CN114983900A - Preparation method of emulsion with antioxidant and repairing effects - Google Patents

Abstract

The invention discloses a preparation method of an emulsion with antioxidant and repairing effects, and belongs to the technical field of cosmetic preparation. The coating comprises the following components in percentage by mass: parthenia italica extract, antibacterial peptide fusion protein Cb-SOD-Cb, isododecane, centella asiatica extract, edelweiss extract, purslane extract, tetrandra extract, tetrahydromethylpyrimidine carboxylic acid, dactylospermum odoratum extract, hydrolyzed collagen, camellia extract, oligopeptide-3, glycerol glucoside, betaine, panthenol, sodium hyaluronate, oligopeptide-5, allantoin, butanediol, squalane, acrylic acid (ester)/beheneth-25 methacrylate copolymer, glycerol, cetyl ethylhexanoate, oat kernel oil, and sorbitan-30 tetraisostearate. The emulsion prepared by the method can enhance the activity of skin cells, remove free radicals, promote and tighten skin, inhibit the accumulation of skin melanin, and build delicate and transparent skin color.

Description

Preparation method of emulsion with antioxidant and repairing effects

Technical Field

The invention belongs to the technical field of cosmetic preparation, and particularly relates to a preparation method of an emulsion with antioxidant and repairing effects.

Background

With the continuous improvement of the living standard of human beings, the demand of people on skin health is higher and higher, especially the protection of the exposed skin, such as the face, the neck, the limbs and the like. When the skin is exposed to ultraviolet radiation for a long time in outdoor environment, the skin may be damaged, and serious people may have skin injury, acne, melanin accumulation, etc.

At present, the main preparation forms of the cosmetics for treating the condition are concentrated in products such as essence water, cream, facial masks and the like, the formula components of the products are complex, the stability of a plurality of active substances after being dissolved is poor, the biological activity is easy to lose, and in addition, some preservatives can also increase stimulus sources, so that the effect of the products is influenced.

Disclosure of Invention

In view of this, the present invention provides a method for preparing an emulsion with antioxidant and repairing effects.

In order to achieve the purpose, the invention provides the following technical scheme:

the emulsion with the effects of resisting oxidation and repairing comprises the following components in percentage by mass:

0.001-0.01% of Parthenia italica extract, 0.001-0.05% of antibacterial peptide fusion protein Cb-SOD-Cb, 0.5-1.5% of isododecane, 0.05-0.2% of centella asiatica extract, 0.01-0.1% of edelweiss extract, 0.005-0.02% of purslane extract, 0.001-0.01% of Stephania tetrandra extract, 0.01-0.1% of tetrahydro-methylpyrimidine carboxylic acid, 0.001-0.01% of Strychnos rupestris extract, 0.05-0.2% of hydrolyzed collagen, 0.005-0.02% of camellia extract, 0. 30.0001-0.001% of oligopeptide-oligopeptide, 0.01-0.2% of glycerol glucoside, 0.1-0.5% of betaine, 0.15% of panthenol, 0.05-0.2% of sodium hyaluronate, 0. 50.0001-0.001% of oligopeptide, 0.01-0.1% of allantoin, 2-7% of butanediol, 1-4% of squalane, 0.25% of acrylic acid (polyether) and 0.6-1.2% of behenyl acrylate copolymer, 1.0-2.0% of glycerin, 1.0-2.0% of cetyl ethyl hexanoate, 0.2-0.4% of oat kernel oil, 0.8-1.2% of sorbitan polyether-30 tetraisostearate and the balance of deionized water.

Further, the emulsion with the antioxidant and repairing effects comprises the following components in percentage by mass:

0.001% of a chamomile extract, 0.001% of an antibacterial peptide fusion protein Cb-SOD-Cb, 1% of isododecane, 0.1% of a centella asiatica extract, 0.05% of a leontopodium alpinum extract, 0.01% of a purslane extract, 0.005% of a tetrandra extract, 0.05% of tetrahydro-methyl pyrimidinecarboxylic acid, 0.002% of a brachycephala extract, 0.2% of hydrolyzed collagen, 0.01% of a camellia japonica extract, 0.8978% of an oligopeptide-30.0002%, 0.01% of glycerol glucoside, 0.2% of betaine, 0.15% of panthenol, 0.1% of sodium hyaluronate, 0.3532% of an oligopeptide-50.0002%, 0.05% of allantoin, 5% of butylene glycol, 3% of squalane, 0.9% of an acrylic acid (ester)/beheneth-25 methacrylate copolymer, 1.5% of glycerol, 1.5% of cetyl ethylhexanoate, 0.3% of oat kernel oil, 1.30% of sorbitan-tetraisostearate, and the balance deionized water.

The invention also provides a preparation method of the emulsion with the effects of oxidation resistance and repair, which comprises the following steps:

heating deionized water to 80-83 deg.C, sequentially adding butanediol, squalane, acrylic acid (ester)/beheneth-25 methacrylate copolymer, glycerol, cetyl ethyl hexanoate, oat kernel oil and sorbitan polyether-30 tetraisostearate, stirring, maintaining the temperature until the raw materials are completely dissolved, and cooling;

cooling to the required temperature, sequentially adding a Parthenium italicum extract, an antibacterial peptide fusion protein Cb-SOD-Cb, isododecane, a centella asiatica extract, a leontopodium alpinum extract, a purslane extract, a tetrandra root extract, tetrahydro-methylpyrimidine carboxylic acid, a streptoverticillium vesiculosum extract, hydrolyzed collagen, a camellia extract, oligopeptide-3, glycerol glucoside, betaine, panthenol, sodium hyaluronate, oligopeptide-5 and allantoin into the solution, stirring, and cooling to room temperature to obtain the emulsion with the effects of resisting oxidation and repairing.

Further, the heat preservation time is 20 min.

Further, the temperature reduction treatment means that the temperature is reduced to 30-35 ℃.

The antibacterial peptide fusion protein Cb-SOD-Cb is a multifunctional polypeptide, has antibacterial property and the activity of superoxide dismutase for removing free radicals, can effectively remove the damage of the free radicals to the skin, and simultaneously inhibits bacteria at the acne part.

Compared with the prior art, the invention has the beneficial effects that:

squalane is a hydrocarbon oil obtained by hydrogenating squalene extracted from deep sea shark liver, and has effects in promoting repair of epidermis and effectively forming natural protective membrane; the plant extracts such as the centella asiatica extract, the alpine edelweiss extract, the purslane extract, the tetrandra root extract and the like have the effects of resisting oxidation, diminishing inflammation and preventing allergy, and peptide molecules such as oligopeptide-3, oligopeptide-5 and the like can effectively promote cell proliferation, promote metabolism of newcastle, activate cell activity, promote the generation of elastin and collagen, and achieve the effects of repairing damaged skin, lightening skin wrinkles, reducing pigmentation and the like. Long-chain hydrophilic molecules such as glycerol glucoside, sodium hyaluronate and the like can better retain water and enhance moisture retention. The effective components are prepared into emulsion, so that fat-soluble and water-soluble functional components can be better fused through an emulsification technology to jointly act on the skin, and meanwhile, the emulsion has a better skin moistening effect and can promote the transdermal absorption of the functional components.

The antioxidant and repairing emulsion prepared by the invention can enhance the activity of skin cells, clear free radicals, promote and tighten skin, inhibit the accumulation of skin melanin, stimulate the generation of skin collagen, maintain the water-oil balance of skin and construct delicate and white skin color.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a schematic diagram of the structure of a Cb-SOD-Cb fusion protein;

FIG. 2 is a diagram showing the structure prediction of a Cb-SOD-Cb fusion protein; wherein the green color: SOD, blue: linker, red: cecropin b;

FIG. 3 is a schematic diagram of the recombinant vector pET 26-Cb-SOD-Cb;

FIG. 4 is a SDS-PAGE image of Cb-SOD-Cb protein and recombinant bacteria crushing precipitation; wherein M: standard molecular weight marker, 1: recombinant Cb-SOD-Cb protein after renaturation (0.5mg/mL), 2: recombinant Cb-SOD-Cb protein after renaturation (0.5mg/mL), 3: crushing the recombinant bacteria, and centrifuging to obtain a precipitate (an inclusion body containing Cb-SOD-Cb);

FIG. 5 is a graph of the effect of different emulsion contents on cell proliferation rate;

FIG. 6 is a comparison of before and after use of the product of example 1 by a patient;

figure 7 is a comparison of before and after patient use of the product of example 1.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, 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 invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

An emulsion with antioxidant and repairing effects comprises the following components in percentage by mass:

0.001-0.01% of extract of Helichrysum ITALICUM (Helichrysum ITALICUM), 0.001-0.05% of antibacterial peptide fusion protein Cb-SOD-Cb, 0.5-1.5% of isododecane, 0.05-0.2% of extract of centella asiatica (CENTELLA ASIATICA), 0.01-0.1% of extract of Leontopodium ALPINUM, 0.005-0.02% of extract of Portulaca OLERACEA, 0.001-0.01% of extract of Stephania tetrandra (STEPHANIA TETRANDRA), 0.01-0.1% of tetrahydromethylpyrimidine carboxylic acid, 0.001-0.01% of extract of Cystoseira olecia, 0.05-0.2% of hydrolyzed collagen, 0.005-0.02% of extract of camellia japonica, 0. 30.0001-0.001%, 0.01-0.2% of glycerol glucoside, 0.1-0.5% of betaine, 0.15.05-0.05% of hydrolyzed collagen, 0.05-0.05% of sodium hyaluronate, 0.01-0.1% of extract of hyaluronic acid, 0.1-0.02% of oligopeptide-0.001%, 0.5% of oligopeptide-0.001% of oligopeptide, 0.05% of sodium hyaluronate, 0.05-0.1% of sodium hyaluronate, 0.1% of oligopeptide-0.5% of oligopeptide, 2-7% of butanediol, 1-4% of squalane, 0.6-1.2% of acrylic acid (ester)/behenyl alcohol polyether-25 methacrylate copolymer, 1.0-2.0% of glycerol, 1.0-2.0% of cetyl alcohol ethyl hexanoate, 0.2-0.4% of oat (AVENA SATIVA) kernel oil, 0.8-1.2% of sorbierite-30 tetraisostearate and the balance of deionized water.

Preferably, the emulsion with the antioxidant and repairing effects comprises the following components in percentage by mass:

0.001% of a chamomile extract, 0.001% of an antibacterial peptide fusion protein Cb-SOD-Cb, 1% of isododecane, 0.1% of a centella asiatica extract, 0.05% of a leontopodium alpinum extract, 0.01% of a purslane extract, 0.005% of a tetrandra extract, 0.05% of tetrahydro-methyl pyrimidinecarboxylic acid, 0.002% of a brachycephala extract, 0.2% of hydrolyzed collagen, 0.01% of a camellia japonica extract, 0.8978% of an oligopeptide-30.0002%, 0.01% of glycerol glucoside, 0.2% of betaine, 0.15% of panthenol, 0.1% of sodium hyaluronate, 0.3532% of an oligopeptide-50.0002%, 0.05% of allantoin, 5% of butylene glycol, 3% of squalane, 0.9% of an acrylic acid (ester)/beheneth-25 methacrylate copolymer, 1.5% of glycerol, 1.5% of cetyl ethylhexanoate, 0.3% of oat kernel oil, 1.30% of sorbitan-tetraisostearate, and the balance deionized water.

The invention also provides a preparation method of the emulsion with the effects of oxidation resistance and repair, which comprises the following steps:

heating deionized water to 80-83 deg.C, sequentially adding butanediol, squalane, acrylic acid (esters)/beheneth-25 methacrylate copolymer, glycerol, cetyl ethyl hexanoate, oat kernel oil and sorbitan polyether-30 tetraisostearate, stirring, maintaining the temperature for 20min until the raw materials are completely dissolved, and cooling to 30-35 deg.C;

cooling to the required temperature, sequentially adding a Parthenium italicum extract, an antibacterial peptide fusion protein Cb-SOD-Cb, isododecane, a centella asiatica extract, a leontopodium alpinum extract, a purslane extract, a tetrandra root extract, tetrahydro-methylpyrimidine carboxylic acid, a streptoverticillium vesiculosum extract, hydrolyzed collagen, a camellia extract, oligopeptide-3, glycerol glucoside, betaine, panthenol, sodium hyaluronate, oligopeptide-5 and allantoin into the solution, stirring, and cooling to room temperature to obtain the emulsion with the effects of resisting oxidation and repairing.

After each component is added, the next component is added after the components are uniformly stirred.

The antibacterial peptide fusion protein Cb-SOD-Cb is a multifunctional polypeptide, has antibacterial property and the activity of superoxide dismutase for removing free radicals, can effectively remove the damage of the free radicals to the skin, and simultaneously inhibits bacteria at the acne part.

The preparation method of the antibacterial peptide fusion protein Cb-SOD-Cb comprises the following steps:

(1) constructing a recombinant Cb-SOD-Cb antioxidant antibacterial protein expression vector: the 3' end of the nucleotide sequence of cecropin B is connected with a nucleotide sequence of a DQYTD flexible peptide segment, then connected with a nucleotide sequence for coding a SOD protein of human zinc-copper superoxide dismutase, then connected with a nucleotide sequence of a DQYTD flexible peptide segment, and then connected with a nucleotide sequence of cecropin B to form a cecropin B-DQYTD-SOD-DQYD-cecropin B cross-connecting structure, which is abbreviated as Cb-SOD-Cb, and the recombinant gene structure is shown in figure 1.

A space structure (figure 2) obtained by 3D structural simulation (https:// zhangggroup. org/I-TASSER /) of the recombinant protein shows that superoxide dismutase SOD and cecropin B at two ends form a free open state under the connection of flexible peptide, and the respective functional regions cannot be influenced, and the recombinant protein can attack the outer membrane of bacteria without damaging the space structure of the SOD, so that the aim of resisting bacteria is fulfilled.

The Cb-SOD-Cb gene sequence is connected to an escherichia coli expression vector pET26b (+) through Nde I and XhoI to obtain a recombinant vector pET26-Cb-SOD-Cb, and the structure of the vector is shown in figure 3. The synthetic gene was made by Shanghai Czeri bioengineering, Inc.

Nucleotide sequence of cecropin B (SEQ ID NO. 1):

AAGTGGAAAGTATTTAAAAAGATAGAAAAAATGGGTCGTAACATTCGTAACGGCATTGTAAAGGCGGGTCCGGCGATCGCCGTTCTGGGCGAAGCTAAAGCTCTG。

nucleotide sequence (SEQ ID NO.2) encoding human zinc-copper superoxide dismutase SOD protein:

GCAACCAAGGCGGTGTGCGTCTTGAAGGGTGACGGTCCGGTGCAGGGTATTATCAACTTCGAGCAGAAAGAGAGCAATGGCCCGGTGAAAGTTTGGGGTTCTATCAAAGGCTTGACCGAAGGTCTGCATGGTTTTCACGTGCACGAGTTCGGCGACAATACCGCGGGCTGCACCAGCGCAGGTCCGCATTTTAACCCGCTTTCGCGCAAACACGGCGGCCCAAAAGATGAGGAGCGCCACGTTGGTGACCTCGGCAACGTGACTGCGGATAAGGACGGCGTTGCCGACGTGTCCATTGAAGATAGCGTTATCAGCCTGTCCGGTGATCATTGCATCATTGGCCGTACGCTGGTTGTCCACGAGAAGGCGGACGATCTGGGAAAGGGCGGTAATGAAGAAAGCACCAAGACGGGTAACGCGGGTTCTCGTCTGGCTTGTGGTGTTATCGGCATCGCACAA。

DQYTD flexible peptide stretch nucleotide sequence (SEQ ID NO. 3): GATCAATATACTGAC are provided.

Cb-SOD-Cb gene sequence (SEQ ID NO. 4):

ATGAAGTGGAAAGTATTTAAAAAGATAGAAAAAATGGGTCGTAACATTCGTAACGGCATTGTAAAGGCGGGTCCGGCGATCGCCGTTCTGGGCGAAGCTAAAGCTCTGGACCAATACACCGATGCAACCAAGGCGGTGTGCGTCTTGAAGGGTGACGGTCCGGTGCAGGGTATTATCAACTTCGAGCAGAAAGAGAGCAATGGCCCGGTGAAAGTTTGGGGTTCTATCAAAGGCTTGACCGAAGGTCTGCATGGTTTTCACGTGCACGAGTTCGGCGACAATACCGCGGGCTGCACCAGCGCAGGTCCGCATTTTAACCCGCTTTCGCGCAAACACGGCGGCCCAAAAGATGAGGAGCGCCACGTTGGTGACCTCGGCAACGTGACTGCGGATAAGGACGGCGTTGCCGACGTGTCCATTGAAGATAGCGTTATCAGCCTGTCCGGTGATCATTGCATCATTGGCCGTACGCTGGTTGTCCACGAGAAGGCGGACGATCTGGGAAAGGGCGGTAATGAAGAAAGCACCAAGACGGGTAACGCGGGTTCTCGTCTGGCTTGTGGTGTTATCGGCATCGCACAAGATCAATATACTGACAAGTGGAAAGTCTTTAAAAAAATCGAGAAAATGGGTCGCAACATCCGTAATGGTATTGTTAAGGCGGGCCCAGCGATCGCGGTGCTCGGCGAGGCGAAGGCGCTGTAA。

amino acid sequence of cecropin B (SEQ ID NO. 5):

KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL。

human zinc-copper superoxide dismutase SOD protein amino acid sequence (SEQ ID NO. 6):

ATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ。

Cb-SOD-Cb amino acid sequence (SEQ ID NO. 7):

MKWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKALDQYTDATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQDQYTDKWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL。

(2) screening and expressing a recombinant protein Cb-SOD-Cb expression strain BL21(DE 3):

the recombinant vector pET26-Cb-SOD-Cb obtained in the step (1) is used for transforming an escherichia coli strain BL21(DE3) by a heat shock method, and positive transformants screened by an LB solid medium plate containing 50 mug/mL kanamycin are cultured in an LB medium until OD is reached ₆₀₀ When the expression level was 0.6 to 0.8, IPTG-induced expression was carried out, and after 3 to 6 hours of induction expression, cells were collected and the protein expression level was examined by SDS-PAGE.

The specific operation is as follows: the inducible positive transformant selected in step (1), i.e., pET26-Cb-SOD-Cb/BL21(DE3), was streaked on an LB plate for 1 day, and a single colony was inoculated into a 5mL centrifuge tube containing 1mL of LB medium containing 50. mu.g/mL kanamycin and cultured overnight at 37 ℃ at 200 rpm. The next day, the cells were inoculated at 1% inoculum size into 500mL of LB medium containing 50. mu.g/mL of kanamycin, and cultured at 37 ℃ and 200rpm to OD ₆₀₀ When the concentration is 0.8, isopropyl thiogalactoside (IPTG) is added to the mixture to a final concentration of 1 mmol, the recombinant protein is induced to express at 37 ℃ and 200rpm for 5 hours, and then the mixture is centrifuged at 4000rpm for 10min to collect the bacterial cells. Wherein the LB culture medium is prepared from 5g of yeast powder, 10g of peptone and 5g of sodium chloride, adding water to 1000mL, and adjusting the pH value to 7.2-7.4.

(3) Preparation of recombinant Cb-SOD-Cb antioxidant antibacterial protein

After the escherichia coli strain with high expression of Cb-SOD-Cb antioxidant antibacterial protein screened in the step (2) is subjected to induction expression, 6000rpm/min is centrifuged for 10min, thalli are collected and placed at the temperature of minus 20 ℃, freeze thawing is carried out repeatedly for three times, lysozyme is added to enable the final concentration of lysozyme to be 100 mu g/mL, incubation is carried out at the temperature of 37 ℃ for 20min, ultrasonic crushing and ultrasonic crushing conditions are as follows: 300w, 4s, 6s, 20 min. Bacteria breaking buffer solution: 20nM PBS (150 nM NaCl), pH 7.4. After the crushing is finished, the mixture is centrifuged at 20000rpm and 4 ℃ for 20min, and the precipitate (most of the inclusion body protein) is left.

Washing impurities by inclusion bodies: the inclusion body pellet was washed sequentially with Wash I (50mM Tris-HCl, 10mM EDTA, 5 wt% glycerol, 1 wt% TritonX-100, 100 mM. beta. -mercaptoethanol, pH 8.0), Wash II (Wash I +0.3M Urea), Wash III (Wash I +0.6M Urea).

Inclusion body solubilization (denaturation): dissolving the inclusion body precipitate according to the mass ratio of 2: 1 adding pure water and uniformly mixing by using a small homogenizer, and mixing uniformly according to the mass ratio of 1: adding 8M urea solution into the mixture 25, placing the mixture on a magnetic stirrer, stirring the mixture for 4 hours to obtain a relatively clear solution, centrifuging the solution at 20000rpm and 4 ℃ for 20min, and taking clear supernatant fluid, namely the denatured liquid of the Cb-SOD-Cb antioxidant antibacterial protein.

Renaturation of inclusion bodies: denaturant and buffer solutions of Cb-SOD-Cb antioxidant antimicrobial protein (50mM Tirs-HCl, 150mM sodium chloride, 1M urea, pH 8.4) were mixed in a ratio of 1: 9, stirring uniformly, refrigerating overnight in a refrigerator at 4 ℃, centrifuging to remove a small amount of precipitate, desalting by using a 10kDa ultrafiltration membrane pack, and concentrating to obtain the recombinant Cb-SOD-Cb antioxidant antibacterial protein.

The result of detecting the recombinant protein by SDS-PAGE is shown in FIG. 4, the recombinant bacterium is broken and then electrophoresed, and a band (lane 3) is evident at the 25kDa position, thus judging that the target protein is successfully expressed by inclusion bodies. After washing and renaturation of the inclusion bodies, the precipitate was electrophoretically detected at different concentrations to show a distinct band at 25kDa (lane 1, 2), which is recombinant Cb-SOD-Cb).

Other raw materials used in the invention are purchased from the market.

Helichrysum italicum extract, centella asiatica extract, Hibiscus alpina extract, Portulaca oleracea extract, Cystolochia aromatica extract, Camellia japonica extract, betaine, oat kernel oil, available from the company foam Biotech (Shanghai).

Oligopeptide-3, oligopeptide-5, hydrolyzed collagen, and radix Stephaniae Tetrandrae extract, available from Hangzhou Huanwei Biotech, Inc.

Tetrahydromethylpyrimidine carboxylic acid and sodium hyaluronate were purchased from Shandong Tian Sanza Biotech Co., Ltd.

Isododecane, squalane, butylene glycol, glycerol, acrylic/beheneth-25 methacrylate copolymers, available from dow chemical (shanghai) ltd.

Allantoin was purchased from Ningbo Xiangshen Biochemical Co., Ltd.

Panthenol was purchased from Hangzhou Xinfukejiu.

Cetyl ethylhexanoate was purchased from new materials, inc.

Glycerol glucoside was purchased from Chineseme Biotech development, Inc. in Qingdao.

Sorbitol polyether-30 tetraisostearate was purchased from Shanghai Dempson assistants, Inc.

The room temperature referred to herein means 25 ℃.

Example 1

The emulsion with the antioxidant and repairing effects comprises the following components:

0.001% of a chamomile extract, 0.001% of an antibacterial peptide fusion protein Cb-SOD-Cb, 1% of isododecane, 0.1% of a centella asiatica extract, 0.05% of a leontopodium alpinum extract, 0.01% of a purslane extract, 0.005% of a tetrandra extract, 0.05% of tetrahydro-methyl pyrimidinecarboxylic acid, 0.002% of a brachycephala extract, 0.2% of hydrolyzed collagen, 0.01% of a camellia japonica extract, 0.8978% of an oligopeptide-30.0002%, 0.01% of glycerol glucoside, 0.2% of betaine, 0.15% of panthenol, 0.1% of sodium hyaluronate, 0.3532% of an oligopeptide-50.0002%, 0.05% of allantoin, 5% of butylene glycol, 3% of squalane, 0.9% of an acrylic acid (ester)/beheneth-25 methacrylate copolymer, 1.5% of glycerol, 1.5% of cetyl ethylhexanoate, 0.3% of oat kernel oil, 1.30% of sorbitan-tetraisostearate, and the balance deionized water.

The preparation method comprises the following steps:

heating deionized water to 82 deg.C, sequentially adding butanediol, squalane, acrylic acid (ester)/beheneth-25 methacrylate copolymer, glycerol, cetyl ethyl hexanoate, oat kernel oil and sorbitan polyether-30 tetraisostearate, stirring, maintaining the temperature for 20min until the raw materials are completely dissolved, and cooling to 32 deg.C;

cooling to the required temperature, sequentially adding a helichrysum italicum extract, an antibacterial peptide fusion protein Cb-SOD-Cb, isododecane, a centella asiatica extract, a leontopodium alpinum extract, a purslane extract, a tetrandra root extract, tetrahydromethylpyrimidine carboxylic acid, a cysticercus vesiculosus extract, hydrolyzed collagen, a camellia flower extract, oligopeptide-3, glycerol glucoside, betaine, panthenol, sodium hyaluronate, oligopeptide-5 and allantoin into the solution, stirring, and cooling to room temperature to obtain the emulsion with the effects of resisting oxidation and repairing.

Example 2

The emulsion with the antioxidant and repairing effects comprises the following components:

0.01% of a chamomile extract, 0.05% of an antibacterial peptide fusion protein Cb-SOD-Cb, 0.5% of isododecane, 0.05% of a centella asiatica extract, 0.01% of a leontopodium alpinum extract, 0.02% of a purslane extract, 0.001% of a tetrandra extract, 0.01% of tetrahydromethylpyrimidine carboxylic acid, 0.01% of a streptococus vesiculosus extract, 0.05% of hydrolyzed collagen, 0.005% of a camellia extract, oligopeptide-30.001%, 0.02% of glycerol glucoside, 0.1% of betaine, 0.15% of panthenol, 0.2% of sodium hyaluronate, oligopeptide-50.001%, 0.01% of allantoin, 7% of butylene glycol, 1% of squalane, 0.6% of an acrylic acid (ester)/beheneth-25 methacrylate copolymer, 1.0% of glycerol, 1.0% of cetyl ethylhexanoate, 0.2% of avellaneth-30% of tetraisostearate, and the balance deionized water.

The preparation method comprises the following steps:

heating deionized water to 80 deg.C, sequentially adding butanediol, squalane, acrylic acid (ester)/beheneth-25 methacrylate copolymer, glycerol, cetyl ethyl hexanoate, oat kernel oil and sorbitan polyether-30 tetraisostearate, stirring, maintaining the temperature for 20min until the raw materials are completely dissolved, and cooling to 30 deg.C;

cooling to the required temperature, sequentially adding a Parthenium italicum extract, an antibacterial peptide fusion protein Cb-SOD-Cb, isododecane, a centella asiatica extract, a leontopodium alpinum extract, a purslane extract, a tetrandra root extract, tetrahydro-methylpyrimidine carboxylic acid, a streptoverticillium vesiculosum extract, hydrolyzed collagen, a camellia extract, oligopeptide-3, glycerol glucoside, betaine, panthenol, sodium hyaluronate, oligopeptide-5 and allantoin into the solution, stirring, and cooling to room temperature to obtain the emulsion with the effects of resisting oxidation and repairing.

Example 3

The emulsion with the antioxidant and repairing effects comprises the following components:

0.001% of a chamomile extract, 0.001% of an antibacterial peptide fusion protein Cb-SOD-Cb, 1.5% of isododecane, 0.2% of a centella asiatica extract, 0.1% of a leontopodium alpinum extract, 0.005% of a purslane extract, 0.01% of a tetrandra extract, 0.1% of tetrahydromethylpyrimidine carboxylic acid, 0.001% of a streptococus vesiculosus extract, 0.2% of hydrolyzed collagen, 0.02% of a camellia japonica extract, oligopeptide-30.0001%, 0.1% of glycerol glucoside, 0.5% of betaine, 0.15% of panthenol, 0.05% of sodium hyaluronate, oligopeptide-50.0001%, 0.1% of allantoin, 2% of butylene glycol, 4% of squalane, 1.2% of an acrylic acid (ester)/beheneth-25 methacrylate copolymer, 2.0% of glycerol, 2.0.0% of cetyl ethylhexanoate, 0.4% of avenin-30% of tetraisostearate, and the balance deionized water.

The preparation method comprises the following steps:

heating deionized water to 80 deg.C, sequentially adding butanediol, squalane, acrylic acid (ester)/beheneth-25 methacrylate copolymer, glycerol, cetyl ethyl hexanoate, oat kernel oil and sorbitan polyether-30 tetraisostearate, stirring, maintaining the temperature for 20min until the raw materials are completely dissolved, and cooling to 30 deg.C;

cooling to the required temperature, sequentially adding a Parthenium italicum extract, an antibacterial peptide fusion protein Cb-SOD-Cb, isododecane, a centella asiatica extract, a leontopodium alpinum extract, a purslane extract, a tetrandra root extract, tetrahydro-methylpyrimidine carboxylic acid, a streptoverticillium vesiculosum extract, hydrolyzed collagen, a camellia extract, oligopeptide-3, glycerol glucoside, betaine, panthenol, sodium hyaluronate, oligopeptide-5 and allantoin into the solution, stirring, and cooling to room temperature to obtain the emulsion with the effects of resisting oxidation and repairing.

Test example 1

The physicochemical indexes are as follows (repeated tests are set in the following experimental processes, and the average value is taken):

1. traits

The emulsion with the antioxidant and repairing effects prepared in the embodiments 1-3 of the invention is milk white, and has moderate viscosity and consistency and higher stability.

2. Examination of pH value

The emulsions prepared in examples 1-3 having antioxidant and repairing effects were measured using a pH meter, which was 7.0.

3. Cold and hot storage test

The emulsions with antioxidant and repairing effects prepared in examples 1-3 were respectively packaged in transparent cosmetic bottles, and refrigerated in a refrigerator at 4 ℃ for 30 days without delamination.

The emulsion with the functions of oxidation resistance and repair is preserved for 72 hours in a constant temperature box at the temperature of 45 ℃, and the prepared emulsion with the functions of oxidation resistance and repair has no phenomena of layering, precipitation, odor change and the like.

4. Centrifugal test

The emulsions with antioxidant and repairing effects prepared in examples 1-3 were respectively placed in centrifuge tubes and centrifuged at 3000rpm for 30min without delamination.

5. Room temperature storage test

The emulsions with antioxidant and repairing effects prepared in examples 1 to 3 were respectively packaged in transparent cosmetic bottles, and left to stand at room temperature for 6 months without delamination, sensory changes and off-flavor.

6. Irritation test, allergy test

The hair on the back of the rabbit was cut off, and the emulsion having antioxidant and repairing effects prepared in examples 1-3 was applied to the cut hair, and then the cut hair was compared with the non-coated hair, resulting in no irritation or allergic reaction.

Test example 2

The antioxidant activity of the emulsion with antioxidant and repairing functions prepared in example 1 is measured by adopting the determination of the autoxidation rate of pyrogallol:

as shown in Table 1, the control tube reagent was incubated in a water bath at 25 ℃ for 20min, rapidly mixed and shaken in the above volumes, immediately poured into a cuvette with an optical path of 1cm, and the absorbance was measured every 60 seconds at a wavelength of 325nm to calculate A/min ₃₂₅ The added value of (A) is the auto-oxidation rate of pyrogallol ₀ 。

Putting the sample tube reagent in water bath at 25 DEG CKeeping the temperature for 20min, rapidly mixing and shaking according to the volume, immediately pouring into a cuvette with optical path of 1cm, measuring light absorption value every 60s at wavelength of 325nm, and calculating A/min ₃₂₅ The value added is the autoxidation rate A of the pyrogallol under the inhibition condition _m 。

TABLE 1 reagents for pyrogallol autoxidation Process

The preparation method of the sample in the sample tube comprises the following steps: the emulsion with antioxidant and repair efficacy prepared in examples 1-3 and Phosphate Buffered Saline (PBS) with pH 8.2 were mixed in a volume ratio of 1: 9 is added into a vortex oscillator to be fully mixed, and a test sample of the emulsion is obtained.

The antioxidant activity of the emulsion with antioxidant and repairing effects prepared in example 1 is determined by the rate of inhibiting the auto-oxidation of pyrogallol:

the enzyme activity was calculated according to the following formula, based on the definition of the enzyme activity unit:

A ₀ : the auto-oxidation rate of pyrogallol; a. the _m : adding an emulsion sample to obtain the autoxidation rate of pyrogallol; total activity (U) ═ unit activity × V total emulsion sample stock.

The test shows that the SOD enzyme activity of the emulsion per milliliter is more than 20000U, and the emulsion can rapidly remove free radicals on the surface of the skin and reduce the skin injury.

Test example 3

The specific method of the experiment for promoting cell proliferation of the emulsion with the effects of oxidation resistance and repair prepared in the embodiment 1 is as follows:

1) the well-grown BALB/3T3 CloneA31 cells were digested and collected, counted, and the cell concentration was adjusted to 1X 10 ⁴ cells/ml, 2000 cells/well in 96-well plates, 100. mu.l/well, medium conditions 10% FBS DMEM (fetal bovine serum medium), 37 ℃,5％CO ₂ culturing under the condition.

2) After 24h, starvation culture of the cells is carried out, namely the culture medium is changed into a 0.4% FBS DMEM maintaining culture solution, other conditions are not changed, and the culture is continued for 24 h.

3) The emulsion was diluted with PBS buffer to the following concentrations (100%, 50%, 25%, 12.5%, 6.25%) and mixed well with a vortex shaker and added stepwise to a 96-well plate at 10. mu.l/well for 48-72 h.

4) After culturing for 72h, an MTT detection kit/CCK 8 kit is adopted to detect the cell proliferation condition, and the experimental result shows that the freeze-dried powder has obvious proliferation promoting activity on cells when the concentration of the freeze-dried powder is 0.1 mg/mL.

The experimental results are shown in FIG. 5. As can be seen from FIG. 5, the dilution containing 6.25% emulsion showed a clear, i.e. most pronounced, cell proliferation promoting activity of BALB/3T3 CloneA31 cells.

Test example 4

The use cases of the effects are as follows:

1. for a sensitive skin patient, the horny layer of the skin is relatively thick, and the skin is easy to be allergic to generate acne and red swelling. The emulsion with the effects of oxidation resistance and repair, which is prepared by the embodiment 1 of the invention, is applied for 2 times a day, 0.3-0.5mL each time, and after the emulsion is used for 2 months, the skin sensitivity is reduced, the redness and swelling disappear, and the skin smoothness is improved. As shown in particular in fig. 6.

2. One subject had dull and lusterless skin and had acne marks on the skin. The emulsion with the effects of resisting oxidation and repairing, which is prepared by the invention in the embodiment 1, is applied for 2 times a day, 0.3-0.5mL each time, and after the emulsion is used for 1 month, the skin is white, the smoothness is improved, and the acne marks obviously disappear. As shown in particular in fig. 7.

Test example 5

80 persons in the age range of 30-60 years are selected as trial subjects by adopting a civil survey grading method, and each person uses the emulsion with the antioxidant and repairing effects prepared in the embodiment 1 of the invention to wear the face 14 times per week (namely 2 times per day), and the using time is two months. The using effects of the ingredients are divided into 5 points: 5 is the highest score, which means better and very satisfactory; 4, the classification is very good; 3 is acceptable; when the score is 3 or less, it is not preferable and is not acceptable. The average scores of the respective items are shown below, and the results are shown in Table 2.

TABLE 2 examination of the comprehensive results

Effects of use	Average score
		Nourishing and nourishing property	4.9
Whitening property	4.9
		Moisture retention	4.8

Comparative example 1

The difference from example 1 is that no component of the antibacterial peptide fusion protein Cb-SOD-Cb was added.

As a result, as shown in table 3, it can be seen that the emulsion prepared in this comparative example has nourishing, moisturizing effects, but the reduction of its antioxidant effect weakens its skin whitening effect.

TABLE 3 examination of the comprehensive results

Effects of use	Average score
		Nourishing and nourishing property	4.6
Whitening property	3.8
		Moisture retention	4.8

Comparative example 2

The difference from example 1 is that no oligopeptide-3 or oligopeptide-5 components are added.

As a result, as can be seen from table 4, the emulsion prepared in this comparative example was found to have whitening and moisturizing effects, but the reduction of its cell proliferation promoting effect reduced its nourishing effect on the skin.

TABLE 4 examination of the comprehensive results

Effect of use	Average score
		Nourishing and nourishing property	3.7
Whitening property	4.5
		Moisture retention	4.8

Comparative example 3

The difference from example 1 is that no glycerol glucoside, sodium hyaluronate component, was added.

As a result, as shown in table 5, it can be seen that the emulsion prepared in this comparative example has nourishing and whitening effects, but lacks the long-chain polysaccharide molecules having strong hydrophilicity, reducing its moisturizing effect on the skin.

TABLE 5 examination of the comprehensive results

Effects of use	Average score
		Nourishing and nourishing property	4.7
Whitening property	4.8
		Moisture retention	3.9

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 and improvements made within the spirit and principle of the present invention are intended to be included therein.

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Claims (5)

1. The emulsion with the effects of oxidation resistance and repair is characterized by comprising the following components in percentage by mass:

0.001-0.01% of Parthenia italica extract, 0.001-0.05% of antibacterial peptide fusion protein Cb-SOD-Cb, 0.5-1.5% of isododecane, 0.05-0.2% of centella asiatica extract, 0.01-0.1% of edelweiss extract, 0.005-0.02% of purslane extract, 0.001-0.01% of Stephania tetrandra extract, 0.01-0.1% of tetrahydro-methylpyrimidine carboxylic acid, 0.001-0.01% of Strychnos rupestris extract, 0.05-0.2% of hydrolyzed collagen, 0.005-0.02% of camellia extract, 0. 30.0001-0.001% of oligopeptide-oligopeptide, 0.01-0.2% of glycerol glucoside, 0.1-0.5% of betaine, 0.15% of panthenol, 0.05-0.2% of sodium sulfonate, 0. 50.0001-0.001% of oligopeptide, 0.01-0.1% of allantoin, 2-7% of butanediol, 1-4% of squalane, 0.25% of acrylic acid (ester) and 0.6-2% of behenyl alcohol copolymer, 1.0-2.0% of glycerin, 1.0-2.0% of cetyl ethyl hexanoate, 0.2-0.4% of oat kernel oil, 0.8-1.2% of sorbitan polyether-30 tetraisostearate and the balance of deionized water.

2. The emulsion with the effects of oxidation resistance and repair according to claim 1, which is characterized by comprising the following components in percentage by mass:

0.001% of a chamomile extract, 0.001% of an antibacterial peptide fusion protein Cb-SOD-Cb, 1% of isododecane, 0.1% of a centella asiatica extract, 0.05% of a leontopodium alpinum extract, 0.01% of a purslane extract, 0.005% of a tetrandra extract, 0.05% of tetrahydro-methyl pyrimidinecarboxylic acid, 0.002% of a brachycephala extract, 0.2% of hydrolyzed collagen, 0.01% of a camellia japonica extract, 0.8978% of an oligopeptide-30.0002%, 0.01% of glycerol glucoside, 0.2% of betaine, 0.15% of panthenol, 0.1% of sodium hyaluronate, 0.3532% of an oligopeptide-50.0002%, 0.05% of allantoin, 5% of butylene glycol, 3% of squalane, 0.9% of an acrylic acid (ester)/beheneth-25 methacrylate copolymer, 1.5% of glycerol, 1.5% of cetyl ethylhexanoate, 0.3% of oat kernel oil, 1.30% of sorbitan-tetraisostearate, and the balance deionized water.

3. A method for preparing the emulsion with antioxidant and repairing effects according to claim 1 or 2, comprising the following steps:

heating deionized water to 80-83 deg.C, sequentially adding butanediol, squalane, acrylic acid (ester)/beheneth-25 methacrylate copolymer, glycerol, cetyl ethyl hexanoate, oat kernel oil and sorbitan polyether-30 tetraisostearate, stirring, maintaining the temperature until the raw materials are completely dissolved, and cooling;

cooling to the required temperature, sequentially adding a Parthenium italicum extract, an antibacterial peptide fusion protein Cb-SOD-Cb, isododecane, a centella asiatica extract, a leontopodium alpinum extract, a purslane extract, a tetrandra root extract, tetrahydro-methylpyrimidine carboxylic acid, a streptoverticillium vesiculosum extract, hydrolyzed collagen, a camellia extract, oligopeptide-3, glycerol glucoside, betaine, panthenol, sodium hyaluronate, oligopeptide-5 and allantoin into the solution, stirring, and cooling to room temperature to obtain the emulsion with the effects of resisting oxidation and repairing.

4. The method according to claim 3, wherein the holding time is 20 min.

5. The preparation method according to claim 3, wherein the temperature reduction treatment is temperature reduction to 30-35 ℃.

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