CN115501162B

CN115501162B - Anti-wrinkle firming skin composition rich in alpine plants and preparation method thereof

Info

Publication number: CN115501162B
Application number: CN202211466982.4A
Authority: CN
Inventors: 魏要武; 朱洪; 杨霞卿; 唐芳勇; 李忠逵; 李雪; 王真史
Original assignee: Guangzhou Cenkon Biochemical Products Co ltd
Current assignee: Guangzhou Cenkon Biochemical Products Co ltd
Priority date: 2022-11-22
Filing date: 2022-11-22
Publication date: 2023-01-31
Anticipated expiration: 2042-11-22
Also published as: CN115501162A

Abstract

The invention discloses an anti-wrinkle firming skin composition rich in alpine plants and a preparation method thereof, aiming at providing an anti-wrinkle firming skin composition which has the functions of preventing ultraviolet sunburn, nourishing skin, activating cell repair and the like and can further reduce skin relaxation and wrinkles; the technical scheme comprises the following components in percentage by mass: 15-20% of the component A, 15-30% of the component B, 15-30% of the component C, 30-40% of the component D, 10-20% of the component E; the component A consists of a seabuckthorn fruit extract A and a glycyrrhiza glabra root extract; the component B consists of carnosine and a seabuckthorn fruit extract B; the component C is Yunnan ginkgo extract and seabuckthorn fruit extract C; the component D comprises hydrolyzed collagen; the component E is bergenia extract; belongs to the technical field of cosmetics.

Description

Anti-wrinkle firming skin composition rich in alpine plants and preparation method thereof

Technical Field

The invention relates to an anti-wrinkle firming skin composition, in particular to an anti-wrinkle firming skin composition rich in alpine plants, and belongs to the field of daily cosmetics.

Background

With the increase of age, environmental pollution and living pressure, the skin aging of people is more and more obvious, and the anti-wrinkle, tightening, beauty and skin care for the middle-aged and the elderly and even young people are more and more valued by people. Skin aging is usually manifested by decreased skin elasticity, increased skin wrinkles, increased skin roughness, decreased moisturizing intensity, and the like. Currently, many proposals for moisturizing and nourishing skin are made to solve this problem, but it is obvious that the mere use of such moisturizing and skin-care in daily life is not enough to prevent skin aging. Other natural factors such as environment, sunlight and pressure can also accelerate skin aging, and skin is easy to have spots, fine lines and the like. Meanwhile, years can cause body aging, skin metabolism is slowed down, collagen and elastic tissue fibers of the skin are changed, texture and content are reduced, the skin gradually loses elasticity, becomes loose, and wrinkles appear.

The anti-aging products on the current market are mostly filled with moisturizing and hyaluronic acid, whiten, brighten and resist skin wrinkles and relaxation and other problems by the scheme of anti-aging, the main dosage form is an essence product, for example, CN202010138595.2 discloses an anti-wrinkle tightening essence and a preparation method thereof, and the technical scheme comprises the following components in parts by weight: 0.5-3 parts of spirulina maxima extract, 0.005-0.1 part of sodium hyaluronate, 0.01-0.05 part of cyclodextrin, 0.01-0.05 part of ubiquinone, 0.1-1 part of avenanthramide, 1-5 parts of yeast extract, 0.1-1 part of fucoidan, 0.01-0.1 part of humic acid, 0.1-3 parts of pyrus ussuriensis extract, 0.05-0.3 part of decapeptide, 0.05-0.3 part of glycoprotein and 0.005-0.05 part of rose oil. Pure plant oil-soluble essential oil products are also available, such as CN201910017107.X, the invention provides anti-aging, anti-wrinkle and firming essential oil which mainly comprises the following components in percentage by weight: 0.1-1% of hypecoum extract, 0.1-1% of samaria extract, 0.1-1% of spilanthol extract and 0.1-1% of undaria pinnatifida extract. After the four components are reasonably compounded, the living environment of local tissues and cells is rapidly improved through the active molecules of the plant extracts with affinity action, the metabolism is accelerated, some problems caused by local metabolic disorders are comprehensively solved, the cell metabolism and cell regeneration functions are promoted, the skin wrinkle problem can be obviously improved, and the skin wrinkle-resisting, anti-aging and tightening effects are obvious.

However, the skin care product adopts a peptide substance and small molecular hyaluronic acid filling scheme aiming at skin wrinkles and sagging skin, or adopts algin and other film forming agents to form a film, has a temporary appearance effect, but does not aim at solving the root problems of metabolic aging of body cells, cell vitamin deficiency and the like caused by sunlight ultraviolet ray invasion and free radical accumulation,

therefore, the natural alpine plant components are deeply developed aiming at the problems, and the natural alpine plant components have great application values of synchronously preventing ultraviolet ray invasion, supplementing skin vitamins, activating cell vitality, maintaining skin collagen secretion, clearing cell free radicals and enhancing cell self-repairing capability. The alpine plants are easier to promote to have more abundant active ingredients due to the fact that the alpine plants have more complex external environments, high ultraviolet rays, long-time illumination, high altitude, low oxygen and other severe environments, the active ingredients can break through the appearance aiming at skin aging, the content of plant source ingredients is low, the effect is mild, and the alpine plants are the priority for long-term skin care.

Disclosure of Invention

Aiming at the defects and shortcomings of the prior art, the anti-wrinkle and skin-firming composition has the functions of preventing sunburn caused by ultraviolet rays, nourishing skin, activating cell repair and the like, and can further reduce skin relaxation and wrinkles.

The second technical scheme of the invention is to provide a preparation method of the anti-wrinkle and firming skin composition rich in alpine plants, which comprises the following steps:

the anti-wrinkle and firming skin composition rich in alpine plants comprises the following components in percentage by mass: 15-20% of the component A, 15-30% of the component B, 15-30% of the component C, 30-40% of the component D and 10-20% of the component E;

the component A consists of a seabuckthorn fruit extract A and a glycyrrhiza glabra root extract;

the component B consists of carnosine and a seabuckthorn fruit extract B;

the component C consists of Yunnan ginkgo extract and seabuckthorn fruit extract C;

the component D comprises hydrolyzed collagen;

the component E is bergenia purpurascens extract.

Further, the anti-wrinkle firming skin composition rich in alpine plants comprises a component D and safflower bee pollen.

Further, the anti-wrinkle firming skin composition rich in alpine plants comprises the following components in percentage by mass: 1-3:1.

Further, the anti-wrinkle and firming skin composition rich in alpine plants comprises the following components in parts by mass: 2-4:1.

Furthermore, the anti-wrinkle firming skin composition rich in alpine plants also comprises 0.7-1% of preservative.

Furthermore, the anti-wrinkle firming skin composition rich in alpine plants is characterized in that the preservative is one of phenoxyethanol, caprylyl glycol, caprylyl hydroxypentanoic acid and menthone.

Further, the anti-wrinkle firming skin composition rich in alpine plants is prepared by the following steps of:

1) Oven drying oil cake of fructus Hippophae, pulverizing to fine powder below 100 meshes, adding 20-50 times of aqueous two-phase extraction solvent, soaking for 30-60min, placing into flash extractor at 2000-2500rpm, performing flash extraction for 3-5 times for 10-26s each time, collecting filtrate, centrifuging, removing residue, collecting filtrate, and layering;

the aqueous two-phase extraction solvent comprises the following components in parts by volume: 15-20% of ethanol, 20-25% of ammonium acetate and pure water, wherein the ethanol, the ammonium acetate and the pure water are added to 100%;

2) Taking supernatant, recovering ethanol under reduced pressure and vacuum, passing the concentrated solution through AB-8 resin at a column feeding speed of 1BV/h and washing with pure water of 4BV and 2BV/h, and collecting pure water samples for later use; decolorizing with 2BV 10% ethanol at a speed of 1BV/h, eluting with 60-80% ethanol, collecting eluate, concentrating under reduced pressure, and redissolving with pure water to obtain fructus Hippophae extract C;

3) Taking the pure water elution sample obtained in the step 2), adjusting the pH value to 10, adjusting the temperature to 45 ℃, adding 10000-24000U/mL of protease, performing enzymolysis for 2h, heating to 90 ℃, inactivating the enzyme for 10min, centrifuging at 5000rpm for 10min to remove precipitates, passing the supernatant through an ultrafiltration membrane of 500-1000Da, and collecting the membrane filtrate to obtain the seabuckthorn fruit extract B.

4) Taking the supernatant: concentrating to 0.1g/mL, adding anhydrous ethanol until the final alcohol degree is 80%, stirring for 15min, refrigerating overnight, centrifuging, collecting precipitate, and dissolving the precipitate with pure water to obtain fructus Hippophae extract A.

The second technical scheme provided by the invention is that the preparation method of the anti-wrinkle and skin-tightening composition rich in alpine plants comprises the following steps:

1) Weighing the components;

2) Uniformly mixing the component D and the component E for later use;

mixing the components A, B and C, adding the standby components prepared in step 2), heating to 50 deg.C, stirring at 300rpm for 15min, mixing, adding antiseptic, stirring for 5min, and filtering with 0.2 μm filter membrane to obtain the final product.

Compared with the prior art, the invention has the beneficial effects that:

firstly, the technical scheme provided by the invention is that a seabuckthorn fruit extract is subjected to secondary resource development by drying oil meal of oil-squeezed seabuckthorn fruits through a double-water-phase combined flash extraction technology, although the double-water-phase technology is a technology for conventionally extracting biological components in plants such as protein, enzyme and the like, multi-component extraction is firstly carried out in the technical scheme, ammonium acetate and ethanol with proper proportion are added into a pure water system to form two systems with large polarity difference, the affinity effect of the ethanol and water on the oil meal can be effectively utilized to soak raw materials to be extracted in the soaking process, and the double-water-phase system is destroyed through rapid extraction such as cavitation and wall breaking on powdery seabuckthorn fruits through the flash extraction technology, so that the polar solvent and the nonpolar solvent can both carry out cavitation extraction on the seabuckthorn fruits, and the high-speed cavitation effect on the plant raw materials and the characteristic that various different polar solvents in the double-water-phase system can be compatible with each other in a similar principle to fully dissolve the multi-components, thereby achieving the optimal extraction effect. After extraction, ammonium acetate and water are gradually formed due to the polarity characteristics of the aqueous two-phase system and distributed on the lower layer, and ethanol and water are distributed on the upper layer, so that the clear layering condition is realized. Concentrating the supernatant to remove ethanol, loading onto resin column, adsorbing pigment and flavone components, passing protein and enzyme components through resin column without adsorption, washing and decolorizing with resin, directionally eluting to obtain flavone component (fructus Hippophae extract C), solubilizing the supernatant directly passing through resin column by adjusting pH, adding alkaline protease for enzymolysis to obtain small-fragment active peptide component, further inactivating enzyme, removing impurities and membrane to obtain active peptide component (fructus Hippophae extract B), concentrating and enriching the lower layer of supernatant, removing impurities and decolorizing with classical ethanol precipitation method, collecting precipitate, and re-dissolving to obtain fructus Hippophae polysaccharide component (fructus Hippophae extract A).

Secondly, the invention provides synergistic interaction of the components, wherein the seabuckthorn fruit extract A and the glabrous greenbrier rhizome extract are compounded to be rich in polysaccharide, the polysaccharide is used for conveying vitality to cells and repairing cell walls and organelles, the polysaccharide has the effects of resisting oxidation and clearing free radicals when being compounded with the Yunnan ginkgo seed extract and the seabuckthorn fruit extract C (flavone), the compound bergenia herb extract is used for preventing ultraviolet ray invasion, hydrolyzed collagen can supplement water and preserve moisture, the safflower bee pollen can promote skin metabolism and improve skin nutrition, and the compound carnosine and the seabuckthorn fruit extract B (active peptide) can activate deep skin cells. Experiments prove that the components of the invention have synergistic interaction.

Thirdly, the anti-wrinkle skin-firming composition rich in alpine plants provided by the invention can coordinate and synergize all components, can remove a large amount of free radicals generated in the natural aging process of an organism, effectively repair damage, delay aging and improve the activity of the organism, and can inhibit photoaging caused by ultraviolet rays by removing active oxygen of cells, increasing type I collagen and inhibiting the content of MMP-1 enzyme.

Fourthly, the essence and cream emulsion of the composition rich in alpine plants and used for resisting wrinkles and tightening skin provided by the invention have the effects of preventing ultraviolet ray invasion, supplementing vitamins of the skin, activating cell vitality, maintaining collagen secretion of the skin and enhancing cell self-repairing capacity.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

Used as follows:

the Glycyrrhiza glabra root extract is from Biotech, inc. of Baihabo, guangzhou;

carnosine, sourced from shenzhen sacral medicine research and development limited company;

the Yunnan semen Ginkgo extract is derived from Guangzhou Baiyu Biotechnology limited;

hydrolyzed collagen is derived from Shenzhen alpha Biotech, inc.;

safflower bee pollen is from Siandengchi Biotech limited;

the extract of bergenia purpurascens is from Sainta Spreng Biotech Co., ltd

The sea-buckthorn fruit extracts A, B and C are all self-made, and the process is as follows:

the aqueous two-phase extraction solvent comprises the following components in parts by volume: 15-20% of ethanol, 20-25% of ammonium acetate and pure water, and the balance is made up to 100%;

2) Taking supernatant, vacuum-recovering ethanol under reduced pressure, passing the concentrated solution through AB-8 resin at a column loading speed of 1BV/h, washing with pure water at a speed of 4 BV/h and 2BV/h, and collecting pure water samples for later use; adding 2BV 10% ethanol for decolorization and impurity removal at the speed of 1BV/h, then eluting with 60-80% ethanol, collecting the eluent, concentrating under reduced pressure, and redissolving with pure water to obtain a seabuckthorn fruit extract C;

Example 1

The anti-wrinkle and firming skin composition rich in alpine plants provided by the embodiment comprises the following components: 10g of seabuckthorn fruit extract A, 5g of glycyrrhiza glabra root extract, 8g of carnosine, 12g of seabuckthorn fruit extract B, 10g of Yunnan ginkgo seed extract, 10g of seabuckthorn fruit extract C, 20g of hydrolyzed collagen, 10g of red flower melissa and 15g of bergenia purpurea extract.

Example 2

The invention provides a wrinkle-resisting and skin-tightening composition rich in alpine plants, which comprises the following components: 10g of seabuckthorn fruit extract A, 10g of glycyrrhiza glabra root extract, 15g of carnosine, 5g of seabuckthorn fruit extract B, 10g of Yunnan ginkgo seed extract, 5g of seabuckthorn fruit extract C, 20g of hydrolyzed collagen, 10g of red flower melissa and 15g of bergenia purpurea extract.

Example 3

The invention provides a wrinkle-resisting and skin-tightening composition rich in alpine plants, which consists of the following components: 7.5g of echinacea extract A, 7.5g of glycyrrhiza glabra root extract, 20g of carnosine, 10g of seabuckthorn fruit extract B, 5g of Yunnan ginkgo seed extract, 5g of seabuckthorn fruit extract C, 25g of hydrolyzed collagen, 10g of red flower melissa and 10g of bergenia purpurea extract.

Example 4

The invention provides a wrinkle-resisting and skin-tightening composition rich in alpine plants, which comprises the following components: 10g of seabuckthorn fruit extract A, 7g of glycyrrhiza glabra root extract, 20g of carnosine, 10g of seabuckthorn fruit extract B, 12g of Yunnan ginkgo seed extract, 8g of seabuckthorn fruit extract C, 23g of hydrolyzed collagen and 10g of bergenia purpurascens extract.

Example 5

An essence containing 1 alpine plant-rich anti-wrinkle and skin-firming composition described in example 1, which comprises the following formula (in percentage by weight): 0.5% of acrylic acid (ester)/C10-30 alkanol acrylate cross-linked polymer, 10% of butanediol, 10% of glycerol, 30% of alpine plant anti-wrinkle and skin-tightening-rich composition in example 1, 4% of PEG-20 methyl glucose sesquistearate, 0.5% of blue silk skin beautifying agent, 0.5% of triethanolamine, 0.1% of octyl glycol and the balance of deionized water.

The preparation process comprises the following steps: adding water into an emulsifying pot, stirring to form a vortex, adding acrylic acid (ester)/C10-30 alkanol acrylate cross-linked polymer, homogenizing and dispersing uniformly, adding butanediol, glycerol and other raw materials needing high-temperature dissolution, stirring and heating to 85 ℃, keeping the temperature for 10min, adding an emulsifying thickener after complete dissolution, cooling to 50 ℃ after homogenizing and emulsifying, adding triethanolamine, the alpine plant anti-wrinkle and skin-firming composition, the blue silk skin-beautifying agent and the caprylyl glycol in the embodiment 1, stirring for 15min, mixing uniformly, and filtering to discharge the material.

Example 6

A cream emulsion containing 1 alpine plant-rich anti-wrinkle and skin-firming composition described in example 1, wherein the formula (in percentage by weight) is as follows: carbomer 0.5%, butylene glycol 10%, glycerin 10%, isononyl isononanoate 5%, behenyl alcohol 3%, caprylic/capric triglyceride 5%, methyl glucose sesquistearate 6%, sodium acrylate 4%, example 2 alpine plant anti-wrinkle firming skin-enriched composition 20%, biopolysaccharide gum 15%, triethanolamine 0.4%, phenoxyethanol 2%, and the balance deionized water.

The preparation process comprises the following steps:

1. adding water into a water phase pot, stirring to form a vortex, adding carbomer, stirring to disperse uniformly, adding butanediol, glycerol and other raw materials needing high-temperature dissolution, stirring, heating to 80 ℃, and keeping the temperature for 15min to dissolve completely.

2. Adding isononyl isononanoate, behenyl alcohol, caprylic/capric triglyceride and methyl glucose sesquistearate into the oil phase pot, stirring, heating to 84 deg.C, and maintaining the temperature for 12min for complete dissolution.

3. Sequentially filtering in an emulsifying pot, adding water phase material and oil phase material, stirring, vacuumizing, homogenizing, emulsifying, adding sodium acrylate, homogenizing, and cooling.

4. Cooling to 50 deg.C, adding triethanolamine, biological polysaccharide gum, the composition rich in alpine plant for resisting wrinkle and tightening skin, and phenoxyethanol, stirring for 15min, mixing well, and filtering to obtain the final product.

Comparative example 1

Seabuckthorn fruit extract A.

Comparative example 2

Glycyrrhiza glabra root extract.

Comparative example 3

Carnosine.

Comparative example 4

And (3) a seabuckthorn fruit extract B.

Comparative example 5

Yunnan gingko biloba extract.

Comparative example 6

And (3) seabuckthorn fruit extract C.

Comparative example 7

Hydrolyzing the collagen.

Comparative example 8

Safflower bee pollen.

Comparative example 9

Rhizoma Seu herba Bergeniae extract.

Comparative example 10

The invention provides a wrinkle-resisting and skin-tightening composition rich in alpine plants, which comprises the following components: 20g of seabuckthorn fruit extract A, 7g of glycyrrhiza glabra root extract, 20g of carnosine, 12g of Yunnan ginkgo seed extract, 8g of seabuckthorn fruit extract C, 23g of hydrolyzed collagen and 10g of bergenia purpurascens extract.

Comparative example 11

The invention provides a wrinkle-resisting and skin-tightening composition rich in alpine plants, which consists of the following components: 7g of glycyrrhiza glabra root extract, 20g of carnosine, 18g of sea buckthorn fruit extract B, 12g of Yunnan ginkgo seed extract, 8g of sea buckthorn fruit extract C, 23g of hydrolyzed collagen and 10g of bergenia cordifolia extract.

Comparative example 12

The invention provides a wrinkle-resisting and skin-tightening composition rich in alpine plants, which comprises the following components: 10g of seabuckthorn fruit extract A, 7g of glycyrrhiza glabra root extract, 20g of carnosine, 12g of Yunnan ginkgo seed extract, 18g of seabuckthorn fruit extract B, 23g of hydrolyzed collagen and 10g of bergenia purpurascens extract.

Effect testing

1. Free radical scavenging experiments

Free radicals are oxygen radicals generated during the aging process of the body and can cause oxidative damage to cells. The free radical scavenging agent can effectively repair cell damage, delay aging and improve the vitality of the organism.

DPPH is a stable nitrogen-centered radical with an absorption maximum at 519 nm. The alcohol solution of DPPH is dark purple, and when the radical scavenger is added to the DPPH solution, the single electron of DPPH is paired to lighten the color, with a consequent decrease in the absorbance at the maximum absorption wavelength. Thus, the ability of a substance to scavenge DPPH radicals can be judged by the extent to which the absorbance is diminished. The hydroxyl radical is another high-activity oxygen radical, has strong oxidizability, and can generate great influence on the structure, the property and the color of a substrate after the hydroxyl radical is generated by reaction in the detection process, so that the detection is carried out by a spectrophotometer.

DPPH radical clearance: the DPPH is prepared into 65.7mg/L DPPH solution working solution by absolute ethyl alcohol. And (3) mixing 2mL of sample to be detected with 4mL of working solution of the PPH solution, reacting for 30min at normal temperature in a dark place, and detecting a light absorption value at a wavelength of 517nm by using an ultraviolet spectrophotometer. DPPH radical clearance (%) = [1- (B-C)/a ] × 100%. A, measuring the light absorption value of a reference solution (absolute ethyl alcohol replaces a sample to be measured); b, the light absorption value measured by the test object; c: absorbance of blank (absolute ethanol instead of DPPH).

Hydroxyl radical clearance rate: 0.5 mL of 0.75 mmol/L O-diazaphenanthrene absolute ethyl alcohol solution is put into a test tube, 1 mL of 0.15 mol/L PBS (pH 7.40) and 0.5 mL of distilled water are sequentially added, after the mixture is fully mixed, 0.5 mL of 0.75 mmol/L FeSO4 solution is added, after the mixture is mixed, 0.5 mL of 0.01% H2O2 solution with volume fraction (the same as below) is added, after the mixture is subjected to water bath at 37 ℃ for 60min, the absorbance of the mixture is measured at 536 nm wavelength to serve as the absorbance of a damaged tube (A damage). The intact tube was treated with 0.5 mL of distilled water instead of 0.5 mL of 0.01% H2O2 solution in the intact tube, and the absorbance of the intact tube at 536 nm was measured (A was intact). Sample tube 0.5 mL of sample solution was used instead of 0.5 mL of distilled water in the damaged tube, and the absorbance of the sample tube at 536 nm wavelength (sample A) was measured by the same procedure as the damaged tube. OH clearance of the sample was calculated as follows:

the samples of examples 1 to 4 and comparative examples 1 to 11 are diluted to 2% concentration for free radical scavenging test, and after the test result data are processed, the free radical scavenging effect of each group of cases is shown in the following table 1:

TABLE 1 radical scavenging Rate

The comparative examples 1 to 9 are single components, and the test results show that the bergenia extract is the best in the effect of scavenging free radicals, and other better components are extracts from alpine plant components such as Yunnan ginkgo extract, seabuckthorn fruit extract B and C, and the collagen, the seabuckthorn fruit extract A and the glabrous greenbrier rhizome extract are poor in effect. In comparative examples 10 to 12 formed by compounding a single extract, the effect of scavenging free radicals is improved to a certain extent relative to a single component, which indicates that a certain synergistic effect may exist in the single extract, but in comparative examples 10 to 12, any combination lacking one of the seabuckthorn fruit extracts A, B and C is adopted, so that the effect of scavenging free radicals is obviously inferior to that in the embodiment, which indicates that the seabuckthorn fruit extracts A, B and C in the anti-wrinkle compact composition can be synergistic with each other, and the other components are compounded to greatly help the synergistic effect of scavenging free radicals of the whole extract.

2. Determination of human skin fibroblast anti-aging related index

Ultraviolet-inducible active oxygen: at the molecular level, uv-activation of reactive oxygen species leads to breakdown of collagen in the extracellular matrix and to the shut-down of new collagen synthesis. Ultraviolet rays can cause photoaging, leading to a decrease in the clearance of cellular active oxygen, a decrease in type i collagen, which maintains skin elasticity, and an increase in MMP-1 enzyme content, and thus the anti-aging efficacy can be evaluated by measuring the level of active oxygen, type i collagen, and MMP-1 content.

Measurement of active oxygen content of human skin fibroblasts: and (4) measuring the content of the sample according to the instruction of the active oxygen detection kit. After the human skin fibroblasts had been treated 24 h after dilution of the test samples to a concentration of 0.1%, they were rinsed 2 times with PBS and incubated in a cell incubator at 37 ℃ for 20 min in a culture medium containing 10 \8197/. Mu.mol/L \8197, 2',7' -dichlorofluorescent yellow diacetate (2 ',7' -dichlorodi-hydrofluorescein 8197, diacetate, DCFH-DA). The cells were washed 3 times with DMEM. After the cells are collected, the fluorescence intensity under the conditions of the excitation wavelength of 488 nm and the emission wavelength of 525 nm is measured by a fluorescence microplate reader. The positive control ROSUP and the natural antioxidant VC matched with the kit are used as controls.

Measurement of human skin fibroblast collagen type i content: and (3) determining the sample according to the instruction of the human I type collagen enzyme-linked immunoassay kit. After the test sample is diluted to 0.1% concentration and human skin fibroblasts are treated for 24 h, cell culture solution supernatant is taken and centrifuged at 1000 Xg for 15min at the temperature of 2-8 ℃, and the supernatant is immediately taken for measurement. Adding a sample or a standard substance into an enzyme-labeled well coated with an antibody in advance, adding a recognition antigen marked by horseradish peroxidase (HRP), incubating for 1 h at 37 ℃, competitively combining the recognition antigen and the solid-phase antigen to form an immune complex, washing by PBST, enabling the combined HRP to catalyze Tetramethylbenzidine (TNB) to turn blue, then turning yellow under the action of acid, measuring absorbance at the wavelength of 450 nm, and calculating the type I collagen content of cells after sample stimulation by contrasting a standard curve.

Measuring MMP-1 content of human skin fibroblasts: and (4) carrying out sample determination according to the MMP-1 enzyme-linked immunoassay kit specification. After the test sample is diluted to 0.1% concentration and human skin fibroblasts are treated for 24 h, cell culture solution supernatant is taken and centrifuged for 15min at the temperature of 2-8 ℃ and at the speed of 1000 Xg, and the supernatant is immediately taken for measurement. A sample or a standard substance, a biotinylated anti-MMP-1 antibody and HRP-marked avidin are sequentially added into a micropore plate coated with a purified MMP-1 antibody, and the mixture is thoroughly washed and then developed by a substrate TMB. TMB is converted to blue by peroxidase and yellow by acid. The shade of the color is positively correlated with the MMP-1 content in the sample. And (3) detecting the absorbance at the wavelength of 450 nm by using a microplate reader, and calculating the MMP-1 content of the sample after stimulation by contrasting with a standard curve.

The test results of the above indexes of the samples of examples 1 to 4 and comparative examples 1 to 11 are shown in the following table 2 after data processing:

TABLE 2 anti-aging related indices of fibroblasts

Analysis on the active oxygen scavenging rate data shows that the test data of the single components in the comparative examples 1 to 9 have similarity to the free radical scavenging experimental data in the active oxygen scavenging effect on fibroblasts, and the single components are all high mountain plant extracts such as bergenia extract, yunnan ginkgo extract, seabuckthorn fruit extract B and C with better scavenging effect, which shows the consistency of data trend of free radical scavenging effect tests and also shows that one of the main effects of the components is scavenging active oxygen. Comparative examples 10 to 12 were partly improved in the active oxygen scavenging effect with respect to the single-component comparative examples 1 to 9, and partly remained unchanged, but were insufficient in the improvement range with respect to the examples, indicating that the scavenging of active oxygen by each single extract needs to be synergistically assisted by a good ratio, and cannot simply depend on an increase in the component ratio.

As can be seen from the data of the increasing rate of the type I collagen content, the main components capable of remarkably stimulating the increase of the expression level of the collagen in the single component are peptide substances and the collagen, wherein the effect of the seabuckthorn fruit extract B is the most remarkable, which shows that the seabuckthorn fruit extract B can effectively stimulate cells to secrete and synthesize the collagen, other single components have no effect on promoting and synthesizing the collagen basically, similar effects are also realized in comparative examples 10-12, the effect is remarkably weakened after the seabuckthorn fruit extract B is lacked, and the effect of stimulating the secretion and synthesis of the collagen is enhanced to a certain extent after the seabuckthorn fruit extract B is added.

MMP-1 enzyme is a typical collagen degrading enzyme, and collagen synthesized in cells is continuously degraded in metabolism, so that the skin is loose and collapsed, and wrinkles are deepened. By analyzing MMP-1 inhibition rate data, the single component with obvious inhibition effect on MMP-1 is the Yunnan ginkgo biloba extract of the comparative example 5, the inhibition effect on the MMP-1 enzyme activity reaches 28.34%, and the effect of other extracts is not obvious. The embodiment and the comparative example show certain differences after multi-component compounding, the embodiment has a strong MMP-1 inhibition effect, the comparative examples 10-12 have weak effects, the result shows that the compounding of the hippophae rhamnoides fruit extract and the Yunnan ginkgo and other extracts has a certain synergistic effect on MMP-1 content inhibition, and the weakest effect is shown when the hippophae rhamnoides fruit extract C is absent, so that the strong synergistic effect exists between the Yunnan ginkgo and the hippophae rhamnoides fruit extract C.

Human body testing

The human body testing process is briefly described as follows:

96 volunteers with wrinkles on canthus, raised lines and wrinkles on face are selected, the age interval is 30-50 years old, and the volunteers are signed with informed consent. Volunteers were randomly divided into 4 groups of 24 persons each, one group of volunteers used the blank cream-based group 1, one group of volunteers used the example 5, one group used the blank cream-based group 2, and another group used the product of example 6, the product was continuously used for 30 days, smeared 1 time in the morning and evening, and the volunteers were given evaluation feedback on the effects of the product by the consumer using a questionnaire. The evaluation results are shown in table 3 below:

table 3 evaluation results

The blank paste base group 1 is the body of the example 5 without adding the alpine plant anti-wrinkle and skin-tightening composition in the example 1, and the results of continuous trial test on a human body for 30 days show that the blank paste base group 1 has no obvious improvement on wrinkles and tightness of the skin, while the example 5 has an obvious effect relative to the blank group, and other facial lines have feedback, which shows that the blank paste base group has a better effect of reducing the facial lines.

The blank cream base group 2 is a body of the example 6 without adding the alpine plant anti-wrinkle skin-firming composition of the example 2, and the results of the continuous trial test on the human body for 30 days show that the blank cream base group 2 has no obvious improvement on wrinkles and firmness of the skin, while the example 6 has an obvious effect relative to the blank group and has positive feedback on the reduction of facial lines, which shows that the blank cream base group has a better skin-firming effect.

The present invention is not limited to the embodiments, and those skilled in the art can modify or replace the technical solution of the present invention within the scope of the present invention, which is disclosed by the present invention, and the present invention is also included in the scope of the present invention.

Claims

1. The anti-wrinkle and firming skin composition rich in alpine plants is characterized by comprising the following components in percentage by mass: 15-20% of the component A, 15-30% of the component B, 15-30% of the component C, 30-40% of the component D and 10-20% of the component E;

the component B consists of carnosine and a seabuckthorn fruit extract B;

the component D comprises hydrolyzed collagen;

the component E is bergenia extract;

the seabuckthorn fruit extracts A, B and C are respectively prepared by the following methods:

1) Oven drying oil cake of fructus Hippophae, pulverizing into fine powder below 100 meshes, adding 20-50 times of aqueous two-phase extraction solvent, soaking for 30-60min, placing into flash extractor at 2000-2500rpm, performing flash extraction for 3-5 times for 10-26s each time, collecting filtrate, centrifuging, removing residue, collecting filtrate, and layering;

3) Taking the pure water elution sample obtained in the step 2), adjusting the pH to 10, adjusting the temperature to 45 ℃, adding 10000-24000U/mL of protease, performing enzymolysis for 2h, heating to 90 ℃, inactivating the enzyme for 10min, centrifuging at 5000rpm for 10min to remove precipitates, passing the supernatant through an ultrafiltration membrane of 500-1000Da, and collecting the filtrate of the filtration membrane to obtain a seabuckthorn fruit extract B;

4) Taking the lower layer clear liquid: concentrating to 0.1g/mL, adding anhydrous ethanol until the final alcohol degree is 80%, stirring for 15min, refrigerating overnight, centrifuging, collecting precipitate, and dissolving the precipitate with pure water to obtain fructus Hippophae extract A.

2. The alpine plant-rich anti-wrinkle and firming skin composition as claimed in claim 1, wherein the D component further comprises safflower bee pollen.

3. The anti-wrinkle firming skin composition rich in alpine plants according to claim 1, wherein the mass ratio of the seabuckthorn fruit extract A to the glycyrrhiza glabra root extract is as follows: 1-3:1.

4. The alpine plant-rich anti-wrinkle and anti-firming skin composition as claimed in claim 1, wherein the mass ratio of the carnosine to the hippophae rhamnoides fruit extract B is: 1-3:1.

5. The anti-wrinkle and firming skin composition rich in alpine plants according to claim 1, wherein the mass ratio of the Yunnan ginkgo extract to the seabuckthorn fruit extract C is as follows: 1-3:1.

6. The alpine plant-rich anti-wrinkle and firming skin composition as claimed in claim 2, wherein the mass ratio of the hydrolyzed collagen to the safflower bee pollen is: 2-4:1.

7. The alpine plant-rich anti-wrinkle and firming skin composition as claimed in claim 1, further comprising 0.7-1% of a preservative.

8. The alpine plant-rich anti-wrinkle firming skin composition as claimed in claim 7, wherein the antiseptic is one of phenoxyethanol, caprylyl glycol, caprylyl hydroxypentanoic acid, and menthone.

9. The preparation method of the alpine plant-rich anti-wrinkle firming skin composition according to claim 1, comprising the following steps in sequence:

1) Weighing the components according to the mass percentage of claim 1;

2) Uniformly mixing the component D and the component E for later use;

3) Mixing the component A, the component B and the component C uniformly, adding the standby component prepared in the step 2), heating to 50 ℃, stirring at 300rpm for 15min, mixing uniformly, adding a preservative, continuously stirring for 5min, and filtering through a 0.2-micron filter membrane to obtain a finished product.