CN113667653A

CN113667653A - Transdermal full-absorption type anti-aging nano functional mask based on superoxide dismutase

Info

Publication number: CN113667653A
Application number: CN202110993092.8A
Authority: CN
Inventors: 赵聪; 司威
Original assignee: Shenzhen Buer Biotechnology Co ltd
Current assignee: Guangzhou Wanqian Vermicelli Cosmetics Co ltd
Priority date: 2021-08-24
Filing date: 2021-08-27
Publication date: 2021-11-19
Anticipated expiration: 2041-08-27
Also published as: CN113667653B

Abstract

The invention provides a novel superoxide dismutase mutant, which shows the functions of improving skin aging and reducing wrinkles and has wide application prospect. Further, the invention also provides a transdermal full-absorption type anti-aging nano functional mask containing the mutant.

Description

Transdermal full-absorption type anti-aging nano functional mask based on superoxide dismutase

Technical Field

The invention belongs to the field of biological enzymes, and particularly relates to mutated superoxide dismutase and a transdermal full-absorption type anti-aging nano functional mask based on the superoxide dismutase.

Background

Two major causes of skin aging are due to sunlight irradiation and the invasion of active oxygen. The active oxygen removes foreign substances by decomposing the foreign substances in the process of removing the foreign substances introduced into the body. However, if too much active oxygen is generated for various reasons while playing a positive role, it not only attacks the own cell tissue but also combines with unsaturated fatty acids in the body to form lipid peroxides. In particular, it binds to proteins and lipids in the skin to form oxides, which causes skin aging, and it acts strongly on the oxidation process of melanin, causing pigmentation.

Superoxide Dismutase (SOD) is an antioxidant metalloenzyme existing in organisms, can catalyze Superoxide anion free radical disproportionation to generate oxygen and hydrogen peroxide, and plays a vital role in the balance of oxidation and antioxidation of the organisms. Generally, SOD can be classified into Cu, Zn-SOD, Mn-SOD and Fe-SOD according to the metal prosthetic group contained in SOD. The supplement of the exogenous SOD has the functions of delaying skin aging, resisting oxidation and removing color spots, and can be widely added into various cosmetics or skin care products.

Since the eighties of the last century in China, the application research of SOD mainly focuses on the extraction and purification of animal and plant SOD, most of domestic SOD products are prepared from animal blood, but the product yield is limited due to the limited raw materials, the sanitary safety and the like, particularly, with the occasional reports of malignant infectious diseases such as mad cow disease, avian influenza, foot and mouth disease and the like all over the world, the risk of animal-derived blood products is increased, the product cost is increased due to the increased purity requirement, particularly, due to the influence of mad cow disease, the European Union stipulates that after the use of bovine blood SOD as a food additive is forbidden from 1 month in 1997, the use of genetic engineering is an effective way of widely opening SOD enzyme sources, reducing the cost and obtaining SOD with natural activity. However, SOD enzymes have problems of low yield of genetic engineering, unstable activity, etc., and it is necessary to increase their stability to a wider range of temperatures for easy transportation when applied to cosmetics, so that it is necessary to develop an SOD enzyme capable of overcoming the above disadvantages.

Disclosure of Invention

In order to solve the above technical problems, the present application provides, in a first aspect, a mutated Cu, Zn-SOD enzyme. More specifically, the amino acid sequence of the mutant Cu, Zn-SOD enzyme is shown in SEQ ID NO. 1.

In a second aspect of the present application, there is provided a use of a mutated Cu, Zn-SOD enzyme in preparing an anti-aging cosmetic, preferably, the cosmetic is a essence or a mask, and more preferably, the essence comprises the following components by mass:

2-5% of mutated Cu, Zn-SOD enzyme, 5-8% of glycerol, 0.01-2% of hyaluronic acid, 0.01-2% of levorotatory vitamin C, 2-5% of vitamin E, 1-2% of urea, 0.5-2% of propylene glycol and the balance of deionized water.

In a third aspect of the application, an anti-aging facial mask containing the mutated Cu, Zn-SOD enzyme is provided, and the preparation method of the facial mask is to soak a facial mask material with the essence or add the essence into the facial mask material. More preferably, the facial mask is a nano facial mask.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1) according to the invention, amino acid mutation is carried out on Cu and Zn-SOD enzymes (NP-012638.1) of Saccharomyces cerevisiae (Saccharomyces cerevisiae), so that the H47A and G86W double-mutation Cu and Zn-SOD enzymes are obtained, and under the condition of activity maintenance, the thermal stability is obviously improved, and the application value and the economic value are wide;

2) the mutant Cu, Zn-SOD enzyme is produced by fermentation through a genetic engineering technology and is prepared into skin care products such as essence, facial masks and the like, and the products can restore the elasticity of the skin and have obvious anti-aging effect.

Drawings

FIG. 1 comparison of enzyme activities of respective mutants before and after heat treatment

Detailed Description

Example 1: thermostability of mutant enzymes

Cu and Zn-SOD enzymes (NP-012638.1) from Saccharomyces cerevisiae (Saccharomyces cerevisiae) are obtained from NCBI, and 4 key amino acid sites (S39G, H47A, G86W and H131Q) on the active site of the Cu and Zn-SOD enzymes are selected for mutation transformation research.

And (3) constructing a Cu, Zn-SOD enzyme site-directed modification mutant by using a one-step reverse PCR method, and transforming the correctly constructed site-directed mutation modification plasmid into a host cell after sequencing verification.

The single colony is separated from the correctly constructed recombinant bacteria of each mutation site (group 1: S39G + H47A, group 2: S39G + G86W, group 3: S39G + H131Q, group 4: H47A + G86W, group 5: H47A + H131Q and group 6: G86W + H131Q) by plate and streak, the single colony is picked up to be cultured in LB culture medium (Kan) for 12H, inoculated to TB fermentation culture medium (Kan) for fermentation for 26H, and fermentation supernatant, namely each mutant recombinase is taken to be subjected to heat preservation and heat treatment in water bath at the temperature of 7.0 and 60 ℃ for 15min, and the enzyme activities before and after the heat treatment are respectively detected. The original enzyme activity without mutation and without heat treatment at 60 ℃ was taken as 100% (control group). As shown in FIG. 1, the residual enzyme activity of the S39G + H47A and H47A + G86W mutants after heat treatment is higher than that of the control bacteria. The H47A + G86W mutant showed a significant increase in thermostability.

Example 2: verification of antioxidant Activity

Test samples: wild type Cu, Zn-SOD enzyme and H47A + G86W double mutation Cu, Zn-SOD enzyme are respectively mixed with ultrapure water and diluted into test solution with mass fraction of 5%.

Hydroxyl radical scavenging experiments: respectively measuring absorbance values of the mixed solutions at 536nm by using a spectrophotometric method conventional in the field, recording As, replacing a sample solution with distilled water As a blank group, recording As Ab, replacing H with distilled water₂O₂And measuring the absorbance value An of the damage group, and calculating the hydroxyl radical clearance of the test sample according to the following formula: hydroxyl radical clearance (%) [ (As-An)/(Ab-An)]X 100%, the average of three measurements was taken. The test results are shown in table 1.

DPPH free radical scavenging experiments: taking 3mL of 0.2mmol/L DPPH-absolute ethanol solution, adding 1mL of test sample, mixing uniformly, keeping out of the sun for 30min at room temperature, measuring the absorbance at the wavelength of 517nm, and recording the absorbance value as A1, adding 3mL of absolute ethanol solution and 1mL of test sample solution, and recording the absorbance values as A2, and adding 3mL of DPPH-absolute ethanol solution and 1mL of absolute ethanol solution, and recording the absorbance values as A3. DPPH radical clearance was calculated as follows: DPPH radical clearance (%) [1- (a1-a2)/A3] × 100%, average of three measurements. The test results are shown in table 1.

TABLE 1

The experimental results in table 1 show that the hydroxyl radical clearance and DPPH radical clearance of the H47A + G86W double-mutation Cu and Zn-SOD enzyme of the invention are improved relative to the wild type, which indicates that the mutant Cu and Zn-SOD enzyme with improved thermal stability can effectively clear away the free radicals and has the effects of oxidation resistance and aging resistance.

Example 3 mutant preparations skin elasticity and moisturization function test

The H47A + G86W double-mutation Cu, Zn-SOD enzyme is prepared into skin care essence, and the skin care essence comprises the following components in percentage by mass:

H47A + G86W double-mutation Cu, 5% of Zn-SOD enzyme, 5% of glycerol, 1.5% of hyaluronic acid, 1% of levorotatory vitamin C, 5% of vitamin E, 1% of urea, 0.5% of propylene glycol and the balance of deionized water.

The control group 1 is prepared by replacing H47A + G86W double-mutation Cu, Zn-SOD enzyme with wild-type Cu, Zn-SOD enzyme.

Control 2 was prepared without addition of Cu, Zn-SOD enzyme.

Testing the population: 60 healthy volunteers with an average age of 40 + -2 were selected, randomized into three groups of 20 individuals, each applied with the above samples earlier and later on the face, and the subjects could not apply any other cosmetic product on the experimental part during the experiment.

The test method comprises the following steps:

testing the change of the moisture content of the skin by using a moisture tester (CorneometerCM825) to evaluate the moisture retention degree of the three samples on the skin; the change in the value of the skin elasticity was measured with a skin elasticity tester (Cutomer dual MPA 580). The results are given in table 2 below (mean of three measurements):

TABLE 2

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

SEQUENCE LISTING

<110> Shenzhen Wen Biotech Limited

<120> mutant superoxide dismutase and application thereof in preparing anti-aging products

<130> 1

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 154

<212> PRT

<213> Artificial sequence

<400> 1

Met Val Gln Ala Val Ala Val Leu Lys Gly Asp Ala Gly Val Ser Gly

1 5 10 15

Val Val Lys Phe Glu Gln Ala Ser Glu Ser Glu Pro Thr Thr Val Ser

20 25 30

Tyr Glu Ile Ala Gly Asn Ser Pro Asn Ala Glu Arg Gly Phe Ala Ile

35 40 45

His Glu Phe Gly Asp Ala Thr Asn Gly Cys Val Ser Ala Gly Pro His

50 55 60

Phe Asn Pro Phe Lys Lys Thr His Gly Ala Pro Thr Asp Glu Val Arg

65 70 75 80

His Val Gly Asp Met Trp Asn Val Lys Thr Asp Glu Asn Gly Val Ala

85 90 95

Lys Gly Ser Phe Lys Asp Ser Leu Ile Lys Leu Ile Gly Pro Thr Ser

100 105 110

Val Val Gly Arg Ser Val Val Ile His Ala Gly Gln Asp Asp Leu Gly

115 120 125

Lys Gly Asp Thr Glu Glu Ser Leu Lys Thr Gly Asn Ala Gly Pro Arg

130 135 140

Pro Ala Cys Gly Val Ile Gly Leu Thr Asn

145 150

Claims

1. A mutated Cu, Zn-SOD enzyme, the amino acid sequence of the mutated Cu, Zn-SOD enzyme is shown in SEQ ID NO. 1.

2. Use of the mutated Cu, Zn-SOD enzyme according to claim 1 for the preparation of anti-aging cosmetic.

3. The use of claim 2, wherein the cosmetic is a serum or a mask.

4. The application of claim 3, wherein the essence comprises the following components in percentage by mass:

the mutated Cu, Zn-SOD enzyme of claim 1, 2-5%, glycerol 5-8%, hyaluronic acid 0.01-2%, L-vitamin C0.01-2%, vitamin E2-5%, urea 1-2%, propylene glycol 0.5-2%, and the balance deionized water.

5. The use according to claim 3, wherein the mask is prepared by soaking the mask material with the essence of claim 4 or adding the essence to the mask material.

6. An anti-aging product, which is a serum or a mask, and the active ingredient of which comprises the mutated Cu, Zn-SOD enzyme of claim 1.

7. The anti-aging product according to claim 6, wherein the product is a nano-mask.