CN118806662A - Anti-aging skin care night composition and preparation method and application thereof - Google Patents
Anti-aging skin care night composition and preparation method and application thereof Download PDFInfo
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- CN118806662A CN118806662A CN202410887677.5A CN202410887677A CN118806662A CN 118806662 A CN118806662 A CN 118806662A CN 202410887677 A CN202410887677 A CN 202410887677A CN 118806662 A CN118806662 A CN 118806662A
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Landscapes
- Cosmetics (AREA)
Abstract
The invention provides an anti-aging skin care night composition, a preparation method and application thereof. Specifically, the invention provides a first active ingredient combination for preparing an anti-aging skin care composition or preparation, which comprises or consists of the following active ingredients in parts by weight: 1 to 5 parts of Matrixyl TM,3000, 1 to 5 parts of supersplit bullet wetting peptide PRO and 0.05 to 1 part of radix scutellariae extract. The invention establishes a circadian rhythm model for collagen assembly and synthesis on human fibroblasts, and specifically screens out an anti-aging skin care night composition based on the circadian rhythm model, and the composition can synergistically promote collagen synthesis at night.
Description
Technical Field
The invention relates to the field of cosmetics, in particular to an anti-aging skin care night composition, a preparation method and application thereof.
Background
Collagen synthesis and assembly is a complex biological process involving multiple steps and regulatory mechanisms within the cell. Studies have shown that collagen synthesis, secretion and degradation are dependent on circadian rhythms. The synthesis and assembly of collagen has a circadian rhythm, namely, the collagen fibers are assembled in the daytime, and the procollagen is synthesized at night. Breaking circadian rhythm can cause collagen fiber to accumulate, structure disorder, elasticity and strength to be greatly reduced, and is unfavorable for skin aging resistance.
The existing cosmetics have the effect of directly regulating circadian rhythm related genes, but how the circadian rhythm genes regulate and promote the solution of related skin problems (such as anti-aging, relieving and the like) are not studied and corresponding product/ingredient solutions are not found. On the other hand, the recommended collocation of the conventional anti-aging night cream is more and more only combined from the aspects of product texture and efficacy appeal, but the relationship between the night cream efficacy appeal and the circadian rhythm and the anti-aging collagen is not clearly disclosed.
Thus, there is an urgent need in the art to develop anti-aging skin care night compositions capable of modulating collagen changes within the synchronized skin's circadian rhythm.
Disclosure of Invention
The invention aims to provide an anti-aging skin care night composition capable of synchronizing the internal circadian rhythm of skin and regulating collagen change.
The first aspect of the present invention provides a first active ingredient combination for preparing an anti-aging skin care composition or formulation comprising or consisting of the following active ingredients:
1-5 parts by weight of Matrixyl TM;
1-5 parts by weight of supermolecule bullet wetting peptide PRO; and
0.05 To 1 weight portion of baical skullcap root extract.
In another preferred embodiment, the first active ingredient combination comprises or consists of the following active ingredients:
1-3 parts by weight of Matrixyl TM;
1-3 parts by weight of supermolecule bullet wetting peptide PRO; and
0.05 To 0.5 weight portion of baical skullcap root extract.
In another preferred embodiment, the first active ingredient combination comprises or consists of the following active ingredients:
Matrixyl TM 3000,3000 parts by weight;
Supersplit bullet wetting peptide PRO 1 weight part; and
0.1 Part by weight of baical skullcap root extract.
In another preferred embodiment, the concentration of each component in the first active ingredient combination is:
20～40mg/ml MatrixylTM3000；
10-30 mg/ml supermolecule elastin peptide PRO;
0.5-2 mg/ml of radix scutellariae extract.
In another preferred example, the radix Scutellariae extract is baicalin-cyclodextrin-vesicle complex, the
The compound comprises the following components in parts by weight:
12-18 parts of baicalin
70-90 Parts of vesicle material
3-7 Parts of cyclodextrin.
In another preferred example, the vesicle material has the following components in parts by weight:
50-60 parts of saccharomycete fermentation product
20-30 Parts of bacillus fermentation products.
In another preferred example, the compound has the following components in parts by weight:
14-16 parts of baicalin
78-83 Parts of vesicle material
4-6 Parts of cyclodextrin.
In another preferred example, the vesicle material has the following components in parts by weight:
54-56 parts of saccharomycete fermentation product
23-25 Parts of bacillus fermentation products.
In a second aspect the present invention provides an anti-ageing skin care composition comprising a combination of the first active ingredients according to the first aspect of the invention, together with a cosmetically or pharmaceutically acceptable carrier.
In another preferred embodiment, the first active ingredient combination comprises 1 to 99% by weight of the total composition.
In another preferred embodiment, the anti-aging skin care composition comprises or consists of the following active ingredients in weight percent:
MatrixylTM3000 1～5％；
Superfractionation bullet wetting peptide PRO 1-5%; and
0.05 To 1 percent of baical skullcap root extract.
In another preferred embodiment, the anti-aging skin care composition comprises or consists of the following active ingredients in weight percent:
MatrixylTM3000 3％；
Superfractionation bullet wetting peptide PRO 1%; and
0.1% Of radix scutellariae extract.
In another preferred embodiment, the anti-aging skin care composition further comprises other anti-aging skin care active ingredients.
In another preferred embodiment, the carrier is selected from the group consisting of: excipients, thickeners or diluents.
In another preferred embodiment, the excipient is selected from the group consisting of:
A165 emulsifier, mixed alcohol, pentaerythritol distearate, shea butter, caprylic/capric triglyceride, macadamia nut oil, silicone oil, synthetic squalane, transparent xanthan gum, glycerin, carbo 940, butylene glycol, disodium EDTA, pentanediol, allantoin, cactus extract, irritation-suppressing factor, sodium hydroxide, methyl chloroisothiazolinone and methyl isothiazolinone (MTI), phenoxyethanol, ethylhexyl glycerol, or the like.
In another preferred embodiment, the formulation of the composition or formulation is selected from the group consisting of: a solid dosage form, a semi-solid dosage form, or a liquid dosage form.
In another preferred embodiment, the formulation of the composition or formulation is selected from the group consisting of: solutions, gels, creams, or emulsions.
In another preferred embodiment, the composition is a pharmaceutical or cosmetic composition, preferably in the form of a topical pharmaceutical dosage form.
In another preferred example, the formulation is an external formulation or a transdermal formulation (e.g., an external solution, an ointment, a patch, etc.).
In another preferred embodiment, the formulation or composition comprises a cosmetic, food, medical device or pharmaceutical product.
In another preferred embodiment, the cosmetic is an efficacy cosmetic.
In a third aspect, the present invention provides a method of preparing an anti-aging skin care composition according to the second aspect of the present invention, the method comprising:
(a) Mixing 1-5 parts by weight of Matrixyl TM 3000,3000, 1-5 parts by weight of supersplit bullet wetting peptide PRO and 0.05-1 part by weight of radix scutellariae extract to obtain a first active ingredient combination;
(b) Mixing the first active ingredient combination obtained in step (a) with a cosmetically, chemically or biologically acceptable carrier, thereby obtaining said anti-aging skin care composition.
In another preferred embodiment, the composition is in the form of a solid, semi-solid, or liquid dosage form, such as a solution, gel, cream, emulsion.
In a fourth aspect, the present invention provides the use of a first combination of active ingredients according to the first aspect of the invention or an anti-ageing skin care composition according to the second aspect of the invention in the preparation of an anti-ageing product.
In another preferred embodiment, the anti-aging product is selected from the group consisting of: an anti-aging essence, anti-aging water, anti-aging facial cream, anti-aging facial cleanser and anti-aging facial mask.
In another preferred embodiment, the anti-aging product is used for: promoting collagen fiber assembly, promoting procollagen synthesis, increasing type I collagen content, increasing skin elasticity, scavenging free radicals, resisting oxidation, increasing skin moisture content, eliminating inflammation, or combinations thereof.
In another preferred embodiment, the anti-aging product is for: promote collagen fiber assembly during the daytime and/or promote procollagen synthesis during the nighttime.
In a fifth aspect, the invention provides a kit comprising a first container and a second container,
The first container contains a first active ingredient combination according to the first aspect of the invention, and a cosmetically, chemically or biologically acceptable carrier;
The second container contains a second anti-aging skin care composition comprising a second active ingredient combination comprising or consisting of the following active ingredients, and a cosmetically, chemically, or biologically acceptable carrier:
Acetyl tetrapeptide-9 1-5 weight portions;
1-5 parts by weight of hexapeptide PRO; and
0.05 To 1 weight portion of baical skullcap root extract.
In another preferred embodiment, the second active ingredient combination comprises or consists of the following active ingredients:
acetyl tetrapeptide-9 1 parts by weight;
1 part by weight of hexapeptide PRO; and
0.05 Part by weight of baical skullcap root extract.
In another preferred embodiment, the second anti-aging skin care composition comprises the following active ingredients in weight percent:
acetyl tetrapeptide-9 1-5%;
1-5% of hexapeptide PRO; and
0.05 To 1 percent of baical skullcap root extract.
In another preferred embodiment, the second anti-aging skin care composition comprises the following active ingredients in weight percent:
acetyl tetrapeptide-9 1-3%;
1-3% of hexapeptide PRO; and
0.05 To 0.5 percent of baical skullcap root extract.
In another preferred embodiment, the second anti-aging skin care composition comprises the following active ingredients in weight percent:
Acetyl tetrapeptide-9 2%;
Hexapeptide PRO 2%; and
0.1% Of radix scutellariae extract.
In another preferred embodiment, the concentration of each component in the second active ingredient combination is:
acetyl tetrapeptide-9 in 10-30 mg/ml;
10-30 mg/ml hexapeptide PRO;
0.5-2 mg/ml of radix scutellariae extract.
In another preferred embodiment, the second active ingredient combination comprises from 1 to 99% by weight of the total weight of the second anti-aging skin care composition.
In another preferred embodiment, the kit further comprises instructions, the instructions describing the steps of: applying the composition in the first container at night; the composition in the second container was applied during the day.
In a sixth aspect the present invention provides a method for preventing and/or slowing down skin ageing comprising the steps of:
(a) Administering to a subject in need thereof:
An anti-aging skin care composition according to the second aspect of the present invention; or (b)
The anti-aging skin care composition according to the second aspect of the present invention comprises a second active ingredient combination, and a cosmetically, chemically or biologically acceptable carrier. In another preferred embodiment, the second active ingredient combination comprises or consists of the following active ingredients:
Acetyl tetrapeptide-9 1-5 weight portions;
1-5 parts by weight of hexapeptide PRO; and
0.05 To 1 weight portion of baical skullcap root extract.
In another preferred embodiment, the application refers to the application of the anti-aging skin care composition according to the second aspect of the present invention at night; the second anti-aging skin care composition is applied during the daytime.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 is a graph showing the results of the preparation of a verified circadian rhythm model in example 1 of the invention. Wherein the orange curve represents the expression profile of the rhythmic gene ATNX L in HSF cells over 48 consecutive hours; the blue curve rhythmic gene CRY1 is expressed in HSF cells for a continuous 48h curve, where ZT refers to the time of the time service factor (Zeitgeber time, ZT, the environmental time ordered by the laboratory, the artificial control of the illumination in rhythmic studies).
FIG. 2 is a graph showing the results of verifying the assembly efficacy of collagen fibers in example 3 of the present invention. Wherein, graph a represents the detection of the relative expression amount of LOX mRNA; panel b shows the relative expression level detection of CTSK mRNA, and Panel c shows the relative expression level detection of MMP1 mRNA; the white portion of the graph is shown as daytime and the shaded portion is shown as nighttime, (ns is no significant difference, P <0.05, P <0.01, P < 0.001).
FIG. 3 is a graph showing the results of verifying the collagen synthesis/secretion efficacy in example 3 of the present invention. Wherein, a graph shows the relative expression amount detection of Pde4d mRNA; panel b shows the relative expression level detection of Mia3mRNA, and panel c shows the relative expression level detection of Vps33b mRNA; the white portion of the graph is shown as daytime and the shaded portion is shown as nighttime, (ns is no significant difference, P <0.05, P <0.01, P < 0.001).
Detailed Description
The inventor of the present invention has conducted intensive studies by establishing a model of a circadian rhythm of collagen assembly and synthesis on human fibroblasts, and on the basis of this, disclosed an anti-aging skin care composition for regulating collagen changes for synchronizing the internal circadian rhythm of skin, which comprises a daily composition and a night composition, wherein the daily composition mainly comprises acetyl tetrapeptide-9; the hexapeptide PRO and the baical skullcap root extract are prepared by a proper preparation method. The night composition mainly comprises Matrixyl TM 3000,3000, supermolecule elastin PRO and radix scutellariae extract, and is prepared by a proper preparation method. The daily composition can synergistically promote collagen fiber assembly in daytime, and the night composition can promote procollagen synthesis at night. The present invention has been completed on the basis of this finding.
Terminology
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 "night cream-collagen TM", the "night composition", the "composition of the invention" and the "anti-aging skin care composition" are used interchangeably.
As used herein, the "day cream-collagen bundles TM", the "daily composition" and the "second anti-aging skin care composition" are used interchangeably.
As used herein, the term "daytime" is used interchangeably with the term "daytime".
As used herein, the term "night" is used interchangeably with the term "night".
As used herein, "anti-aging skin care" includes: promoting collagen fiber assembly during daytime, promoting procollagen synthesis at night, increasing type I collagen content, increasing skin elasticity, scavenging free radicals, resisting oxidation, increasing skin moisture content, and eliminating inflammation.
As used herein, the terms "comprising" or "containing" include "comprising," consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …" and "consisting of … …" are under the notion of "containing", "having" or "including".
As used herein, the term "cosmetically, chemically or biologically acceptable" ingredients are substances that are suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation and allergic response), i.e., commensurate with a reasonable benefit/risk ratio.
As used herein, the term "cosmetically, chemically, or biologically acceptable carrier" includes various excipients, thickeners, or diluents. The term refers to such vectors: they are not per se essential active ingredients and are not overly toxic after administration. Suitable vectors are well known to those of ordinary skill in the art. The carrier may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as fillers, lubricants, glidants, wetting or emulsifying agents, flavoring agents, pH buffering substances and the like may also be present in these carriers. Some preferred carriers are, for example but not limited to: a165 emulsifier, mixed alcohol, pentaerythritol distearate (PES), shea butter, caprylic/capric triglyceride (GTCC), macadamia nut oil, silicone oil, synthetic squalane, transparent xanthan gum, glycerol, carbopol 940, butylene glycol, disodium EDTA, pentylene glycol, allantoin, cactus extract, irritation-suppressing factors, sodium hydroxide, methyl chloroisothiazolinone and methyl isothiazolinone (MTI), phenoxyethanol and ethylhexyl glycerol (PEHG), or analogs thereof.
As used herein, the terms "vesicle," "microcapsule material," "vesicle material," are used interchangeably, and refer to vesicle material of microbial origin, in the present invention, the outer shell portion of a baicalin-cyclodextrin-vesicle complex, encapsulating a cyclodextrin nanoparticle comprising baicalin.
Baicalin-cyclodextrin-vesicle complex
As used herein, the terms "baicalin microcapsule", "natural vesicle technology modified baicalin", "baicalin-cyclodextrin-vesicle complex", "inventive complex" are used interchangeably, and refer to a baicalin extract having high transdermal efficiency, high bioavailability, and high drug loading prepared by the inventive method.
The baicalin-cyclodextrin-vesicle complex is formed by wrapping baicalin by using a biomembrane vesicle structure formed by saccharomycetes and bacillus fermentation products. The biomembrane vesicle structure not only can solve the problems of the application of raw materials such as baicalin solubility and stability, but also has a very similar structure with human cell membranes, and compared with the traditional liposome wrapping mode, the biomembrane vesicle structure is more beneficial to improving the transdermal and cytoblast capacities of baicalin, so that the baicalin plays a better role in resisting oxidation and aging in a skin care product system.
The baicalin-cyclodextrin-vesicle compound is a compound with uniform particle size and good safety, and has the structure that: the vesicle material wraps cyclodextrin nanoparticles including baicalin, the vesicle material is composed of a saccharomycete fermentation product and a bacillus fermentation product, and the cyclodextrin nanoparticles including baicalin are formed by the way that baicalin is included in molecular cavity gaps of cyclodextrin.
In the present invention, the baicalin-cyclodextrin-vesicle complex comprises:
(i) An active ingredient, wherein the active ingredient is baicalin;
(ii) A vesicle material comprising a yeast fermentation product and a bacillus fermentation product; and (iii) cyclodextrin; and
The cyclodextrin inclusion (ii) of (i) an active ingredient forms nanoparticles;
The (iii) vesicle material encapsulates the nanoparticle to form a baicalin-cyclodextrin-vesicle complex.
In another preferred example, the mass ratio of the active ingredient to the vesicle material to the cyclodextrin is 12-18:70-90:3-7.
In another preferred embodiment, the cyclodextrin is selected from the group consisting of alpha cyclodextrin, beta cyclodextrin, gamma cyclodextrin, or a combination thereof.
The baicalin-cyclodextrin-vesicle complex has uniform particle size of 50-500 nm and a polymer dispersibility index of 0.05-0.3. The Zeta potential of the baicalin-cyclodextrin-vesicle compound is-10-0 mV, the potential distribution is uniform, the deviation range is small, no obvious particles or material fragments are dispersed, and the safety is good.
The microstructure of the baicalin-cyclodextrin-vesicle complex is that baicalin is included in cyclodextrin, and a vesicle material wraps the cyclodextrin including the baicalin.
The baicalin-cyclodextrin-vesicle compound has excellent skin permeation effect, the permeation depth of the baicalin-cyclodextrin-vesicle compound in the external skin tissue is P1, and compared with the permeation depth P0 of the baicalin in the external skin tissue, the ratio (P1/P0) of the baicalin-cyclodextrin-vesicle compound to the baicalin-cyclodextrin-vesicle compound is more than or equal to 3, preferably more than or equal to 4, and more preferably more than or equal to 5; compared with the penetration depth P2 of the liposome-coated baicalin in the skin tissue in vitro, the ratio (P1/P2) of the liposome-coated baicalin to the liposome-coated baicalin is more than or equal to 1.5, preferably more than or equal to 2.
The baicalin-cyclodextrin-vesicle complex has excellent transdermal release rate. In another preferred embodiment, the excellent transdermal release rate means no obvious burst phenomenon within 0 to 8 hours. In another preferred embodiment, the cumulative release is greater than or equal to 80%, preferably 85%, at 36 hours; and/or, the cumulative release is greater than or equal to 45%, preferably 50%, at 8 hours.
Vesicle material
The vesicle material of the present invention includes vesicle materials of microbial origin. The microorganisms include strains of the genus Saccharomyces and strains of the genus Bacillus.
In another preferred embodiment, the strain of Saccharomyces is selected from the group consisting of: saccharomyces cerevisiae, saccharomyces cerevisiae yeast, or a combination thereof. In another preferred embodiment, the strain of bacillus is selected from the group consisting of: bacillus subtilis.
The vesicle materials of the present invention are commercially available or can be prepared by conventional methods. Typically, fermentation may be employed to prepare the vesicle material. A specific preparation method of a representative vesicle material is disclosed in patent WO2022055250.
Baicalin
Baicalin (baicalin) is a flavonoid extracted and separated from the dry root of radix Scutellariae (Scutellaria baicalensis Georgi), and is hardly dissolved in common cosmetic solvents such as water and ethanol. Baicalin has remarkable biological activity, has antibacterial, diuretic, antiinflammatory, antiallergic and spasmolytic effects, and has strong physiological effects such as anticancer effect. Has taken an important role in clinical medicine. Baicalin can absorb ultraviolet rays, remove oxygen free radicals and inhibit melanin generation, so that the baicalin can be used for medicines and cosmetics, and is a cosmetic raw material with good application prospect.
Human fibroblasts (HSF)
Human fibroblasts (HSF) are one of the main constituents of skin tissue, and the fiber density, fiber thickness and fiber orientation of the fibroblasts can reflect the state and aging degree of cells to a great extent, while UV light attenuates collagen fibers of cells to accelerate cell aging.
Collagen fiber assembly related gene
Comprises lysyl oxidase (lysyl oxidase, LOX), cathepsin K (CATHEPSIN K, CTSK), matrix metalloproteinase 1 (Matrix metalloproteinase 1, MMP 1)
Lysyl oxidase (lysyl oxidase, LOX): is responsible for covalent cross-linking of collagen and elastin in the ECM. The function is to initiate oxidative deamination by catalyzing epsilon-amino groups of lysine and/or hydroxylysine. This catalytic process results in oxidation of the nitrogen atoms in the amino acids, yielding highly reactive intermediates which in turn participate in the formation of covalent crosslinks. This oxidative deamination is a critical step in the covalent cross-linking of collagen and elastin. Through the action of LOX, covalent connection is formed between collagen and elastin molecules which are originally linearly arranged, and a network structure is formed. This network structure imparts strong elasticity and toughness to the tissue, making it resistant to hydrolysis by non-specific proteases. Thus, by this covalent cross-linking, extracellular collagen and elastin are able to form a stable fibrous network, providing support and structure, maintaining the morphology and function of the tissue, and are also critical for skin to maintain elasticity.
Cathepsin K (CATHEPSIN K, CTSK): a member belonging to the papain superfamily: lysosomal cysteine proteases are an important extracellular matrix degrading enzyme found in recent years in lysosomal intracellular proteases.
Matrix metalloproteinase 1 (Matrix metalloproteinase 1, mmp 1): is the most important enzyme for degrading type I and type III collagen. When cathepsin K and MMP1 are overexpressed, extracellular matrix components are specifically degraded, disrupting the normal structure of collagen fibers and elastic fibers.
Collagen synthesis-related genes: the collagen plays a very important supporting role in skin, and can maintain the compactness and elasticity of the skin, and particularly, the type I collagen (College I), which is the collagen with the highest content in the skin, accounts for about 90% of the total collagen content, comprises Mia3, pde4d and Vps33 b. The mRNA of Collagen I.alpha.1 and Collagen I.alpha.2 (encoding Collagen. Alpha.1 (I) and. Alpha.2 (I), respectively) specifically targets the SEC61 epitope. In eukaryotes, SEC61 a binds to one β subunit and one γ subunit to form a heterotrimeric complex SEC61 a βγ, thereby forming a protein transduction pore through which the newly synthesized Collagen I enters the lumen of the endoplasmic reticulum. Pde4d is functionally related to Pde7A1, pde7A1 protein regulating protein transport from the endoplasmic reticulum to the golgi apparatus, a function necessary for procollagen secretion. Thus, it can be demonstrated that Pde4d is involved in the procollagen secretion process. Mia3 gene encodes TANGO1S and TANGO1L, both of which are involved in the selective transport of procollagen by the Golgi apparatus. Vps33b plays a role in post-translational modification of extra-endoplasmic reticulum collagen.
In summary, mia3 (encoding tengo 1, believed to promote protein loading at the endoplasmic reticulum exit site), pde4d (Pde 4d is functionally related to another phosphodiesterase Pde7A1, pde7A1 is believed to be important in regulating the transport of procollagen I in COS7 cells), and Vps33b (Vps 33b is important in post-translational modification of collagen IV) control the synthetic secretion of collagen, the expression levels of which are able to reflect the synthetic secretion levels of collagen.
Various conventional carriers or auxiliary materials such as filling agents, flavoring agents, antioxidants, coating materials and the like required by preparing different formulations can be added into the anti-aging skin care composition.
As a preferred mode of the invention, in the combination, each component is individually packaged in a bag, a box or other various containers. The combination can be a kit, a packaging box, a packaging bag, a cosmetic box and the like. The kit, the packaging box, the packaging bag or the cosmetic box can also be directly filled with the extracts of the raw materials so as to be convenient to use. The kit, pack, packaging bag or cosmetic case of the present invention may be directly filled with the composition of the present invention.
The kit, the packaging box, the packaging bag or the cosmetic box can also comprise instructions, wherein the application method of each component is described.
The technical scheme of the invention has the following main advantages:
The invention utilizes a circadian rhythm model for collagen assembly and synthesis on human fibroblasts, and specifically screens out an anti-aging skin care night composition, wherein each active ingredient in the night composition can promote collagen synthesis at night and has a synergistic effect.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents involved in the present invention are available from commercial sources unless otherwise specified.
Example 1 preparation and validation of Human Skin Fibroblast (HSF) rhythmic model
The purpose of this example was to examine whether HSF cells were rhythmic during the culture every 4h, within 0-48 h. The expression levels of the rhythmic gene ATNX, L, CRY1 and the internal reference gene GAPDH were detected by a real-time fluorescent quantitative PCR method (QPCR method). The specific process is as follows:
1.1 experimental grouping:
HSF cells were prepared for subsequent testing after 0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48h incubation, respectively, for a total of 13 groups.
1.2 Experimental procedure:
1) The HSF cell suspension was inoculated into a petri dish.
2) After the cells had grown on the dishes, the medium was removed, 2mL of complete medium containing 100nM dexamethasone was added, and the culture was continued in a 37℃cell incubator containing 5% CO 2 for 30min, after which the complete medium was replaced and continued for 24h.
3) And (3) sample collection: the cells were observed to grow without abnormalities under a mirror, washed by adding 2mL of PBS to the petri dish, lysed by adding 1mL of Trizol after removal of PBS, and lysates were collected in the corresponding labeled EP tubes. And respectively collecting cell sample lysates of 0h, 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h and 48h at each sample collection interval, and storing in a refrigerator at-80 ℃.
4) Extraction of sample RNA: 200 mu L of chloroform is added into a sample EP tube, and the mixture is mixed for 15 to 30 seconds by intense vortex vibration; centrifuging at 12000rpm at 4deg.C for 15min, separating the sample into 3 layers, and collecting 400 μl supernatant; adding 500 μl of isopropanol into each tube, mixing for 6-8 times upside down, centrifuging at 4deg.C and 10000rpm for 10min, and discarding supernatant; the precipitate was washed 2 times with 75% glacial ethanol: centrifuging at 4deg.C and 10000rpm for 10min, discarding supernatant, and repeating the centrifugation once again to remove residual liquid in the tube; finally, dissolving RNA precipitate by using 20 mu LDEPC water; taking 1 mu L of RNA concentration, and storing and using the rest in a refrigerator at the temperature of minus 80 ℃; using DEPC water as blank control; a260/280 is 1.8-2.0.
5) Reverse transcription reaction: performing RNA reverse transcription reaction using Takara reverse transcription kit; before using, 5X PRIME SCRIPT RT Mix is separated to the bottom of the tube, and the mixture is used after being mixed by a pipette repeatedly and slowly; preparing a reaction system: the system is required to be carried out on ice; the total volume of the system was 10. Mu.L; the reaction system is shown in table 1 below:
TABLE 1
The reaction conditions of the Takara reverse transcription kit are shown in table 2 below:
TABLE 2
6) Real-time fluorescent quantitative PCR reaction: the reaction solution obtained by the reverse transcription is diluted 4 times by EASY Dilution and then used; the SYBR Green method was employed. The quantitative reaction reagent was TRANSSTART TOP GREEN QPCR Supermix and the reaction was performed using an ABI ViiA7PCR instrument. The reaction system is shown in tables 3 and 4 below:
TABLE 3 Table 3
TABLE 4 Table 4
The real-time fluorescent quantitative PCR reaction conditions are shown in Table 5 below:
TABLE 5
7) Statistical analysis: experimental data are expressed as Mean standard deviation (mean±sd), data processing was performed using Graphpad prism9.0.0 statistical software, and One-way ANOVA with Dunn's m μ Ltiple comparison test statistical analysis was performed.
Experimental results as shown in fig. 1, during the incubation of HSF cells with dexamethasone, the inventors periodically collected mRNA samples every 4 hours, and detected the expression levels of rhythmic genes ATNX L and CRY1 using QPCR. Analysis by fitting curves revealed that the mRNA transcripts of ATNX L and CRY1 exhibited a nearly sinusoidal periodic variation trend. From this, the inventors succeeded in preparing a node model based on HSF cells.
As can be seen from fig. 1, the inventors can select a time point of 8h as a daytime detection point, and 20h or 24h as a nighttime detection point.
Example 2 cell safety concentration screening of active ingredient
The CCK-8 kit (Cell Counting Kit, abbreviated as CCK-8) is a rapid, high-sensitivity and non-radioactive colorimetric detection kit which is widely applied to cell proliferation and cytotoxicity detection depending on WST-8 (2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazolium monosodium salt). WST-8 can be reduced into a highly water-soluble orange yellow formazan product (formazan) by some dehydrogenases in mitochondria under the action of an electron coupling carrier 1-method PMS, and the color depth of the generated formazan is in direct proportion to cell proliferation and in inverse proportion to cytotoxicity. Absorbance was measured at a wavelength of 450nm using an enzyme-labeled instrument, which indirectly reflects the number of living cells.
This example uses the CCK-8 method to detect the safe concentration of active peptide in Human Skin Fibroblasts (HSF).
The specific process is as follows:
2.1 experimental grouping:
4 kinds of active peptides and 1 kind of active ingredients are sequentially as follows: acetyl tetrapeptide-9, hexapeptide PRO, matrixyl TM 3000,3000, supermolecule elastin PRO, radix Scutellariae extract, each set 9 concentration gradients.
Wherein the radix Scutellariae extract is baicalin-cyclodextrin-vesicle complex, and the preparation method comprises the following steps:
10 g of cyclodextrin is weighed and put into a colloid mill for grinding, 30 g of baicalin is weighed and put into the colloid mill, a large amount of deionized water is added after full mixing and grinding, the uniformly mixed cyclodextrin baicalin powder is fully dissolved, the solution is homogenized by a high-pressure micro-jet homogenizer at 30MPa homogenizing pressure, the solution is collected after being homogenized for several times, and the solution is put into a freeze dryer for removing the solvent at the temperature of minus 80 ℃ to obtain the baicalin cyclodextrin inclusion nanoparticle.
The lyophilized Bacillus subtilis powder was dissolved with nutrient broth liquid (NB) medium. Transferring the dissolved bacterial liquid into a sterile test tube with a certain amount of liquid culture medium, and uniformly mixing and culturing. Inoculating the activated strain into an conical flask filled with NB medium, and shake culturing under appropriate conditions. The bacillus thallus is transferred into NB liquid culture medium containing a certain amount of yeast powder for culture. After the culture is finished, collecting fermentation liquor, centrifuging to obtain bacterial precipitate, adding phosphate buffer solution into the precipitate according to a certain volume ratio, re-suspending, and homogenizing and crushing the buffer solution containing bacterial with a high-pressure micro-jet homogenizer at 30MPa homogenizing pressure. The solution containing the thallus fragments is passed through a solid chromatographic column, and vesicle fractions are collected by separation and purification.
Weighing a proper amount of 80 g vesicle fraction, diluting with phosphoric acid buffer solution, weighing 20g baicalin cyclodextrin inclusion nanoparticles, uniformly mixing the two, putting the mixture into an ultrasonic instrument for ultrasonic treatment, putting the liquid into a high-pressure extruder, taking nitrogen as a pressure source, extruding through a polycarbonate film, centrifuging, collecting bottom sediment, drying, and grinding to obtain baicalin microcapsules, which are also called as baicalin extract in the invention.
2.2 Experimental procedure:
1) HSF cell suspensions were seeded and incubated in 96-well plates (4000/100. Mu.L/well) for 24h while blank and control groups were set.
2) The whole medium was changed and each well was added with different concentrations of active peptide, 6 replicates per group and incubation in incubator was continued for 24h.
3) Mu.L of CCK-8 was added to each well and incubated in an incubator for 2h.
4) The absorbance at 450nm was measured by a microplate reader.
5) The calculation formula is as follows:
Percent cell viability = [ (absorbance of experimental group-blank)/(absorbance of control group-blank) ] ×100%
6) Statistical analysis: experimental data are expressed as Mean standard deviation (mean±sd) and data processing experimental results using Graphpad prism9.0.0 statistical software are shown in table 6:
TABLE 6
The above cell viability results indicate that: acetyl tetrapeptide-9 and hexapeptide PRO are below 20mg/mL, matrixyl TM is below 30mg/mL, supermolecule elastin PRO is below 10mg/mL, and radix Scutellariae extract is below 1mg/mL, which have good biological safety.
Example 3 efficacy study of day cream-collagen bundles TM and night cream-collagen TM on collagen fiber Assembly and collagen Synthesis secretion-related genes
This example mainly explores the function of day cream-collagen bundles TM to promote collagen fiber assembly, and the efficacy of night cream-collagen TM to promote collagen synthesis and secretion. The expression of the following genes was detected by a real-time fluorescent quantitative PCR method (QPCR method):
a) Fiber assembly-related genes: LOX, CTSK, MMP1;
b) Collagen synthesis-related genes: mia3, pde4d, vps33b
C) Reference gene: GAPDH
D) Day cream-collagen bundles TM comprise the following components in mass ratio:
Acetyl tetrapeptide-9: hexapeptide PRO: radix Scutellariae extract = 20:20:1
The night cream-collagen TM comprises the following components in percentage by mass:
matrixyl TM 3000,3000: supermolecule elastin PRO: radix Scutellariae extract=30:10:1.
3.1 Experimental grouping:
The groups were divided into 12 major groups, each containing 4 time points, for a total of 48 minor groups, as shown in table 7 below.
1) Daytime control group (0, 4, 8, 12 h);
2) Daytime + acetyl tetrapeptide-9 group (0, 4, 8, 12 h);
3) Daytime + hexapeptide PRO group (0, 4, 8, 12 h);
4) Daytime + baical skullcap root extract group (0, 4, 8, 12 h);
5) Day + day cream collagen bundle TM group (0, 4, 8, 12 h);
6) Day + night frost collagen TM group (0, 4, 8, 12 h);
7) Night control (12, 16, 20, 24 h);
8) Night + Matrixyl TM group 3000 (12, 16, 20, 24 h);
9) The black night + supramolecular elastin PRO group (12, 16, 20, 24 h);
10 Black night + scutellaria root extract group (12, 16, 20, 24 h);
11 Night + day cream-collagen bundles TM group (12, 16, 20, 24 h).
12 Night + night-collagen TM group (12, 16, 20, 24 h).
TABLE 7
Note that: daytime and night time refer to the daytime checkpoint (8 h) and the night time checkpoint (20 or 24 h), respectively, of example 1
3.2 Experimental procedure:
1) HSF cell suspensions were seeded in 6-well plates.
2) After the cells had grown to adhere, the medium in the 6-well plate was removed, 1mL of complete medium containing 100nM dexamethasone was added, and the cells were incubated in a 37℃cell incubator containing 5% CO 2 for 30min, and the complete medium was replaced for further culture for 24h.
3) And respectively incubating the active ingredients in the daytime and the night according to the time points, collecting samples of each group, and collecting samples in the daytime for 0h, 4h, 8h and 12h and collecting samples in the night for 12h, 16h, 20h and 24h.
4) Extraction of 48 sample RNAs: 200 mu L of chloroform is added into a sample EP tube, and the mixture is mixed for 15 to 30 seconds by intense vortex vibration; centrifuging at 12000rpm at 4deg.C for 15min, separating the sample into 3 layers, and collecting 400 μl supernatant; adding 500 μl of isopropanol into each tube, mixing for 6-8 times upside down, centrifuging at 4deg.C and 10000rpm for 10min, and discarding supernatant; the precipitate was washed 2 times with 75% glacial ethanol: centrifuging at 4deg.C and 10000rpm for 10min, discarding supernatant, and repeating the centrifugation once again to remove residual liquid in the tube; finally, dissolving RNA precipitate by using 20 mu LDEPC water; taking 1 mu L of RNA concentration, and storing and using the rest in a refrigerator at the temperature of minus 80 ℃; using DEPC water as blank control; a260/280 is 1.8-2.0. The subsequent steps are as described in example 1.
To examine whether day cream-collagen bundles TM and night cream-collagen TM can promote collagen synthesis and assembly, the inventors treated HSF cell models with day cream-collagen bundles TM and night cream-collagen TM, respectively. The results are shown in FIGS. 2-3 and Table 8, table 8
Wherein, the mRNA expression level of each gene is expressed as relative transcription level (%) with respect to the Control group.
The results in table 8 show that:
(1) During the daytime (or daytime), the use of 20mg/ml of acetyl tetrapeptide-9 alone or 20mg/ml of acetohexapeptide PRO alone reduced the transcript level of LOX mRNA by 17% and 16%, respectively, and the use of 1mg/ml of Scutellaria baicalensis root extract alone increased the transcript level of LOX mRNA by 34.66%, respectively, compared to the control group.
Whereas day cream-collagen bundles TM combining the three were able to increase LOX mRNA transcription levels by 69.65% during the day, it is evident (-17%) + (-16%) +34.66% = 1.66% <69.65%, indicating that day cream-collagen bundles TM have unexpected synergistic effects on promoting collagen fiber assembly during the day; whereas night cream-collagen TM increased LOX mRNA transcription by 19.42% during the day (fig. 2 a).
Day cream-collagen bundles TM reduced transcription of CTSK and MMP1mRNA by 46.74% and 39.11%, respectively, during the day (fig. 2b and 2 c); whereas night cream-collagen TM reduced transcription of CTSK and MMP1mRNA by 12.1% and 17.76%, respectively, during the day (fig. 2b and 2 c).
It follows that day cream-collagen bundles TM are capable of promoting collagen fiber assembly by promoting transcription of collagen fiber assembly key genes and inhibiting transcription of collagen fiber degradation genes during the daytime to maintain collagen fibers in an assembled state at a high level, thereby promoting elasticity and toughness of skin. In contrast, night cream-collagen TM is significantly weaker than day cream-collagen bundles TM, and thus is more suitable for day use with day cream-collagen bundles TM.
(2) At night (or at night), compared with the control group, the transcription level of Vps33mRNA can be reduced by 20.1%, 5.47 or 12.1% respectively after 30mg/ml Matrixyl TM 3000,10 mg/ml supersplit bullet wetting peptide PRO or 1mg/ml radix Scutellariae extract is used alone; the night cream-collagen TM combining the three can increase the transcription of Mia3mRNA by 66.53% at night; clearly (-20.1%) + (-5.47) +(-12.1%) = (-37.67) <66.53%, indicating that night cream-collagen TM has an unexpected synergistic effect on promoting collagen synthesis at night.
Similarly, night cream-collagen TM increased mRNA transcription by 100.01%, 126.67% and 66.53% at night for Pde4d (fig. 3 a), mia3 (fig. 3 b) and Vps33b (fig. 3 c), respectively;
Day cream-collagen bundles TM increased Mia3mRNA transcription level by 21.23% at night, with no significant effect on Vps33b and Pde4d mRNA transcription levels.
Therefore, the effect of the night cream-collagen TM on promoting the synthesis and secretion of the collagen is obviously higher than that of day cream-collagen bundle TM, and the night use of the night cream-collagen TM can rapidly promote the synthesis and secretion of the skin collagen, so that the night use of the night cream-collagen TM is more suitable.
Discussion of the invention
The synthesis and assembly of collagen in the dermis of the skin has an objective law of circadian rhythm, namely, the assembly of collagen fibers in the daytime and the synthesis of procollagen at night. Therefore, the design of the skin care product should follow the rule, the product used in the daytime should promote the assembly of collagen fibers, and the product used at night should assist in accelerating the synthesis of procollagen.
The combination of actives screened on the circadian model for the appropriate daytime (application in day cream) significantly promotes collagen fiber assembly during the "day" period of the model, compared to use alone, but day cream compositions do not have similar effects during night use, and night cream compositions do not have similar effects during daytime and night use. That is, not any active substance stack can accord with the objective circadian rhythm of collagen in skin, and has the effect of promoting the assembly of collagen fibers in daytime and anti-aging effect.
Commercially available circadian rhythm skin care products generally have the function of regulating circadian rhythm or directly promoting collagen synthesis through superposition of active ingredients. However, it has been found by the present invention that some ingredients are used in an unsuitable period of time and do not function as ingredients themselves and do not have skin care effects. The invention has the advantages that the effective component proportion combination corresponding to the time period is found through the screening result of the active components in the circadian rhythm model, and the skin is protected in the accurate time period (different components are applied to 2 products of early night cream), so that the active components in the skin care product play a better role in resisting aging.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Claims (10)
1. A first active ingredient combination for use in preparing an anti-aging skin care composition or formulation, comprising or consisting of the following active ingredients:
1-5 parts by weight of Matrixyl TM;
1-5 parts by weight of supermolecule bullet wetting peptide PRO; and
0.05 To 1 weight portion of baical skullcap root extract.
2. The first active ingredient combination of claim 1, wherein the first active ingredient combination comprises or consists of the following active ingredients:
Matrixyl TM 3000,3000 parts by weight;
Supersplit bullet wetting peptide PRO 1 weight part; and
0.1 Part by weight of baical skullcap root extract.
3. The first active ingredient combination of claim 1, wherein the scutellaria root extract is a baicalin-cyclodextrin-vesicle complex having the following components in parts by weight:
12-18 parts of baicalin
70-90 Parts of vesicle material
3-7 Parts of cyclodextrin.
4. An anti-aging skin care composition comprising the first active ingredient combination of claim 1 and a cosmetically or pharmaceutically acceptable carrier.
5. A method of preparing the anti-aging skin care composition of claim 4, comprising:
(a) Mixing 1-5 parts by weight of Matrixyl TM 3000,3000, 1-5 parts by weight of supersplit bullet wetting peptide PRO and 0.05-1 part by weight of radix scutellariae extract to obtain a first active ingredient combination;
(b) Mixing the first active ingredient combination obtained in step (a) with a cosmetically, chemically or biologically acceptable carrier, thereby obtaining said anti-aging skin care composition.
6. Use of the first active ingredient combination of claim 1 or the anti-aging skin care composition of claim 4 in the preparation of an anti-aging product.
7. The use according to claim 6, wherein the anti-ageing product is selected from the group consisting of:
an anti-aging essence, anti-aging water, anti-aging facial cream, anti-aging facial cleanser and anti-aging facial mask.
8. The use according to claim 6, wherein said anti-aging product is for: promoting collagen fiber assembly, promoting procollagen synthesis, increasing type I collagen content, increasing skin elasticity, scavenging free radicals, resisting oxidation, increasing skin moisture content, eliminating inflammation, or combinations thereof.
9. A medicine box is characterized by comprising a first container and a second container,
The first container contains the first active ingredient combination of claim 1, and a cosmetically, chemically, or biologically acceptable carrier;
The second container contains a second anti-aging skin care composition comprising a second active ingredient combination comprising or consisting of the following active ingredients, and a cosmetically, chemically, or biologically acceptable carrier:
Acetyl tetrapeptide-9 1-5 weight portions;
1-5 parts by weight of hexapeptide PRO; and
0.05 To 1 weight portion of baical skullcap root extract.
10. The kit of claim 9, further comprising instructions for use of the kit as follows: applying the composition in the first container at night; the composition in the second container was applied during the day.
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