conjugated structure of the imidazole group, the electron cloud density is high, and the lone pair electrons contained in N enable the imidazole group to easily obtain protons and easily remove the protons. Can rapidly capture hydroxyl free radical, singlet oxygen, peroxy radical, etc. Thus, carnosine, decarboxylated carnosine or acetyl carnosine have a very strong antioxidant capacity.

The polypeptide composition with the functions of resisting oxidation and repairing, disclosed by the invention, has a preparation selected from the following components: essence, powder, tablet, capsule, emulsion, ointment, cream or gel.

In another aspect of the invention, the use of a polypeptide composition having antioxidant and repair effects in the preparation of an antioxidant cosmetic or pharmaceutical composition is provided.

In another aspect of the invention, there is provided a use of a polypeptide composition having antioxidant and repair effects in the preparation of a cosmetic or pharmaceutical composition for treating, preventing or repairing skin aging or photoaging.

The treatment, prevention or repair of skin aging or photoaging is the promotion of collagen production, reduction, prevention or treatment of facial wrinkles.

Compared with the prior art, the invention has the advantages that:

1. aiming at different aging mechanisms, the invention adopts the polypeptide for promoting the endogenous synthesis of coenzyme Q10, improving the skin energy and the polypeptide for inhibiting lipid peroxidation damage, the two are compounded according to the ratio of 1:4, and the polypeptide combination of different targets plays a synergistic effect, thereby effectively resisting oxidation and being used for reducing the oxidative damage of protein and lipid; remarkably promotes the generation of collagen, and can be used for improving skin, slowing down skin aging and repairing aged skin.

2. Pentapeptide-34 trifluoroacetate exhibits significant cytotoxicity at low doses, which is often overlooked in the prior art when the polypeptides are used in combination. From the safety aspect, the pentapeptide-34 trifluoroacetate with a safe dose is used for compounding, and the obtained composition has excellent safety while exerting the effects of oxidation resistance and repair.

Drawings

FIG. 1 is a graph showing the effect of test samples on HaCaT cell activity.

Fig. 2 shows the DPPH radical scavenging results (n-4) for each sample at different concentrations.

FIG. 3 is a microphotograph of sirius red stained cells.

Detailed Description

For a better understanding of the present invention, the following detailed description of the invention is given in conjunction with examples, however, it should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

The compounding proportion of each polypeptide in the composition of the embodiment of the invention is as follows:

name of raw materials	Composition 1	Composition 2	Composition 3
				Pentapeptide-34 trifluoroacetate salt (hereinafter referred to as peptide A)	1	2.5	4
Decarboxyme carnosine (hereinafter referred to as peptide B)	4	2.5	1

Note: at 100ppm of composition, as peptide a: peptide B ═ 1:4, peptide A was 20ppm and peptide B was 80 ppm.

Example 1 cell Activity assay

1.1 reagents and materials

Thiazole blue (MTT) (Sigma), dimethyl sulfoxide (DMSO) (Sigma), high-glucose medium (DMEM) (Gibco), fetal bovine serum (Gibco).

1.2 instruments

Enzyme-linked immunosorbent assay (American MD), CO₂Incubator (Shanghai Yi Heng), super clean bench (Suzhou purification).

1.3 cell lines

Human keratinocytes (HaCaT) were purchased from kunming cell banks, typical culture collection committee of chinese academy of sciences.

1.4 samples to be tested

Administration group:

peptide A, tested at concentrations of 12.5ppm, 25ppm, 50ppm, 100ppm, respectively;

peptide B, tested at concentrations of 12.5ppm, 25ppm, 50ppm, 100ppm, respectively;

composition 1, tested at concentrations of 12.5ppm, 25ppm, 50ppm, 100ppm, respectively;

composition 2, tested at concentrations of 12.5ppm, 25ppm, 50ppm, 100ppm, respectively;

composition 3, tested at concentrations of 12.5ppm, 25ppm, 50ppm, 100ppm, respectively;

the administration group was dissolved with 0.5% DMSO solution.

control group: 0.5% DMSO.

Positive control group: 2% DMSO.

1.5 Experimental methods

Culturing frozen HaCaT cells according to the ratio of 1: passage 2 to about 5, and selecting the cells with better growth as experimental objects. Mixing cell 200Inoculating 0 cell/well in 96-well plate, diluting according to multiple ratio after cell adherence, adding administration group, control group and positive control group samples, supplementing culture medium to 200 μ L, standing at 37 deg.C and 5% CO₂Incubate in incubator for 72 h.

Then 22. mu.L of 5mg/mL MTT per well was added, continuing at 37 ℃ with 5% CO₂Incubate in incubator for 4 h. The stock solution was discarded and 150. mu.L/well of DMSO was added. After 5min, reference OD values at 490nm and 630nm wavelength were read using a microplate reader.

1.6 results

The MTT method is a method for detecting cell survival and growth, and takes a control group as a reference ratio, 4 dosing concentrations of each sample are set from high to low, a cell activity detection experiment is carried out on HaCaT cells, and the measured OD value is in direct proportion to the cell activity.

FIG. 1 is a graph showing the effect of test samples on HaCaT cell activity. The results show that peptide A showed significant cytotoxicity at concentrations above 50ppm and peptide B was not toxic to cells at concentrations in the range of 100ppm, compared to the control group. Peptide a and peptide B were mixed at different ratios to give composition 1 (peptide a: peptide B: 1:4), composition 2 (peptide a: peptide B: 2.5), and composition 3 (peptide a: peptide B: 4:1), respectively. Composition 1 is not toxic to HaCaT cells and significantly promotes cell proliferation at high concentrations of 100 ppm; while the composition 2 generates toxicity to HaCaT cells at 100ppm, and the cell activity is obviously reduced; composition 3 was cytotoxic at both 50ppm and 100ppm concentrations.

From the above, it is found that when peptide a and peptide B are combined at different ratios and the ratio of the two is 1:4, the proliferation promoting effect on HaCaT cells is dose-dependent in the range of 100ppm, and the cytotoxicity of peptide a (pentapeptide-34 trifluoroacetate) can be avoided, so that the use concentration of composition 1 can be increased to achieve a more excellent technical effect while satisfying safety.

Example 2 Oxidation resistance test

2.1 samples to be tested

A peptide A; a peptide B; composition 1.

A sample to be tested is weighed, dissolved by 95% methanol to the concentration of L mg/mL, and then diluted by multiple times, wherein the diluted volume is 100 mu L, so that the final test concentrations are 1ppm, 10ppm, 50ppm, 100ppm and 200ppm respectively.

2.2 formulation of DPPH System

12.5mg of DPPH is weighed, dissolved in 95% methanol and transferred to a 50ml brown volumetric flask, the volume is constant, 250. mu.g/ml, and the mixture is stored away from light.

DPPH was diluted to 200 μ g/ml with 95% methanol at a dilution ratio (DPPH: 95% methanol-4: 1).

2.3 measuring light absorption

Taking 100 mu L of methanol solution of a sample to be detected and 100 mu L of 200 mu g/mL DPPH standard mixed solution, reacting for 90min in a dark place, and measuring absorbance Abs at 490nm_sampl. Absorbance at 490nm was measured using 100. mu.L methanol and 100. mu.L of 200. mu.g/mL DPPH standard as controls_control. Absorbance determination at 490nm with 100. mu.L of test substance and 100. mu.L of methanol as blank_blank。

The DPPH clearance of each sample was calculated as follows:

clearance rate avalaue (%) (Abs)_control-Abs_sampl)/(Abs_control-Abs_blank)*100％

2.4 test results

DPPH is a free radical, the solution is blue, and when DPPH is reduced, DPPH becomes yellow, so that the antioxidant property of the test sample can be quantitatively and qualitatively analyzed according to the change, and the test results are shown in table 1 and fig. 2 below.

Table 1 results of DPPH radical scavenging at different concentrations for each sample (n ═ 4)

The results show that composition 1 has a greater DPPH radical scavenging capacity than peptides A and B alone at the same concentration in the range of 10-200ppm and is dose dependent. Therefore, the composition 1 has a better antioxidant effect by compounding the peptide A and the peptide B according to the proportion of 1:4, and can play a synergistic effect.

Example 3 cell-promoting collagen production experiment

3.1 reagents and materials

Fetal bovine serum, DMEM medium, penicillin, streptomycin and sirius red staining kit.

3.2 instruments

Fluorescence microscope, CO₂An incubator.

3.3 cell lines

HSF human skin fibroblasts.

3.4 samples to be tested

Administration group:

peptide A, tested at 25ppm, 50ppm, respectively;

peptide B, tested at 25ppm, 50ppm, respectively;

composition 1, tested at 25ppm and 50ppm, respectively;

the administration group was dissolved with 0.5% DMSO solution.

control group: 0.5% DMSO.

3.5 Experimental methods

Taking a bottle of cells in an exponential growth phase, adding 0.25% trypsin digestion solution, digesting to make adherent cells fall off, and counting by 1-4 multiplied by 10⁵And (4) preparing cell suspension per mL. 10000 cells/well of cell suspension are taken out and inoculated on a 96-well plate after being properly diluted, and when the cells grow to be about 80 percent, a UV light aging model is established. The control group was not UV irradiated; the UV group and the administration group were repeatedly washed with an appropriate amount of PBS until colorless, and 50. mu.L PBS was added at 80J/cm³The UV lamp was used for irradiation, and the distance between the lamp source and the culture flask was 15 cm. After irradiation, PBS was discarded, the UV group was not dosed, and the dosing group was dosed with culture medium and drug diluted to 200. mu.L. The control group, UV group and administration group were continued at 37 ℃ with 5% CO₂Incubate in incubator for 24 h.

Dyeing with sirius red: after the incubated cells were fixed with 4% paraformaldehyde for 15min, they were washed with PBS. And adding a staining solution A and a staining solution B according to the operation instruction by using a sirius red staining kit, washing away floating color by using PBS, and observing under a microscope.

3.6 results

The sirius red can stain collagen of cells or tissues, the collagen can be stained red, and skin fibroblasts are rich in collagen, so that the qualitative analysis of the collagen and the morphological analysis of the cells can be performed.

The microphotograph of sirius red stained cells is shown in fig. 3. The results show that the control group of cells appeared distinctly fibrous, regularly arranged, densely (as shown in the dark part of FIG. 3); after UV irradiation, the cell density is greatly reduced. The phenomenon of cell density thinning caused by UV radiation can be improved to a certain extent by 25ppm of peptide A and peptide B. The 25ppm composition 1 can obviously increase the number of cells per unit area and promote the cells to generate more collagen. 50ppm of peptide A showed no improvement, even at a lower cell density than the UV group. 50ppm of peptide B can increase the unit collagen content, and 50ppm of composition 1 can obviously improve the cell density and has stronger capability of promoting cells to generate collagen.

In conclusion, the peptide A and the peptide B are compounded according to the proportion of 1:4, and the peptide A and the peptide B play a synergistic effect, so that the composition 1 has better light aging resistance than the single peptide A and the single peptide B, can obviously promote the generation of collagen, and effectively improves the skin quality, slows down the skin aging and repairs the aged skin.

The foregoing is a more detailed description of the present invention in connection with specific preferred embodiments thereof, and is not intended to limit the invention to the particular forms disclosed. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Reference documents:

[1]Daniel Schniertshauer et al.Accelerated Regeneration of ATP Level after Irradiation in Human Skin Fibroblasts by Coenzyme Q10.Photochemistry and Photobiology,2016,92:488–494.

[2]Inui M,et al.Mechanisms of inhibitory effects of CoQ10 on UVB-induced wrinkle formation in vitro and in vivo[J].Biofactors,2008,32(1-4):237-243.

[3]Narda M,et al.A novel water-based anti-aging suncare formulation provides multifaceted protection and repair against environmental aggressors:evidence from in vitro,ex vivo,and clinical studies[J].Clinical,cosmetic and investigational dermatology,2019,12:533.

Claims

1. a polypeptide composition having antioxidant and reparative effects, comprising:

2. The polypeptide composition having antioxidant and reparative effects according to claim 1, wherein the preparation of the polypeptide composition is selected from the group consisting of: essence, powder, tablet, capsule, emulsion, ointment, cream or gel.

3. Use of the polypeptide composition with antioxidant and repair action according to claim 1 for the preparation of an antioxidant cosmetic or pharmaceutical composition.

4. Use of the polypeptide composition with antioxidant and repair action according to claim 1 for the preparation of a cosmetic or pharmaceutical composition for the treatment, prevention or repair of skin ageing or photoaging.

5. The use according to claim 4, wherein the treatment, prevention or repair of skin aging or photoaging is the promotion of collagen production, reduction, prevention or treatment of facial wrinkles.