CN109692130B - Mask liquid containing defensins and preparation method thereof - Google Patents

Abstract

The invention discloses a defensin-containing mask liquid and a preparation method thereof, wherein the mask liquid comprises the following components in percentage by weight: 4.0 to 6.0 percent of glycerin, 0.3 to 0.6 percent of defensin, 3.0 to 5.0 percent of 1, 3-butanediol, 0.1 to 0.2 percent of hyaluronic acid, 1.5 to 2.5 percent of arbutin, 1.5 to 2.5 percent of polyethylene glycol, 0.4 to 0.6 percent of carbomer resin, 0.4 to 0.6 percent of panthenol, 4.0 to 6.0 percent of vitamin E liposome, 2.0 to 4.0 percent of vitamin C liposome, 0.3 to 0.5 percent of triethanolamine, 0.2 to 0.4 percent of PEG (60) hydrogenated castor oil, 0.1 to 0.5 percent of preservative, 0.3 to 0.6 percent of essence and the balance of deionized water. The components of the facial mask liquid have a synergistic effect, can repair skin in multiple ways, and is safe and non-irritant; and the preparation method is simple, can be used for industrial production, and is beneficial to popularization and application.

Description

Mask liquid containing defensins and preparation method thereof

Technical Field

The invention relates to the technical field of daily cosmetics, in particular to a mask liquid containing defensins and a preparation method thereof.

Background

With the development of society and the progress of science and technology, the pressure of people on work and life is higher and higher, and the problem of environmental pollution is also higher and higher. The long-term life of the skin care product in the environment with fast pace, irregular diet and great pollution causes the continuous weakening of the skin tolerance of people, the sensitive and damaged skin conditions are more and more serious, and more people have sub-health skin. Therefore, skin care has gradually become an indispensable part of human life.

The facial mask is one of beauty products, has the advantages of remarkable skin improving effect and convenient use, and is popular with consumers. When the mask is used, the non-woven fabric mask soaked with the mask liquid is covered on the face for 15-20 minutes, so that the face can be temporarily isolated from outside air and pollution, the skin temperature is increased, skin pores are expanded, sweat gland secretion and metabolism are promoted, the oxygen content of the skin is increased, products of epidermal cell metabolism and accumulated grease substances are favorably removed from the skin, and the mask liquid in the mask permeates into the horny layer of the skin epidermis to ensure that the skin becomes soft, naturally bright and elastic.

Defensins (defensins) are small cysteine-rich polypeptides, typically consisting of 29-54 amino acids, including 6-8 conserved cysteines. The defensin has broad-spectrum antimicrobial activity, can effectively kill viruses, bacteria, fungi, spirochetes and the like, and also has cytotoxic effect on tumor cells. At present, the application of defensins in the preparation of facial mask liquid is not reported.

Disclosure of Invention

In view of the prior art, the invention aims to provide a mask liquid containing defensins and a preparation method thereof. The facial mask liquid can quickly repair the skin with thin, damaged and dark yellow epidermis; the skin care product has the advantages of effectively inhibiting bacteria, eliminating red and swollen, eliminating skin toxin, smoothing capillary vessels, softening cutin of epidermis, balancing oil secretion, shrinking pores, and being beneficial to removing blackheads and acnes; enhancing the resistance of the skin and preventing scar hyperplasia; meanwhile, the skin care product has the effects of mildly sterilizing, diminishing inflammation, regulating sebum secretion, promoting the growth of epidermal cells and enabling the damaged skin to heal without pigment and scars; it also has effects in regulating skin pH, balancing oil and fat, killing bacteria, relieving inflammation, shrinking pores, and preventing wrinkle and acne.

In order to realize the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, there is provided the use of a defensin in the preparation of a facial mask fluid.

Preferably, the amino acid sequence of the defensin is: CYCRIPACIAGERRYGTCIYGGRLWAFCC, respectively; (SEQ ID NO.1)

More preferably, the defensin comprises one or more D-type amino acids in the amino acid sequence; specifically, the amino acid sequence is as follows:

CYCRIPACIX1GERRYGX2CIYX3GRLWAX4CC；

wherein X1 is Ala or D-Ala;

x2 is Thr or D-Thr;

x3 is Gly or D-Gly;

x4 is Phe or D-Phe;

wherein at least one of X1, X2, X3, or X4 is a D amino acid.

The second aspect of the invention provides a defensin-containing mask liquid, which comprises the following components in percentage by weight:

4.0 to 6.0 percent of glycerin, 0.3 to 0.6 percent of defensin, 3.0 to 5.0 percent of 1, 3-butanediol, 0.1 to 0.2 percent of hyaluronic acid, 1.5 to 2.5 percent of arbutin, 1.5 to 2.5 percent of polyethylene glycol, 0.4 to 0.6 percent of carbomer resin, 0.4 to 0.6 percent of panthenol, 4.0 to 6.0 percent of vitamin E liposome, 2.0 to 4.0 percent of vitamin C liposome, 0.3 to 0.5 percent of triethanolamine, 0.2 to 0.4 percent of PEG (60) hydrogenated castor oil, 0.1 to 0.5 percent of preservative, 0.3 to 0.6 percent of essence and the balance of deionized water.

Preferably, the preservative is a plant extract preservative; more preferably, the plant extract preservative is cactus extract and is prepared by the following method:

cleaning fresh radix et caulis Opuntiae Dillenii, pulping, vacuum freeze drying, and pulverizing to obtain radix et caulis Opuntiae Dillenii extract.

Preferably, the essence is a sea buckthorn extract and is prepared by the following method:

drying fructus Hippophae, pulverizing, sieving with 40-60 mesh sieve, and supercritical CO₂The extraction process comprises extracting at 25-30MPa and 35-45 deg.C with CO₂The flow rate is 20-25L/h, the entrainer is 95% ethanol, the entrainer is 6-8% of the raw material weight, and the extraction time is 1-2h, thus obtaining the sea buckthorn extract.

In a third aspect of the present invention, there is provided a method for preparing the defensin-containing facial mask liquid, comprising the steps of:

(1) adding glycerol, 1, 3-butanediol, polyethylene glycol, carbomer resin, panthenol, triethanolamine, PEG (60) hydrogenated castor oil and deionized water into a stirring pot, heating to 70-80 deg.C, and stirring for 10-20 min to obtain mixed solution A;

(2) cooling the mixed solution A to 30-40 deg.C, adding defensin, hyaluronic acid, arbutin, vitamin E liposome, vitamin C liposome, antiseptic and essence, stirring for 5-10 min, and homogenizing for 5-10 min.

Preferably, in the step (1) and the step (2), the stirring speed is 40-60 r/min.

The invention has the beneficial effects that:

(1) the mask liquid is prepared by taking the defensin as a raw material for the first time, has high antibacterial property and extremely strong bacteriostatic and bactericidal effects on drug-resistant bacteria; and has no toxic and side effects, no residue and no bacterial drug resistance.

(2) The mask liquid disclosed by the invention has good compatibility among raw materials, so that the product is more uniform and fine, and the affinity of the product to the skin is improved.

(3) According to the facial mask liquid, the natural plant extract is used as the preservative and the essence, so that excessive chemical substances are prevented from being added, and the components are more natural; and the natural plant extract can be used as a preservative and essence, and can generate a synergistic promotion effect with defensin, so that the sterilization effect is improved, and the cell growth can be promoted and the wound healing can be accelerated.

(4) The components of the facial mask liquid have a synergistic effect, can repair skin multiple times, and is safe and non-irritant; and the preparation method is simple, can be used for industrial production, and is beneficial to popularization and application.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 application belongs.

As introduced in the background section, defensins have broad-spectrum antimicrobial activity and remarkable bactericidal effect, and are widely applied in the fields of plant disease-resistant genetic engineering, biopharmaceuticals and the like. However, the stability, hydrolysis resistance and biological activity of defensins still need to be further improved, and therefore, structural modification of defensins is a hot spot of research.

The introduction of non-natural D-amino acid is one of the methods for modifying the structure of defensin, and the stability, hydrolysis resistance and biological activity of defensin can be improved by properly introducing D-amino acid. However, the type, position and amount of the introduced D-amino acid are critical to the structural modification effect of the defensin, and if the type, position or amount of the introduced D-amino acid is not appropriate, the stability, hydrolysis resistance and biological activity of the defensin are reduced.

In the present invention, the amino acid composition of the defensin used is: CYCRIPACIAGERRYGTCIYGGRLWA FCC, respectively; (SEQ ID NO. 1).

On the basis, the invention firstly carries out structural modification on the defensin, and a large number of experiments show that the defensin has better effect when modified according to the following amino acid composition:

CYCRIPACIX1GERRYGX2CIYX3GRLWAX4CC；

wherein X1 is Ala or D-Ala;

x2 is Thr or D-Thr;

x3 is Gly or D-Gly;

x4 is Phe or D-Phe;

wherein at least one of X1, X2, X3, or X4 is a D-form amino acid.

The defensins can be prepared by conventional methods in the prior art such as a chemical synthesis method or a genetic engineering method.

The method for modifying the structure of the defensin can directly mix D-type amino acid in chemical synthesis; or D-amino acid is added into the culture medium in the process of gene engineering expression.

On the basis of carrying out structural modification research on the defensin, the invention also uses the defensin in the preparation of facial mask liquid for the first time. In one embodiment of the invention, the mask liquid is prepared from the following raw materials:

4.0 to 6.0 percent of glycerin, 0.3 to 0.6 percent of defensin, 3.0 to 5.0 percent of 1, 3-butanediol, 0.1 to 0.2 percent of hyaluronic acid, 1.5 to 2.5 percent of arbutin, 1.5 to 2.5 percent of polyethylene glycol, 0.4 to 0.6 percent of carbomer resin, 0.4 to 0.6 percent of panthenol, 4.0 to 6.0 percent of vitamin E liposome, 2.0 to 4.0 percent of vitamin C liposome, 0.3 to 0.5 percent of triethanolamine, 0.2 to 0.4 percent of PEG (60) hydrogenated castor oil, 0.1 to 0.5 percent of preservative, 0.3 to 0.6 percent of essence and the balance of deionized water.

Considering that the effects of the chemical preservative and the essence are single, and under the background of advocating nature nowadays, the mask liquid contains too many chemical components which are not easy to be accepted by consumers. Based on the above, the invention adopts the cactus extract as the preservative and adopts the seabuckthorn extract as the essence; wherein, the cactus extract has obvious bacteriostasis and antioxidation, and the sea buckthorn extract can release a pleasant aromatic smell. The cactus extract and the sea buckthorn extract can be used as a preservative and essence, and can generate a synergistic promotion effect with defensins, improve the sterilization effect, promote cell growth and accelerate wound healing.

Vitamin E has effects of resisting oxidation, dilating blood vessel, and preventing pachylosis; vitamin C can synthesize collagen and mucopolysaccharide, reduce free radical damage to skin, delay aging, inhibit skin abnormal pigment deposition and tyrosinase activity, and reduce melanin formation. However, vitamin E and vitamin C are easy to oxidize and deteriorate, and if the vitamin E and the vitamin C are added into a mask substrate by a common method, the vitamin E and the vitamin C are easy to oxidize and deteriorate and lose effects; in addition, vitamin C is water-soluble and does not easily permeate into the stratum corneum of the skin, and thus cannot fully exert its effect.

The liposome is a novel biological carrier, has the good characteristics of slow release, permeability and the like, and can effectively improve the utilization rate of vitamin E and vitamin C by encapsulating vitamin E and vitamin C with the liposome to be used as raw materials for preparing the mask liquid.

For the vitamin E liposome and the vitamin C liposome, commercially available products can be purchased, or the vitamin E liposome and the vitamin C liposome can be prepared by the existing method. For example:

preparing blank liposome from lecithin and cholesterol at a mass ratio of 6:1, mixing the blank liposome and vitamin E/vitamin C at a mass ratio of 9:2, performing ultrasonic treatment for 3min, and performing stabilization treatment and centrifugal separation to obtain vitamin E or vitamin C liposome.

In the mask liquid, all raw material components are an organic whole, and the raw materials have a synergistic promotion effect. Wherein, the defensins, the cactus extract, the sea buckthorn extract and the arbutin are used in a compatibility manner, have a synergistic promotion effect, can improve the sterilization effect, and can promote cell growth and accelerate wound healing; the hyaluronic acid, the panthenol, the triethanolamine and the glycerol have good intermiscibility, and the compatibility of the product to the skin can be improved by matching the hyaluronic acid, the panthenol, the triethanolamine and the glycerol, and the water retention of the skin can be improved; the vitamin E liposome and the vitamin C liposome are added, so that the stability of the vitamin E and the vitamin C can be improved, skin pigmentation can be reduced, color spots and freckles can be removed, and the effects of sterilization and inflammation diminishing can be achieved.

In the test process, raw material components such as defensins, cactus extracts, sea buckthorn extracts and the like are removed in sequence to prepare the facial mask liquid. And then, carrying out an antibacterial test on the prepared mask liquid, and finding that compared with the mask liquid disclosed by the invention, the antibacterial effect of the mask liquid can be reduced by deleting the raw material components.

On the basis of the raw materials of the facial mask liquid, one or more common raw materials for preparing the facial mask liquid, such as glyceryl monostearate, quercetin, houttuynine sodium bisulfite and the like, are added to prepare the facial mask liquid, and the obtained facial mask liquid is also used for bacteriostatic tests.

In conclusion, the facial mask liquid is prepared by optimizing the selection and the dosage of the raw materials. The facial mask liquid provided by the invention has multiple effects: can quickly repair the skin with thin, damaged and dark yellow epidermis; the skin care product has the advantages of effectively inhibiting bacteria, eliminating red and swollen, eliminating skin toxin, smoothing capillary vessels, softening cutin of epidermis, balancing oil secretion, shrinking pores, and being beneficial to removing blackheads and acnes; enhancing the resistance of the skin and preventing scar hyperplasia; meanwhile, the skin care product has the effects of mildly sterilizing, diminishing inflammation, regulating sebum secretion, promoting the growth of epidermal cells and enabling the damaged skin to heal without pigment and scars; it also has effects in regulating skin pH, balancing oil and fat, killing bacteria, relieving inflammation, shrinking pores, and preventing skin wrinkle and acne.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples and comparative examples of the present invention are those conventional in the art and commercially available.

Example 1: preparation of facial mask liquid

1. The raw materials comprise (by weight percent):

5.0% of glycerin, 0.5% of defensin, 4.0% of 1, 3-butanediol, 0.1% of hyaluronic acid, 2.0% of arbutin, 15002.0% of polyethylene glycol, 0.5% of carbomer resin, 0.5% of panthenol, 5.0% of vitamin E liposome, 3.0% of vitamin C liposome, 0.4% of triethanolamine, 0.3% of PEG (60) hydrogenated castor oil, 0.3% of preservative, 0.4% of essence and the balance of deionized water.

Wherein the amino acid sequence of the defensin is as follows:

CYCRIPACIX1GERRYGX2CIYX3GRLWAX4CC；

wherein X1 is D-Ala; x2 is D-Thr; x3 is D-Gly; x4 is D-Phe.

The defensin is obtained by introducing D-type amino acid into corresponding position according to designed substitution site by Fmoc solid phase synthesis method, and extending peptide chain in sequence.

The preservative is a cactus extract and is prepared by the following method:

cleaning fresh radix et caulis Opuntiae Dillenii, pulping, vacuum freeze drying, and pulverizing to obtain radix et caulis Opuntiae Dillenii extract.

The essence is a sea buckthorn extract and is prepared by the following method:

drying fructus Hippophae, pulverizing, sieving with 40 mesh sieve, and supercritical CO₂The extraction process is used for extraction, the extraction pressure is 28MPa, the extraction temperature is 40 ℃, and CO is added₂The flow rate is 25L/h, the entrainer is ethanol with the volume concentration of 95%, the use amount of the entrainer is 7% of the weight of the raw materials, and the extraction time is 2h, so that the sea buckthorn extract is obtained.

2. The preparation method comprises the following steps:

(1) adding glycerol, 1, 3-butanediol, polyethylene glycol, carbomer resin, panthenol, triethanolamine, PEG (60) hydrogenated castor oil and deionized water into a stirring pot, heating to 75 ℃, and stirring for 15 minutes to obtain a mixed solution A;

(2) cooling the mixed solution A to 35 deg.C, adding defensin, hyaluronic acid, arbutin, vitamin E liposome, vitamin C liposome, antiseptic and essence, stirring for 10 min, and homogenizing for 5 min.

Example 2: preparation of facial mask liquid

1. The raw materials comprise (by weight percent):

4.0% of glycerin, 0.6% of defensin, 3.0% of 1, 3-butanediol, 0.2% of hyaluronic acid, 1.5% of arbutin, 15002.5% of polyethylene glycol, 0.4% of carbomer resin, 0.6% of panthenol, 4.0% of vitamin E liposome, 4.0% of vitamin C liposome, 0.3% of triethanolamine, 0.4% of PEG (60) hydrogenated castor oil, 0.1% of preservative, 0.6% of essence and the balance of deionized water.

Wherein the amino acid sequence of the defensin is as follows:

CYCRIPACIX1GERRYGX2CIYX3GRLWAX4CC；

wherein, X1 is Ala; x2 is D-Thr; x3 is Gly; x4 is D-Phe.

The defensin is obtained by introducing D-type amino acid into corresponding position according to designed substitution site by Fmoc solid phase synthesis method, and extending peptide chain in sequence.

The preservative is a cactus extract prepared in the same manner as in example 1.

The essence is a sea buckthorn extract, and the preparation method is the same as example 1.

2. The preparation method comprises the following steps:

(1) adding glycerol, 1, 3-butanediol, polyethylene glycol, carbomer resin, panthenol, triethanolamine, PEG (60) hydrogenated castor oil and deionized water into a stirring pot, heating to 70 ℃, and stirring for 20 minutes to obtain a mixed solution A;

(2) cooling the mixed solution A to 30 deg.C, adding defensin, hyaluronic acid, arbutin, vitamin E liposome, vitamin C liposome, antiseptic and essence, stirring for 5 min, and homogenizing for 10 min.

Example 3: preparation of facial mask liquid

1. The raw materials comprise the following components in percentage by weight:

6.0% of glycerin, 0.3% of defensin, 5.0% of 1, 3-butanediol, 0.1% of hyaluronic acid, 2.5% of arbutin, 15001.5% of polyethylene glycol, 0.6% of carbomer resin, 0.4% of panthenol, 6.0% of vitamin E liposome, 2.0% of vitamin C liposome, 0.5% of triethanolamine, 0.2% of PEG (60) hydrogenated castor oil, 0.5% of preservative, 0.3% of essence and the balance of deionized water.

Wherein the amino acid sequence of the defensin is as follows:

CYCRIPACIX1GERRYGX2CIYX3GRLWAX4CC；

wherein, X1 is Ala; x2 is D-Thr; x3 is Gly; x4 is Phe.

The defensin is obtained by introducing D-type amino acid into corresponding position according to designed substitution site by Fmoc solid phase synthesis method, and extending peptide chain in sequence.

The preservative is a cactus extract, and the preparation method is the same as that of example 1.

The essence is a sea buckthorn extract, and the preparation method is the same as example 1.

2. The preparation method comprises the following steps:

(1) adding glycerol, 1, 3-butanediol, polyethylene glycol, carbomer resin, panthenol, triethanolamine, PEG (60) hydrogenated castor oil and deionized water into a stirring pot, heating to 80 deg.C, and stirring for 10 min to obtain mixed solution A;

(2) cooling the mixed solution A to 40 deg.C, adding defensin, hyaluronic acid, arbutin, vitamin E liposome, vitamin C liposome, antiseptic and essence, stirring for 10 min, and homogenizing for 10 min.

Comparative example 1: preparation of facial mask liquid

1. The raw materials comprise (by weight percent):

5.0% of glycerin, 4.0% of 1, 3-butanediol, 0.1% of hyaluronic acid, 15002.0% of polyethylene glycol, 0.5% of carbomer resin, 0.5% of panthenol, 5.0% of vitamin E liposome, 3.0% of vitamin C liposome, 0.4% of triethanolamine, 0.3% of PEG (60) hydrogenated castor oil, 0.3% of preservative, 0.4% of essence and the balance of deionized water.

Wherein the preservative is imidazolidinyl urea; the essence is sea buckthorn extract (prepared as in example 1).

2. The preparation method comprises the following steps:

(1) adding glycerol, 1, 3-butanediol, polyethylene glycol, carbomer resin, panthenol, triethanolamine, PEG (60) hydrogenated castor oil and deionized water into a stirring pot, heating to 75 ℃, and stirring for 15 minutes to obtain a mixed solution A;

(2) and (3) when the temperature of the mixed solution A is reduced to 35 ℃, adding hyaluronic acid, vitamin E liposome, vitamin C liposome, preservative and essence, stirring for 10 minutes, and then homogenizing for 5 minutes to obtain the hyaluronic acid-vitamin E liposome-vitamin C liposome-vitamin E.

Comparative example 2:

1. the raw materials comprise (by weight percent):

5.0% of glycerin, 4.0% of 1, 3-butanediol, 0.1% of hyaluronic acid, 15002.0% of polyethylene glycol, 0.5% of carbomer resin, 0.5% of panthenol, 5.0% of vitamin E liposome, 3.0% of vitamin C liposome, 0.4% of triethanolamine, 0.3% of PEG (60) hydrogenated castor oil, 0.3% of preservative, 0.4% of essence and the balance of deionized water.

Wherein the preservative is cactus extract (prepared as in example 1); the essence is emulsified essence.

2. The preparation method comprises the following steps:

(1) adding glycerol, 1, 3-butanediol, polyethylene glycol, carbomer resin, panthenol, triethanolamine, PEG (60) hydrogenated castor oil and deionized water into a stirring pot, heating to 75 ℃, and stirring for 15 minutes to obtain a mixed solution A;

(2) and (3) when the temperature of the mixed solution A is reduced to 35 ℃, adding hyaluronic acid, vitamin E liposome, vitamin C liposome, preservative and essence, stirring for 10 minutes, and then homogenizing for 5 minutes to obtain the vitamin A.

And (3) safety test:

the facial mask liquids prepared in examples 1 to 3 were tested according to the microbiological examination method, toxicological test method and human safety examination method in technical specifications for cosmetic safety (2015), and the results all meet the requirements of the specifications, thus indicating that the facial mask liquid of the present invention is safe to use.

Test example 1: bacteriostasis test

1. Test materials: facial mask solutions prepared in example 1 and comparative examples 1-2.

2. Preparation of test bacteria:

respectively inoculating Escherichia coli, Staphylococcus aureus, Bacillus thuringiensis and Bacillus subtilis to the beef extract culture solution in a sterile operating room, inoculating Aspergillus, cerevisiae Fermentum and Candida to the bean sprout juice culture solution, and culturing at 28-30 deg.C for 1-3 days.

3. The test method comprises the following steps:

adopting an agar punching diffusion method, dividing an aseptic culture dish into 4 groups, wherein each group comprises 4, pouring the sterilized beef extract culture solution into the aseptic culture dish, each dish is 15-20 mL, sucking 0.1mL of test bacteria such as escherichia coli, staphylococcus aureus, bacillus thuringiensis and bacillus subtilis to the aseptic culture dish by using an aseptic pipette after the beef extract culture solution is solidified, and uniformly coating the test bacteria onto the aseptic culture dish, wherein different strains are added into each culture dish of each group.

The method comprises the steps of dividing an aseptic culture dish into 4 groups, wherein each group comprises 3, pouring sterilized bean sprout juice culture solution into the aseptic culture dish, each dish is 15-20 mL, sucking 0.1mL of aspergillus, brewer's yeast and candida test bacteria to be added onto the aseptic culture dish by using an aseptic pipette after the culture dish is solidified, and coating the test bacteria uniformly, wherein different strains are added into each culture dish of each group.

After the coating, the culture medium was perforated with 4 equidistant holes using a sterilized stainless steel perforator having an outer diameter of 4 mm. mu.L of the facial mask solutions prepared in example 1, comparative example 1 and comparative example 2 were each pipetted by a micro-sampler, and an equal amount of sterile water was added to one well of the center to serve as a control for labeling. The bacteria were incubated at 37 ℃ for 24h, and the molds and yeasts were incubated at 28 ℃ for 72 h. The diameter of the inhibition zone (in mm) is measured, the test is repeated for 5 times, and the average value of 5 times is taken as the diameter of the inhibition zone.

4. And (3) test results:

the test results are shown in table 1.

Table 1: data of bacteriostatic test

As can be seen from Table 1, the facial mask liquid prepared by the invention has obvious inhibition effect on escherichia coli, staphylococcus aureus, bacillus thuringiensis, bacillus subtilis, aspergillus, beer yeast and candida; in the facial mask liquids prepared in comparative examples 1-2, the defensins, the cactus extract and the sea buckthorn extract are absent, so that the inhibition effect on the bacteria is remarkably reduced, and the defensins, the cactus extract and the sea buckthorn extract can synergistically enhance the antibacterial effect.

Test example 2: fibroblast proliferation assay

1. Test samples: facial mask solutions prepared in example 1 and comparative examples 1-2.

The above test samples were dried to obtain powders, respectively, and then prepared into test sample solutions with a concentration of 0.5mg/ml using PBS.

2. The test method comprises the following steps:

preparing the prepared suspension of human fibroblasts (obtained from the residual normal skin after operation, generation 4-10) into a suspension with the concentration of 2.5 multiplied by 10⁴Inoculating each/ml of the cells into a 96-well plate, adding culture medium with corresponding volume into the blank control group without inoculating the cells, adding 20 μ l PBS into each well of the negative control group after the cells are attached to the wall, adding 20 μ l test sample solution (prepared by PBS) into each well of the sample group, setting 6 multiple wells in each group, and placing in an incubator (37 ℃, 5% CO)₂) And then added with human MTT (purchased from Sigma, lot number: 08797HJ) solution (in PBS) 20. mu.l, continued in CO₂Incubator (37 ℃, 5% CO)₂) And (4) incubating. The 96-well plate was removed, the medium in each well carefully aspirated, DMSO added, and mixed well. And measuring the absorbance value at 570nm by using an enzyme-linked immunosorbent assay detector, and comparing with a negative control group to obtain the relative proliferation rate of the sample group.

In the formula: t-sample well Abs; c-negative control well Abs; c0-blank control well Abs.

The test results are shown in Table 2.

Table 2: fibroblast proliferation assay results

The change of the structure of the dermis is the main cause of skin aging, and fibroblasts are the main cells constituting the dermis layer. The number of fibroblasts in the dermis layer gradually decreases as aging occurs and the skin thickness gradually thins. Therefore, the promotion of fibroblast proliferation is an important way to solve the problem of skin aging. As can be seen from Table 2, the facial mask liquid of the invention has the effect of synergistically promoting the proliferation of human fibroblasts, and the anti-aging effect of the facial mask liquid of the invention is proved.

Test example 3: wound healing test

1. Test materials:

the facial mask solution, HACAT cells, prepared in example 1 and comparative examples 1-2 was purchased from Shanghai Bogu Biotech Co., Ltd, under the designation BG 183.

2. The test method comprises the following steps:

taking HACAT cells in logarithmic growth phase, inoculating the HACAT cells in a 6-hole culture plate, and drawing equidistant transverse lines on the back of the 6-hole plate by using a pen when the HACAT cells are 100% fused. The cultured cell wound model was created by scoring the cell monolayer longitudinally using a 100uL tip. The control and test groups 1-3 were randomly grouped after scratching, the control group was treated with the same amount of cell maintenance solution, and the test groups 1-3 were treated with the facial solutions prepared in example 1 and comparative examples 1-2, respectively. And after the treatment, observing the change condition near the intersection point of the scratch and the drawn transverse line, respectively photographing for 0 hour, 6 hours and 12 hours, and measuring the width of the scratch under a fluorescence microscope.

3. And (3) test results:

the test results are shown in Table 3.

Table 3: scratch width measured under fluorescent microscope

As can be seen from Table 3, the facial mask fluid of the invention can effectively promote migration and fusion of HACAT cells, and proves that the facial mask fluid of the invention can promote healing of wounds.

Test example 4: test for inhibiting melanogenesis

1. Test samples:

facial mask solutions prepared in example 1 and comparative examples 1-2.

2. The test method comprises the following steps:

human skin melanoma cells SK-MEL-1 were placed in minimal essential medium containing 10% bovine embryo serum and incubated at 37 ℃ and 5% CO₂Culturing under the conditions of (1). The cultured cells were smeared on the bottom of a 75-well flask, and the number of cells was 3X 10⁵Wells, left overnight, to allow cells to adhere to the wall. After confirmation, each medium was changed to a new medium containing 10ppm of the test sample. The control group used DMSO (dimethyl sulfoxide) -containing medium.

In the above manner, each medium was changed to a new medium containing the test sample once every 2 days until the cultured cells were sufficient to fill the flask. Then, the medium fluid was taken out, washed with PBS (phosphate buffered saline), and dissolved in 1N NaOH, and the Optical Density (OD) at 500 nm was measured, and the inhibition rate of melanin formation was calculated according to the following formula 1, and the results are shown in table 4.

Formula 1: melanin formation inhibition (%) {1- (OD of each experimental sample/OD of control group) } × 100.

Table 4: results of the test for inhibiting melanogenesis

Test sample	Melanin formation inhibition (%)
		Example 1	50.6
Comparative example 1	21.8
		Comparative example 2	14.5

As can be seen from Table 4, the facial mask solution of the present invention significantly inhibited the formation of melanin and exhibited a whitening effect on the skin, as compared to comparative examples 1 and 2.

Test example 5: test for resisting water loss in dry environment

1. Test samples:

example 1 and comparative examples 1-2.

2. The test method comprises the following steps:

to simulate skin conditions, a 25.4X 76.2mm glass slide was selected and taped with 3M adhesive tape. Approximately 200mg of sample is weighed, evenly spread on a slide and placed in a constant temperature and humidity instrument. The experiment was carried out at room temperature (25 ℃) using a saturated ammonium sulphate solution to control a constant humidity environment of 80%. Weighing after a certain time (30, 60, 90, 120, 240 minutes); and (3) calculating the water loss rate of the sample by taking water as a blank control, comparing the moisture retention rate according to the water loss rate, wherein the smaller the water loss rate is, the better the moisture retention effect is. The results are shown in Table 5.

Water loss (%) (M2-M3)/(M2-M1) × 100%;

wherein, M1-weight of glass slide (g), M2-total weight after smearing before placement (g), and M3-total weight after smearing after placement (g).

Table 5: test result for resisting water loss in dry environment

As can be seen from Table 5, the facial mask liquid provided by the invention can resist drying damage in a dry environment, and has the effects of moisturizing and beautifying the skin.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Weihai Tongfeng ocean Biotechnology Limited

<120> mask liquid containing defensins and preparation method thereof

<130> 2019

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 29

<212> PRT

<213> defensins

<400> 1

Cys Tyr Cys Arg Ile Pro Ala Cys Ile Ala Gly Glu Arg Arg Tyr Gly

1 5 10 15

Thr Cys Ile Tyr Gly Gly Arg Leu Trp Ala Phe Cys Cys

20 25

Claims (1)

1. The mask liquid containing defensins is characterized by comprising the following raw materials in percentage by weight:

5.0% of glycerin, 0.5% of defensin, 4.0% of 1, 3-butanediol, 0.1% of hyaluronic acid, 2.0% of arbutin, 15002.0% of polyethylene glycol, 0.5% of carbomer resin, 0.5% of panthenol, 5.0% of vitamin E liposome, 3.0% of vitamin C liposome, 0.4% of triethanolamine, 0.3% of PEG (60) hydrogenated castor oil, 0.3% of preservative, 0.4% of essence and the balance of deionized water;

wherein the amino acid sequence of the defensin is as follows:

CYCRIPACIX1GERRYGX2CIYX3GRLWAX4CC；

wherein X1 is D-Ala; x2 is D-Thr; x3 is D-Gly; x4 is D-Phe;

the defensin is obtained by introducing D-type amino acid into corresponding positions according to designed substitution sites by adopting an Fmoc solid-phase synthesis method and then extending a peptide chain in sequence;

the preservative is a cactus extract and is prepared by the following method:

cleaning fresh radix et caulis Opuntiae Dillenii, pulping, vacuum freeze drying, and pulverizing to obtain radix et caulis Opuntiae Dillenii extract;

the essence is a sea buckthorn extract and is prepared by the following method:

drying fructus Hippophae, pulverizing, sieving with 40 mesh sieve, and supercritical CO₂The extraction process is used for extraction, the extraction pressure is 28MPa, the extraction temperature is 40 ℃, and CO is added₂The flow rate is 25L/h, the entrainer is ethanol with the volume concentration of 95%, the dosage of the entrainer is 7% of the weight of the raw materials, and the extraction time is 2h, so that the sea buckthorn extract is obtained;

the vitamin E liposome and the vitamin C liposome are prepared by the following method:

preparing a blank liposome from lecithin and cholesterol according to a mass ratio of 6:1, mixing the blank liposome and vitamin E/vitamin C according to a mass ratio of 9:2, carrying out ultrasonic treatment for 3min, and carrying out stabilization treatment and centrifugal separation to obtain a vitamin E or vitamin C liposome;

the mask liquid is prepared by the following method:

(1) adding glycerol, 1, 3-butanediol, polyethylene glycol, carbomer resin, panthenol, triethanolamine, PEG (60) hydrogenated castor oil and deionized water into a stirring pot, heating to 75 ℃, and stirring for 15 minutes to obtain a mixed solution A;

(2) cooling the mixed solution A to 35 deg.C, adding defensin, hyaluronic acid, arbutin, vitamin E liposome, vitamin C liposome, antiseptic and essence, stirring for 10 min, and homogenizing for 5 min.