CN117084951A

CN117084951A - Compact anti-wrinkle composition and preparation method thereof

Info

Publication number: CN117084951A
Application number: CN202310644683.3A
Authority: CN
Inventors: 孙涛; 王秋玥; 韩广权
Original assignee: Dalian Fullerene Pharmaceutical Co ltd
Current assignee: Dalian Fullerene Pharmaceutical Co ltd
Priority date: 2023-06-02
Filing date: 2023-06-02
Publication date: 2023-11-21

Abstract

The invention belongs to the technical field of cosmetics, and particularly relates to a compact anti-wrinkle composition and a preparation method thereof. The invention provides a compact anti-wrinkle composition mainly comprising: butanediol, cyclopentadimethicone, cyclohexasiloxane, isononyl isononanoate, soy isoflavone, PEG-10 dimethicone, acetyl hexapeptide-8, oligopeptide-1, palmitoyl tripeptide-5, palmitoyl tetrapeptide-7, caprylyl glycol, ethylhexyl glycerol, mannitol, squalane, caprylic/capric triglyceride, resveratrol, tocopherol, citrus peel oil, sweet almond oil, hydrogenated vegetable oil in combination with centella asiatica, licorice root, star anise fruit, ampelopsis grossedentata, cactus, licorice root, radix scutellariae, kuh-seng root, angelica root, wild chrysanthemum, lily flower, witch hazel bark/twig, calendula, extracts of long soft hair yam root, and 1, 2-hexanediol. The composition provided by the invention has remarkable anti-wrinkle and tightening effects.

Description

Compact anti-wrinkle composition and preparation method thereof

Technical Field

The invention belongs to the technical field of cosmetics, and particularly relates to a compact anti-wrinkle composition and a preparation method thereof.

Background

It is known that skin aging is classified into natural aging and aging due to environmental factors, and generally over 25 years of age, and especially female skin is gradually slowed down in metabolism, and the stratum corneum gradually becomes thicker. In addition, besides the skin condition can be influenced by age, with the acceleration of life rhythm, sleep deficiency and diet unhealthy caused in vivo hormonal changes due to work and life pressure, environmental pollution, ultraviolet radiation and improper skin care mode can all influence skin health, so that a large number of free radicals are generated, and the active molecular radicals can trigger a series of free radical reactions to cause cell damage and even death, thereby accelerating skin aging. Research shows that the skin care product can effectively delay the skin aging to a certain extent after long-term use.

At present, the skin care products with the functions of resisting aging and tightening are declared to be various in types and good in quality in the market, and the effects of resisting aging and tightening after most products are used are not obvious.

Disclosure of Invention

The invention aims to provide a compact anti-wrinkle composition and a preparation method thereof, and the compact anti-wrinkle composition provided by the invention can obviously remove Reactive Oxygen Species (ROS) and has an anti-oxidation effect; meanwhile, the collagen type I regeneration promoting agent has the effect of promoting the regeneration of the collagen type I, thereby having remarkable anti-wrinkle and tightening effects.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a compact anti-wrinkle composition, which comprises the following components in percentage by mass:

4.8 to 11.5 percent of butanediol, 3 to 7.3 percent of cyclopentadimethicone, 1.6 to 4 percent of cyclohexasiloxane, 3.6 to 8.4 percent of isononyl isononanoate, 3 to 8 percent of glycerol, 41 to 2.5 percent of polyethylene glycol, 0.14 to 0.35 percent of soybean isoflavone, 0.9 to 2.1 percent of PEG-10 dimethicone, 0.002 to 0.002 percent of acetyl hexapeptide-80.001, 42 to 0.0000005 percent of oligopeptide-10.0000001, 50.0001 to 0.0002 percent of palmitoyl tripeptide, 0.004 to 0.002 percent of palmitoyl tetrapeptide-70.00001 to 0.00005 percent, 0.001 to 0.005 percent of emulsifying agent, 0.004 to 0.005 percent of octal, 0.005 to 0.005 percent of octal, 0.04 to 0.005 percent of orange peel extract, 0.002 to 0.04 percent of orange peel extract, 0.001 to 0.002 percent of orange peel extract, 0.004 to 0.002 percent of orange peel extract, 0.002 to 0.04 percent of orange peel extract, 0.04 to 0.04 percent of orange peel extract.

Preferably, the preservative comprises phenoxyethanol, methylparaben, sodium benzoate, potassium sorbate, p-hydroxyacetophenone, and propylparaben.

Preferably, the preservative comprises, in mass percent of the tightening and anti-wrinkle composition of 100: 0.0004 to 0.00045 percent of phenoxyethanol, 0.12 to 0.2 percent of methylparaben, 0.0005 to 0.0008 percent of sodium benzoate, 0.0003 to 0.0004 percent of potassium sorbate, 0.0005 to 0.001 percent of p-hydroxyacetophenone and 0.05 to 0.1 percent of propylparaben.

Preferably, the filler is magnesium sulfate.

Preferably, the emulsion stabilizer is disteardimonium hectorite.

Preferably, the emulsifier is polysorbate-20.

Preferably, the skin penetrating agent is a pepper extract.

The invention provides a preparation method of the compact anti-wrinkle composition, which comprises the following steps:

heating cyclopentadimethicone, cyclohexasiloxane, isononyl isononanoate, part of glycerol, part of soy isoflavone, PEG-10 dimethicone, squalane, emulsion stabilizer, caprylic/capric triglyceride and part of preservative to a first temperature for first mixing to obtain a first mixture;

Heating water, butanediol, residual glycerol, polyethylene glycol-4, residual soybean isoflavone, filler, tocopherol and other part of preservative to a first temperature for second mixing to obtain a second mixture;

homogenizing the first mixture and the second mixture under a first temperature condition to obtain a homogenized material; the first temperature is more than or equal to 80 ℃;

cooling the homogenized material to a second temperature, and performing a third mixing with acetyl hexapeptide-8, oligopeptide-1, palmitoyl tripeptide-5, palmitoyl tetrapeptide-7, an emulsifier, caprylyl glycol, ethylhexyl glycerol, a residual preservative, mannitol, resveratrol, centella asiatica extract, licorice root extract, star anise fruit extract, ampelopsis grossedentata extract, opuntia ficus-indica extract, licorice root extract, scutellaria root extract, sophorae radix extract, angelica root extract, wild chrysanthemum extract, lily extract, witch hazel bark/tender branch extract, calendula extract, 1, 2-hexanediol, dioscorea longa root extract, essence and skin penetrating agent to obtain a third mixture; the second temperature is more than or equal to 45 ℃;

and cooling the third mixture to obtain the compact anti-wrinkle composition.

Preferably, the homogenizing rotating speed is 6000-8000 r/min, and the homogenizing heat preservation time is 15-20 min.

Preferably, the first temperature is 80-85 ℃, and the second temperature is 45-50 ℃.

The application provides a compact anti-wrinkle composition, which comprises the following components in percentage by mass: 4.8 to 11.5 percent of butanediol, 3 to 7.3 percent of cyclopentadimethicone, 1.6 to 4 percent of cyclohexasiloxane, 3.6 to 8.4 percent of isononyl isononanoate, 3 to 8 percent of glycerol, 41 to 2.5 percent of polyethylene glycol, 0.14 to 0.35 percent of soybean isoflavone, 0.9 to 2.1 percent of PEG-10 dimethicone, 0.002 to 0.002 percent of acetyl hexapeptide-80.001, 42 to 0.0000005 percent of oligopeptide-10.0000001, 50.0001 to 0.0002 percent of palmitoyl tripeptide, 0.004 to 0.002 percent of palmitoyl tetrapeptide-70.00001 to 0.00005 percent, 0.001 to 0.005 percent of emulsifying agent, 0.004 to 0.005 percent of octal, 0.005 to 0.005 percent of octal, 0.04 to 0.005 percent of orange peel extract, 0.002 to 0.04 percent of orange peel extract, 0.001 to 0.002 percent of orange peel extract, 0.004 to 0.002 percent of orange peel extract, 0.002 to 0.04 percent of orange peel extract, 0.04 to 0.04 percent of orange peel extract. The tightening and anti-wrinkle composition provided by the application realizes remarkable anti-wrinkle and tightening effects through the combination and proper mass ratio. Test results of examples: the zebra fish type I collagen gene expression promotion test experiment shows that: the composition sample provided by the application can obviously promote the expression of the zebra fish col1a1a gene and has the effect of promoting the regeneration of the type I collagen; the test experiment of reducing the movement distance of the zebra fish embryo shows that: the composition sample provided by the application can obviously shorten the movement distance of zebra fish embryos; the test of scavenging of active oxygen (ROS) from zebra fish embryo shows that: the composition sample provided by the application can obviously remove ROS in zebra fish embryos and has an antioxidation effect, so that the composition provided by the application has obvious anti-wrinkle and tightening effects.

Drawings

FIG. 1 is a histogram of the relative expression level of zebra fish type I collagen gene of test example 1 (p < 0.05);

FIG. 2 is a representative graph of test results of the reduction of the movement distance of the zebra fish embryo in test example 2;

FIG. 3 is a bar graph of test example 2 showing the reduction of the embryo movement distance of zebra fish (p < 0.05);

FIG. 4 is a representative graph of test results of test example 3, zebra fish embryo Reactive Oxygen Species (ROS) clearance;

FIG. 5 is a bar graph of test results of test example 3 zebra fish embryo Reactive Oxygen Species (ROS) clearance (< 0.05);

fig. 6 is a plot of test example 3 zebra fish embryo Reactive Oxygen Species (ROS) measurement area, ROS labeled green fluorescence.

Detailed Description

In the present invention, all preparation materials/components are commercially available products well known to those skilled in the art unless specified otherwise.

In the present invention, the preservative preferably includes phenoxyethanol, methylparaben, sodium benzoate, potassium sorbate, p-hydroxyacetophenone, and propylparaben. The invention preferably adopts phenoxyethanol, methylparaben, sodium benzoate, potassium sorbate, p-hydroxyacetophenone and propylparaben as preservative, and can realize the preservative effect, and can also be compatible with other components in the composition, thereby improving the anti-wrinkle and skin tightening effects of the composition.

In the present invention, the preservative preferably comprises, at 100% by mass of the tightening and anti-wrinkle composition: 0.0004 to 0.00045 percent of phenoxyethanol, 0.12 to 0.2 percent of methylparaben, 0.0005 to 0.0008 percent of sodium benzoate, 0.0003 to 0.0004 percent of potassium sorbate, 0.0005 to 0.001 percent of p-hydroxyacetophenone and 0.05 to 0.1 percent of propylparaben; further preferably, it comprises phenoxyethanol 0.00041 to 0.00045%, methylparaben 0.15 to 0.2%, sodium benzoate 0.0006 to 0.0008%, potassium sorbate 0.00035 to 0.0004%, p-hydroxyacetophenone 0.0006 to 0.001%, propylparaben 0.06 to 0.1%, most preferably, phenoxyethanol 0.00045%, methylparaben 0.2%, sodium benzoate 0.0008%, potassium sorbate 0.0004%, p-hydroxyacetophenone 0.001%, propylparaben 0.1%.

In the present invention, the filler is particularly preferably magnesium sulfate. In the present invention, the magnesium sulfate functions as an anti-inflammatory agent and a viscosity controlling agent in addition to the filler.

In the present invention, the emulsion stabilizer is particularly preferably disteardimonium hectorite. In the present invention, the disteardimonium hectorite acts as a suspending agent in addition to an emulsion stabilizer.

In the present invention, the emulsifier is particularly preferably polysorbate-20. In the present invention, the polysorbate-20 functions as a dispersant, a solubilizer, and a stabilizer in addition to the emulsifier.

In the present invention, the skin penetrating agent is particularly preferably a pepper extract.

In the present invention, the tightening and anti-wrinkle composition preferably comprises the following components in percentage by mass: 5 to 10 percent of butanediol, 3.5 to 6.5 percent of cyclopentadimethicone, 2 to 3.5 percent of cyclohexasiloxane, 4 to 8 percent of isononyl isononanoate, 3.5 to 7.5 percent of glycerin, 0.5 to 2 percent of polyethylene glycol-41.5 to 0.32 percent of soybean isoflavone, 1 to 2 percent of PEG-10 dimethicone, 80.0011 percent of acetyl hexapeptide, oligopeptide-10.0000002 to 0.0000005 percent, palmitoyl tripeptide-50.00012 percent, 70.00002 to 0.00004 percent of palmitoyl tetrapeptide-350.005 percent, 0.002 to 0.005 percent of emulsifying agent, 0.0042 to 0.0045 percent of octaethylene glycol, 0.0006 to 0.0009 percent of ethylhexyl glycerol, 0.172 to 0.305 percent of preservative, 0.0002 to 0.0005 percent of mannitol, 0.5 to 1 percent of filling agent, 0.4 to 0.6 percent of squalane, 0.3 to 0.4 percent of emulsion stabilizer, 0.2 to 0.4 percent of caprylic acid/capric triglyceride, 0.02 to 0.03 percent of adsorbing agent, 0.0021 to 0.008 percent of resveratrol, 0.00228 percent of tocopheryl alcohol, 0.042-0.048% of sweet almond oil, 0.042-0.045% of hydrogenated vegetable oil, 0.0006-0.001% of centella asiatica extract, 0.0006-0.001% of licorice root extract, 0.0012-0.0017% of star anise fruit extract, 0.0012-0.0025% of ampelopsis grossedentata extract, 0.0015-0.0025% of Cactus extract, 0.002-0.0035% of licorice root extract, 0.0032-0.0045% of radix scutellariae root extract, 0.0025-0.0035% of radix sophorae flavescentis root extract, 0.0045-0.0055% of angelica root extract, 0.0045-0.0055% of wild chrysanthemum flower extract, 0.0045-0.0055% of white lily flower extract, 0.0045-0.5% of golden camellia bark/tender branch extract, 0.0045-0.5% of calendula extract, 0.0012-0.0012% of 1, 2-hexanediol, 0.0016% of radix polygoni multiflori extract and 0.03-0.02% of hair penetrating agent; further preferably comprises the following components in percentage by mass: 7 to 9 percent of butanediol, 4 to 5.5 percent of cyclopentadimethicone, 2.5 to 3 percent of cyclohexasiloxane, 5 to 6 percent of isononyl isononanoate, 4 to 7 percent of glycerin, 0.6 to 1.8 percent of polyethylene glycol, 0.2 to 0.3 percent of soybean isoflavone, 1.2 to 1.6 percent of PEG-10 dimethicone, 0.045 percent of acetyl hexapeptide-80.0011 percent, oligopeptide-10.0000005 percent, palmitoyl tripeptide-50.00012 percent, palmitoyl tetrapeptide-70.00004 percent, 0.005 percent of emulsifying agent, 0.00425 percent of octaethylene glycol, 0.0008 percent of ethylhexyl glycerin, 0.3022 percent of preservative 0.3022 percent of mannitol, 0.6 to 0.9 percent of bulking agent, 0.45 to 0.55 percent of squalane, 0.35 percent of emulsion stabilizer, 0.2 percent of caprylic/capric triglyceride, 0.03 percent of adsorbent, 0.0024 percent of resveratrol, 0.00025 percent of tocopherol, 0.01 percent of orange peel oil, 0.045 percent of sweet almond oil, 0.001 percent of hydrogenated vegetable oil 0.04475 percent of centella asiatica extract, 0.001 percent of licorice extract, 0.003 percent of licorice extract, 0.005 percent of extract of white paeony root extract, 0.005 percent of herba polygoni multiflori extract, 0.002 percent of herba polygoni multiflori extract, 0.005 percent of herba polygoni multiflori extract, 0.002 percent of herba polygoni multiflori extract, and the balance of herba polygoni multiflori extract, 0.0.0.002 percent of extract, 0.005 percent of herba polygoni multiflori extract, 0.0.0.002 percent of extract, and the rest, 0.0.0.0.002 percent of extract of herba polygoni multifloric 1 percent of extract, and 1 percent of herba Officinalis prepared.

cooling the homogenized material to a second temperature, and performing a third mixing with acetyl hexapeptide-8, oligopeptide-1, palmitoyl tripeptide-5, palmitoyl tetrapeptide-7, polysorbate-20, caprylyl glycol, ethylhexyl glycerol, remaining preservative, mannitol, resveratrol, centella asiatica extract, licorice root extract, star anise fruit extract, ampelopsis grossedentata extract, opuntia ficus-indica extract, licorice root extract, scutellaria root extract, kuh-seng root extract, angelica root extract, wild chrysanthemum extract, lily extract, witch hazel bark/tender branch extract, calendula extract, 1, 2-hexanediol, dioscorea longifolia root extract, essence and skin penetrating agent to obtain a third mixture; the second temperature is more than or equal to 45 ℃;

And cooling the third mixture to obtain the compact anti-wrinkle composition.

According to the invention, cyclopentadimethicone, cyclohexasiloxane, isononyl isononanoate, part of glycerol, part of soybean isoflavone, PEG-10 dimethicone, squalane, emulsion stabilizer, caprylic/capric triglyceride and part of preservative are heated to a first temperature for first mixing, so as to obtain a first mixture. In the present invention, the partial preservative is preferably methylparaben. The first temperature is preferably 80 to 85 ℃, more preferably 80 to 84 ℃. The first mixing is carried out under stirring, preferably in an oil phase pan.

The invention heats water, butanediol, residual glycerol, polyethylene glycol-4, residual soybean isoflavone, filling agent, tocopherol and other part of preservative to a first temperature for second mixing to obtain a second mixture. In the present invention, the other part of the preservative is preferably propyl paraben. The second mixing is carried out under stirring, preferably in an emulsifying pot.

After the first mixture and the second mixture are obtained, the first mixture and the second mixture are homogenized under the first temperature condition to obtain a homogenized material. In the invention, the uniform rotating speed is preferably 6000-8000 r/min, more preferably 6000r/min; the incubation time for the homogenization is preferably 15 to 20min, more preferably 15min.

After obtaining the homogenized material, the present invention cools the homogenized material to a second temperature, and performs a third mixing with acetyl hexapeptide-8, oligopeptide-1, palmitoyl tripeptide-5, palmitoyl tetrapeptide-7, polysorbate-20, caprylyl glycol, ethylhexyl glycerol, remaining preservative, mannitol, resveratrol, centella asiatica extract, licorice root extract, star anise fruit extract, ampelopsis grossedentata extract, opuntia ficus-indica extract, licorice root extract, baical skullcap root extract, kuh-seng root extract, angelica root extract, wild chrysanthemum extract, lily bulb extract, witch hazel bark/twig extract, calendula extract, 1, 2-hexanediol, long soft-hair yam root extract, essence and skin penetrating agent, to obtain a third mixture. In the present invention, the second temperature is 45℃or higher, preferably 45 ℃. The third mixing is preferably carried out under stirring.

After the third mixture is obtained, the third mixture is cooled down, and the compact anti-wrinkle composition is obtained. In the present invention, the temperature of the resulting compact anti-wrinkle composition after cooling is preferably 25 to 30 ℃. The cooling is carried out under the condition of stirring.

The technical solutions provided by the present invention are described in detail below in conjunction with examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.

Example 1

This example uses the materials and the mass percentages of the materials in table 1 to prepare a compact anti-wrinkle composition.

TABLE 1 raw materials used in example 1 and mass percent of each raw material

Note that: the (daily) essence used in the embodiment is AFLC06480 Di sweet heart essence, which is purchased from Guangzhou Aifu essence technology Co.

The preparation method comprises the following steps:

(1) Adding the raw materials of No. 3, no. 4, no. 5, no. 6, no. 8, no. 9, no. 20-23 and No. 29-31 into an oil phase pot, starting stirring, heating to 80-84 ℃, and stirring and mixing completely until a uniform material body is obtained, so as to obtain phase A for constant temperature standby;

(2) Adding the raw materials of No. 1, no. 2, no. 6, no. 7, no. 8, no. 19, no. 28 and No. 49 into an emulsifying pot, starting stirring, heating to 80-84 ℃, stirring and mixing completely to a uniform material body, and preserving the temperature for 30 minutes (the process is continuously stirred) to obtain a phase B for standby;

(3) Adding phase A into phase B at 80-84 ℃, homogenizing at high speed (6000 rpm) for 15 minutes to obtain a homogeneous material;

(4) Starting to cool (continuously stirring in the process), cooling the homogenized material to 45 ℃, sequentially adding the No. 10-18, no. 24-27, no. 32-48 and No. 50 raw materials into the homogenized material, and stirring to obtain a uniform material body;

(5) And (5) starting cooling (stirring continuously in the process), cooling to 30 ℃, stopping stirring, and discharging to obtain the compact anti-wrinkle composition.

Example 2

This example uses the materials and the mass percentages of the materials in table 2 to prepare a compact anti-wrinkle composition.

TABLE 2 raw materials used in example 2 and mass percentages of the raw materials

The preparation method is the same as in example 1.

Example 3

This example uses the materials and the mass percentages of the materials in table 3 to prepare a compact anti-wrinkle composition.

TABLE 3 raw materials used in EXAMPLE 3 and mass percent of each raw material

The preparation method is the same as in example 1.

The product prepared in example 1 of the present invention was subjected to performance test by silver (International) biosciences in water (address: biosciences center 2, 516, 11, science and technology, university of hong Kong academy of sciences). The test results are shown in test examples 1 to 3. The products of examples 2 to 3 were similar to the product performance test results of example 1.

Test example 1

The composition prepared in example 1 was tested. Collagen is the highest content of extracellular matrix proteins in humans, with type I collagen being the most abundant protein in the skin. Zebra fish have the same type I collagen distribution as human and show a high degree of conservation with humans, their innate expression drops significantly at 6 days post fertilization and becomes very low between 10 and 12 days post fertilization. The zebra fish type I collagen genes (col 1a1a, col1a1b and col1a 2) are tested for expression, the relative expression amount changes of the zebra fish type I collagen genes in the treatment group and the blank control group are compared, and the anti-wrinkle and tightening effects of the raw materials or products can be evaluated by calculating the type I collagen gene expression promotion rate.

1 apparatus and device. 1.1 zebra fish farming equipment: the equipment is provided with a temperature control device, a water circulation device and a filtering device, and the culture container is made of glass or common food-grade PC materials. A general zebra fish culture device can be adopted or a fish tank (such as 45cm in length and width and 45cm in length and 30 cm) can be arranged according to the requirements of a laboratory. 1.2 spawning box/jar: made of glass, stainless steel or other inert material, equipped with a mesh screen (mesh size 2mm x 2mm, made of stainless steel or materials to protect roe). 1.3 Water quality monitoring device: pH meter, dissolved oxygen meter, salinity meter (conductivity meter). 1.4 analytical balance: precision 0.1mg.1.5 microscope: with the camera system, the maximum magnification should be greater than 80 times of the whole microscope. 1.6 test vessel: test container: glass or polystyrene test vessels (e.g., 6-well plates, petri dishes). For example, where the test substance may be adsorbed to a polystyrene container (e.g., a nonpolar chemical), an inert material (e.g., glass) should be selected to reduce adsorption. 1.7 incubator: precision ± 1 ℃.1.8 brine shrimp incubator: the equipment is provided with a hatching barrel, an air pipe, an air flow regulating valve, an air pump stop switch and a check valve device. 1.9 other conventional test instruments and equipment: pipette, centrifuge tube, glass container (e.g., beaker, volumetric flask, etc.), vortex mixer, ultrasonic water bath, centrifuge, cryogenic refrigerator, adjustable pipettor, and pipette tip.

2 main reagents and consumables. 2.1 water, according to GB/T6682. 2.2 cosolvents such as methanol, dimethyl sulfoxide, ethanol, etc., analytical grade. 2.3 parent fish culture water: weighing 4g of sea salt, dissolving in 10L of water to prepare the sea salt, wherein the salinity is 0.25-0.50 per mill, the conductivity is 500-800 mu S/cm, the saturation of dissolved oxygen is 80%, the pH value is 6.5-8.5, and the hardness is 30-300 mg/L (calculated by calcium carbonate). 2.4 brine shrimp (Artemia salina) egg brine shrimp resting eggs need to be dried and refrigerated in the dark. Weighing about 15g sea salt, dissolving in 1L water to prepare brine for hatching brine shrimp eggs, wherein the density is preferably not more than 4g/L brine shrimp eggs. 2.5 fish embryo culture: 2940mg of anhydrous calcium chloride, 1233mg of magnesium sulfate heptahydrate, 630mg of sodium bicarbonate and 55mg of potassium chloride are weighed and dissolved in 10L of water to prepare the calcium chloride. The pH value is 6.5-8.5. Chemicals were all analytically pure grade. 2.6 Phosphate Buffered Saline (PBS) solution. 2.7RNAlater solution (Invitrogen; AM 7020), stored frozen. 2.8TRIzol reagent (Invitrogen; 15596018), stored cold. 2.9 chloroform. 2.10 isopropanol, stored frozen. 2.11 ethanol, 37.5mL ethanol was weighed and dissolved in 12.5mL ultrapure distilled water, and stored frozen. PrimerScript RT kit for DNA Eraser, 2.12g (Takara; cat No. RR047A). 2.13 real-time PCR kit: SYBR Premix Ex Taq (Takara; cat No. RR420A) type 2.14 collagen type I gene primers are shown in Table 4:

TABLE 4 collagen type I Gene primer

3 experimental methods. 3.1 test organisms: the oviposition test of wild type AB strain zebra fish (Danio rerio) with reliable sources (such as the China national zebra fish resource center) is applied. The parent fish should have a good reproductive capacity-the fish age is optimal for 6-12 months. And the lateral cross is used for passaging as much as possible to keep genetic diversity, and a new batch of parent fish needs to be replaced after the parent fish of the pure strain breeds for 5 generations. Parent fish should not have obvious visible infection and disease characteristics and have not undergone drug treatment for 2 months. Parent fish should be domesticated for more than 14 days after introduction into the laboratory prior to breeding and spawning tests. 3.2 cultivation requirements. 3.2.1 the water temperature of the cultivation is preferably controlled between 26 and 28.5 ℃, and the indoor temperature is recommended to be controlled between 20 and 25 ℃.3.2.2 the cultivation density is preferably controlled at 1-2 fish per liter of water and the illumination is fixed every day for 12-16 hours, and a good filtration system is required to be maintained. 3.2.3 feeds at least 2 times per day, including at least 1 brine shrimp (Artemia salina) larvae, at intervals of more than 3 hours, avoiding excessive feeds affecting water quality and cleanliness. 3.3 spawning requirements. 3.3.1 for example, fish eggs were collected with spawning cylinders and male and female fish were treated with 2: the ratio of 1 is that the egg is put into an egg jar 1-2 hours before the lamp is turned off in the day before egg laying. Since zebra fish occasionally do not spawn, it is recommended to prepare multiple spawning cylinders for use at the same time. 3.3.2 to avoid genetic bias, fish eggs collected in a minimum of 3 spawning cylinders were mixed and selected for use. If the fish eggs are collected by the culture fish tank, the egg collecting box is put into the culture fish tank for collecting the fish eggs before turning off the lamp on the day before spawning or before turning on the lamp on the day before spawning. 3.3.3 to avoid feeding of the roe by adult fish, the spawn collection box is covered with an inert mesh. 3.3.4 mating, spawning and fertilization are completed within about 30 minutes after the lights are turned on, at which time the egg collection box may be removed from the aquarium. 3.3.5 after the roe is taken out of the roe collecting box, it is recommended to clean the fish embryo with the culture solution of the fish embryo. 3.3.6 healthy zebra fish embryos are selected and cultured at a density of no more than 1 fish embryo in 200 μl fish embryo culture broth at 28+ -1deg.C for 6 days after fertilization.

4 preparation of test object. 4.1 cosmetic raw materials: 4.1.1 water-soluble raw materials: the test solution can be prepared by directly dissolving the test object with the fish embryo culture solution. 4.1.2 non-water soluble raw materials: optionally, a solvent such as methanol, dimethyl sulfoxide, ethanol, etc. is used. The test object and the organic solvent are mixed according to the following ratio of 1:1 (weight g: volume mL), and then adding the fish embryo culture solution to prepare the required concentration, shaking for 30s, and centrifuging at 6500 Xg for 10min. The supernatant was taken and tested. 4.2 cosmetic products: 4.2.1 Water-soluble product: the test solution can be prepared by directly dissolving the test object with the fish embryo culture solution. 4.2.2 non-water soluble products: optionally, a solvent such as methanol, dimethyl sulfoxide, ethanol, etc. is used. The test object and the organic solvent are mixed according to the following ratio of 1:1 (weight g: volume mL), and then adding the fish embryo culture solution to prepare the required concentration, shaking for 30s, and centrifuging at 6500 Xg for 10min. The supernatant was taken and tested. 4.3 notes: the concentration of the test solution of the test substance should ensure a mortality rate of 10% for the zebra fish embryos (embryos without heartbeat features are defined as dead). Multiple concentration groups can be set according to the requirement during testing.

5 testing steps. Healthy 6-day-old zebra fish after fertilization were selected. 5.1 test packets: the test requires a blank (fish embryo culture) and a test (test) group. If the organic solvent is used for dissolution, the concentration of the solvent in each group of solution needs to be the same. 5.1.1 blank settings: 36 zebra fish were randomly selected and transferred to 24-well plates, each well containing 12 zebra fish and 2.5mL of fish embryo culture broth. 5.1.2 treatment of test substance: 36 zebra fish were randomly selected and transferred to 24-well plates, each well containing 12 zebra fish and 2.5mL of test solution. Culturing in an incubator at 28+ -1deg.C for 24+ -1 h. The 12 zebra fish in each well were collected in 1.5mL tubes, the solution was removed, 0.5mL RNAlater solution was added, and frozen for storage. 5.2RNA extraction: the RNAlater solution was removed and washed 3 times with PBS solution. The solution was placed in an ice bath, the PBS solution was removed, and 500. Mu.L of TRIzol reagent was added. The embryos were homogenized with a particle pestle and placed in an ice bath for 10min. 100 μl of chloroform was added, vortexed for 1min, and placed in an ice bath for 5min. The sample was centrifuged for 20min, the supernatant was transferred to a 1.5mL centrifuge tube, 250. Mu.L of isopropanol was added, vortexed for 1min, placed in an ice bath for 10min, and the sample was centrifuged for 20min. All supernatant was removed, 500 μl ethanol was added, vortexed for 1min, the samples were centrifuged for 5min, and placed in an ice bath for 10min. This step was repeated 3 times. The ethanol was removed, the samples were centrifuged for 5min, and the samples were air dried in a hood with the lid opened for 10min. 10. Mu.L of DNase/RNase-free ultrapure distilled water was added and heated at 55℃for 15min.5.3cDNA Synthesis: RNA samples were diluted to 1000 ng/7. Mu.L with DNase/RNase free ultrapure distilled water. RNA was synthesized into cDNA using PrimerScript RT kit containing gDNAEras (Takara; catno. RR047A) and stored frozen. 5.4 real-time RT-PCR: a primer mix and a real-time PCR master mix were prepared for each primer. cDNA samples were diluted 10-fold with DNase/RNase free ultrapure distilled water. Each well of PCR was filled with 9. Mu.LPCR master mix and 1. Mu.L of diluted cDNA sample using 96 well plates. The PCR was performed by sealing the 96-well plate with an optical film, centrifuging the plate for 5min, and performing real-time PCR amplification. 5.5 data and results calculation: real-time PCR data was collected. Ct was used as the amplification result, and the relative expression amounts of each gene (col 1a1a, col1a1b and col1a 2) were calculated as the test results, using the beta-actin gene amplification amount as the housekeeping gene.

6 results calculation

Calculating the collagen gene expression promotion rate:

promotion rate= (T-C)/c×100% formula (1); in the formula (1): t-the relative expression level of the collagen gene of the zebra fish treated by the test substance; c, the relative expression quantity of zebra fish collagen genes in a blank control group; and carrying out double-tail T test on the relative expression quantity of the zebra fish collagen genes of the test object treatment group and the relative expression quantity of the zebra fish collagen genes of the blank control group to obtain a p value.

And 7, testing validity verification. 7.1 test determination: at least 90% of fish embryos survive each test group after exposure is complete, otherwise the corresponding test group results are not valid.

8 results. 8.1 promotion rate of collagen Gene expression the test substance promotes the expression of collagen genes at the corresponding test concentration. 8.2 evaluation of test substances for their ability to promote collagen Gene expression, test substances were analyzed for statistical differences (p < 0.05) in the detection values from the test substance-treated group and the blank group.

9 result correlation interpretation. In the case that the collagen gene expression promotion rate is positive and has a statistical difference (p < 0.05), the test result can be used as a test object to have supporting evidence for promoting collagen regeneration, and supporting anti-wrinkle and tightening effects are declared.

The test results of this test example are shown in Table 5 and FIG. 1. As can be seen from table 5 and fig. 1: the composition samples prepared in example 1 had an acceleration rate of 12% (p=0.019), -43% (p=0.00036), -12% (p=0.12) for the expression of the genes of the zebra fish col1a1a, col1a1b and col1a2, respectively, at a test concentration of 5 g/L. Namely, the composition sample prepared in the embodiment 1 of the invention can obviously promote the expression of the zebra fish col1a1a gene, has the effect of promoting the regeneration of the type I collagen, and has the effects of wrinkle resistance and compactness.

TABLE 5 test example 1 test results

Test example 2

The composition prepared in example 1 was tested. Zebra fish has the same neural medium and regulatory mechanism as human body, and thus has been widely used in research and test of neuroactive drugs. Some components in the cosmetics such as conopeptide, hexapeptide, snake venom peptide, etc. can effectively block nerve ending signal transmission of skin to achieve the effects of instantly relieving wrinkles and tightening. The motion distance of the zebra fish embryos is measured by using instrument records and software through zebra fish embryo behaviours, the motion distance changes of the zebra fish embryos of a test object treatment group and a blank control group are compared, and the motion distance reduction rate is calculated to evaluate the anti-wrinkle and tightening effects of raw materials or products.

1 the apparatus and equipment are essentially the same as in test example 1, except that: the motion recorder for the zebra fish is additionally arranged, so that the motion track of the zebra fish embryo can be recorded by infrared rays, and the track can be measured and analyzed. Such as DanioVision (Noldus Information Technology).

2 main reagents and consumables. 2.1 water, according to GB/T6682. 2.2 cosolvents such as methanol, dimethyl sulfoxide, ethanol, etc., analytical grade. 2.3 parent fish culture water: weighing 4g of sea salt, dissolving in 10L of water to prepare the sea salt, wherein the salinity is 0.25-0.50 per mill, the conductivity is 500-800 mu S/cm, the saturation of dissolved oxygen is 80%, the pH value is 6.5-8.5, and the hardness is 30-300 mg/L (calculated by calcium carbonate). 2.4 brine shrimp (Artemia salina) egg brine shrimp resting eggs need to be dried and refrigerated in the dark. Weighing about 15g sea salt, dissolving in 1L water to prepare brine for hatching brine shrimp eggs, wherein the density is preferably not more than 4g/L brine shrimp eggs. 2.5 fish embryo culture: 2940mg of anhydrous calcium chloride, 1233mg of magnesium sulfate heptahydrate, 630mg of sodium bicarbonate and 55mg of potassium chloride are weighed and dissolved in 10L of water to prepare the calcium chloride. The pH value is 6.5-8.5. Chemicals were all analytically pure grade. 6.6 tricaine solution: 0.13g of cocaine was weighed out and dissolved in 50mL of water to prepare a 10mM stock solution. And (5) refrigerating. The working solution is prepared by diluting the stock solution with fish embryo culture solution by 100 times and fresh before use.

The experimental method is the same as the base cloth of test example 1, except that: 3.3.6 healthy zebra fish embryos are selected and cultured at a density of no more than 1 fish embryo in 200 μl fish embryo culture broth at 28+ -1deg.C until 5 days after fertilization.

4 test pieces were prepared in the same manner as in test example 1.

5 testing steps. The following test procedure was 96-well plate test guidelines. Healthy zebra fish embryos 5 days after fertilization were selected. 5.1 test packets: the test requires setting a blank control group (fish embryo culture liquid), a positive control group (tricaine working liquid) and a test object group (test object). If the organic solvent is used for dissolution, the concentration of the solvent in each group of solution needs to be the same. 5.1.1 blank settings: 16 fish embryos are randomly selected and transferred to 96-well plates, each well containing one fish embryo and 0.2mL of fish embryo culture medium. 5.1.2 positive control settings: 16 fish embryos are randomly selected and transferred to 96-well plates, each well containing one fish embryo and 0.2mL of tricaine working solution. 5.1.3 treatment of test substance: 16 fish embryos are randomly selected and transferred to 96-well plates, each well containing one fish embryo and 0.2mL of test solution. Placing in a constant temperature cabinet at 28+ -1deg.C for 2h. Then transferring the fish to a zebra fish motion recorder, and recording the swimming track of each zebra fish embryo in 5 minutes in a dark environment by using infrared tracking software. 0.18mL of the solution in each well is replaced by a fish embryo culture solution, and then the fish embryo culture solution is placed in a constant temperature box at 28+/-1 ℃ for 24+/-1 h, and the survival rate of the fish embryo is counted.

6 data and results calculation

The pixel intensity of each well was read as the distance of motion of each fish embryo.

7 result calculation

Calculating a motion distance reduction rate:

reduction ratio= (C-T)/c×100% formula (2). In the formula (2): t-average value of embryo movement distance of the test object treated group fish; c-average value of fish embryo movement distance of blank control group; and (3) performing double-tail T test on the movement distance of the zebra fish in the test object treatment group and the movement distance of the zebra fish in the blank control group to obtain a p value.

And 8, testing validity verification. 8.1 test determination: at least 90% of fish embryos survive each test group after exposure is complete, otherwise the corresponding test group results are not valid. 8.2 test requirements: each batch of test needs to be provided with a positive control group, and the fish embryo movement distance reduction rate of each batch of test positive control group is required to be positive, and p is less than 0.05.

9 results. 9.1 reduction of the movement distance: the reduction rate of the test object on the movement distance of the zebra fish embryo under the corresponding test concentration. 9.2 evaluation of the ability of the test substance to shorten the distance traveled by the embryos of zebra fish, analysis of whether the test substance-treated group and the control group were statistically different (p < 0.05).

10 result correlation interpretation. In the case of a positive reduction in the distance of movement with a statistical difference (p < 0.05), the test results can be used as supporting evidence that the test object has anti-wrinkle and tightening effects, which are declared.

The test results of this test example are shown in Table 6 and FIGS. 2 to 3. As can be seen from table 6 and fig. 2 to 3: the composition samples prepared in example 1 had a 50% reduction in the distance traveled by the zebra fish embryos at a test concentration of 5g/L (p=0.00013). The hunger composition samples prepared in example 1 can significantly reduce the movement distance of zebra fish embryos, supporting the claims of anti-wrinkle and tightening efficacy.

TABLE 6 test example 2 test results

Test example 3

The composition prepared in example 1 was tested. The production of wrinkles is associated with the inactivation of proteins, nucleic acids, lipids and other molecules by oxidation caused by Reactive Oxygen Species (ROS). In daily life, various reasons such as sunlight, compound stimulation and the like may cause the increase of oxidative stress damage of skin, and the skin is aged abnormally, so that the skin loses elasticity and wrinkles are generated. The ROS regulation mechanism in the zebra fish body is the same as that of the human body. The fluorescent staining reagent can be used for efficiently and specifically marking the ROS in the zebra fish embryo cells, and the fluorescence intensity of the ROS is positively correlated with the ROS level in the cells. The application software analysis quantitatively reads the green fluorescent signal in the zebra fish embryo which is enhanced along with the increase of ROS, and the test compares the change of the ROS level of fish embryos in the treatment group and the blank control group, and calculates the ROS clearance rate to evaluate the anti-wrinkle and tightening efficacy of the raw materials or products.

1 apparatus and device. The same as in test example 1.

2 main reagents and consumables. 2.1 Water complies with GB/T6682 specifications. 2.2 cosolvents such as methanol, dimethyl sulfoxide, ethanol, etc. analytical grade. 2.3 parent fish culture water. Weighing 4g of sea salt, dissolving in 10L of water to prepare the sea salt, wherein the salinity is 0.25-0.50 per mill, the conductivity is 500-800 mu S/cm, the saturation of dissolved oxygen is 80%, the pH value is 6.5-8.5, and the hardness is 30-300 mg/L (calculated by calcium carbonate). 2.4 brine shrimp (Artemia salina) egg brine shrimp resting eggs need to be dried and refrigerated in the dark. Weighing about 15g sea salt, dissolving in 1L water to prepare brine for hatching brine shrimp eggs, wherein the density is preferably not more than 4g/L brine shrimp eggs. 2.5 fish embryo culture solution is prepared by weighing 2940mg of anhydrous calcium chloride, 1233mg of magnesium sulfate heptahydrate, 630mg of sodium bicarbonate and 55mg of potassium chloride, and dissolving in 10L of water. The pH value is 6.5-8.5. Chemicals were all analytically pure grade. 2.6 glutathione solution 1g glutathione powder was weighed and dissolved in 10mL water to make 100g/L stock solution. And (5) refrigerating the mixture in small branches. Is effective within 3 months. The working solution is prepared by diluting the stock solution to 0.1g/L with fish embryo culture solution and fresh before use. 2.7 Reactive Oxygen Species (ROS) staining solution. 0.1g of H2DCFDA is weighed and dissolved in 10mL of dimethyl sulfoxide to prepare a storage solution. Split into 0.1mL vials of brown glass and stored frozen. Is effective within 3 years. The working solution was prepared by diluting the stock solution with fish embryo culture solution to 10. Mu.M solution and fresh before use.

3 experimental methods. Substantially the same as in test example 1, except that: 3.3.6 healthy zebra fish embryos are selected and cultured at a density of no more than 1 fish embryo in 200 μl fish embryo culture broth at 28+ -1deg.C until 2 days post fertilization.

4 preparation of test object. The same as in test example 1.

5 testing steps. The following test procedure was 96-well plate test guidelines. Healthy zebra fish embryos 2 days after fertilization were selected. 5.1 test packets. The test requires setting a blank control group (fish embryo culture liquid), a positive control group (glutathione working liquid) and a test object group (test object). If the organic solvent is used for dissolution, the concentration of the solvent in each group of solution needs to be the same. 5.1.1 blank settings. 24 fish embryos are randomly selected and transferred to 96-well plates, each well containing one fish embryo and 0.2mL of fish embryo culture medium. 5.1.2 positive control settings. 24 fish embryos are randomly selected and transferred to 96-well plates, each well containing one fish embryo and 0.2mL glutathione working solution. 5.1.3 treatment of the test substance. 24 fish embryos are randomly selected and transferred to 96-well plates, each well containing one fish embryo and 0.2mL of test solution. Culturing in an incubator at 28+ -1deg.C for 72+ -1 h after fertilization. Transfer to 24-well plates, each containing 12 fish embryos and 2mL of Reactive Oxygen Species (ROS) test solution, and place in a 28+ -1deg.C incubator for 2+ -1 h.5.2 microscopic analysis of samples. And placing the fish embryo sideways. And photographing the fish embryo under a fluorescence stereo microscope according to uniform photographing parameters. 5.3 data and results calculation the photographs were opened with analysis software such as Image J, the yolk area of each fish embryo (see fig. 6) was marked, then the "measured average intensity" in the "measurement" column was selected, and the "average signal intensity" in the test results was selected as ROS signal intensity.

6 results calculation

Reactive Oxygen Species (ROS) clearance was calculated:

clearance= (C-T)/c×100% formula (3), formula (3): t-average value of "average signal intensity" of the ROS of the test object treated group fish embryo; average value of "average signal intensity" of ROS in fish embryos of C-blank; and (3) performing double-tail T test on the ROS signal intensity of the fish embryo of the test object treatment group and the ROS signal intensity of the blank control group to obtain a p value.

And 7, testing validity verification. 7.1 test determination. At least 90% of fish embryos survive each test group after exposure is complete, otherwise the corresponding test group results are not valid. 7.2 test requirements. Each batch of test requires a positive control group, which requires positive control group fish embryo ROS clearance to be positive and p <0.05.

8 results. 8.1ROS elimination rate. The test substance has a clearance rate of ROS at the corresponding test concentration. 8.2 evaluation. Test subjects were evaluated for their ability to promote ROS scavenging, and test subjects were analyzed for statistical differences (p < 0.05) in detection values from the control group.

9 result correlation interpretation. In the case of positive ROS clearance with statistical differences (p < 0.05), the test results may be used as supporting evidence that the test object has antioxidant efficacy, supporting the claims of anti-wrinkle and tightening efficacy.

The test results of this test example are shown in Table 7 and FIGS. 4 to 5. As can be seen from table 7 and fig. 4 to 5: the composition samples prepared in example 1 had a ROS clearance of 99% (p= 0.000000000000000000013) for zebrafish embryos at a test concentration of 5 g/L. The composition sample prepared in the embodiment 1 of the invention can obviously remove ROS in zebra fish embryos, has an antioxidant effect, and supports the claims of anti-wrinkle and tightening effects.

TABLE 7 test example 3 test results

Although the foregoing embodiments have been described in some, but not all embodiments of the invention, other embodiments may be obtained according to the present embodiments without departing from the scope of the invention.

Claims

1. The tightening anti-wrinkle composition is characterized by comprising the following components in percentage by mass:

2. The compact anti-wrinkle composition according to claim 1, wherein the preservative comprises phenoxyethanol, methylparaben, sodium benzoate, potassium sorbate, p-hydroxyacetophenone, and propylparaben.

3. The tightening anti-wrinkle composition according to claim 2, wherein the preservative comprises, in mass percent of the tightening anti-wrinkle composition being 100%: 0.0004 to 0.00045 percent of phenoxyethanol, 0.12 to 0.2 percent of methylparaben, 0.0005 to 0.0008 percent of sodium benzoate, 0.0003 to 0.0004 percent of potassium sorbate, 0.0005 to 0.001 percent of p-hydroxyacetophenone and 0.05 to 0.1 percent of propylparaben.

4. The tightening and anti-wrinkle composition according to claim 1, wherein the filler is magnesium sulfate.

5. The compact anti-wrinkle composition according to claim 1, wherein the emulsion stabilizer is distearyldimethylammonium hectorite.

6. The compact anti-wrinkle composition according to claim 1 or 5, wherein the emulsifier is polysorbate-20.

7. The tightening anti-wrinkle composition according to claim 1, wherein the skin penetrating agent is a pepper extract.

8. The method for preparing a compact anti-wrinkle composition according to any one of claims 1 to 7, comprising the steps of:

And cooling the third mixture to obtain the compact anti-wrinkle composition.

9. The method according to claim 8, wherein the homogenizing speed is 6000 to 8000r/min, and the homogenizing time is 15 to 20min.

10. The method of claim 8, wherein the first temperature is 80-85 ℃ and the second temperature is 45-50 ℃.