CN112043632A - Application of grape seed extract double inclusion - Google Patents
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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
- A61K8/96—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
- 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
- A61K8/11—Encapsulated compositions
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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
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/4993—Derivatives containing from 2 to 10 oxyalkylene groups
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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
- A61K8/55—Phosphorus compounds
- A61K8/553—Phospholipids, e.g. lecithin
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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
- A61K8/63—Steroids; Derivatives thereof
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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
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/738—Cyclodextrins
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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
- A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
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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
- A61Q19/08—Anti-ageing preparations
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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
- A61K2800/56—Compounds, absorbed onto or entrapped into a solid carrier, e.g. encapsulated perfumes, inclusion compounds, sustained release forms
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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
Abstract
The invention provides application of a grape seed extract double-coated body in preparing a skin care product for enhancing skin barrier and/or resisting skin photoaging, and mainly relates to application in preparing a skin care product for improving TEER value and Ki67 content in skin and reducing CPD expression level in skin. The invention also provides a preparation method of the grape seed extract double inclusion body, which can prepare the grape seed extract cyclodextrin liposome double inclusion body which is complete in inclusion, good in stability, difficult to precipitate and light in color, solves the problems of poor solubility and deep color of the grape seed extract, and improves the bioavailability of the grape seed extract, thereby exerting the skin care effect of the grape seed extract to the maximum extent, ensuring that the grape seed extract can exert obvious effect at lower concentration, reducing the production cost, being used for preparing anti-aging skin care products, and realizing the functions of enhancing the skin barrier, resisting the photo-aging of the skin and the like by improving the TEER value and the Ki67 content in the skin and reducing the CPD expression amount in the skin.
Description
Technical Field
The invention relates to the technical field of skin care products, in particular to application of a grape seed extract double inclusion.
Background
Skin aging is a complex physiological phenomenon affected by a variety of factors, and is divided into intrinsic aging and extrinsic aging, which are commonly accompanied. Intrinsic aging mainly refers to aging caused by gene or age, and is manifested by dry skin, fine wrinkles caused by insufficient subcutaneous fat, and the like. External aging is the phenomenon of aging caused by external environment, including aging caused by light, pollution, chemicals and the like.
Skin barrier function refers to the physical barrier function of the epidermis, especially the stratum corneum. The skin care product is generally composed of a brick wall structure formed by a sebum membrane, keratin of a horny layer and lipid, and mucopolysaccharides in dermis, mucopolysaccharides and the like, so that a healthy skin barrier can resist skin invasion by external harmful substances, irritants and sunlight, and has moisturizing and regulating effects. However, due to environmental, dietary and improper skin care reasons, the skin barrier is easily damaged, making the skin self-defense insufficient, leading to skin problems such as skin sensitivity, aging, dryness, desquamation, etc. In daily life, the skin shows aging manifestations such as sagging, wrinkles and loss of elasticity due to the effects of ultraviolet rays, pollution and chemicals, and is accompanied by a decrease in the barrier function of the skin. Therefore, the study on how to reduce skin permeability, increase cell proliferation and resist photodamage is of great significance for delaying skin aging and barrier repair.
The grape is one of the fruits with the largest yield in the world, the annual yield is about 6500 ten thousand tons, the annual yield of the grapes in China is about more than 200 ten thousand tons, wherein the grape seeds account for about 7-10 percent of the total weight of the fresh fruits, and the sources are sufficient. The grape seeds contain rich linoleic acid, protein, procyanidine and other components, and the grape seeds are proved to have remarkable activities of resisting oxidation and removing free radicals, can effectively eliminate superoxide anion free radicals and hydroxyl free radicals, can repair injured collagen and elastic fibers, tighten the skin and prevent wrinkles, can make the skin smooth and elastic after long-term use, have the effects of maintaining beauty and keeping young, and have great application prospects. However, the grape seed extract has poor water solubility and oil solubility, low bioavailability and dark color, so that the application of the grape seed extract in cosmetics is limited.
At present, grape seed extracts sold and applied in markets at home and abroad are mainly prepared by a separation and purification method combining solvent extraction and column chromatography, and are extremely difficult to apply to cosmetics due to poor water solubility and oil solubility (only can reach slightly soluble standards). Although some improved processes solve the problem of solubility of the product by adding a cosolvent, original components of grape seeds are damaged, the pH value of the product is influenced, and the safety and the efficacy of the product are also influenced. For example, CN110664684A discloses a preparation method of an anti-aging and barrier repair essence. The cosmetic additive comprises: grape seed extract, butanediol, water, etc. The grape seed extract in the invention has antioxidant effect, but because the solubility is poor, the grape seed extract needs to be dissolved in a mixed solvent of butanediol and water, and the higher content of butanediol can cause stimulation to sensitive skin and influence the effect of the grape seed extract.
CN108813609A discloses a sulfobutyl ether-beta-cyclodextrin inclusion compound of grape seed extract, but the grape seed extract is only wrapped by cyclodextrin and is not easy to be completely wrapped, and simultaneously, the grape seed extract wrapped by cyclodextrin has partial water solubility improved, but the fat solubility is still poor, thereby influencing the bioavailability. CN105125435B discloses liposome coating of dendrobium officinale extract and grape seed extract, but the grape seed extract is separately coated with liposome, which is prone to unstable coating, the grape seed extract is separated out, the color is still deep, the particle size of the inclusion is also large, and the bioavailability is still not high. CN111449187A discloses a carvacrol/beta-cyclodextrin liposome modified by lactobacillus buchneri S-layer protein, a preparation method and an antibacterial application thereof. However, due to the characteristics of insolubility, deep color and the like of the grape seed extract, problems of incomplete and unstable coating, easy precipitation and the like are easy to occur in the process of carrying out cyclodextrin liposome double coating, so that a method which can carry out complete cyclodextrin liposome double coating, stable double inclusion and difficult precipitation on the grape seed extract is urgently needed to be found, and therefore the problems of insolubility, deep color, low bioavailability and the like of the grape seed extract are really solved.
Transmembrane resistance (TEER), Cyclobutane Pyrimidine Dimer (CPD) and nucleoprotein (Ki67) are commonly used indicators to verify barrier repair and anti-aging in 3D skin tests. The transmembrane resistance value (TEER) can indicate cell integrity and compactness. A high TEER value indicates low skin permeability, reflecting better skin barrier integrity. Cyclobutane Pyrimidine Dimer (CPD) is one of the characteristic photoproducts produced after uv damage to cells. Ultraviolet irradiation can form a dimer between two adjacent thymine bases of the same strand in a deoxyribonucleic acid (DNA) molecule, the double helix structure of the DNA is influenced, the copying and transcription functions of the DNA are hindered, and therefore photoaging is caused. Ki67 is a nuclear protein involved in cell proliferation and RNA transcription and is commonly used as a marker for proliferating cells. If the active substance can increase cells expressing Ki67, namely keratinocyte with proliferation capacity, new keratinocyte can be continuously supplemented to keep skin young and active and resist aging.
Therefore, the research on how to improve the Ki67 content and the TEER value in the skin under the ultraviolet irradiation and reduce the CPD expression level in the skin has important significance for researching barrier repair and anti-aging. At present, no skin care product for repairing skin barriers and resisting aging is prepared by using grape seed extract double-inclusion body, and particularly, relevant reports that the Ki67 content and the TEER value in the skin can be improved under the irradiation of ultraviolet light and the CPD expression amount in the skin is reduced exist.
Disclosure of Invention
In view of the above problems, the present invention provides the use of a double-coated grape seed extract for preparing a skin care product for increasing TEER value and Ki67 content in the skin and reducing CPD expression level in the skin, thereby being useful for preparing a skin care product for enhancing skin barrier and protecting the skin against photoaging. The grape seed extract cyclodextrin liposome double inclusion body which is complete in encapsulation, good in stability, difficult to precipitate and very light in color is prepared, the problems of poor solubility and deep color of the grape seed extract are solved, the bioavailability of the grape seed extract is improved, the skin care effect of the grape seed extract is exerted to the greatest extent, the grape seed extract cyclodextrin liposome double inclusion body is used for preparing an anti-aging skin care product, and the functions of enhancing the skin barrier, resisting skin photoaging and the like are realized by improving the TEER value and the Ki67 content in the skin and reducing the CPD expression amount in the skin.
The grape seed extract of the present invention can be prepared by the method of example 8 of U.S. granted patent US11/890278, which comprises selecting grape seeds without any impurities, extracting with 600 ml of 100% ethanol at room temperature for 18 hours, filtering the ethanol extract through Whatman filter paper, removing the ethanol through a rotary evaporator to obtain a concentrated primary extract, repeating the above operations on the primary extract, drying in a vacuum drying oven, further concentrating to obtain a final extract, and grinding to obtain the grape seed extract.
In one aspect, the invention provides the use of a grape seed extract double inclusion body in the preparation of a skin care product for enhancing the skin barrier and/or resisting skin photoaging, wherein the grape seed extract double inclusion body is prepared by coating a grape seed extract with cyclodextrin and liposome.
Further, the preparation method of the grape seed extract double inclusion comprises the following steps:
(1) adding grape seed extract into cyclodextrin, ultrasonic treating, and stirring for a long time to obtain inclusion S1
(2) S1 was added to liposomes, cholesterol, sorbitol ester-80 and homogenized under high pressure to obtain grape seed extract double inclusion bodies.
In another aspect, the invention provides a use of a grape seed extract double inclusion body in the preparation of a skin care product for improving the TEER value in the skin, wherein the grape seed extract double inclusion body is formed by wrapping a grape seed extract by cyclodextrin and liposome.
The transmembrane resistance value (TEER) can indicate cell integrity and compactness. A high TEER value indicates low skin permeability, reflecting better skin barrier integrity. Researches prove that the grape seed extract double-inclusion body provided by the invention can obviously improve the TEER value of skin under ultraviolet irradiation, furthest exert the skin care effect of the grape seed extract, realize the function of enhancing the skin barrier and be used for preparing skin care products for improving the TEER value of the skin.
Further, the preparation method of the grape seed extract double inclusion comprises the following steps:
(1) adding grape seed extract into cyclodextrin, ultrasonic treating, and stirring for a long time to obtain inclusion S1
(2) S1 was added to liposomes, cholesterol, sorbitol ester-80 and homogenized under high pressure to obtain grape seed extract double inclusion bodies.
In another aspect, the invention provides an application of a grape seed extract double inclusion body in preparing a skin care product for reducing the expression level of CPD in skin, wherein the grape seed extract double inclusion body is formed by wrapping a grape seed extract by cyclodextrin and liposome.
Cyclobutane Pyrimidine Dimer (CPD) is one of the characteristic photoproducts produced after uv damage to cells. Ultraviolet irradiation can form a dimer between two adjacent thymine bases of the same strand in a deoxyribonucleic acid (DNA) molecule, the double helix structure of the DNA is influenced, the copying and transcription functions of the DNA are hindered, and therefore photoaging is caused. Researches prove that the grape seed extract double-inclusion body provided by the invention can obviously reduce the expression quantity of CPD in the skin under ultraviolet irradiation, reduce photodamage, furthest exert the skin care effect of the grape seed extract, realize the functions of resisting skin photoaging and the like, and can be used for preparing skin care products for reducing the expression quantity of CPD in the skin.
Further, the preparation method of the grape seed extract double inclusion comprises the following steps:
(1) adding grape seed extract into cyclodextrin, ultrasonic treating, and stirring for a long time to obtain inclusion S1
(2) S1 was added to liposomes, cholesterol, sorbitol ester-80 and homogenized under high pressure to obtain grape seed extract double inclusion bodies.
In another aspect, the invention provides an application of a grape seed extract double inclusion body in preparing a skin care product for increasing Ki67 content in skin, wherein the grape seed extract double inclusion body is formed by wrapping a grape seed extract by cyclodextrin and liposome.
Ki67 is a nuclear protein involved in cell proliferation and RNA transcription and is commonly used as a marker for proliferating cells. If the active substance can increase cells expressing Ki67, namely keratinocyte with proliferation capacity, new keratinocyte can be continuously supplemented to keep skin young and active and resist aging.
Researches prove that the grape seed extract double-inclusion body provided by the invention can obviously improve the Ki67 content in skin under ultraviolet irradiation, promote cell proliferation, reduce light injury, furthest exert the skin care effect of the grape seed extract, realize the functions of resisting skin photoaging and the like, and can be used for preparing skin care products for improving the Ki67 content in skin.
Further, the preparation method of the grape seed extract double inclusion comprises the following steps:
(1) adding grape seed extract into cyclodextrin, ultrasonic treating, and stirring for a long time to obtain inclusion S1
(2) S1 was added to liposomes, cholesterol, sorbitol ester-80 and homogenized under high pressure to obtain grape seed extract double inclusion bodies.
In another aspect, the present invention provides a use of a dual inclusion body of grape seed extract for preparing a skin care product for enhancing skin barrier and/or preventing skin photoaging, the dual inclusion body of grape seed extract is prepared by:
(1) adding grape seed extract into cyclodextrin, ultrasonic treating, and stirring for a long time to obtain inclusion S1
(2) S1 was added to liposomes, cholesterol, sorbitol ester-80 and homogenized under high pressure to obtain grape seed extract double inclusion bodies.
The polysorbate-80 is added as a solubilizer, which can help the grape seed extract to be solubilized, and plays a role in assisting the cyclodextrin liposome and enhancing the stability of the double inclusion body of the grape seed extract. The addition of polysorbate-80 does not affect the activity of grape seed extract.
Cholesterol has the effect of regulating membrane fluidity and may be referred to as a liposomal "fluidity buffer", and the addition of cholesterol during the preparation of double-encapsulated grape seed extract may help form more stable liposomes. The addition of cholesterol does not affect the activity of the grape seed extract.
Further, said enhancing the skin barrier refers to increasing the TEER value in the skin.
Further, said protection against skin photoaging means increasing the amount of Ki67 in the skin and/or decreasing the expression of CPD in the skin.
Further, the cyclodextrin is selected from one or more of hydroxypropyl-beta-cyclodextrin, methyl-beta-cyclodextrin, sulfobutyl ether-beta-cyclodextrin, triacetyl-beta-cyclodextrin, hepta-6-bromo-6-deoxy-beta-cyclodextrin, (2-hydroxyethyl) -beta-cyclodextrin, 6-O-alpha-maltosyl-beta-cyclodextrin, mono- (6-azido-6-deoxy) -beta-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin.
The cyclodextrin has hydrophilic outer edge and hydrophobic inner cavity, so that the solubility of the grape seed extract with poor water solubility can be increased, and the stability and bioavailability of the grape seed extract can be improved.
Further, the cyclodextrin is hydroxypropyl-beta-cyclodextrin.
The hydroxypropyl-beta-cyclodextrin is very easy to dissolve in water, has better water solubility and higher safety, and can form an inclusion compound with the grape seed extract, thereby improving the water solubility, stability, oxidation resistance and photolysis resistance of the grape seed extract, enhancing the corneal permeability of the grape seed extract and improving the bioavailability.
Further, the liposome is selected from one or more of soybean lecithin, egg yolk lecithin, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and distearoylphosphatidylcholine.
By double wrapping of cyclodextrin and liposome, the prepared grape seed extract has light color, almost colorless after being diluted to 1% concentration, good water solubility and lipid solubility, can be conveniently used for preparing skin care products, and remarkably improves the bioavailability of the grape seed extract.
Further, the liposome is soybean lecithin.
The soybean lecithin is used as the wrapping film material of the grape seed extract, so that the double wrapping bodies have lower phase transition temperature, good fluidity and low viscosity.
Further, the mass ratio of the grape seed extract and the hydroxypropyl-beta-cyclodextrin added in the step (1) is 0.001-5: 10-50, and the balance is water; s1, the adding mass ratio of the soybean lecithin, the cholesterol and the polysorbate-80 is 10-50: 2-10, and the balance is water.
Further, the stirring in the step (1) for a long time is continuously stirred for more than 40 hours, and after the stirring is finished, the mixture is micronized and homogenized by a high-pressure homogenizer to prepare the small-particle-size double inclusion body containing the grape seed extract.
The grape seed extract double-coated body has the beneficial effects that the grape seed extract double-coated body is used for preparing the skin care product for improving the TEER value and the Ki67 content in the skin and reducing the CPD expression amount in the skin, so that the grape seed extract double-coated body can be used for preparing the skin care product for enhancing the skin barrier and resisting the skin photoaging. The invention also provides a preparation method of the grape seed extract double inclusion body, which can prepare the grape seed extract cyclodextrin liposome double inclusion body which is complete in inclusion, good in stability, difficult to precipitate and light in color, solves the problems of poor solubility and deep color of the grape seed extract, and improves the bioavailability of the grape seed extract, thereby exerting the skin care effect of the grape seed extract to the maximum extent, ensuring that the grape seed extract can exert obvious effect at lower concentration, reducing the production cost, being used for preparing anti-aging skin care products, and realizing the functions of enhancing the skin barrier, resisting the photo-aging of the skin and the like by improving the TEER value and the Ki67 content in the skin and reducing the CPD expression amount in the skin.
Drawings
FIG. 1 is a graph of the TEER change in skin upon UVB irradiation for each active group of example 4
FIG. 2 is a photograph comparing microscopic examination of the effect of the actives of each group of example 5 on UVB radiation induced impairment of skin barrier function
FIG. 3 is a photograph of the change in CPD content of the skin caused by exposure of the actives of each group of example 6 to UVB radiation
FIG. 4 is a graph comparing the change in the CPD positivity of the skin to UVB radiation for each active group of example 6
FIG. 5 is a photograph of the microscopic examination of the effect of the active groups of example 7 on Ki67 positive cells in skin after UVB irradiation
FIG. 6 is a graph comparing the effect of the actives of each group of example 7 on Ki67 positive cells in skin after UVB irradiation
Detailed Description
The present invention is described in further detail below with reference to examples, which are intended to facilitate the understanding of the present invention without limiting it in any way. The reagents used in this example were all known products and were obtained by purchasing commercially available products.
Example 1 double inclusion of grape seed extract provided by the invention
The grape seed extract of this example was prepared according to the extraction method of example 8 of US11/890278 by extracting grape seeds without any impurities with 600 ml of 100% ethanol at room temperature for 18 hours, filtering the ethanol extract through Whatman filter paper, removing the ethanol by a rotary evaporator to obtain a concentrated primary extract, repeating the above operations on the primary extract, drying in a vacuum drying oven, further concentrating to obtain a final extract, and grinding to obtain a grape seed extract, which is effective in increasing PP2A activity.
Adding 5g of grape seed extract into 50g of hydroxypropyl-beta-cyclodextrin and 50ml of water, performing ultrasonic treatment and continuously stirring for more than 40h to obtain an inclusion S1, adding the obtained S1 into 50g of soybean lecithin, 10g of cholesterol, 10g of sorbitol ester-80 and 330ml of water, and performing micronization and homogenization by using a high-pressure homogenizer to obtain the 1% double-inclusion of the grape seed extract, which is completely wrapped, has light color and uniform particle size.
EXAMPLE 2 Effect of polysorbate-80 and cholesterol
The method for producing a grape seed extract of the present example refers to example 1. Respectively taking 5g of grape seed extract, 50g of hydroxypropyl-beta-cyclodextrin and 50g of soybean lecithin for preparing 1% double inclusion of the grape seed extract, and dividing the two inclusion into four groups, wherein the first group is not added with cholesterol or polysorbate-80, the second group is added with 10g of cholesterol, the third group is added with polysorbate-8010 g, the fourth group is added with 10g of cholesterol and polysorbate-8010 g, mixing and continuously stirring for 40h, and then carrying out micronization and homogenization by using a high-pressure homogenizer to prepare the 1% double inclusion of the grape seed extract. The appearance of the prepared double inclusion of four groups of grape seed extracts was observed, and the stability was examined by standing at 40 ℃ for 15 days. The results are shown in Table 1.
TABLE 1 Effect of Polysorbate-80 and Cholesterol on the preparation of Dual Inclusion of grape seed extract
| Group of | Composition (I) | Appearance of the product | Stability of |
| First group | / | The color is dark due to incomplete wrapping | Deepening the color and causing precipitation |
| Second group | Cholesterol | Slightly darker in color | Deepening the color and causing precipitation |
| Third group | Polysorbate-80 | Light color | The color is slightly deepened and slightly separated out |
| Fourth group | Cholesterol + polysorbate-80 | Very light in color | Very light color without precipitation |
As shown in table 1, as shown in the first group, when there is no cholesterol and polysorbate-80 in the system, the obtained grape seed extract double inclusion body has poor stability, is prone to partial precipitation, and is prone to cause a dark color due to incomplete inclusion, which is not suitable for cosmetic applications; as shown in the second group, when only cholesterol is present in the system and polysorbate-80 is not present, the prepared grape seed extract double inclusion body has slightly enhanced stability compared with the first group, but the problem of slightly darker color still exists, and the problems of incomplete wrapping and dark color can not be solved as the stability of the prepared grape seed extract double inclusion body can not be supported only by cholesterol; as shown in the third group, when only polysorbate-80 was present in the system without cholesterol, the prepared dual wraps of grape seed extract had significantly enhanced stability compared to the first and second groups, and it was found that polysorbate-80 plays a very important role in the stability of the entire system; as shown in the fourth group, when the cholesterol and the polysorbate-80 are both in the system, the stability is best, the color is very light, the diluted grape seed extract is basically colorless, and the prepared grape seed extract double inclusion body completely solves the problem of dark color under the stable support of the polysorbate-80 and the cholesterol to the whole system, is very stable and completely meets the preparation requirement of skin care products.
Example 3 protection against UVB radiation induced impairment of skin Barrier function
Recombinant three-dimensional skin ArchSkinTMConstruction: human primary skin keratinocytes (purchased from Shanghai Eji Biotech Co., Ltd.) were cultured in an incubator at 37 ℃ containing 5% carbon dioxide, maintaining a humidity of 95%. When the cells reached 80% confluence, the cells were seeded in a Transwell chamber and the lower chamber was incubated with the addition of appropriate media. After another 7-10 days, the three-dimensional skin ArchSkin is recombinedTMCan be used for experimental research.
Taking recombinant three-dimensional skin ArchSkinTMUVB-induced stimulation experiments were performed, and the experiments were divided into Normal (NT), UV and experimental groups (UV + samples), each set of three replicates. On the day of the experiment, 3. mu.L of the sample was first applied to the ArchSkin groupTMSurface, followed by UVB (500 mJ/cm)2) The UV group and experimental group were irradiated to the skin. After the irradiation treatment was completed, the skin was placed in an incubator and cultured for another 24 hours. The specific experimental information is shown in Table 2, wherein the 1% sunflower seed oil unsaponifiable matter is selected as the control in group 3, and the skin barrier repair and light damage resistance are improved by increasing the skin lipid content due to the rich phytosterols and tocopherols in the sunflower seed oil unsaponifiable matter (European patent EP1707189A2), so that the 1% sunflower seed oil unsaponifiable matter is used as the control in the present invention, which shows the excellent barrier repair of grape seed extractAnd resistance to light damage.
TABLE 2 Experimental groups
After the experiment was completed, the Transwell chamber with skin was transferred to a new sterile cell plate, 500. mu.l PBS was added to the upper chamber, 1ml PBS was added to the lower chamber, and then TEER value measurements were performed on each group of skin using an electric resistance meter to evaluate the barrier function of the skin, the results of which are shown in FIG. 1.
Figure 1 shows that UVB irradiation significantly caused a reduction in TEER values for the restructured three-dimensional skin, ArchSkinTM, suggesting that skin TEER values were reduced after some light damage to skin barrier function. From the experimental results, compared with the UVB group and the 0.001% grape seed extract, the double-coated body of the 0.001% grape seed extract can more obviously prevent the TEER value from being reduced caused by ultraviolet radiation after the skin is treated under different conditions, and the effect is equivalent to that of the unsaponifiable matter of the 1% sunflower seed oil. Therefore, the double inclusion of the grape seed extract can obviously improve the function of the grape seed extract for enhancing the skin barrier.
Example 4 Effect on UVB radiation induced structural damage to skin
UVB-induced recombinant three-dimensional skin ArchSkin according to example 3TMAnd (4) stimulation experiment. After the experiment was completed, ArchSkin was grasped with a surgical bladeTMThe skin was carefully cut and peeled from the cell culture chamber. Collected ArchSkinTMThe skin was stored in tissue fixative and fixed overnight at 4 ℃. The next day, all skin samples were dehydrated with different concentrations of ethanol, embedded in paraffin, and sectioned. On the day of the HE staining experiment, paraffin sections were sequentially placed in xylene I (10min) -xylene II (10min) -xylene III (10min) -absolute ethanol I (5min) -absolute ethanol II (5min) -95% ethanol (5min) -90% ethanol (5min) -80% ethanol (5min) -70% ethanol (5min), and then washed with distilled water for 5 min. The sections were then stained in Mayer's hematoxylin stain for 5min and washed in tap water to return to blue. The sections were stained in 1% water-soluble eosin stain for 5min and rinsed with tap water for 30 s. 95% alcohol(30s) -95% ethanol (1min) -absolute ethanol I (5min) -absolute ethanol II (5min) -xylene I (5min), air-dried, and sealed with neutral gum, and finally examined under the microscope. The results of the experiment are shown in FIG. 2.
The research result of figure 2 shows that UVB irradiation can remarkably induce recombination of three-dimensional skin ArchSkinTMStructural damage, further H&The E staining results show that the number of viable cell layers is reduced and the skin thickness is thinner. After the skin is treated under different conditions, compared with a UVB group and a 0.001% grape seed extract, the skin treated by the double inclusion body of the 0.001% grape seed extract is obviously the thickest and has the effect equivalent to that of an unsaponifiable matter of 1% sunflower seed oil, and the result shows that the double inclusion body of the 0.001% grape seed extract has an obvious protective effect on the reduction of the number of the skin living cells caused by ultraviolet irradiation treatment, and the effect has an obvious synergistic effect compared with the non-wrapped grape seed extract.
EXAMPLE 5 protection of UVB irradiation induced DNA photodamage in skin cells
UVB-induced recombinant three-dimensional skin ArchSkin according to example 4TMAnd (4) stimulation experiment. After the experiment was completed, ArchSkin was grasped with a surgical bladeTMThe skin was carefully cut and peeled from the cell culture chamber. Collected ArchSkinTMThe skin was stored in tissue fixative and fixed overnight at 4 ℃. The next day, all skin samples were dehydrated with different concentrations of ethanol, embedded in paraffin, and sectioned. On the day of CPD antibody staining experiment, the section to be stained is dewaxed according to the steps, and then the section is added into a beaker of sodium citrate buffer solution, and antigen retrieval is carried out after microwave heating. The sections were then placed in a wet box, blocked with 1% BSA in a blocking solution at room temperature for 2h, followed by dropwise addition of primary anti-CPD and incubation at 4 ℃ overnight. The next day, sections were washed three times with PBS containing 1% Triton-X100 for 5min each. Then, a secondary antibody is added dropwise to continue incubation for 1h at room temperature, and after washing again, coloration is carried out by using a DAB kit.
The microscopic examination result is shown in fig. 3, and the result shows that the UVB irradiation can obviously induce the recombinant three-dimensional skin ArchSkinTM skin DNA photodamage, the black spots (CPD) in the cross section of the skin are obviously increased, which is reflected in the expression of an enhanced DNA damage marker CPD, after the treatment of the double inclusion body of the 0.001 percent grape seed extract, the CPD number is obviously reduced, and the reduction amplitude is obviously larger than the treatment result of the 0.001 percent grape seed extract in the 5 th group.
The CPD cell positive rate is calculated by using the ratio of the number of positive cells to the cross-sectional area of the epidermis, the percentage of decrease in the CPD cell positive rate after treatment under different conditions is calculated by taking the CPD cell positive rate of the UVB group as 100%, and the result of quantitative analysis data is shown in FIG. 4. Fig. 4 shows that the CPD cell positivity was significantly increased in the UVB group, decreased by 18.7% in skin treated with the 0.001% double inclusion of grape seed extract, by a magnitude significantly higher than 6.5% in the UVB and grape seed extracts, and by 10.2% in the sunflower seed oil unsaponifiable group after treatment with different actives, indicating that the double inclusion of grape seed extract can significantly resist the aging caused by photodamage and has a significantly better effect than the grape seed extract without the inclusion treatment.
Example 6 Effect on proliferation of basal cells of skin after UVB irradiation
UVB-induced recombinant three-dimensional skin ArchSkin according to example 4TMAnd (4) stimulation experiment. After the experiment was completed, ArchSkin was grasped with a surgical bladeTMThe skin was carefully cut and peeled from the cell culture chamber. Collected ArchSkinTMThe skin was stored in tissue fixative and fixed overnight at 4 ℃. The next day, all skin samples were dehydrated with different concentrations of ethanol, embedded in paraffin, and sectioned. On the day of Ki67 antibody staining experiment, the section to be stained was dewaxed according to the above procedure, and then the section was added into a beaker of sodium citrate buffer solution, and subjected to microwave heating for antigen retrieval. The sections were then placed in a wet box, blocked with 1% BSA in a blocking solution at room temperature for 2h, followed by dropwise addition of primary anti-Ki 67 and incubation at 4 ℃ overnight. The next day, sections were washed three times with PBS containing 1% Triton-X100 for 5min each. Then, a secondary antibody is added dropwise to continue incubation for 1h at room temperature, and after washing again, coloration is carried out by using a DAB kit.
The microscopic examination result is shown in fig. 5, and the result shows that the UVB irradiation can remarkably destroy the recombined three-dimensional skin ArchSkinTMProliferation of basal cells of the skin, expressed as significantly reduced Ki67 positive cells compared to the normal group, in contrast to 0.001% of grape seed extractThe inclusion and the skin treated by the unsaponifiable 1% sunflower seed oil still can see normally proliferated basal keratinocytes, but the skin treated by the grape seed extract does not have normally proliferated basal keratinocytes, so that the grape seed extract propylene glycol solution which is not subjected to the inclusion treatment has no obvious effect on improving Ki67 in the skin, probably because the grape seed extract has weak permeability, the effect of promoting the proliferation capability of the skin basal cells cannot be exerted, and the bioavailability is not high.
The number of Ki67 in the staining results of grape seed extract inclusion and grape seed extract was scored and evaluated, and the results are shown in fig. 6. As can be seen from fig. 6, the 0.001% grape seed extract double inclusion and 1% sunflower seed oil unsaponifiable had comparable efficacy in increasing Ki67 in skin after UVB irradiation, while the 0.001% grape seed extract without inclusion had essentially no effect in increasing Ki67 in skin. Therefore, the double inclusion bodies of the 0.001 percent grape seed extract have exact protective effect on the reduction of the proliferation capacity of basal cells after UVB irradiation.
In conclusion, the double inclusion of the grape seed extract with the concentration of 0.001% can obviously protect the skin from being damaged by ultraviolet rays, and exert the efficacy of the grape seed extract to the maximum extent, thereby repairing the skin barrier and improving the skin aging problem. The double-wrapping effect of the grape seed extract is obviously superior to that of a 0.001 percent propylene glycol solution of the grape seed extract, and the double-wrapping technology is mainly adopted to improve the grape seed extract, so that the problems of deeper solubility and color are solved, the permeability is increased, the bioavailability of the grape seed extract is improved, and the grape seed extract can achieve the effect equivalent to that of 1 percent sunflower seed oil unsaponifiable matter at lower concentration.
Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. The application of the grape seed extract double inclusion body in preparing the skin care product for enhancing the skin barrier and/or resisting the skin photoaging is characterized in that the grape seed extract double inclusion body is formed by wrapping a grape seed extract by cyclodextrin and liposome.
2. The application of the grape seed extract double inclusion in preparing the skin care product for improving the TEER value in the skin is characterized in that the grape seed extract double inclusion is formed by wrapping a grape seed extract by cyclodextrin and liposome.
3. The application of a grape seed extract double inclusion body in preparing a skin care product for reducing the CPD expression level in the skin and/or increasing the Ki67 content in the skin is characterized in that the grape seed extract double inclusion body is formed by wrapping a grape seed extract by cyclodextrin and liposome.
4. The application of the grape seed extract double inclusion body in preparing the skin care product for enhancing the skin barrier and/or resisting the skin photoaging is characterized in that the preparation method of the grape seed extract double inclusion body comprises the following steps:
(1) adding grape seed extract into cyclodextrin, ultrasonic treating, and stirring for a long time to obtain inclusion S1
(2) S1 was added to liposomes, cholesterol, sorbitol ester-80 and homogenized under high pressure to obtain grape seed extract double inclusion bodies.
5. Use according to claim 4, wherein said enhancing the skin barrier is increasing the TEER value in the skin; the anti-skin photoaging means that the expression level of CPD in the skin is reduced and/or the content of Ki67 in the skin is increased.
6. The use according to any one of claims 4 to 5, wherein the cyclodextrin is selected from any one or more of hydroxypropyl- β -cyclodextrin, methyl- β -cyclodextrin, sulfobutyl ether- β -cyclodextrin, triacetyl- β -cyclodextrin, hepta-6-bromo-6-deoxy- β -cyclodextrin, (2-hydroxyethyl) - β -cyclodextrin, 6-O- α -maltosyl- β -cyclodextrin, mono- (6-azido-6-deoxy) - β -cyclodextrin, γ -cyclodextrin.
7. The use according to claim 6, wherein the cyclodextrin is hydroxypropyl- β -cyclodextrin.
8. The use according to any one of claims 4 to 5, wherein the liposome is selected from any one or more of soybean lecithin, egg yolk lecithin, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and distearoylphosphatidylcholine.
9. The use of claim 8, wherein said liposome is soy lecithin.
10. The use according to any one of claims 4 to 8, wherein the grape seed extract and the hydroxypropyl-beta-cyclodextrin are added in a mass ratio of 0.001-5: 10-50 in the step (1), and the balance is water; s1, the adding mass ratio of the soybean lecithin, the cholesterol and the polysorbate-80 is 10-50: 2-10, and the balance is water.
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