CN111228210A - Composition for repairing skin barrier and preparation method and application thereof - Google Patents
Composition for repairing skin barrier and preparation method and application thereof Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/494—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom
- A61K8/4953—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom containing pyrimidine ring derivatives, e.g. minoxidil
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- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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- A61K8/9717—Rhodophycota or Rhodophyta [red algae], e.g. Porphyra
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
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- A—HUMAN NECESSITIES
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
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Abstract
The invention provides a composition for repairing skin barrier, a preparation method and application thereof. The composition comprises a hypsizygus marmoreus callus culture filtrate, a tetrahydro-methyl pyrimidine carboxylic acid and a hydrolyzed red algae extract. The composition for repairing the skin barrier can strengthen the skin barrier, improve the anti-allergy potential of the skin, improve the skin micro-ecology, and has the effects of moisturizing, brightening the skin, repairing and regenerating, resisting aging, removing wrinkles, resisting oxidation, protecting, resisting allergy, relieving and improving the skin immunity.
Description
Technical Field
The invention belongs to the technical field of cosmetics, and particularly relates to a composition for repairing a skin barrier, and a preparation method and application thereof.
Background
Healthy skin can effectively retain moisture, thereby maintaining good elastic and shiny appearance of the skin. However, the skin is affected by the living environment and physiological conditions, and skin problems such as sensitivity, dry desquamation, excessive oil secretion, color spots, skin relaxation, aging, red blood streak and the like may occur on the skin, and the most important reason is that the skin barrier is damaged. The skin barrier is a protective structure which is constructed by the cuticle and the sebum membrane together, can buffer physical and chemical stimulation, effectively inhibit bacterial breeding, block the harm of bacteria, viruses and fungi to the skin, lock moisture and prevent the moisture from evaporating, thereby realizing the function of keeping moisture. Skin barrier damage causes skin to be attacked by bacteria, viruses and fungi, and causes unscrupulous evaporation of moisture in the skin, thereby causing various problems in the skin.
At present, a commonly used means for repairing a skin barrier is sufficient water supplement, most of moisture in the traditional cosmetics only exists on the surface layer of the skin, the evaporation speed of the moisture on the surface layer of the skin is far higher than the absorption speed, the amount of the moisture capable of being absorbed by the skin is limited, the speed of repairing the skin barrier is low, the time consumption is long, and the simple water supplement has no effective improvement effect on the color spot problem caused by skin pigment deposition.
CN102349867A discloses a moisturizing repair cosmetic and a preparation method and application thereof. The cosmetic comprises a moisturizing repair freeze-dried powder and a moisturizing repair basic liquid. The main components of the freeze-dried powder comprise grape seed extract, hydrolyzed red algae extract, hyaluronic acid with small molecular weight, hydrolyzed collagen, recombinant human keratinocyte growth factor, recombinant human epidermal cell growth factor and low molecular weight heparin sodium. The moisturizing and repairing cosmetic can be used for daily skin care, and has the beautifying effects of moisturizing skin, repairing water-deficient damaged cells and enhancing skin elasticity.
CN110638740A discloses a composition for regulating skin microecology and its application, wherein the composition for regulating skin microecology comprises a schizosaccharomyces cerevisiae fermentation product lysate, a yeast fermentation product filtrate, a lactobacillus/algae extract fermentation product, a giant kelp extract and seawater. The composition with the function of regulating the skin microecology can selectively protect probiotics and inhibit pathogenic bacteria, has a good relieving effect when being applied to cosmetics, but has a poor effect of repairing a skin barrier, and has a limited moisture amount absorbed by the skin, so that the speed of repairing the skin barrier is slow, and the time consumption is long.
Therefore, the development of a skin care product capable of comprehensively repairing the skin barrier is the focus of current research.
Disclosure of Invention
The invention aims to provide a composition for repairing skin barrier and a preparation method and application thereof. The composition for repairing the skin barrier can strengthen the skin barrier, improve the anti-allergy potential of the skin, improve the skin micro-ecology, and has the functions of moisturizing, brightening the skin, repairing and regenerating, resisting aging, removing wrinkles, resisting oxidation, protecting, resisting allergy, relieving and improving the skin immunity.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a composition for repairing skin barrier comprising a hypsizygus marmoreus callus culture filtrate, a tetrahydro-methyl pyrimidine carboxylic acid and a hydrolyzed red algae extract.
In the invention, the hypecoum vulgare callus culture filtrate (CRITMUM MARITIMUM) is prepared from hypecoum vulgare, the root of the hypecoum vulgare must absorb nutrients between coasts to face harsh land environment, so that a unique life system is developed, the hypecoum vulgare callus culture filtrate contains abundant vitamin C and mineral substances, can regulate the maladjustment of skin melanin, restore the protective capability of skin antioxidation, promote the healing of skin wounds, regulate the functions of keratinocyte division, transfer, shedding and the like in an epidermal layer, improve the appearance of skin, increase the skin whitening effect, stimulate the generation of cutin, promote the metabolism of the epidermis, improve the self-healing capability of the skin, reduce the invasion of free radicals, improve the aging of the skin, improve the moisturizing performance of the skin, lighten pigmentation and improve the luster of the skin.
In the invention, the tetrahydro-methyl pyrimidine carboxylic acid is derived from desert halophilic bacteria growing in Egyptian desert salt lake, inhibits UVA-induced mitochondrial gene mutation, inhibits UVA-induced nuclear DNA damage, protects keratinocytes from UVB damage, protects Langerhans cells from UV damage, and inhibits inflammatory reaction caused by UVA. The tetrahydro-methyl pyrimidine carboxylic acid can also protect cell membranes against surface activity damage, enhance the stability of the cell membranes, enhance the barrier function of stratum corneum and reduce TEWL. In addition, the charge distribution promotes the formation of an ice-like water structure, the water activity is reduced, the moisture is preserved for a long time, and the ice-like water structure is used for keeping the skin fresh.
In the invention, the polysaccharide active substance in the hydrolyzed red algae extract can be combined with skin protein to form a moisturizing gel, and a protective film can be formed on the surface of skin through hydration, so that water evaporation is prevented, and the skin moisturizing state is enhanced and recovered. Meanwhile, the polysaccharide active substance in the hydrolyzed red algae extract has the effects of diminishing inflammation, resisting allergy and tightening the skin. Can promote skin tissue regeneration, accelerate skin metabolism, promote skin cell repair, fill skin basal tissue, smooth wrinkles from inside to outside, and care skin.
In the invention, the hypsizygus marmoreus callus culture filtrate, the tetrahydro-methylpyrimidine carboxylic acid and the hydrolyzed red algae extract are matched with each other to realize synergistic interaction, so that the skin barrier is comprehensively repaired. By strengthening the microbial barrier, the physicochemical barrier and the immune barrier of the skin, harmful invasion factors are isolated outside the skin, and a complete and healthy skin defense line is constructed, so that various skin problems are fundamentally solved. In addition, the composition has anti-inflammatory and soothing effects, and can quickly and effectively soothe inflammatory reaction and discomfort symptoms of the skin, and maintain the steady state and basic health of the skin.
Preferably, the mass ratio of the hypsizygus marmoreus callus culture filtrate to the tetrahydro-methyl pyrimidine carboxylic acid to the hydrolyzed red algae extract is (3-9): (2-5): 1-3);
wherein, 3-9 can be 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, etc.;
wherein, 2-5 can be 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8, 5, etc.;
wherein "1 to 3" may be 1, 1.2, 1.4, 1.5, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, etc.
Preferably, the hypecoum vulgare callus culture filtrate is prepared by the following method:
(1) callus induction culture: inoculating the hypecoum erectum explant to an induction culture medium for callus induction culture to obtain callus;
(2) suspension culture of callus particles: inoculating the callus obtained in the step (1) into a liquid culture medium for shake culture to obtain a proliferation callus;
(3) expanding and culturing the callus tissue: inoculating the proliferated callus obtained in the step (2) into a sterile cell bag for amplification culture to obtain the hypecoum erectum callus culture;
(4) separation: and (4) filtering and drying the hypecoum vulgare callus culture obtained in the step (3) to obtain the fennel callus culture filtrate.
Preferably, the hypecoum erectum of step (1) is a sterilized hypecoum erectum.
Preferably, the induction medium of step (1) is an agar medium.
Preferably, the temperature of the induction culture in step (1) is 15 to 30 ℃, for example, 15 ℃, 17 ℃, 19 ℃, 21 ℃, 23 ℃, 25 ℃, 27 ℃, 29 ℃, 30 ℃ and the like, and the time of the induction culture is 12 to 16 days, for example, 12 days, 13 days, 14 days, 15 days, 16 days and the like.
Preferably, the liquid medium in step (2) is MS medium.
Preferably, the temperature of the shake culture in step (2) is 15-30 ℃, for example, 15 ℃, 17 ℃, 19 ℃, 21 ℃, 23 ℃, 25 ℃, 27 ℃, 29 ℃, 30 ℃ and the like, and the time of the shake culture is 12-16 days, for example, 12 days, 13 days, 14 days, 15 days, 16 days and the like.
Preferably, the shaking culture in the step (2) is carried out in a shaking culture box, and the rotation speed of the shaking culture box is 50-200 r/min, such as 50r/min, 60r/min, 70r/min, 80r/min, 90r/min, 100r/min, 120r/min, 140r/min, 160r/min, 180r/min and 200 r/min.
Preferably, the temperature of the amplification culture in the step (3) is 15 to 30 ℃, for example, 15 ℃, 17 ℃, 19 ℃, 21 ℃, 23 ℃, 25 ℃, 27 ℃, 29 ℃, 30 ℃ and the like, and the time of the amplification culture is 20 to 25 days, for example, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days and the like.
Preferably, the filtration of step (4) is performed by gauze.
Preferably, the pore size of the gauze is 80-100 meshes, for example, 80 meshes, 82 meshes, 84 meshes, 86 meshes, 88 meshes, 90 meshes, 92 meshes, 94 meshes, 96 meshes, 98 meshes, 100 meshes.
Preferably, the drying temperature in step (4) is 20-30 deg.C, such as 20 deg.C, 21 deg.C, 22 deg.C, 23 deg.C, 24 deg.C, 25 deg.C, 26 deg.C, 27 deg.C, 28 deg.C, 29 deg.C, 30 deg.C.
Preferably, the hypecoum vulgare callus culture filtrate is prepared by the following method:
(1) callus induction culture: inoculating the hypecoum erectum explant into an agar culture medium for callus induction culture, wherein the temperature of the induction culture is 15-30 ℃, and the time of the induction culture is 12-16 days, so as to obtain a callus;
(2) suspension culture of callus particles: inoculating the callus obtained in the step (1) into an MS culture medium, and performing shake culture in a shake culture box at a rotating speed of 50-200 r/min, wherein the temperature of the shake culture is 15-30 ℃, and the time of the shake culture is 12-16 days, so as to obtain a proliferation callus;
(3) expanding and culturing the callus tissue: inoculating the proliferated callus obtained in the step (2) into a sterile cell bag for amplification culture, wherein the temperature of the amplification culture is 15-30 ℃, and the time of the amplification culture is 20-25 days, so as to obtain the hypecoum vulgare callus culture;
(4) separation: filtering the hypecoum vulgare callus culture obtained in the step (3) through gauze of 80-100 meshes, and drying at 20-30 ℃ to obtain the fennel callus culture filtrate.
Preferably, the tetrahydro-methyl-pyrimidine-carboxylic acid is prepared by the following method:
(A) culturing desert halophilic bacteria: inoculating desert halophilic bacteria into a liquid culture medium for culture to obtain desert halophilic bacteria thalli;
(B) osmotic shock: suspending the desert halophilic bacteria thallus obtained in the step (A) in a high osmotic pressure solution for soaking and filtering to obtain a precipitated desert halophilic bacteria thallus;
(C) electrodialysis: and (C) carrying out electrodialysis treatment on the desert halophilic bacteria thallus obtained in the step (B) to obtain the tetrahydro-methyl pyrimidine carboxylic acid.
Preferably, the liquid medium of step (a) is SCDLP liquid medium.
Preferably, the cultivation in step (A) is carried out in a shaking incubator, the rotation speed of the shaking incubator is 800-1000 r/min, such as 800r/min, 820r/min, 840r/min, 860r/min, 880r/min, 900r/min, 920r/min, 940r/min, 960r/min, 980r/min, 1000r/min, etc.
Preferably, the temperature of the culture in step (A) is 15-30 ℃, for example, 15 ℃, 17 ℃, 19 ℃, 21 ℃, 23 ℃, 25 ℃, 27 ℃, 29 ℃, 30 ℃ and the like, and the time of the culture is 45-55 h, for example, 45h, 46h, 47h, 48h, 49h, 50h, 51h, 52h, 53h, 54h, 55h and the like.
Preferably, the high osmotic pressure solution in the step (B) comprises the following components in percentage by mass: 10 to 20% (for example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc.) of sucrose, 0.05 to 0.2% (for example, 0.05%, 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18%, 0.2%, etc.) of EDTA, and the balance of water.
Preferably, the soaking time in the step (B) is 0.5-2 h, for example, 0.5h, 0.6h, 0.8h, 1h, 1.2h, 1.4h, 1.6h, 1.8h, 2h, etc.
Preferably, the tetrahydro-methyl-pyrimidine-carboxylic acid is prepared by the following method:
(A) culturing desert halophilic bacteria: inoculating desert halophilic bacteria into an SCDLP liquid culture medium, and culturing in a shaking incubator at the rotating speed of 800-1000 r/min at the temperature of 15-30 ℃ for 45-55 h to obtain desert halophilic bacteria thallus;
(B) osmotic shock: resuspending the desert halophilic bacteria thallus obtained in the step (A) in a high osmotic pressure solution (the high osmotic pressure solution comprises, by mass, 10-20% of sucrose, 0.05-0.2% of EDTA and the balance of water), soaking for 0.5-2 h, and filtering to obtain a precipitate desert halophilic bacteria thallus;
(C) electrodialysis: and (C) carrying out electrodialysis treatment and chromatographic purification on the desert halophilic bacteria thallus obtained in the step (B) to obtain the tetrahydro-methyl pyrimidine carboxylic acid.
Preferably, the hydrolyzed red algae extract is prepared by the following method:
(a) extraction: adding a solvent into red algae powder for extraction, standing and taking precipitate to obtain a red algae polysaccharide crude material;
(b) hydrolysis: dissolving the red algae polysaccharide crude material obtained in the step (a) in water, adding a hydrolyzing agent, mixing and stirring to obtain a hydrolyzed red algae extracting solution;
(c) and (3) ultrafiltration: and (c) treating the hydrolyzed red algae extracting solution obtained in the step (b) by an ultrafiltration membrane, concentrating and desalting to obtain a hydrolyzed red algae extract.
Preferably, step (a) is preceded by a step of pre-treatment.
Preferably, the step of pre-treating is: cleaning red algae, pulverizing, and sieving to obtain red algae powder.
Preferably, the mesh number of the sieve is 100-200 meshes, for example, 100 meshes, 120 meshes, 140 meshes, 160 meshes, 180 meshes, 200 meshes and the like can be provided.
Preferably, the red algae is selected from any one or combination of at least two of Durvillea, thallus Porphyrae, Eucheuma Gelatinosum, sea weed, Beauveria nodosa, and Corallium japonicum Thunb, preferably Durvillea.
Preferably, the specific steps of the extraction in the step (a) are as follows: adding water into red algae powder for primary extraction to obtain primary extract, filtering the primary extract, concentrating, adding ethanol water solution for secondary extraction, standing to obtain precipitate to obtain red algae polysaccharide coarse material.
Preferably, the mass ratio of the water to the red algae powder is (20-30): 1, and can be, for example, 20:1, 22:1, 24:1, 26:1, 28:1, 30:1 and the like.
Preferably, the temperature of the primary extraction is 60-80 deg.C, such as 60 deg.C, 62 deg.C, 64 deg.C, 66 deg.C, 68 deg.C, 70 deg.C, 72 deg.C, 74 deg.C, 76 deg.C, 78 deg.C, 80 deg.C, etc., and the time of the primary extraction is 8-12h, such as 8h, 9h, 10h, 11h, 12h, etc.
Preferably, the concentration of the ethanol aqueous solution by volume ratio is 70 to 90%, and may be, for example, 70%, 72%, 74%, 75%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, or the like.
Preferably, the temperature of the secondary extraction is 50-70 deg.C, such as 50 deg.C, 52 deg.C, 54 deg.C, 56 deg.C, 58 deg.C, 60 deg.C, 62 deg.C, 64 deg.C, 66 deg.C, 68 deg.C, 70 deg.C, etc., and the time of the secondary extraction is 5-8h, such as 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h, etc.
Preferably, the hydrolytic agent of step (b) is 20-40 wt% (for example, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%) of aqueous hydrogen peroxide solution.
Preferably, the mixing and stirring time in the step (b) is 1 to 2 hours, such as 1 hour, 1.2 hours, 1.4 hours, 1.6 hours, 1.8 hours, 2 hours and the like, and the mixing and stirring temperature is 30 to 60 ℃, such as 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃ and the like.
Preferably, the time of ultrafiltration in step (c) is 20-40min, such as 20min, 22min, 24min, 26min, 28min, 30min, 32min, 34min, 36min, 38min, 40min, etc., and the temperature of ultrafiltration is 30-40 deg.C, such as 30 deg.C, 31 deg.C, 32 deg.C, 33 deg.C, 34 deg.C, 35 deg.C, 36 deg.C, 37 deg.C, 38 deg.C, 39 deg.C, 40 deg.C, etc.
Preferably, the hydrolyzed red algae extract is prepared by the following method:
(a) extraction: adding water into red algae powder to perform primary extraction at the temperature of 60-80 ℃, wherein the primary extraction time is 8-12h, the mass ratio of the water to the red algae powder is (20-30): 1, so as to obtain a primary extracting solution, filtering and concentrating the primary extracting solution, adding an ethanol water solution with the volume ratio concentration of 70-90% to perform secondary extraction, standing, and taking a precipitate, so as to obtain a red algae polysaccharide coarse material;
(b) hydrolysis: dissolving the red algae polysaccharide crude material obtained in the step (a) in water, adding 20-40 wt% of hydrogen peroxide water solution, mixing and stirring at 30-60 ℃ for 1-2 h to obtain hydrolyzed red algae extracting solution;
(c) and (3) ultrafiltration: and (c) treating the hydrolyzed red algae extract obtained in the step (b) by an ultrafiltration membrane, concentrating and desalting, wherein the ultrafiltration time is 20-40min, and the ultrafiltration temperature is 30-40 ℃ to obtain the hydrolyzed red algae extract.
In a second aspect, the present invention provides a method for preparing a composition for repairing a skin barrier according to the first aspect, the method comprising: mixing the hypecoum vulgare callus culture filtrate, the tetrahydro-methyl pyrimidine carboxylic acid and the hydrolyzed red algae extract to obtain the composition for repairing skin barrier.
Preferably, the mixing temperature is 20 ~ 40 ℃, for example can be 20 ℃, 22 ℃, 24 ℃, 26 ℃, 28 ℃, 30 ℃, 32 ℃, 34 ℃, 36 ℃, 38 ℃, 40 ℃ etc..
Preferably, the mixing time is 10-30 min, such as 10min, 12min, 14min, 16min, 18min, 20min, 22min, 24min, 26min, 28min, 30min, etc.
Preferably, the composition for repairing skin barrier is prepared by: mixing the hypsizygus marmoreus callus culture filtrate, the tetrahydro-methyl pyrimidine carboxylic acid and the hydrolyzed red algae extract at 20-40 ℃ for 10-30 min to obtain the composition for repairing the skin barrier.
In a third aspect, the present invention provides the use of a composition for repairing a skin barrier according to the first aspect in a skin care product.
Compared with the prior art, the invention has the following beneficial effects:
(1) the composition for repairing the skin barrier comprises hypsizygus marmoreus callus culture filtrate, tetrahydro-methylpyrimidine carboxylic acid and a hydrolyzed red algae extract, all components are matched with each other and have synergistic interaction, harmful invasion factors are isolated outside the skin by strengthening a microbial barrier, a physicochemical barrier and an immune barrier of the skin, a complete and healthy skin defense line is constructed, and various skin problems are fundamentally solved.
(2) The composition for repairing the skin barrier also has the effects of resisting inflammation and relieving, quickly and effectively soothing the inflammatory reaction and the uncomfortable symptoms of the skin, and maintaining the steady state and the basic health of the skin.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Preparation example 1
The preparation example prepares a hypsizygus marmoreus callus culture, and the hypsizygus marmoreus callus culture filtrate is prepared by the following method:
(1) callus induction culture: inoculating the hypecoum erectum explant into an agar culture medium for callus induction culture, wherein the induction culture temperature is 20 ℃, and the induction culture time is 14 days, so as to obtain callus;
(2) suspension culture of callus particles: inoculating the callus obtained in the step (1) into an MS culture medium, and performing shake culture in a shake culture box at a rotating speed of 100r/min, wherein the temperature of the shake culture is 20 ℃, and the time of the shake culture is 14 days, so as to obtain a proliferation callus;
(3) expanding and culturing the callus tissue: inoculating the proliferated callus obtained in the step (2) into a sterile cell bag for amplification culture at the temperature of 25 ℃ for 24 days to obtain the hypecoum erectum callus culture;
(4) separation: and (4) filtering the hypecoum vulgare callus culture obtained in the step (3) through gauze of 100 meshes, and then performing vacuum drying at 25 ℃ to obtain the fennel callus culture filtrate.
Preparation example 2
This preparation example prepared a tetrahydro-methyl-pyrimidine-carboxylic acid prepared by the following method:
(A) culturing desert halophilic bacteria: inoculating desert halophilic bacteria into an SCDLP liquid culture medium, and culturing in a shaking incubator at the rotating speed of 1000r/min, wherein the culturing temperature is 20 ℃, and the culturing time is 48h, so as to obtain desert halophilic bacteria thallus;
(B) osmotic shock: resuspending the desert halophilic bacteria thallus obtained in the step (A) in a high osmotic pressure solution (the high osmotic pressure solution comprises 18% of sucrose, 0.1% of EDTA and the balance of water by mass percent), soaking for 1h, and filtering to obtain a precipitate desert halophilic bacteria thallus;
(C) electrodialysis: and (C) carrying out electrodialysis treatment on the desert halophilic bacteria thallus obtained in the step (B) to obtain the tetrahydro-methyl pyrimidine carboxylic acid.
Preparation example 3
The preparation example prepares a hydrolyzed red algae extract, and the preparation method of the hydrolyzed red algae extract comprises the following steps:
(a) extraction: cleaning Duerweili algae, pulverizing, and sieving with 100-fold 200-mesh sieve to obtain red algae powder; adding water into red algae powder, extracting at 70 ℃ for the first time, wherein the first extraction time is 10 hours, the mass ratio of the water to the red algae powder is 20:1, obtaining a first extracting solution, filtering the first extracting solution, concentrating, adding an ethanol water solution with the volume ratio concentration of 80%, extracting for the second time, standing, and taking a precipitate to obtain a red algae polysaccharide coarse material;
(b) hydrolysis: dissolving the red algae polysaccharide crude material obtained in the step (a) in water, adding 30 wt% of aqueous hydrogen peroxide, and mixing and stirring at 50 ℃ for 1h to obtain hydrolyzed red algae extracting solution;
(c) and (3) ultrafiltration: and (c) treating the hydrolyzed red algae extracting solution obtained in the step (b) by using an ultrafiltration membrane, concentrating and desalting, wherein the ultrafiltration time is 30min, and the ultrafiltration temperature is 30 ℃ to obtain a hydrolyzed red algae extract.
Example 1
The present example provides a composition for repairing a skin barrier, the composition comprising: sea fennel callus culture filtrate 0.5g, tetrahydro methyl pyrimidine carboxylic acid 0.4g and hydrolyzed red algae extract 0.3 g.
The preparation method of the composition for repairing the skin barrier comprises the following steps: mixing 0.5g hypecoum vulgare callus culture filtrate, 0.4g tetrahydro-methylpyrimidine carboxylic acid and 0.3g hydrolyzed red algae extract at 30 deg.C for 20min to obtain the composition for repairing skin barrier.
Example 2
The present example provides a composition for repairing a skin barrier, the composition comprising: sea fennel callus culture filtrate 0.8g, tetrahydro methyl pyrimidine carboxylic acid 0.2g and hydrolyzed red algae extract 0.2 g.
The preparation method of the composition for repairing the skin barrier comprises the following steps: mixing 0.8g hypecoum vulgare callus culture filtrate, 0.2g tetrahydro-methyl pyrimidine carboxylic acid and 0.2g hydrolyzed red algae extract at 20 deg.C for 30min to obtain the composition for repairing skin barrier.
Example 3
The present example provides a composition for repairing a skin barrier, the composition comprising: sea fennel callus culture filtrate 0.6g, tetrahydro methyl pyrimidine carboxylic acid 0.3g and hydrolyzed red algae extract 0.3 g.
The preparation method of the composition for repairing the skin barrier comprises the following steps: mixing 0.6g hypecoum vulgare callus culture filtrate, 0.3g tetrahydro-methyl pyrimidine carboxylic acid and 0.3g hydrolyzed red algae extract at 40 deg.C for 10min to obtain the composition for repairing skin barrier.
Example 4
This example provides a composition for repairing a skin barrier, differing from example 1 in that the composition comprises: sea fennel callus culture filtrate 0.9g, tetrahydro methyl pyrimidine carboxylic acid 0.2g and hydrolyzed red algae extract 0.1g, and the skin barrier repairing composition was prepared in the same manner as in example 1.
Example 5
This example provides a composition for repairing a skin barrier, differing from example 1 in that the composition comprises: sea fennel callus culture filtrate 0.3g, tetrahydro methyl pyrimidine carboxylic acid 0.5g and hydrolyzed red algae extract 0.3g, and the skin barrier repairing composition was prepared in the same manner as in example 1.
Example 6
This example provides a composition for repairing a skin barrier, differing from example 1 in that the composition comprises: sea fennel callus culture filtrate 0.1g, tetrahydro methyl pyrimidine carboxylic acid 0.6g and hydrolyzed red algae extract 0.5g, and the skin barrier repairing composition was prepared in the same manner as in example 1.
Example 7
This example provides a composition for repairing a skin barrier, differing from example 1 in that the composition comprises: sea fennel callus culture filtrate 0.6g, tetrahydro methyl pyrimidine carboxylic acid 0.1g and hydrolyzed red algae extract 0.5g, and the skin barrier repairing composition was prepared in the same manner as in example 1.
Example 8
This example provides a composition for repairing a skin barrier, differing from example 1 in that the composition comprises: sea fennel callus culture filtrate 0.6g, tetrahydro methyl pyrimidine carboxylic acid 0.55g and hydrolyzed red algae extract 0.05g, and the skin barrier repairing composition was prepared in the same manner as in example 1.
Comparative example 1
This comparative example provides a composition for repairing skin barrier, which is different from example 1 in that it does not contain hypsizygus marmoreus callus culture filtrate, the content of the tetrahydro-methylpyrimidine carboxylic acid is increased to 0.65g, the content of the hydrolyzed red algae extract is increased to 0.55g, and the composition for repairing skin barrier is prepared in the same manner as in example 1.
Comparative example 2
This comparative example provides a composition for repairing skin barrier, which is different from example 1 in that it does not contain tetrahydro-methyl-pyrimidine-carboxylic acid, the hypsizygus marmoreus callus culture filtrate content is increased to 0.7g, the hydrolyzed red algae extract content is increased to 0.5g, and the composition for repairing skin barrier is prepared in the same manner as in example 1.
Comparative example 3
This comparative example provides a composition for repairing skin barrier, which is different from example 1 in that the composition does not contain hydrolyzed red algae extract, the hypsizygus marmoreus callus culture filtrate content is increased to 0.65g, the tetrahydro-methylpyrimidine carboxylic acid content is increased to 0.55g, and the composition for repairing skin barrier is prepared in the same manner as in example 1.
Test example 1
Safety performance testing
The compositions for repairing skin barriers provided in examples 1 to 8 and the compositions for repairing skin barriers provided in comparative examples 1 to 3 were subjected to a safety performance test by the following method:
(1) haemolysis test of erythrocytes
Preparation of erythrocyte suspension: selecting healthy rabbits, taking 9mL of blood from heart, adding 1mL of 2% potassium oxalate solution, centrifuging, discarding supernatant, diluting the precipitate to 20mL with 20mmol/L PBS solution, and storing at 4 ℃ for later use. Select samples were diluted with PBS solution to different concentrations, with 5 concentration gradients set for each sample. Adding 200 μ L of the above erythrocyte suspension (final concentration of the sample is controlled to be 5, 10, 20, 50, 100mg/mL respectively) into 10mL of diluent of the sample to be tested, taking distilled water as total blood-dissolving control, taking PBS solution as negative control, mixing gently, incubating at 37 deg.C for 30min, centrifuging at 2000r/min for 10min, collecting supernatant, and testing its absorbance at 560nm with spectrophotometer (A)560) Calculating the hemolysis rate according to the following formula;
drawing a standard curve of hemolysis rate to sample concentration, and calculating the sample concentration (HD) when 50% of erythrocytes are hemolyzed50)。
(2) Protein denaturation experiments:
diluting the sample to 10g/L with PBS solution, collecting 10mL diluted solution of sample to be tested, adding 200 μ L of the above erythrocyte suspension, using distilled water as blank control, 1mg/mL Sodium Dodecyl Sulfate (SDS) solution as positive control, and gently dilutingMixing, incubating at 37 deg.C for 30min, centrifuging at 2000r/min for 10min, collecting supernatant, and measuring absorbance A at 540nm and 575nm with spectrophotometer540And A575Calculating a protein Denaturation Index (DI) according to the following formula;
wherein R is1Blank control group a575Blank control group A540，R2Experimental group a575Experimental group A540，R3Positive control group A575Positive control group A540。
Evaluating the irritation of the sample to be tested according to the L/D value, wherein the L/D value is HD50DI, erythrocyte hemolysis assay irritation grading criteria are shown in Table 1 below:
TABLE 1
| L/D | Grading |
| >100 | Has no irritation |
| 10<L/D≤100 | Micro-stimulation property |
| 1<L/D≤10 | Mild irritation |
| 0.1<L/D≤1 | Moderate irritation |
The results of the above-described hemolysis test and protein denaturation test are shown in Table 2 below:
TABLE 2
As can be seen from the composition for repairing skin barriers and performance tests provided by 1-8, the irritation of the skin care composition provided by the invention is classified as no irritation, which shows that the skin care composition provided by the invention has the characteristics of being mild and no irritation.
Test example 2
Test for moisture retention
The compositions for repairing skin barriers provided in examples 1 to 8 and comparative examples 1 to 3 were subjected to a moisturizing performance test by the following method:
(1) rate of change of percutaneous water loss: the apparatus is a Tewameter TM300 from CK, Germany, testing the external environment: room temperature 25 ℃ and humidity 60%. 110 healthy 18-40-year-old volunteers were selected, half of the male and female proportion, and randomly divided into 11 groups of 5 males and 5 females, and the skin barrier repairing compositions provided in examples 1-8 and comparative examples 1-3 were used, respectively. Each subject was tested for change in transepidermal water loss (based on 100% prior to use) three times before the skin composition was used and 4 days after the consecutive use, and after averaging, the average value for each group was calculated and one decimal point was retained.
(2) Moisture retention ratio: the instrument is of the German Courage + Khazaka electronic type: derma UnitSSC, test temperature 25 ℃, humidity 60%. 110 healthy 18-40-year-old volunteers were selected, half of the male and female proportion, and randomly divided into 11 groups of 5 males and 5 females, and the skin barrier repairing compositions provided in examples 1-8 and comparative examples 1-3 were used, respectively. The skin moisture content (based on 100% before use) of each subject was measured three times before use and 4h after use, and after averaging, the average value of each group was calculated, and one decimal point was retained.
(3) In-vitro weighing method moisturizing performance test: weighing 0.2g of sample, respectively and uniformly coating the sample on a 5cm × 5cm glass plate stuck with the microporous breathable adhesive tape, putting the glass plate into a dryer with constant temperature and constant humidity, respectively weighing the mass of the glass plate after being placed for 4 hours, and calculating the moisture retention rate. The moisture retention rate calculation formula is as follows: moisture retention rate/% (M)2～M0)/(M1～M0)×100％。
Wherein M is0Is the mass/g, M, of the glass sheet1For the mass/g, M of the glass plate after sample application2The glass sheet mass/g after standing in the desiccator for several hours.
The specific test results are shown in table 3:
TABLE 3
| Sample (I) | Percutaneous rate of change of water loss/%) | Water retention ratio/%) | Moisture retention/degree |
| Example 1 | 90.85 | 94.90 | 94.90 |
| Example 2 | 91.54 | 92.18 | 93.30 |
| Example 3 | 92.42 | 90.33 | 93.42 |
| Example 4 | 95.21 | 89.29 | 90.13 |
| Example 5 | 95.52 | 87.61 | 89.30 |
| Example 6 | 98.54 | 85.74 | 87.90 |
| Example 7 | 97.95 | 84.97 | 85.27 |
| Example 8 | 100.07 | 84.31 | 85.55 |
| Comparative example 1 | 128.53 | 79.40 | 75.92 |
| Comparative example 2 | 119.42 | 78.90 | 80.12 |
| Comparative example 3 | 124.61 | 79.72 | 82.00 |
From the above test data, it can be seen that the skin barrier repairing compositions provided in embodiments 1 to 8 have a percutaneous water loss change rate of 100% or less, a water retention rate of 84.31% or more, and a moisture retention rate of 85% or more measured by an in vitro weighing method, which fully indicates that the skin barrier repairing composition of the present invention includes a hypsizygus marmoreus callus culture filtrate, a tetrahydro methylpyrimidine carboxylic acid, and a hydrolyzed red algae extract, and the components cooperate with each other to synergistically cooperate with skin protein to form a moisture-retaining gel, and can form a protective film on the skin surface through hydration, thereby preventing water evaporation, and helping to strengthen and restore the skin moisture-retaining state. Promote the formation of the ice-like crystal water structure, reduce the water activity, keep moisture for a long time, and keep the skin fresh by the ice-like crystal water structure.
Test example 3
Test for Oxidation resistance
(1) Evaluation of superoxide anion radical scavenging ability
4.5mL of 0.05mol/L Tris-HCl buffer solution with pH 8.2 was preheated in a 25 ℃ water bath for 30 min. Then adding 1mL of sample and 0.4mL of 25mol/L pyrogallol solution, mixing uniformly, reacting in a water bath at 25 ℃ for 5min, and adding 1.0mL of 8mol/L HCl to terminate the reaction. Absorbance values were measured at 299nm with Tris-HCl buffer as reference. Blank control 1mL of sample in solvent was used instead of sample. The superoxide anion radical clearance rate (%) ([ 1- (A)2/A1)]X 100% in the formula1Absorbance values for the blank; a. the2The absorbance values for the corresponding samples.
(2) Evaluation of hydroxyl radical scavenging ability
2mmol/L FeSO are added into a 25mL cuvette in turn43mL，1mmol/L H2O23mL, shaking up, then adding 3mL of salicylic acid with the concentration of 6mmol/L, shaking up, heating in a water bath at 37 ℃ for 15min, taking out, and measuring the absorbance; adding the solution to be tested, shaking, heating in water bath for 15min, and taking out to test its absorbance. (ii) the hydroxyl radical clearance (%) ═ a0-Ax-(A0-Ax0)]/A0X 100% in the formula0The absorbance value of the reaction system before adding the sample is obtained; a. thexRemoving hydroxyl radicals from the sample to obtain an absorbance value of the system; a. thex0Absorbance values for the system after scavenging hydroxyl radicals for the blank control.
The specific test results are shown in table 4:
TABLE 4
| Sample (I) | Superoxide anion radical scavenging rate% | Hydroxy radical clearance/%) |
| Example 1 | 94.88 | 94.20 |
| Example 2 | 95.12 | 95.00 |
| Example 3 | 94.86 | 93.28 |
| Example 4 | 93.31 | 92.88 |
| Example 5 | 91.12 | 92.00 |
| Example 6 | 92.00 | 91.56 |
| Example 7 | 91.74 | 92.24 |
| Example 8 | 92.79 | 91.20 |
| Comparative example 1 | 80.90 | 85.21 |
| Comparative example 2 | 82.75 | 82.34 |
| Comparative example 3 | 84.03 | 85.01 |
From the above test data, the compositions for repairing skin barrier provided in embodiments 1 to 8 have superoxide anion radical scavenging rate of 91% or more and hydroxyl radical scavenging rate of 91% or more. The composition for repairing the skin barrier comprises the hypsizygus marmoreus callus culture filtrate, the tetrahydro-methylpyrimidine carboxylic acid and the hydrolyzed red algae extract, and all the components are matched with each other, so that the composition has a good effect of removing free radicals in a synergistic manner, and the antioxidant protection capability of the skin is recovered.
Test example 4
Anti-allergic Performance test
Hyaluronidase is a participant of anaphylactic reaction, has strong correlation with inflammation and allergy, and various medicaments for releasing histamine from mast cells can regulate the activity of hyaluronidase and inhibit the activity of hyaluronidase to be used as indexes for researching antiallergic effect. Therefore, the Elson-Morgan method was used for the hyaluronidase in vitro inhibition experiment. 0.1ml of 0.25mmol/LCaCl is taken2The solution and 0.5mL of hyaluronidase solution are cultured for 20min at 37 ℃ in an insulation way; adding 0.5mL of the Chinese medicinal composition, and culturing at 37 deg.C for 20 min; adding 0.5mL sodium hyaluronate solution, keeping the temperature at 37 ℃ for 30min, and standing at normal temperature for 5 min; adding 0.1mL0.4mol/LNaOH solution and 0.5mL acetylacetone solution, heating in a boiling water bath for 15min, and immediately cooling for 5min with ice water; adding 1.0mL of Ellisib reagent, diluting with 3.0mL of anhydrous ethanol, standing for 20min for color development, and measuring the absorbance value with a spectrophotometer. Calculating the formula: hyaluronidase inhibition (%) [ ((a-B) - (C-D))/a-B]X 100%, wherein: a represents the ABS value of the control solution, B represents the ABS value of the control blank solution, C represents the ABS value of the sample solution, and D represents the ABS value of the sample blank solution.
The specific test results are shown in table 5:
TABLE 5
As can be seen from the above test data, the hyaluronidase inhibition rate of the composition for repairing skin barrier provided in embodiments 1 to 8 is above 62%, which fully indicates that the composition for repairing skin barrier of the present invention includes hypsizygus marmoreus callus culture filtrate, tetrahydro-methylpyrimidine carboxylic acid, and hydrolyzed red algae extract, and the components cooperate with each other, so as to achieve synergistic interaction and good hyaluronidase inhibition rate, and the composition for repairing skin barrier also has anti-inflammatory and soothing effects, and is capable of rapidly and effectively soothing inflammatory reaction and discomfort symptoms of skin, and maintaining skin homeostasis and basic health.
Test example 5
Test of whitening Effect
Selecting the same generation B16 melanocyte of logarithmic growth phase, blowing and beating into single cell suspension after conventional pancreatin digestion treatment, counting and quantitatively inoculating in 96-well plate, removing supernatant after 24 hr, respectively adding RPMI1640 culture solution containing 10% sample, and adding CO with volume fraction of 5% at 37 deg.C2Culturing under saturated wet condition, adding RPMI1640 culture solution (containing water) into control well, culturing for three days, and replacing culture solution once in the middle; discarding the supernatant, counting the cell density after the conventional pancreatin digestion treatment, and centrifuging to obtain cell sediment; washing twice with PBS, centrifuging to remove supernatant, quantitatively adding 1mol/L NaOH solution containing 10% DMSO, water bathing at 80 deg.C for 50min, and measuring light absorption value at 450nm of microplate reader; melanin production inhibition (%) × (absorbance of each test material/absorbance of control group) × 100%.
The specific test results are shown in table 6:
TABLE 6
From the above test data, it can be seen that the melanin production inhibition rate of the composition for repairing a skin barrier provided in embodiments 1 to 8 is above 34%, which fully indicates that the composition for repairing a skin barrier of the present invention includes a hypsizygus marmoreus callus culture filtrate, a tetrahydro-methylpyrimidine carboxylic acid, and a hydrolyzed red algae extract, and the components are mutually matched to achieve a synergistic effect, so that the composition has a good melanin production inhibition rate, and has the effects of whitening and brightening.
Test example 6
Efficacy test for anti-aging and wrinkle-removing
Experiment for promoting elastin production: human fibroblasts were arranged at 2X 104The cells were inoculated on a 96-well plate at a density per well, and cultured in an incubator at 37 ℃ for 24 hours with a fresh medium, and after the cells adhered to the wall, the fresh medium was replaced, and the skin barrier repairing compositions provided in examples 1 to 8 and comparative examples 1 to 3 (the final concentration of the sample was controlled to 50mg/mL) were added, respectively, with a 20mmol/L PBS solution as a blank, and then cultured in the incubator at 37 ℃ for 2 days.
After the culture, the cells were eluted with a cell lysis solution and disrupted by sonication. Detecting the content of the elastin by adopting a Biocolor elastin detection kit: treating a sample with an elastin precipitator to precipitate elastin, centrifuging, removing supernatant, adding a dye and a reaction solution into the precipitate to react to form an elastin-dye complex, centrifuging and washing with a PBS solution for three times to remove unbound dye, adding a dye releasing agent to release the dye in the elastin-dye complex, and measuring the absorbance of the sample at 513nm by using a spectrophotometer. The expression amount of elastin was calculated from the absorbance-dye concentration standard curve (elastin expression amount in examples and comparative examples is relative expression amount to blank, blank expression amount is 1), and the test results are shown in table 7.
TABLE 7
| Sample (I) | Amount of elastin expressed |
| Example 1 | 7.5 |
| Example 2 | 7.3 |
| Example 3 | 7.3 |
| Example 4 | 6.7 |
| Example 5 | 6.8 |
| Example 6 | 6.3 |
| Example 7 | 6.5 |
| Example 8 | 6.2 |
| Comparative example 1 | 3.0 |
| Comparative example 2 | 3.4 |
| Comparative example 3 | 2.2 |
As can be seen from the test results in Table 7, the elastin expression levels of the skin barrier repairing compositions provided in examples 1-8 are above 6.2, while those of the skin barrier repairing compositions provided in comparative examples 1-3 are much lower. The composition for repairing the skin barrier comprises the hypsizygus marmoreus callus culture filtrate, the tetrahydro-methylpyrimidine carboxylic acid and the hydrolyzed red algae extract, and all the components are matched with each other to realize synergistic interaction, so that the cell growth is promoted, the skin barrier is repaired, the skin elasticity is increased, and the composition has good anti-aging and wrinkle-removing effects.
Test example 7
Detection of cell regeneration effect after sun exposure
Cell migration assay (cell migration assay) experiments were performed to test the regeneration effect of the suncured cells of the compositions for repairing skin barrier provided in examples 1 to 8 and comparative examples 1 to 3, respectively. The specific test method comprises the following steps: after culturing the skin keratinocytes HaCaT purchased from ATCC in a petri dish (dish) so that 95% or more thereof was obtained, the skin keratinocytes HaCaT were irradiated with ultraviolet rays to form a wound (wound). The plates with the wound (wound) were treated with 2% FBS (fetal bovine serum (fetalbovene serum), welgee) and quantified by measuring the degree of recovery with time by moving distance (blank control, recovery by cells themselves without any of the above compositions). The moving distance was measured using a microscope on the medium, and the same site was measured and compared by the Toupview program maintaining a ratio of 100, and the longer the moving distance (recovery distance/nm) was, the better the recovery degree of cell healing was, and the better the cell regeneration effect was, and the specific test results are shown in Table 8.
TABLE 8
From the above test data, the compositions for repairing skin barrier provided in examples 1 to 8 repaired keratinocytes after uv sunburn, and the recovery distance was over 610nm, whereas the recovery ability of the comparative examples 1 to 3 was slightly increased compared to the cell itself. It is fully demonstrated that the composition for repairing skin barrier of the present invention comprises the hypsizygus marmoreus callus culture filtrate, the tetrahydro-methylpyrimidine carboxylic acid and the hydrolyzed red algae extract, and the components are mutually matched, have synergistic effect, and have good capability of repairing cell regeneration.
The applicant states that the present invention is illustrated by the above examples of the composition for repairing skin barrier of the present invention and the preparation method and application thereof, but the present invention is not limited to the above detailed method, i.e. it does not mean that the present invention must be implemented by the above detailed method. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. A composition for repairing skin barriers, said composition comprising a hypsizygus marmoreus callus culture filtrate, a tetrahydro-methyl pyrimidine carboxylic acid and a hydrolyzed red algae extract.
2. The composition for repairing a skin barrier according to claim 1, wherein the mass ratio of the hypsizygus marmoreus callus culture filtrate, the tetrahydro-methylpyrimidine carboxylic acid and the hydrolyzed red algae extract is (3-9): (2-5): (1-3).
3. A skin barrier repairing composition according to claim 1 or 2 wherein said hypecoum vulgare callus culture filtrate is prepared by the following method:
(1) callus induction culture: inoculating the hypecoum erectum explant to an induction culture medium for callus induction culture to obtain callus;
(2) suspension culture of callus particles: inoculating the callus obtained in the step (1) into a liquid culture medium for shake culture to obtain a proliferation callus;
(3) expanding and culturing the callus tissue: inoculating the proliferated callus obtained in the step (2) into a sterile cell bag for amplification culture to obtain the hypecoum erectum callus culture;
(4) separation: and (4) filtering and drying the hypecoum vulgare callus culture obtained in the step (3) to obtain the fennel callus culture filtrate.
4. The composition for repairing a skin barrier according to claim 3, wherein the induction medium of step (1) is an agar medium;
preferably, the temperature of the induction culture in the step (1) is 15-30 ℃, and the time of the induction culture is 12-16 days;
preferably, the liquid medium in step (2) is MS medium;
preferably, the temperature of the shake culture in the step (2) is 15-30 ℃, and the shake culture time is 12-16 days;
preferably, the shake culture in the step (2) is carried out in a shake culture box, and the rotation speed of the shake culture box is 50-200 r/min;
preferably, the temperature of the amplification culture in the step (3) is 15-30 ℃, and the time of the amplification culture is 20-25 days;
preferably, the filtration of step (4) is performed by gauze;
preferably, the aperture of the gauze is 80-100 meshes;
preferably, the drying temperature in the step (4) is 20-30 ℃.
5. A composition for repairing a skin barrier according to any of claims 1-4, wherein said tetrahydro-methyl-pyrimidine-carboxylic acid is prepared by:
(A) culturing desert halophilic bacteria: inoculating desert halophilic bacteria into a liquid culture medium for culture to obtain desert halophilic bacteria thalli;
(B) osmotic shock: suspending the desert halophilic bacteria thallus obtained in the step (A) in a high osmotic pressure solution for soaking and filtering to obtain a precipitated desert halophilic bacteria thallus;
(C) electrodialysis: and (C) carrying out electrodialysis treatment on the desert halophilic bacteria thallus obtained in the step (B) to obtain the tetrahydro-methyl pyrimidine carboxylic acid.
6. The composition for repairing a skin barrier according to claim 5, wherein said liquid medium of step (A) is SCDLP liquid medium;
preferably, the culture in the step (A) is carried out in a shaking incubator, and the rotation speed of the shaking incubator is 800-1000 r/min;
preferably, the temperature of the culture in the step (A) is 15-30 ℃, and the culture time is 45-55 h;
preferably, the high osmotic pressure solution in the step (B) comprises the following components in percentage by mass: 10-20% of sucrose, 0.05-0.2% of EDTA and the balance of water;
preferably, the soaking time in the step (B) is 0.5-2 h.
7. The composition for repairing skin barriers according to any one of claims 1 to 6, wherein the hydrolyzed red algae extract is prepared by the following method:
(a) extraction: adding a solvent into red algae powder for extraction, standing and taking precipitate to obtain a red algae polysaccharide crude material;
(b) hydrolysis: dissolving the red algae polysaccharide crude material obtained in the step (a) in water, adding a hydrolyzing agent, mixing and stirring to obtain a hydrolyzed red algae extracting solution;
(c) and (3) ultrafiltration: and (c) treating the hydrolyzed red algae extracting solution obtained in the step (b) by an ultrafiltration membrane, concentrating and desalting to obtain a hydrolyzed red algae extract.
8. The skin barrier repairing composition according to claim 7, further comprising a step of pre-treatment before step (a);
preferably, the step of pre-treating is: cleaning red algae, pulverizing, and sieving to obtain red algae powder;
preferably, the mesh number of the sieving is 100-200 meshes;
preferably, the red algae is selected from any one or combination of at least two of Durvillea, thallus Porphyrae, Eucheuma Gelatinosum, sea weed, Beauveria nodosa, and Corallium japonicum Thunb, preferably Durvillea japonica;
preferably, the specific steps of the extraction in the step (a) are as follows: adding water into red algae powder for primary extraction to obtain primary extract, filtering the primary extract, concentrating, adding ethanol water solution for secondary extraction, standing to obtain precipitate to obtain red algae polysaccharide coarse material;
preferably, the mass ratio of the water to the red algae powder is (20-30): 1;
preferably, the temperature of the primary extraction is 60-80 ℃, and the time of the primary extraction is 8-12 h;
preferably, the volume ratio concentration of the ethanol water solution is 70-90%;
preferably, the temperature of the secondary extraction is 50-70 ℃, and the time of the secondary extraction is 5-8 h;
preferably, the hydrolytic agent in the step (b) is 20-40 wt% of aqueous hydrogen peroxide solution;
preferably, the mixing and stirring time in the step (b) is 1-2 hours, and the mixing and stirring temperature is 30-60 ℃;
preferably, the ultrafiltration time of step (c) is 20-40min, and the ultrafiltration temperature is 30-40 ℃.
9. The method for preparing a composition for repairing a skin barrier as claimed in any one of claims 1 to 8, wherein the method for preparing the composition for repairing a skin barrier comprises: mixing the hypecoum vulgaris callus culture filtrate, the tetrahydro-methyl pyrimidine carboxylic acid and the hydrolyzed red algae extract to obtain the composition for repairing the skin barrier;
preferably, the mixing temperature is 20-40 ℃;
preferably, the mixing time is 10-30 min.
10. Use of a composition for repairing a skin barrier according to any one of claims 1 to 8 in a skin care product.
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| CN111956514A (en) * | 2020-09-12 | 2020-11-20 | 梁其文 | Essence lotion for repairing sensitive skin and preparation method thereof |
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| CN112716855A (en) * | 2021-01-27 | 2021-04-30 | 科丝美诗(中国)化妆品有限公司 | Composition with barrier repairing and anti-aging effects and preparation method and application thereof |
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| CN112263540B (en) * | 2020-11-02 | 2023-03-10 | 浙江英树生物科技有限公司 | Whitening composition and preparation method and application thereof |
| CN112716855A (en) * | 2021-01-27 | 2021-04-30 | 科丝美诗(中国)化妆品有限公司 | Composition with barrier repairing and anti-aging effects and preparation method and application thereof |
| CN112941010A (en) * | 2021-01-30 | 2021-06-11 | 湖北嘉弘福科技有限责任公司 | Plant callus efficacy filtrate and exosome extraction method |
| CN114533645A (en) * | 2022-03-07 | 2022-05-27 | 广州市万千粉丝化妆品有限公司 | Application of substrate liquid of activated algae like bactosporium in skin care product and preparation method |
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