CN116763709A - Recombinant mussel mucin cream for nursing damaged skin barrier and preparation method thereof - Google Patents
Recombinant mussel mucin cream for nursing damaged skin barrier and preparation method thereof Download PDFInfo
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- CN116763709A CN116763709A CN202310953006.XA CN202310953006A CN116763709A CN 116763709 A CN116763709 A CN 116763709A CN 202310953006 A CN202310953006 A CN 202310953006A CN 116763709 A CN116763709 A CN 116763709A
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- mussel mucin
- skin
- cream
- recombinant mussel
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- 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
- A61K8/97—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
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9794—Liliopsida [monocotyledons]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
- A61K8/04—Dispersions; Emulsions
- A61K8/06—Emulsions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- 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
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/34—Alcohols
- A61K8/345—Alcohols containing more than one hydroxy group
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- 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
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/37—Esters of carboxylic acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- 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
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/37—Esters of carboxylic acids
- A61K8/375—Esters of carboxylic acids the alcohol moiety containing more than one hydroxy group
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- 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
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- 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
- A61K8/97—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
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/59—Mixtures
- A61K2800/592—Mixtures of compounds complementing their respective functions
- A61K2800/5922—At least two compounds being classified in the same subclass of A61K8/18
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/82—Preparation or application process involves sonication or ultrasonication
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
Abstract
The invention provides a recombinant mussel mucin cream for nursing damaged skin barrier and a preparation method thereof, wherein the recombinant mussel mucin cream comprises recombinant mussel mucin, sorbitol, buckwheat mash fermentation extract, acid auxiliary agent and skin conditioning penetrating agent; the fermented extract of semen Fagopyri Esculenti is prepared by fermenting semen Fagopyri Esculenti and fructus zizaniae caduciflorae with distiller's yeast and extracting with ethanol; the acid auxiliary agent comprises citric acid and/or ursolic acid; skin conditioning penetrants include isopropyl palmitate, caprylic/capric triglyceride, isopropyl isostearate and isopropyl myristate. The recombinant mussel mucin emulsifiable paste provided by the invention is proved to be safe and nontoxic by an in vitro cytotoxicity test, a rabbit skin stimulation test and a guinea pig skin sensitization test; the skin barrier repair test shows that the skin barrier repair agent can effectively regulate skin lipid components, promote balance of skin barrier function repair, effectively repair skin barrier function, improve facial care effect of acne, moisten and supplement water and control grease content, especially cheek and nasal side parts.
Description
Technical Field
The invention relates to the field of recombinant mussel mucin cream for nursing damaged skin barriers and a preparation method thereof.
Background
The skin is the organ with the largest area of the human body and is the first barrier of the body. The skin is composed of epidermis, dermis and subcutaneous tissue, while the epidermal barrier is a powerful guarantee against external aggressions. Acne is the most common clinical skin disease, is a chronic inflammatory skin disease affecting the pilosebaceous glands, is mainly concentrated on the face, is usually represented by acne, red pimple, papulopus herpes and the like as skin lesions, and causes the skin barrier function to be damaged. In the case of improper facial care, the condition is further aggravated, resulting in a vicious circle. Currently, facial acne occurring after 25 years of age is clinically referred to as adult acne, and in recent years female patients have a higher incidence of acne than male patients. Adult acne is different from juvenile acne in terms of location of onset and type of lesions, and therefore, an effective treatment should be provided for adult acne.
Disclosure of Invention
In view of the above, the present invention aims to provide a recombinant mussel mucin cream for damaged skin barrier care and a preparation method thereof.
The technical scheme of the invention is realized as follows:
a recombinant mussel mucin cream for impaired skin barrier care comprising recombinant mussel mucin, sorbitol, buckwheat mash fermented extract, acid adjuvant and skin conditioning penetrant;
the fermented extract of semen Fagopyri Esculenti is prepared by fermenting semen Fagopyri Esculenti (Fagopyrum esculentum Moench.) and fructus Zizaniae Caduciflorae with distiller's yeast and extracting with ethanol;
the acid auxiliary agent comprises citric acid and/or ursolic acid;
skin conditioning penetrants include isopropyl palmitate, caprylic/capric triglyceride, isopropyl isostearate and isopropyl myristate.
Further, the preparation method of the buckwheat mash fermentation extract comprises the following steps: mixing semen Fagopyri Esculenti and fructus Zizaniae Caduciflorae, pulverizing, adding water, steaming, cooling, adding distiller's yeast, and saccharifying to obtain fermented product of semen Fagopyri Esculenti; filtering the fermented product of semen Fagopyri Esculenti, centrifuging, collecting supernatant, adding ethanol solution, performing ultrasonic extraction, centrifuging, collecting precipitate, adding water, dissolving again, removing ethanol, and vacuum freeze drying to obtain fermented extract of semen Fagopyri Esculenti.
Further, the mass ratio of the buckwheat to the black rice is 8-10: 2 to 3; according to the mass ratio of the feed liquid of 1: 4-5, adding water into the buckwheat and black rice for cooking.
Further, the inoculation amount of distiller's yeast is 1% -2%; the saccharification temperature is 32-36 ℃, and the saccharification time is 40-50 h.
Further, according to the volume ratio of 1: 2-3, adding ethanol solution into the supernatant, wherein the volume concentration of the ethanol solution is 70-75%;
the temperature of ultrasonic extraction is 70-75 ℃, and the time of ultrasonic extraction is 6-8 h.
Further, the material comprises the following raw materials in percentage: 0.03 to 0.08 percent of recombinant mussel mucin, 0.05 to 0.15 percent of sorbitol, 0.05 to 0.12 percent of buckwheat mash fermentation extract, 0.005 to 0.015 percent of acid auxiliary agent, 3.20 to 6.80 percent of skin conditioning penetrating agent, 4 to 8 percent of glycerol, 3 to 5 percent of emulsifying agent and the balance of water;
or 0.03 to 0.08 percent of recombinant mussel mucin, 0.05 to 0.15 percent of sorbitol, 0.05 to 0.12 percent of buckwheat mash fermentation extract, 0.005 to 0.015 percent of acid auxiliary agent, 3.20 to 6.80 percent of skin conditioning penetrating agent, 2.0 to 3.5 percent of simethicone, 4 to 8 percent of glycerol, 0.03 to 0.07 percent of thickening agent, 3 to 5 percent of emulsifying agent, 0.02 to 0.07 percent of film forming agent, 1 to 3 percent of lubricating agent and the balance of water.
Further, the acid auxiliary agent comprises the following components in percentage by mass of 1-2: 1-2 of citric acid and ursolic acid;
the skin conditioning penetrating agent comprises the following components in percentage by mass of 1-2: 2-3: 1-2: isopropyl palmitate, caprylic/capric triglyceride, isopropyl isostearate and isopropyl myristate 1-2.
Still further, the thickener comprises hydroxyethylcellulose, the emulsifier comprises behenpolyether-25 and/or glyceryl monostearate, the film former comprises polyvinyl alcohol, and the lubricant comprises cetostearyl alcohol.
The invention provides a preparation method of the recombinant mussel mucin cream for nursing damaged skin barrier, which comprises the following preparation steps:
s1, oil phase preparation: according to the formula amount, an emulsifier is selected to mix the skin conditioning penetrant with simethicone and a lubricant, and after heating and stirring, the mixture is kept warm and stirred to obtain an oil phase solution;
s2, preparation of an aqueous phase: weighing water, adding sorbitol and acid auxiliary agent, heating, adding glycerol, thickener and film forming agent, stirring at constant temperature, adding recombinant mussel mucin and buckwheat mash fermentation extract, and stirring continuously to obtain aqueous phase solution;
s3, emulsification: heating and stirring the aqueous phase solution, slowly adding the oil phase solution into the aqueous phase solution, preserving heat and stirring, vacuumizing, and homogenizing to obtain the recombinant mussel mucin cream.
In the step S1, the heating temperature is 75-85 ℃, the heat preservation temperature is 77-83 ℃, the heat preservation time is 15-25 min, and the stirring speed is 300-400 r/min;
in the step S2, the heating temperature is 80-90 ℃, the heat preservation time is 10-15 min, and the stirring speed is 300-400 r/min;
in the step S3, the heating temperature is 80-90 ℃, the heat preservation time is 15-20 min, and the stirring speed is 20-40 r/min; the vacuumizing pressure is 0.05-0.08 MPa; the homogenizing speed is 1200-2500 r/min, and the homogenizing time is 5-10 min.
Compared with the prior art, the invention has the beneficial effects that:
the recombinant mussel mucin cream for nursing damaged skin barriers is prepared by adding the recombinant mussel mucin, sorbitol and buckwheat mash fermentation extract and combining the components such as an acid auxiliary agent, a skin conditioning penetrating agent and the like in a scientific component proportion, has a good repairing effect on the skin barrier function of an adult acne patient, and can improve the facial microenvironment to relieve the symptoms such as facial irritation, itching and the like.
The recombinant mussel mucin emulsifiable paste provided by the invention is proved to be safe and nontoxic by an in vitro cytotoxicity test, a rabbit skin stimulation test and a guinea pig skin sensitization test; the skin barrier repair test shows that the skin barrier repair agent can effectively regulate skin lipid components, promote balance of skin barrier function repair, effectively repair skin barrier function, improve facial care effect of acne, moisten and supplement water and control grease content, especially cheek and nasal side parts.
Detailed Description
In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.
The experimental methods used in the embodiment of the invention are conventional methods unless otherwise specified.
Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified.
The distiller's yeast is Angel sweet distiller's yeast, angel Yeast stock Co., ltd; recombinant mussel mucin, saminohaisi medical science and technology limited.
EXAMPLE 1 buckwheat mash fermentation extract
1. Buckwheat mash fermented product
Buckwheat and black rice are mixed according to a mass ratio of 8:3, mixing, crushing, adding 4 times of water by weight for steaming, cooling, adding distiller's yeast with the addition amount of 1% of the weight of the buckwheat and black rice, fully and uniformly stirring, and saccharifying at 34+/-2 ℃ for 45h to obtain the buckwheat mash fermented product.
2. Extraction of
Firstly, adopting four layers of gauze to carry out coarse filtration on the buckwheat mash fermentation product, taking filtrate, centrifuging at 4000rpm/min for 10min, taking supernatant, and mixing the supernatant with the volume ratio of 1:2, adding 75% v/v ethanol solution into the supernatant, extracting with ultrasound at 70deg.C for 6h, centrifuging at 4000rpm/min for 10min, collecting precipitate, adding water for redissolution, removing ethanol solution by rotary evaporation, and vacuum freeze drying to obtain semen Fagopyri Esculenti mash fermentation extract.
EXAMPLE 2 recombinant mussel mucin cream
The formula of the cream is as follows: 0.05% of recombinant mussel mucin, 0.10% of sorbitol, 0.08% of buckwheat mash fermentation extract, 0.01% of acid auxiliary agent, 4.80% of skin conditioning penetrating agent, 6% of glycerol, 4% of emulsifying agent and the balance of water;
wherein, the acid auxiliary agent is prepared from the following components in mass ratio 1:1, citric acid ursolic acid;
the skin conditioning penetrant is prepared from the following components in percentage by mass: 3:1.5:2 isopropyl palmitate, caprylic/capric triglyceride, isopropyl isostearate and isopropyl myristate;
the mass ratio of the emulsifier is 1:1 behenyl alcohol polyether-25 and glyceryl monostearate.
The preparation method of the cream comprises the following steps:
s1, oil phase preparation: mixing skin conditioning penetrant according to formula amount by selecting emulsifier, starting heating and stirring modes, setting heating temperature to 80 ℃, stirring rotation speed to 350r/min, and carrying out heat preservation stirring for 20min at 80+/-3 ℃ and 350r/min to obtain oil phase solution;
s2, preparation of an aqueous phase: weighing purified water, adding into a water kettle, starting a stirring mode (350 r/min), adding sorbitol and an acid auxiliary agent, heating to 85 ℃, adding glycerol, carrying out heat preservation and stirring at 85+/-5 ℃ and 350r/min for 15min until the purified water is completely dissolved, adding recombinant mussel mucin and buckwheat mash fermentation extract, and continuing stirring for 5min, wherein the solution is uniform slightly viscous liquid, thus obtaining aqueous phase solution;
s3, emulsification: pumping the aqueous phase solution into a main pot, starting a stirring mode (30 r/min), heating to 85+/-5 ℃, slowly adding the oil phase into the aqueous phase, keeping the temperature (85 ℃) for stirring (30 r/min) for 15 minutes, vacuumizing (0.05-0.08 MPa), starting homogenizing (1800 r/min) for 8 minutes, stirring (30 r/min) and maintaining the vacuum degree for 20 minutes. And (3) starting reflux for cooling, stopping stirring and vacuumizing when the temperature is reduced to 40 ℃, and obtaining the recombinant mussel mucin cream.
EXAMPLE 3 recombinant mussel mucin cream
The formula of the cream is as follows: 0.08% of recombinant mussel mucin, 0.15% of sorbitol, 0.12% of buckwheat mash fermentation extract, 0.015% of citric acid, 6.80% of skin conditioning penetrant, 3.5% of simethicone, 8% of glycerol, 0.07% of hydroxyethyl cellulose, 5% of emulsifier, 0.07% of polyvinyl alcohol, 3% of cetostearyl alcohol and the balance of water;
wherein the skin conditioning penetrant is prepared from the following components in percentage by mass: 3:1.5:2 isopropyl palmitate, caprylic/capric triglyceride, isopropyl isostearate and isopropyl myristate;
the mass ratio of the emulsifier is 1:1 behenyl alcohol polyether-25 and glyceryl monostearate.
The preparation method of the cream comprises the following steps:
s1, oil phase preparation: according to the formula amount, an emulsifier is selected to mix the skin conditioning penetrant with simethicone and the lubricant, a heating and stirring mode is started, the heating temperature is set to 80 ℃, the stirring speed is 350r/min, and the heat preservation and stirring are carried out for 20min at 80+/-3 ℃ and 350r/min to obtain an oil phase solution;
s2, preparation of an aqueous phase: weighing purified water, adding into a water kettle, starting a stirring mode (350 r/min), adding sorbitol and an acid auxiliary agent, heating to 85 ℃, adding a thickener (hydroxyethyl cellulose) and a film forming agent (polyvinyl alcohol) which are well dispersed by glycerin, carrying out heat preservation and stirring at 85+/-5 ℃ and 350r/min for 15min until the mixture is completely dissolved, adding recombinant mussel mucin and a buckwheat mash fermentation extract without insoluble large particles, and continuing stirring for 5min, wherein the solution is uniform slightly viscous liquid, thus obtaining aqueous phase solution;
s3, emulsification: pumping the aqueous phase solution into a main pot, starting a stirring mode (30 r/min), heating to 85+/-5 ℃, slowly adding the oil phase into the aqueous phase, keeping the temperature (85 ℃) for stirring (30 r/min) for 15 minutes, vacuumizing (0.05-0.08 MPa), starting homogenizing (1800 r/min) for 8 minutes, stirring (30 r/min) and maintaining the vacuum degree for 20 minutes. And (3) starting reflux for cooling, stopping stirring and vacuumizing when the temperature is reduced to 40 ℃, and obtaining the recombinant mussel mucin cream.
EXAMPLE 4 recombinant mussel mucin cream
The formula of the cream is as follows: 0.05% of recombinant mussel mucin, 0.10% of sorbitol, 0.08% of buckwheat mash fermentation extract, 0.01% of acid auxiliary agent, 4.80% of skin conditioning penetrating agent, 2.5% of simethicone, 6% of glycerol, 0.05% of hydroxyethyl cellulose, 4% of emulsifying agent, 0.05% of polyvinyl alcohol, 2% of cetostearyl alcohol and the balance of water;
wherein, the acid auxiliary agent is prepared from the following components in mass ratio 1:1, citric acid ursolic acid;
the skin conditioning penetrant is prepared from the following components in percentage by mass: 3:1.5:2 isopropyl palmitate, caprylic/capric triglyceride, isopropyl isostearate and isopropyl myristate;
the mass ratio of the emulsifier is 1:1 behenyl alcohol polyether-25 and glyceryl monostearate.
The preparation method of the cream comprises the following steps:
s1, oil phase preparation: according to the formula amount, an emulsifier is selected to mix the skin conditioning penetrant with simethicone and the lubricant, a heating and stirring mode is started, the heating temperature is set to 80 ℃, the stirring speed is 350r/min, and the heat preservation and stirring are carried out for 20min at 80+/-3 ℃ and 350r/min to obtain an oil phase solution;
s2, preparation of an aqueous phase: weighing purified water, adding into a water kettle, starting a stirring mode (350 r/min), adding sorbitol and an acid auxiliary agent, heating to 85 ℃, adding a thickener (hydroxyethyl cellulose) and a film forming agent (polyvinyl alcohol) which are well dispersed by glycerin, carrying out heat preservation and stirring at 85+/-5 ℃ and 350r/min for 15min until the mixture is completely dissolved, adding recombinant mussel mucin and a buckwheat mash fermentation extract without insoluble large particles, and continuing stirring for 5min, wherein the solution is uniform slightly viscous liquid, thus obtaining aqueous phase solution;
s3, emulsification: pumping the aqueous phase solution into a main pot, starting a stirring mode (30 r/min), heating to 85+/-5 ℃, slowly adding the oil phase into the aqueous phase, keeping the temperature (85 ℃) for stirring (30 r/min) for 15 minutes, vacuumizing (0.05-0.08 MPa), starting homogenizing (1800 r/min) for 8 minutes, stirring (30 r/min) and maintaining the vacuum degree for 20 minutes. And (3) starting reflux for cooling, stopping stirring and vacuumizing when the temperature is reduced to 40 ℃, and obtaining the recombinant mussel mucin cream.
EXAMPLE 5 recombinant mussel mucin cream
The difference between this example and example 4 is that the ratio and composition of the acid adjuvant and skin conditioning penetrant are adjusted as follows:
0.05% of recombinant mussel mucin, 0.10% of sorbitol, 0.08% of buckwheat mash fermentation extract, 0.01% of acid auxiliary agent, 4.80% of skin conditioning penetrating agent, 2.5% of simethicone, 6% of glycerol, 0.05% of hydroxyethyl cellulose, 4% of emulsifying agent, 0.05% of polyvinyl alcohol, 2% of cetostearyl alcohol and the balance of water;
wherein, the acid auxiliary agent is 3:1, citric acid ursolic acid;
the skin conditioning penetrant is prepared from the following components in percentage by mass: 1 isopropyl palmitate and caprylic/capric triglyceride;
the mass ratio of the emulsifier is 1:1 behenyl alcohol polyether-25 and glyceryl monostearate.
The preparation method of the cream is the same as in example 4.
Comparative example 1
The difference between this comparative example and example 4 is that the fermented extract of buckwheat mash is replaced with the extract of buckwheat, and the specific formulation is as follows:
0.05% of recombinant mussel mucin, 0.10% of sorbitol, 0.08% of buckwheat extract, 0.01% of acid auxiliary agent, 4.80% of skin conditioning penetrating agent, 2.5% of simethicone, 6% of glycerol, 0.05% of hydroxyethyl cellulose, 4% of emulsifying agent, 0.05% of polyvinyl alcohol, 2% of cetostearyl alcohol and the balance of water;
wherein, the acid auxiliary agent is prepared from the following components in mass ratio 1:1, citric acid ursolic acid;
the skin conditioning penetrant is prepared from the following components in percentage by mass: 3:1.5:2 isopropyl palmitate, caprylic/capric triglyceride, isopropyl isostearate and isopropyl myristate;
the mass ratio of the emulsifier is 1:1 behenol polyether-25 and glyceryl monostearate;
the preparation method of the buckwheat extract comprises the following steps: crushing buckwheat according to the mass ratio of 1:6, adding 75% v/v ethanol solution, extracting with 70 ℃ ultrasonic wave for 6h, centrifuging at 4000rpm/min for 10min, collecting supernatant, removing ethanol solution by rotary evaporation, and vacuum freeze drying to obtain semen Fagopyri Esculenti extract.
The preparation method of the cream is the same as in example 4.
Test example 1 in vitro cytotoxicity test
The biological response of the test samples to L-929 mammalian fibroblasts was evaluated according to the in vitro cytotoxicity test GB/T16886.5-2017.
1. Sample preparation
(1) Agar medium: 2% agar (autoclaved at 121 ℃ C., 30 min) was mixed with 2 XMEM medium (filter sterilized) containing 10% FBS at 48 ℃ C. In equal volume.
(2) Sample: the sample was directly used, squeezed onto an agar layer, round with a diameter of about 1.0cm, and aseptically handled.
(3) Negative control: cutting high-density polyethylene into 1.0cm diameter discs, ultraviolet irradiating for 30min, and aseptic processing.
(4) Positive control: the sterile filter paper was cut into 3 plates each having a diameter of 1.0cm, and the medium containing 0.1% ZDEC (manufacturer: tokyo chemical industry Co., ltd., specification: 25g, lot number: DUDQG-JF) was blotted during the test.
2. Test method
The test procedure was performed aseptically.
L-929 cells were cultured in MEM medium containing 10% fetal bovine serum at 37℃with 5% CO 2 Culturing in an incubator. Dispersing cells in fresh medium to adjust to 2.5X10 5 cell/ml cell suspension was inoculated into 6-well plate, 2 ml/well, and placed at 37℃in 5% CO 2 Culturing in an incubator for 24 hours.
The stock medium was discarded, 2ml of agar medium (2% sterilized agar was mixed with 2 XMEM medium containing 10% FBS in equal volume at 48 ℃) was added to each well, and the mixture was allowed to stand at room temperature for solidification.
Lightly placing the prepared sample or leaching solution, negative control and positive control on solidified agar layer, sticking tightly, and continuously placing at 37deg.C and 5% CO 2 Culturing in an incubator for 24 hours.
The 6-well plate is taken out, the position of the sample is marked at the bottom by a marker pen, and then the sample is discarded. 0.01% neutral red dye solution is added into each hole, incubated for 1h at 37 ℃ in a dark place, neutral red is removed, and the solution is observed under a microscope.
3. Evaluation criteria
Cytotoxicity response was rated according to criteria.
TABLE 1 cytotoxicity reaction fractionation
| Level of | Degree of reaction | Reaction area observations |
| 0 | Without any means for | No reaction area was observed around and under the sample |
| 1 | Slight | Some of the abnormal or degenerated cells under the test sample |
| 2 | Mild and mild | The reaction area is limited to the area below the sample |
| 3 | Moderate degree | The reaction area exceeded the sample size to 1.0cm |
| 4 | Heavy weight | The reaction area exceeds the sample by more than 1.0cm |
Note that: a classification of greater than 2 is considered to have cytotoxic effects
4. Test results
The results of the cytotoxic reaction scale are as follows.
TABLE 2 cytotoxicity response grade
| Group of | Evaluation of cell damage |
| Negative control | 0 |
| Positive control | 3 |
| Sample of | 1 |
As seen in Table 2, the test samples showed a level 1 toxic response to L-929 cells, with slight cytotoxicity.
Test example 2 skin irritation test
The potential of samples to produce skin irritation reactions under the test conditions was evaluated in accordance with the irritation and skin sensitization test GB/T16886.10-2017 using New Zealand rabbits.
1. Sample preparation
The medical gauze dressing was cut to a size of about 2.5cm×2.5cm, and a test sample and a control sample (0.9% sodium chloride injection) were impregnated, respectively, as patches for the test group and the control group.
2. Test method
The animal was given a dorsal spinal column with both sides removed (about 10 cm. Times.15 cm) 16 hours prior to the test as a test and observation site.
The patches of the test and control samples were applied directly to the corresponding areas of the dorsal skin on either side of the rabbit spinal column, and then fixed with a semi-occlusive bandage for 4 hours. The patch was removed after the contact period was over.
3. Observation of results
The reaction of patch parts and surrounding skin tissues, including erythema, edema, necrosis and the like, was observed 1h, 24h, 48h and 72h after the patch was removed. The scores were rated as 0, 1, 2, 3, 4 standard scores according to the occurrence of erythema and edema, and are shown in Table 3.
TABLE 3 skin irritation response score criteria
Calculations were performed using 24h, 48h and 72h observation data.
The primary stimulation score was calculated for each animal by summing the erythema and edema stimulation scores for each prescribed time and dividing by the total number of observations of 6 (2 test points x 3 observation times).
The average number of primary irritation scores of the test animals is the primary irritation index.
And calculating a control primary stimulus score, and subtracting the score from the primary stimulus score of the test sample to obtain the primary stimulus score. This value is the primary stimulation index of the test sample.
4. Evaluation criteria
TABLE 4 Rabbit stimulation response type
5. Test results
The skin stimulation response of the test group to rabbits did not exceed that of the control group; the primary stimulation index to rabbit skin is 0, and the test sample does not cause skin stimulation response on rabbit skin.
Test example 3 skin sensitization test
According to the stimulus and skin sensitization test GB/T16886.10-2017, a guinea pig closed patch test is adopted to evaluate the potential skin sensitization response of the sample.
1. Sample preparation
Cutting the medical gauze dressing into about 8cm 2 Size, impregnated with test and control samples, respectively, as patches for test and control groups.
2. Test method
(1) Induction phase
The animals were thoroughly shaved before the test began.
The prepared test sample patch is applied to the left upper back dehairing part of each animal and then fixed by a sealing bandage. After 6 hours the bandages and patches were removed. The control group was operated in the same manner as the negative control sample. This procedure was repeated for 3 consecutive days in 1 week for 3 consecutive weeks. During the test, if the animal is covered by the skin again after regrowth of the hair, the animal needs to shave again.
(2) Excitation stage
All test animals and control animals were challenged with test samples 13 days after the last induction application. The test samples were applied topically to the unhairing, untested portion of each animal and secured with a occlusive tape. After 6 hours the bandages and patches were removed.
3. Observation of results
24h and 48h after removal of the samples, the skin conditions at the excitation sites of the animals in the test group and the control group were observed respectively. Skin erythema and edema reactions were described and graded for each site of excitation and for each observation time according to the Magnusson and Kligman grading criteria of table 1.
TABLE 5Magnusson and Kligman fractionation
4. Evaluation criteria
Animals in the control group were rated less than 1, whereas those in the test group were rated greater than or equal to 1-generally suggesting sensitization.
Animals in the control group were rated greater than or equal to 1, and animals in the test group were considered sensitized when the response exceeded the most severe response in the control group.
If the reaction is suspected, the first excitation result is confirmed by performing the excitation again.
5. Test results
The positive test results (skin sensitization positive test, performed once a month) were: the control group guinea pigs have the grade of 0 after 24h and 48h after the patch is removed, the positive incidence rate after the patch is 0%, the test starting weight range is 340.8-367.0 g, the test ending weight range is 496.2-528.3 g, and no abnormal appearance is caused outside skin reaction; the test groups are respectively 3 and 2 in grades 24h and 48h after the patch is removed, the positive incidence rate after the patch is stimulated is 100%, the weight range of the test is 335.2-376.5 g, the weight range of the test is 467.5-500.8 g, and no abnormal appearance is caused outside skin reaction.
The above results indicate that the test sample did not cause skin sensitization, and the sensitization positive rate was 0%.
Test example 4 skin barrier repair test
1. Object(s)
Female volunteers between 25 and 35 years of age, meeting the acne diagnosis criteria, were selected and randomized into 5 groups of 10 persons each.
2. Application method
After the skin is cleaned by clear water in the morning and evening every day, the sample is uniformly smeared on the affected part until the skin is absorbed, and the skin is continuously used for 7 days, and the affected part is regulated to be in proper time, not extruded and frequently touched.
3. Measurement index
The central part of forehead, the tip of nose, the lower jaw, the left and right parts of cheek (intersection point of perpendicular lines of outer canthus of both sides and level of tip of nose) and the like are selected as measuring points, and the measurement is carried out under the environment of 20-25 ℃ and relative humidity of 40-60%.
TEWL value: after cleaning the face, sitting still for 30min, measuring TEWL value by using TM300 percutaneous water loss measuring instrument, taking stable average result asTEWL value (g.h) -1 ·m -2 )。
Skin moisture content (left cheek): after cleaning the face with clear water, sitting still for 30min, measuring the skin moisture content by using a Corneometer CM825 skin moisture tester, wherein the probe and the measuring site are perpendicular, and taking the average result as the skin moisture content (au).
Sebum amount: after cleansing, sitting still for 60min, measuring oil content with skin oil tester Sebumeter SMS10, pressing with pressure of 10N for 30s, and averaging to obtain sebum (μg cm) -2 )。
TABLE 6 test results of skin barrier repair
One of the functions of the epidermis barrier is to prevent a large loss of moisture from the skin, so that when the epidermis is lost or incomplete, the loss of moisture from the epidermis is accelerated, the TEWL value is increased, and usually, the skin barrier function of the face of the acne patient is damaged, and the TEWL value is higher. As seen in Table 6, the TEWL values of the patient's faces varied to varying degrees, with the average values of the TEWL for each group of cheeks being 13.88, 13.47, 12.73, 14.54 and 15.07 g.h, respectively -1 ·m -2 Among them, the TEWL average value of example 4 is lower, and the repair of skin barrier function is better; from the aspect of skin moisture content, the moisture content of the skin of the cheek is improved after 7d of application, so that the moisture-keeping capability of the facial skin is improved; from the sebum level, the left cheek and the left nose side showed uneven secretion of grease, the nasal sebum level was higher than the cheek sebum level, and the acne patient had not only a low skin moisture content but also a high cheek and nose side grease, and after 7d use, the cheek and nose side sebum levels of example 4 were 98.72 and 104.81 μg cm, respectively -2 The oil state of the skin is improved, which shows that the skin lipid component can be effectively regulated by adding the recombinant mussel mucin, sorbitol, buckwheat mash fermentation extract, and the components such as acid auxiliary agent and skin conditioning penetrating agent which are combined with scientific component proportion, and the balance of promoting the repair of skin barrier function is improved, and the skin lipid component is a natural skin extractThe skin barrier function is effectively restored, the facial nursing effect of acne is improved, and the effects of moisturizing and supplementing water and controlling the content of grease, especially cheek and nasal side parts, are achieved.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. A recombinant mussel mucin cream for impaired skin barrier care, comprising recombinant mussel mucin, sorbitol, buckwheat mash fermented extract, acid adjuvant and skin conditioning penetrant;
the buckwheat mash fermentation extract is obtained by fermenting buckwheat and black rice with distiller's yeast and extracting with ethanol;
the acid auxiliary agent comprises citric acid and/or ursolic acid;
the skin conditioning penetrants include isopropyl palmitate, caprylic/capric triglyceride, isopropyl isostearate, and isopropyl myristate.
2. The recombinant mussel mucin cream for use in the care of damaged skin barrier according to claim 1, wherein the preparation method of the fermented extract of buckwheat mash is: mixing semen Fagopyri Esculenti and fructus Zizaniae Caduciflorae, pulverizing, adding water, steaming, cooling, adding distiller's yeast, and saccharifying to obtain fermented product of semen Fagopyri Esculenti; filtering the fermented product of semen Fagopyri Esculenti, centrifuging, collecting supernatant, adding ethanol solution, performing ultrasonic extraction, centrifuging, collecting precipitate, adding water, dissolving again, removing ethanol, and vacuum freeze drying to obtain fermented extract of semen Fagopyri Esculenti.
3. The recombinant mussel mucin cream for damaged skin barrier care according to claim 2, wherein the mass ratio of buckwheat to black rice is 8-10: 2 to 3; according to the mass ratio of the feed liquid of 1: 4-5, adding water into the buckwheat and black rice for cooking.
4. The recombinant mussel mucin cream for impaired skin barrier care according to claim 2, wherein the inoculation amount of distiller's yeast is 1% -2%; the saccharification temperature is 32-36 ℃, and the saccharification time is 40-50 h.
5. The recombinant mussel mucin cream for use in barrier care of damaged skin of claim 2, wherein the ratio by volume is 1: 2-3, adding ethanol solution into the supernatant, wherein the volume concentration of the ethanol solution is 70-75%;
the temperature of ultrasonic extraction is 70-75 ℃, and the time of ultrasonic extraction is 6-8 h.
6. Recombinant mussel mucin cream for damaged skin barrier care according to claim 1 or 2, characterized in that it comprises the following percentage of raw materials: 0.03 to 0.08 percent of recombinant mussel mucin, 0.05 to 0.15 percent of sorbitol, 0.05 to 0.12 percent of buckwheat mash fermentation extract, 0.005 to 0.015 percent of acid auxiliary agent, 3.20 to 6.80 percent of skin conditioning penetrating agent, 4 to 8 percent of glycerol, 3 to 5 percent of emulsifying agent and the balance of water;
or 0.03 to 0.08 percent of recombinant mussel mucin, 0.05 to 0.15 percent of sorbitol, 0.05 to 0.12 percent of buckwheat mash fermentation extract, 0.005 to 0.015 percent of acid auxiliary agent, 3.20 to 6.80 percent of skin conditioning penetrating agent, 2.0 to 3.5 percent of simethicone, 4 to 8 percent of glycerol, 0.03 to 0.07 percent of thickening agent, 3 to 5 percent of emulsifying agent, 0.02 to 0.07 percent of film forming agent, 1 to 3 percent of lubricating agent and the balance of water.
7. Recombinant mussel mucin cream for damaged skin barrier care according to claim 1 or 2, characterized in that the acid adjuvant comprises the following components in mass ratio 1-2: 1-2 of citric acid and ursolic acid;
the skin conditioning penetrating agent comprises the following components in percentage by mass of 1-2: 2-3: 1-2: isopropyl palmitate, caprylic/capric triglyceride, isopropyl isostearate and isopropyl myristate 1-2.
8. A reconstituted mussel mucin cream for use in barrier care of damaged skin according to claim 6, wherein the thickening agent comprises hydroxyethylcellulose, the emulsifying agent comprises behenyl alcohol polyether-25 and/or glyceryl monostearate, the film former comprises polyvinyl alcohol, and the lubricant comprises cetostearyl alcohol.
9. The method for preparing a recombinant mussel mucin cream for damaged skin barrier care according to any one of claims 1 to 8, comprising the following preparation steps:
s1, oil phase preparation: according to the formula amount, an emulsifier is selected to mix the skin conditioning penetrant with simethicone and a lubricant, and after heating and stirring, the mixture is kept warm and stirred to obtain an oil phase solution;
s2, preparation of an aqueous phase: weighing water, adding sorbitol and acid auxiliary agent, heating, adding glycerol, thickener and film forming agent, stirring at constant temperature, adding recombinant mussel mucin and buckwheat mash fermentation extract, and stirring continuously to obtain aqueous phase solution;
s3, emulsification: heating and stirring the aqueous phase solution, slowly adding the oil phase solution into the aqueous phase solution, preserving heat and stirring, vacuumizing, and homogenizing to obtain the recombinant mussel mucin cream.
10. The method for preparing a recombinant mussel mucin cream for the care of damaged skin barrier according to claim 9, wherein in step S1, the heating temperature is 75-85 ℃, the heat preservation temperature is 77-83 ℃, the heat preservation time is 15-25 min, and the stirring speed is 300-400 r/min;
in the step S2, the heating temperature is 80-90 ℃, the heat preservation time is 10-15 min, and the stirring speed is 300-400 r/min;
in the step S3, the heating temperature is 80-90 ℃, the heat preservation time is 15-20 min, and the stirring speed is 20-40 r/min; the vacuumizing pressure is 0.05-0.08 MPa; the homogenizing speed is 1200-2500 r/min, and the homogenizing time is 5-10 min.
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