CN111228136A - Anti-aging wrinkle-removing composite nano preparation and preparation method and application thereof - Google Patents
Anti-aging wrinkle-removing composite nano preparation and preparation method and application thereof Download PDFInfo
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- 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
- A61K8/67—Vitamins
- A61K8/671—Vitamin A; Derivatives thereof, e.g. ester of vitamin A acid, ester of retinol, retinol, retinal
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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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
- A61K8/14—Liposomes; Vesicles
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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
- A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
- A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/18—Antioxidants, e.g. antiradicals
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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
- A61K2800/41—Particular ingredients further characterized by their size
- A61K2800/413—Nanosized, i.e. having sizes below 100 nm
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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
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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
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
Abstract
The invention provides an anti-aging wrinkle-removing composite nano preparation, a preparation method and application thereof, and particularly relates to the nano preparation which contains hydroxypropyl tetrahydropyrane triol, retinol retinoic acid ester and soybean isoflavone as active ingredients and shows obvious anti-aging and wrinkle-removing effects.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to an anti-aging wrinkle-removing composite nano preparation as well as a preparation method and application thereof.
Background
Skin aging includes endogenous skin aging and exogenous skin aging. Endogenous aging is determined by genetic factors of the body, is a naturally occurring process which is difficult to prevent and is manifested by dry skin, laxity, fine lines, sagging, thinning of the skin, and a decrease in subcutaneous fat (causing pits and depressions in the cheeks, etc.). Extrinsic skin aging is a cumulative damage of the skin caused by environmental factors (ultraviolet rays, PM2.5, air pollution, bacterial infection, etc.) or lifestyle. Extrinsic aging is not universal and can be prevented to a certain extent, mainly occurring in exposed parts of the skin, such as the face, neck, back of the hand and forearm, and showing deeper wrinkles, age spots, telangiectasia and the like.
In general, skin aging is the result of the combined action of physiological mechanisms inherent to the human body and external environmental damage. Skin aging is usually manifested by a decrease in skin elasticity and the development of skin wrinkles. It is thought that the reduction of collagen and elastin leads to a reduction in skin elasticity and the generation of wrinkles.
The skin is composed of three layers, epidermis, dermis and subcutaneous tissue. The epidermis is the outermost layer of the skin and consists of, from the inside out, the basal layer, the spinous layer, the granular layer and the stratum corneum. The cells that make up the epidermis are predominantly keratinocytes and, as these cells mature, migrate upward to form different layers of the epidermis until desquamation, for a period of about 26-42 days. Certain substances, such as vitamin a, can accelerate the cycle, resulting in a younger, smoother skin appearance.
With the increasing living standard and aesthetic standard of people, more and more people begin to pay attention to the problem of skin aging. The anti-aging and wrinkle-removing cosmetics become products which are urgently needed by consumers. The skin aging problem can be effectively prevented and improved by removing excessive free radicals in vivo, enhancing the oxidation resistance of the organism and improving the skin microcirculation through the active ingredients in the cosmetics. However, many skin care products employ different chemicals as additives in order to seek a quick and direct effect, resulting in poor safety and frequent side effects.
Therefore, those skilled in the art are working on the development of skin care products with better efficacy and better safety.
Disclosure of Invention
The invention aims to provide an anti-aging wrinkle-removing composite nano preparation as well as a preparation method and application thereof.
In a first aspect of the present invention, there is provided an anti-aging wrinkle-removing composite nano-preparation, which is a liposome nano-preparation and comprises:
hydroxypropyl tetrahydropyran triol, retinol retinoic acid ester, and soy isoflavones.
In another preferred embodiment, the average particle size of the anti-aging wrinkle-removing compound nano preparation is 10-50 nm.
In another preferred embodiment, the anti-aging wrinkle-removing compound nano-preparation comprises:
1-10 parts of hydroxypropyl tetrahydropyrane triol;
0.5-2 parts by weight of soybean isoflavone; and
0.5-2 parts by weight of retinol retinoic acid ester.
In another preferred embodiment, the anti-aging wrinkle-removing compound nano-preparation comprises:
5-10 parts of hydroxypropyl tetrahydropyrane triol;
1-2 parts by weight of soybean isoflavone; and
1-2 parts of retinol retinoic acid ester.
In a second aspect of the present invention, there is provided a cosmetic composition comprising the anti-aging wrinkle-removing complex nano-formulation according to the first aspect of the present invention.
In another preferred embodiment, the anti-aging wrinkle-removing compound nano preparation is contained in the cosmetic composition in an amount of 0.01% -30%.
In another preferred embodiment, the anti-aging wrinkle-removing compound nano preparation is contained in the cosmetic composition in an amount of 1% -20%.
In another preferred embodiment, the anti-aging wrinkle-removing compound nano preparation is contained in the cosmetic composition in an amount of 5% -15%.
In another preferred embodiment, the cosmetic is in the form of a solid, semi-solid, or liquid dosage form, such as a solution, gel, cream, lotion, ointment, cream, paste, cake, powder, patch, and the like.
In a third aspect of the present invention, there is provided a method for preparing an anti-aging wrinkle-removing composite nano-formulation, the method comprising the steps of:
(1) dissolving hydroxypropyl tetrahydropyrane triol, retinol retinoic acid ester and soybean isoflavone in ethanol to obtain a mixed solution I;
(2) dissolving the membrane material in the mixed solution I, and removing ethanol in the solution by vacuum evaporation to obtain a mixed solution II;
(3) adding the mixed solution II into a PBS buffer solution to obtain a mixed solution III;
(4) and (3) transferring the mixed solution III to a high-pressure homogenizer for homogenization, and filtering and sterilizing through a 0.22-micrometer microporous filter membrane to obtain the nano liposome, namely the anti-aging wrinkle-removing composite nano preparation.
In another preferred example, the mixed solution of step (1) includes:
1-10 parts of hydroxypropyl tetrahydropyrane triol;
0.5-2 parts by weight of soybean isoflavone; and
0.5-2 parts by weight of retinol retinoic acid ester.
In another preferred example, the mixed solution of step (1) includes:
5-10 parts of hydroxypropyl tetrahydropyrane triol;
1-2 parts by weight of soybean isoflavone; and
1-2 parts of retinol retinoic acid ester.
In a fourth aspect of the invention, the invention provides the use of the anti-aging wrinkle-removing composite nano preparation in the first aspect of the invention for preparing cosmetics, health products or medicines.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the results of cell survival assays.
FIG. 2 shows the detection results of superoxide dismutase activity.
FIG. 3 shows the results of mouse tissue sections.
Fig. 4 shows the result of the fishtail variation.
Fig. 5 shows the result of the change in fine lines under the eye.
Detailed Description
The inventor obtains an anti-aging wrinkle-removing composite nano preparation and a preparation method thereof through extensive and intensive research, and the nano preparation prepared by the invention contains hydroxypropyl tetrahydropyrane triol, retinol retinoic acid ester and soybean isoflavone as active ingredients, and shows remarkable anti-aging and wrinkle-removing effects.
Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
The invention provides a stable nano liposome preparation containing hydroxypropyl tetrahydropyrane triol, retinol retinoic acid ester and soybean isoflavone as active ingredients.
The liposome (Liposomes) is prepared by coating phospholipid, cholesterol, etc. as membrane material. Phospholipids are dispersed in water to form multilamellar vesicles, each of which is a lipid bilayer separated by water, which are liposomes. Liposomes can be divided into unilamellar liposomes and multilamellar liposomes. Because the basic structure of the plasma membrane of the organism is also the phospholipid bilayer membrane, the liposome has a structure similar to that of the organism cell, and therefore, the liposome has good biocompatibility.
Phospholipids are the main chemical components constituting liposomes, and the most representative of them is lecithin. The lecithin is mainly from egg yolk and soybean, has low preparation cost and stable property, and belongs to neutral phospholipid. Phosphatidylcholine is the main component forming many cell membranes and is also the main raw material for preparing liposomes.
The membrane material selected by the nano-liposome provided by the invention can be phospholipid and cholesterol, wherein the phospholipid comprises but is not limited to soybean lecithin and egg yolk lecithin.
Cholesterol is also another important constituent of liposomes, which is an important component of many natural biological membranes, does not form a membrane structure itself, but can be inserted into phospholipid membranes. The addition of cholesterol can alter the phase transition temperature of the lipid membrane, thereby affecting the permeability and fluidity of the membrane. Thus cholesterol has the effect of stabilizing the phospholipid bilayer membrane.
Hydroxypropyl tetrahydropyran triol (boscalid) originally derived from beech extract, is useful in cosmetics, and in the present invention is available from Puri-Xylane, pu rui, inc.
The retinol retinoic acid ester is obtained by esterification reaction of hydroxyl of retinol and retinoic acid, retains ring terminal group and side chain alkenyl, still has bioactivity of retinol and retinoic acid, and reduces irritation of retinol and retinoic acid.
Soy isoflavones are flavonoids, a class of secondary metabolites formed during the growth of soybeans, and are bioactive substances. Since it is extracted from plants and has a structure similar to that of estrogen, soy isoflavones are also called phytoestrogens.
The main advantages of the invention are:
1) the nano preparation prepared by the invention contains hydroxypropyl tetrahydropyrane triol, retinol retinoic acid ester and soybean isoflavone as active ingredients, and shows remarkable synergistic effect.
2) The nanometer preparation prepared by the invention has no stimulation to skin, and shows obvious anti-aging and wrinkle-removing effects.
The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
EXAMPLE 1 preparation of Nanodiulation
Reference is made to the method in patent document CN1411368A for the preparation of nano-formulations. 8g of hydroxypropyl tetrahydropyrane triol, 1.5g of retinol retinoic acid ester and 0.5g of soybean isoflavone were dissolved in ethanol to give a total of 100mL of a mixed solution. 50g of egg yolk lecithin was dissolved in the mixed solution, heated to 40 ℃ and the ethanol solvent in the solution was removed under 1330 Pa. The remaining liquid was added to 500ml of PBS buffer, and dispersed with stirring.
The above dispersion solution was emulsified by a high pressure emulsifier, and the emulsified dispersion was filtered through a 0.22 μm microporous membrane for sterilization. 15g of the filtrate were filled into freeze-drying bottles which were then placed in a freeze-dryer and run at room temperature at 4000Pa for gas degassing. Degassing, and concentrating at 40 deg.C and 1330Pa to obtain concentrated solution. The concentrate was cooled to-40 ℃ and the solution was then foamed by raising the temperature of the solution to 40 ℃ under high vacuum (1.33 Pa). Vacuum drying the bubbling solution at 30 ℃ under the condition of 13.3Pa, pressurizing the bubbling solution to atmospheric pressure by using nitrogen after vacuum drying, and sealing a freeze-drying bottle to obtain the liposome nano preparation named as nano preparation A.
Liposome nano-formulations (named nano-formulation B) containing only hydroxypropyl tetrahydropyrane triol were prepared by the above-described methods, respectively; a liposomal nano-formulation comprising hydroxypropyl tetrahydropyranyl triol and retinol retinoic acid ester (designated as nano-formulation C); a liposomal nano-formulation comprising hydroxypropyl tetrahydropyrane triol and soy isoflavones (designated as nano-formulation D); and, a liposome nano-formulation comprising retinol retinoic acid ester and soy isoflavone (named nano-formulation E); wherein the amount of each active substance is the same as above.
Example 2 protective Effect on human skin fibroblasts
Human skin fibroblasts (purchased from ATCC, cat # PCS-201-012), were placed in a common DMEM medium (containing 10% (V/V) fetal bovine serum) in 5% CO2Culturing at 37 ℃ in an incubator, carrying out subculture after trypsinization when 80% of cells are fused, and taking 3 rd generation healthy cells for experiment.
After digesting the cells with pancreatin, the cell concentration was adjusted to 5X 104Inoculating to 96-well plate, culturing until cell adheres to wall, removing culture solution, washing with PBS buffer solution for 2 times, placing in medium-wave ultraviolet light, and placing 96-well plate at a distance of 15cm from ultraviolet lamp (ultraviolet intensity of 1 mW/cm)2) And irradiating for 5 min. After the ultraviolet irradiation is finished, different cell culture media are added for culture. In each experimental group, 100mg/L of the nano-preparation a, the nano-preparation B, the nano-preparation C, the nano-preparation D, and the nano-preparation E prepared in example 1 were added to the cell culture medium, and the cell culture medium corresponds to the experimental group a, the experimental group B, the experimental group C, the experimental group D, and the experimental group E. The control group had no uv irradiation. Each set was provided with 5 parallel wells.
After 48h of culture, serum-free medium containing 10% CCK-8 and 5% CO was added to each well of a 96-well plate2Culturing for 2h at 37 ℃ in an incubator, then measuring the absorbance value of each hole by a 450nm microplate reader, and calculating the survival rate of each group of cells according to the absorbance.
The activity of the intracellular superoxide dismutase is detected by adopting a total superoxide dismutase (SOD) determination kit (WST-1 method, Beijing Baiolaboke technologies, Ltd.).
The cell survival rate test results are shown in fig. 1, and the results show that the experimental group a can significantly improve the survival rate of human skin fibroblasts damaged by ultraviolet irradiation, and compared with the model group, the cell survival rate is improved by about 35%.
The detection result of the activity of the superoxide dismutase is shown in figure 2, the result shows that the activity of the superoxide dismutase in the cells can be obviously improved by the experimental group A, and the activity of the superoxide dismutase in the cells treated by the nano preparation A is far higher than that of the model group and 40% higher than that of the control group, so that the nano preparation A can greatly stimulate the expression of the superoxide dismutase in the cells, and further promote the self-repair of the damaged cells. But also shows that the combination of the hydroxypropyl tetrahydropyrane triol, the retinol retinoic acid ester and the soybean isoflavone has a remarkable synergistic effect.
Example 3 test for promoting expression of mucopolysaccharide (hyaluronic acid) in vivo
1. Laboratory animal
Healthy male SPF (specific pathogen free) mice of 6-8 weeks old are 23 +/-2 g in weight. Raising at 25 + -2 deg.C and humidity of 40 + -10%.
2. Establishment, grouping and administration of animal models
Mice were randomly divided into blank, model, matrix, experimental I, experimental II, experimental I II, and experimental IV groups of 6 mice each.
After the mice were acclimatized for one week, they were depilated with 6% sodium sulfide to expose 3cm × 3cm of skin. In addition to blank groups, each group is referred to [ ZHONG W, LIU N, XIE Y, et a1. Antixidant and antimageingactivities of mycelial polysaccharides from Lepitta sordd [ J ]. Int J biol Macromol, 2013, 60 (6): 355-359 ] A model of skin aging was established by subcutaneous injection of D-galactose 1.09/(kg.d) per day.
The blank group was injected subcutaneously with normal saline of the same volume as D-galactose every day for 10 days;
coating conventional skin care emulsion matrix 10mg/cm on the matrix group after molding2；
Experimental group I applied with 10% of nano preparation A skin care emulsion matrix 10mg/cm2；
Experimental group II applied skin care emulsion base containing 10% of nano preparation B at a concentration of 10mg/cm2；
Experimental group III applied skin care lotion base containing 10% of nano preparation C10 mg/cm2；
Experimental group IV applied with 10% nano-preparation D containing skin care emulsion matrix 10mg/cm2。
The model and blank groups were left untreated. After 28 days of continuous application, fasting is carried out for 24 hours, after the skin of the mouse is cleaned, the mouse is killed by cervical dislocation, and the skin is cut off rapidly and stored in a frozen manner. The skin irritation group mouse 7 was also used for the skin irritation test.
The skin care emulsion matrix comprises the following components: 1.5% of citric acid monoglyceride, 2% of jojoba oil, 3% of isopropyl palmitate, tween-603%, 3% of beeswax, 3% of liquid paraffin, 9410.1% of carbomer, 3% of glycerol, 0.1% of hydroxyethyl cellulose, 0.01% of disodium EDTA, 0.01% of triethanolamine and the balance of distilled water, wherein the total amount of the components is 100%.
Acute multiple skin irritation test
The back side of the mice after shaving and cleaning of the skin stimulation groups was divided into two areas (2.5cm × 2.5cm), 0.5g of skin care cream of each group was applied to the left side, a blank control was applied to the right side, the mice were covered with gauze and fixed with adhesive tape, the skin was cleaned after 24h, and the skin reaction of the applied part was observed after 1h, 24h and 48 h.
After 14 consecutive days of application, the skin reaction at the application site was observed and recorded. The skin was evaluated for irritation response and irritation intensity with reference to "evaluation procedure and method for cosmetic safety".
The results show that the skin of the experimental groups I, II, III and IV has no phenomena of edema and erythema compared with the skin without the skin care lotion, and the skin care lotion is proved to have no irritation to the skin. After the skin care lotion is smeared on the back skin of a mouse for a plurality of times for a long time, compared with the blank skin, the smeared skin has no phenomena of erythema and edema, and the skin care lotion is proved to have no irritation to the skin after being smeared for a long time.
Observation of apparent characteristics of mouse skin
The appearance forms of the color, smoothness, wrinkles and the like of the bare skin on the back of each group of mice were observed.
The appearance of the skin of the mice was observed and the blank group was found to have smooth skin, a smooth skin tone and no wrinkles.
The model group mice had dry, loose skin, dark color and wrinkles.
Compared with the model group, the matrix group has insignificant improvement effect.
The skin condition of the mice in the other experimental groups is improved compared with that of the model group, wherein the skin condition of the experimental group I is obviously reduced compared with that of the other groups, and the skin color is obviously biased to be normal.
Measurement of Hyaluronic Acid (HA) content in mouse skin
0.5g of mouse skin tissue was taken, 10% homogenate was prepared using PBS buffer solution, centrifuged, and the supernatant was taken, and the HA content in the mouse skin was measured by following the procedures of a mouse Hyaluronic Acid (HA) assay kit (purchased from Shanghai Mingsheng Biochemical reagent, Ltd.). The results of the experiments are shown in the following table:
TABLE 1
| Group of | HA content (mg/g) |
| Blank group | 2.73 |
| Model set | 1.61 |
| Substrate group | 2.03 |
| Experimental group I | 3.16 |
| Experimental group II | 2.27 |
| Experimental group III | 2.32 |
| Experimental group IV | 2.16 |
The result shows that the experiment group I can remarkably promote the expression of hyaluronic acid in a mouse body, and after the experiment group I is continuously used, the content of hyaluronic acid in skin is higher than that of a blank group, and the nano preparation prepared by combining hydroxypropyl tetrahydropyrane triol, retinol retinoic acid ester and soybean isoflavone has remarkable synergistic effect.
When observing the epidermis layer and the dermis layer in the tissue section of the mouse by microscopy, the hyaluronic acid is combined with the protein (HABP) to cause staining reaction, and partial results are shown in FIG. 3, and it can be obviously observed that the content of the hyaluronic acid in the section of the experimental group I is obviously higher than that in the tissue section of the blank group. In the model group, the hyaluronic acid content was significantly lower.
Example 4 efficacy testing
1. Purpose of testing
The efficacy of the product was examined by using the test product for 6 subjects for 4 weeks, front to back control, combined with instrumental test analysis, to improve crow's feet, fine lines under the eyes, skin elasticity and skin gloss. The product was also evaluated by the test subjects according to the questionnaire after 4 weeks of use.
TABLE 2 test sample information
2. Test subject
1) Chinese female;
2) the facial skin is loose, and the fishtail lines are visible;
3) an existing user of facial skin care cosmetics, who uses the cosmetic for at least 3 days per week and 2 times per day;
4) primary decision makers for skin care product purchase;
5) no clinical and consumer tests have been enrolled for the last 3 months without other general exclusions from market research;
6) there were no other exclusions common to market research.
3. Test flow
3.1 design of the experiment
Left in place for 4 weeks and the instrument tests for photographs. The product removal effect was evaluated by photographing the face with Visia CR at W0 (before product use), W2 (after 2 weeks product use), and W4 (after 4 weeks product use), respectively, and analyzing the fishtail and fine grain area under the eyes before and after the test product use with IPP software. Skin elasticity and firmness, skin gloss were also tested to assess the overall skin improvement effect of the product.
3.2 test instruments and materials
1) Skin elasticity tester (Cutomer, MPA580, Courage and Khazaka, Germany)
2) Skin gloss tester (Glossimeter, GL200, Courage and Khazaka, Germany)
3) Facial digital imaging System (Visia-CR, Canfield, USA)
4) IPP analysis software
5) Non-scrap absorbent dry paper towel
4. Test results
4.1 area of fishtail line
The following is the change in area of the crow's feet, with lower values indicating less crow's feet. Taking the average of both sides and calculating.
TABLE 3 area of crow's feet
| Number of times | Point in time | Average fishtail lineNoodle | Mean rate of improvement |
| 1 | W0 | 5.43 | - |
| 2 | W2 | 4.61 | 14.98％ |
| 3 | W4 | 3.50 | 35.47％ |
Figure 4 shows the results of typical subject crow's feet changes.
4.2 area of fine lines under the eye
The following is the change in the area of the fine lines under the eye, with lower values indicating fewer fine lines. Taking the average of both sides and calculating.
TABLE 4 area of fine lines
| Number of times | Point in time | Average fine grain area | Mean rate of improvement |
| 1 | W0 | 3.36 | - |
| 2 | W2 | 2.36 | 29.82％ |
| 3 | W4 | 2.13 | 36.57％ |
Figure 5 shows the results of fine line changes under the eyes of a typical subject.
Through the comparative analysis of the skin around the face and eyes of 6 subjects after using the product for 4 weeks, the data of the crow's feet area change, the fine line area change, the skin uniqueness, the compactness, the glossiness and the like are combined.
After using the test sample for 2 weeks, some wrinkle improvement efficacy was exhibited.
The area of the fishtail line is reduced by 14.98 percent on average; the fine line area under the eye decreased by 29.82% on average.
After using the test sample for 4 weeks, significant wrinkle improvement efficacy was exhibited.
The area of the crow's feet is reduced by 35.47% on average; the average reduction in the fine line area under the eye was 36.57%.
Meanwhile, the skin monotonicity, the firmness and the glossiness are also obviously improved.
EXAMPLE 5 preparation of Nanodisclosed
10g of hydroxypropyl tetrahydropyrane triol, 0.5g of retinol retinoic acid ester and 0.5g of soybean isoflavone were dissolved in ethanol to give a total of 100mL of a mixed solution. 50g of egg yolk lecithin was dissolved in the mixed solution, heated to 40 ℃ and the ethanol solvent in the solution was removed under 1330 Pa. The remaining liquid was added to 500ml of PBS buffer, and dispersed with stirring.
The above dispersion solution was emulsified by a high pressure emulsifier, and the emulsified dispersion was filtered through a 0.22 μm microporous membrane for sterilization. 15g of the filtrate were filled into freeze-drying bottles which were then placed in a freeze-dryer and run at room temperature at 4000Pa for gas degassing. Degassing, and concentrating at 40 deg.C and 1330Pa to obtain concentrated solution. The concentrate was cooled to-40 ℃ and the solution was then foamed by raising the temperature of the solution to 40 ℃ under high vacuum (1.33 Pa). Vacuum drying the bubbling solution at 30 ℃ under the condition of 13.3Pa, pressurizing to atmospheric pressure by using nitrogen after vacuum drying, and sealing a freeze-drying bottle to obtain the lipidosome nano preparation, namely the anti-aging wrinkle-removing composite nano preparation.
EXAMPLE 6 preparation of Nanodisclosed
3g of hydroxypropyl tetrahydropyrane triol, 2g of retinol retinoic acid ester and 2g of soybean isoflavone were dissolved in ethanol to give a total of 100mL of a mixed solution. 50g of egg yolk lecithin was dissolved in the mixed solution, heated to 40 ℃ and the ethanol solvent in the solution was removed under 1330 Pa. The remaining liquid was added to 500ml of PBS buffer, and dispersed with stirring.
The above dispersion solution was emulsified by a high pressure emulsifier, and the emulsified dispersion was filtered through a 0.22 μm microporous membrane for sterilization. 15g of the filtrate were filled into freeze-drying bottles which were then placed in a freeze-dryer and run at room temperature at 4000Pa for gas degassing. Degassing, and concentrating at 40 deg.C and 1330Pa to obtain concentrated solution. The concentrate was cooled to-40 ℃ and the solution was then foamed by raising the temperature of the solution to 40 ℃ under high vacuum (1.33 Pa). Vacuum drying the bubbling solution at 30 ℃ under the condition of 13.3Pa, pressurizing to atmospheric pressure by using nitrogen after vacuum drying, and sealing a freeze-drying bottle to obtain the lipidosome nano preparation, namely the anti-aging wrinkle-removing composite nano preparation.
EXAMPLE 7 preparation of Nanodisclosed
6g of hydroxypropyl tetrahydropyrane triol, 1.5g of retinol retinoic acid ester and 1.5g of soybean isoflavone were dissolved in ethanol to give a total of 100mL of a mixed solution. 50g of egg yolk lecithin was dissolved in the mixed solution, heated to 40 ℃ and the ethanol solvent in the solution was removed under 1330 Pa. The remaining liquid was added to 500ml of PBS buffer, and dispersed with stirring.
The above dispersion solution was emulsified by a high pressure emulsifier, and the emulsified dispersion was filtered through a 0.22 μm microporous membrane for sterilization. 15g of the filtrate were filled into freeze-drying bottles which were then placed in a freeze-dryer and run at room temperature at 4000Pa for gas degassing. Degassing, and concentrating at 40 deg.C and 1330Pa to obtain concentrated solution. The concentrate was cooled to-40 ℃ and the solution was then foamed by raising the temperature of the solution to 40 ℃ under high vacuum (1.33 Pa). Vacuum drying the bubbling solution at 30 ℃ under the condition of 13.3Pa, pressurizing to atmospheric pressure by using nitrogen after vacuum drying, and sealing a freeze-drying bottle to obtain the lipidosome nano preparation, namely the anti-aging wrinkle-removing composite nano preparation.
EXAMPLE 8 preparation of Nanodisclosed
5g of hydroxypropyl tetrahydropyrane triol, 1g of retinol retinoic acid ester and 1g of soybean isoflavone were dissolved in ethanol to give a total of 100mL of a mixed solution. 50g of egg yolk lecithin was dissolved in the mixed solution, heated to 40 ℃ and the ethanol solvent in the solution was removed under 1330 Pa. The remaining liquid was added to 500ml of PBS buffer, and dispersed with stirring.
The above dispersion solution was emulsified by a high pressure emulsifier, and the emulsified dispersion was filtered through a 0.22 μm microporous membrane for sterilization. 15g of the filtrate were filled into freeze-drying bottles which were then placed in a freeze-dryer and run at room temperature at 4000Pa for gas degassing. Degassing, and concentrating at 40 deg.C and 1330Pa to obtain concentrated solution. The concentrate was cooled to-40 ℃ and the solution was then foamed by raising the temperature of the solution to 40 ℃ under high vacuum (1.33 Pa). Vacuum drying the bubbling solution at 30 ℃ under the condition of 13.3Pa, pressurizing to atmospheric pressure by using nitrogen after vacuum drying, and sealing a freeze-drying bottle to obtain the lipidosome nano preparation, namely the anti-aging wrinkle-removing composite nano preparation.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (10)
1.A composite nano-formulation, wherein the composite nano-formulation is a liposomal nano-formulation and comprises:
hydroxypropyl tetrahydropyran triol, retinol retinoic acid ester, and soy isoflavones.
2. The composite nano-formulation of claim 1, wherein the composite nano-formulation comprises:
1-10 parts of hydroxypropyl tetrahydropyrane triol;
0.5-2 parts by weight of soybean isoflavone; and
0.5-2 parts by weight of retinol retinoic acid ester.
3. The composite nano-formulation of claim 1, wherein the composite nano-formulation comprises:
5-10 parts of hydroxypropyl tetrahydropyrane triol;
1-2 parts by weight of soybean isoflavone; and
1-2 parts of retinol retinoic acid ester.
4. A cosmetic composition comprising the complex nano-formulation of claim 1.
5. The cosmetic composition of claim 4, wherein the composite nano-formulation is present in an amount of 0.01% to 30% by weight of the cosmetic composition.
6. The cosmetic composition of claim 5, wherein the composite nano-formulation is present in an amount of 1% to 20% by weight of the cosmetic composition.
7. The cosmetic composition of claim 5, wherein the composite nano-formulation is present in an amount of 5% to 15% by weight of the cosmetic composition.
8. The cosmetic composition of claim 4, wherein the cosmetic is in the form of a solid, semi-solid, or liquid dosage form, such as a solution, gel, cream, lotion, ointment, cream, paste, cake, powder, patch, or the like.
9. A method for preparing a composite nano-formulation, the method comprising the steps of:
(1) dissolving hydroxypropyl tetrahydropyrane triol, retinol retinoic acid ester and soybean isoflavone in ethanol to obtain a mixed solution I;
(2) dissolving the membrane material in the mixed solution I, and removing ethanol in the solution by vacuum evaporation to obtain a mixed solution II;
(3) adding the mixed solution II into a PBS buffer solution to obtain a mixed solution III;
(4) and (3) transferring the mixed solution III to a high-pressure homogenizer for homogenization, and filtering and sterilizing through a microporous filter membrane to obtain the nano liposome, namely the composite nano preparation.
10. Use of the composite nano-formulation of claim 1 for preparing cosmetics, health products or drugs.
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