CN113667653B

CN113667653B - Transdermal total-absorption anti-aging nano-functional mask based on superoxide dismutase

Info

Publication number: CN113667653B
Application number: CN202110993092.8A
Authority: CN
Inventors: 赵聪; 司威
Original assignee: Guangzhou Wanqian Vermicelli Cosmetics Co ltd
Current assignee: Guangzhou Wanqian Vermicelli Cosmetics Co ltd
Priority date: 2021-08-24
Filing date: 2021-08-27
Publication date: 2024-01-05
Anticipated expiration: 2041-08-27
Also published as: CN113667653A

Abstract

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

Description

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

Technical Field

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

Background

Two main 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 during the removal of the foreign substances entering the body. However, while playing a positive role, if excessive active oxygen is generated for various reasons, it not only invades the own cell tissues but also combines with unsaturated fatty acids in the body to form lipid peroxide. In particular, the skin is aged by being combined with proteins and lipids to form oxides, and the oxides have a strong effect on the oxidation process of melanin to cause pigmentation.

Superoxide dismutase (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 organism oxidation and antioxidant balance. Depending on the metal prosthetic groups contained in SOD, it can be generally classified into Cu, zn-SOD, mn-SOD and Fe-SOD. The supplement of 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, the research on the application of SOD has been mainly focused on the extraction and purification of animal and plant SOD, most of domestic SOD products are prepared from animal blood, but due to the limited raw materials and the health safety, the product yield is limited, especially as the risk of malignant infectious diseases such as mad cow disease, avian influenza, foot-and-mouth disease and the like in the world is reported from time to time, the increase of the purity requirement also increases the cost of the product, especially due to the influence of mad cow disease, the EU prescribes that after the use of bovine blood SOD as a food additive is forbidden from 1 month in 1997, the utilization of genetic engineering is an effective way for widely opening SOD enzyme sources, reducing the cost and obtaining SOD with natural activity. However, SOD enzymes have problems of low genetic engineering yield, unstable activity and the like, and when applied to cosmetics, the SOD enzymes need to be increased in stability against a wider range of temperatures so as to facilitate transportation, so that development of SOD enzymes capable of overcoming the adverse factors is very necessary.

Disclosure of Invention

To solve the above technical problems, a first aspect of the present application provides a mutant Cu, zn-SOD enzyme. More specifically, the amino acid sequence of the mutant Cu, zn-SOD enzyme is shown as SEQ ID NO. 1.

In a second aspect of the present application, there is provided the use of a mutated Cu, zn-SOD enzyme for the preparation of an anti-ageing cosmetic, preferably a cosmetic which is a serum or a mask, more preferably a serum comprising the following components in mass percent:

the mutant Cu, zn and SOD enzyme is 2-5%, glycerin is 5-8%, hyaluronic acid is 0.01-2%, L-vitamin C is 0.01-2%, vitamin E is 2-5%, urea is 1-2%, propylene glycol is 0.5-2%, and the balance is deionized water.

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

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

1) According to the invention, the double mutation Cu, zn-SOD enzyme of H47A, G86W is obtained by carrying out amino acid mutation on the Cu, zn-SOD enzyme (NP-012638.1) of Saccharomyces cerevisiae (Saccharomyces cerevisiae), so that the thermal stability is obviously improved under the condition of keeping the activity, and the method has wide application value and economic value;

2) The invention ferments and produces the mutant Cu, zn-SOD enzyme by genetic engineering technology, and prepares the mutant Cu, zn-SOD enzyme into skin care products such as essence, mask and the like, and the products can restore the skin to a flexible young state and have obvious anti-aging effect.

Drawings

Comparison of enzyme Activity of the mutants of FIG. 1 before and after Heat treatment

Detailed Description

Example 1: mutant enzyme thermostability

The 4 key amino acid sites (S39G, H47A, G86W, H131Q) from Saccharomyces cerevisiae (Saccharomyces cerevisiae) Cu, zn-SOD enzyme (NP 012638.1) were selected for mutation engineering studies from NCBI.

And constructing Cu, zn-SOD enzyme site-directed transformation mutants by a one-step inverse PCR method, and transforming the correctly constructed site-directed mutation transformation plasmid into host cells after sequencing verification.

The 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) which are constructed correctly are subjected to streaking to separate single colonies, the single colonies are picked up to an LB culture medium (Kan) for culturing for 12 hours, inoculated to a TB fermentation culture medium (Kan) for fermentation culturing for 26 hours, and fermentation supernatants, namely, the mutant recombinases, are subjected to water bath heat preservation at a temperature of pH7.0 and a temperature of 60 ℃ for 15 minutes, and enzyme activities before and after heat treatment are detected respectively. The original enzyme activity which was not mutated and which was not heat-treated at 60℃was taken as 100% (control group). As shown in FIG. 1, the residual enzyme activity of the S39G+H243 47A, H47A+G86W mutant was higher than that of the control bacterium after heat treatment. The h47a+g86W mutant showed a significant increase in thermostability.

Example 2: verification of antioxidant Activity

Test sample: the wild Cu, zn-SOD enzyme and H47A+G86W double mutation Cu, zn-SOD enzyme are respectively mixed with ultrapure water to be diluted into a test solution with the mass fraction of 5 percent.

Hydroxyl radical scavenging experiments: the absorbance values of each group of mixed solutions were measured at 536nm using spectrophotometry conventional in the art, and were recorded As, the absorbance values were measured with distilled water instead of the sample solution As a blank group, as Ab, and distilled water instead of H ₂ O ₂ As a damage group, its absorbance value An was measured, and the hydroxyl radical clearance of the test sample was calculated according to the following formula: hydroxyl radical clearance (%) = [ (As-An)/(Ab-An)]X 100% and taking the average of three measurements. The test results are shown in Table 1.

DPPH radical scavenging experiment: taking 3mL of DPPH-absolute ethyl alcohol solution with the concentration of 0.2mmol/L in a cuvette, adding 1mL of test sample, mixing uniformly, keeping away from light at room temperature for 30min, measuring absorbance at 517nm wavelength, marking the absorbance value as A1, adding 3mL of absolute ethyl alcohol solution and 1mL of test sample solution, marking the absorbance value as A2, and adding 3mL of DPPH-absolute ethyl alcohol solution and 1mL of absolute ethyl alcohol solution, marking the absorbance value as A3. DPPH radical scavenging was calculated as follows: DPPH radical clearance (%) = [1- (A1-A2)/A3 ] ×100%, the average of three measurements was taken. The test results are shown in Table 1.

TABLE 1

From the experimental results in Table 1, the H47A+G86W double mutant Cu, zn-SOD enzyme has improved hydroxy free radical clearance and DPPH free radical clearance compared with wild type, which shows that the mutant Cu, zn-SOD enzyme with improved thermal stability can effectively remove free radicals and has the effects of oxidation resistance and aging resistance.

EXAMPLE 3 skin elasticity and moisturizing Performance test of mutant preparations

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+g168w double mutation Cu, zn-sodase 5%, glycerol 5%, hyaluronic acid 1.5%, l-vitamin C1%, vitamin E5%, urea 1%, propylene glycol 0.5%, and the balance deionized water.

Control group 1 was a double mutant Cu, zn-SOD enzyme with H47A+G86W, replaced with wild Cu, zn-SOD enzyme.

Control group 2 was not added with Cu, zn-SOD enzyme.

Test population: 60 healthy volunteers with an average age of 40.+ -.2 were selected and randomly divided into three groups of 20 persons each, each of which was applied earlier than the face, and the subjects were not able to apply any other cosmetics at the experimental site during the experiment.

The testing method comprises the following steps:

the change in skin moisture content was tested using a moisture tester (corneometer cm 825) to evaluate the degree of moisture retention of the skin by the three samples; the change in skin elasticity value was measured with a skin elasticity tester (Cutometer dual MPA, 580). The results are shown in Table 2 below (average of three measurements):

TABLE 2

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

SEQUENCE LISTING

<110> Shenzhen City not two Biotech Co., ltd

<120> a mutant superoxide dismutase and use thereof in the preparation of 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 mutant Cu, zn-SOD enzyme, wherein the amino acid sequence of the mutant Cu, zn-SOD enzyme is shown as SEQ ID NO. 1.

2. Use of a mutated Cu, zn-SOD enzyme according to claim 1 for the preparation of anti-ageing cosmetics.

3. The use according to claim 2, wherein the cosmetic product is a serum or a mask.

4. The use according to claim 3, wherein the serum comprises the following components in percentage by mass:

the mutant 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%, 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, wherein the product is a serum or a mask, and the active ingredient of the product comprises the mutated Cu, zn-SOD enzyme of claim 1.

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