CN116473883A - Anti-aging composition and preparation method and application thereof - Google Patents
Anti-aging composition and preparation method and application thereof Download PDFInfo
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- CN116473883A CN116473883A CN202310576615.8A CN202310576615A CN116473883A CN 116473883 A CN116473883 A CN 116473883A CN 202310576615 A CN202310576615 A CN 202310576615A CN 116473883 A CN116473883 A CN 116473883A
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9706—Algae
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- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
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- A61K8/9783—Angiosperms [Magnoliophyta]
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
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- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
Abstract
The invention relates to an anti-aging composition and a preparation method and application thereof, wherein the anti-aging composition comprises, by mass, 0.03% -2.0% of chlorella extract, 0.01% -1.0% of Fucus vesiculosus extract, 0.01% -1.0% of kelp palm extract, 0.5% -1% of tea extract, 0.5% -2% of yeast fermentation product filtrate, 3% -5% of nicotinamide, 0.005% -0.5% of beta-glucan, 1% -6% of butanediol, 0.05% -2% of glycerol, 0.5% -1% of preservative and the balance of deionized water, wherein the preservative is one or more selected from 1, 2-hexanediol, 1, 2-pentanediol, octaethylene glycol, sodium benzoate, phenoxyethanol, ethylhexyl glycerol, p-hydroxyacetophenone or octaethylene glycol; the mass ratio of the chlorella extract to the Fucus vesiculosus extract to the kelp palm extract is 3:1:1. According to the invention, through the synergistic effect of the components, the composition can effectively remove free radicals and promote the synthesis of collagen, and can be applied to cosmetics or skin care products to lighten skin color, nourish skin and have the effects of resisting wrinkles and tightening.
Description
Technical Field
The invention relates to the technical field of cosmetics, in particular to an anti-aging composition, a preparation method and application thereof.
Background
Aging of the skin is synchronous with aging of the body, and the skin is located at the outermost layer of the body and is more susceptible to exogenous stimulus, so that aging of the skin is the result of the combined action of endogenous and exogenous factors.
With age, the skin gradually ages, and natural aging of the skin affected by endogenous factors, also called natural physiological aging, is also called. The specific expression is that the proportion of the Collagen I to the Collagen III is obviously changed, the elastin synthesis is obviously reduced, the quantity of elastic fibers is reduced, and the skin is caused to have fine and shallow wrinkles; the exogenous factor is mainly ultraviolet rays in sunlight, so exogenous aging is also called skin photoaging, and in order to solve the photoaging problem, we need to remove excessive free radicals generated by photoaging. The free radicals in the human body mainly comprise semiquinone free radicals, oxygen free radicals, carbon, nitrogen and sulfur center free radicals; wherein the superoxide anion radical (. O) 2 - ) The most efficient, the most damaging of the hydroxyl radicals (.OH).
Chinese patent CN113559006A discloses a cosmetic composition for scavenging free radicals and resisting aging, which can scavenge free radicals, reduce oxidative damage, resist cell oxidative damage and cell aging by the synergistic effect of imidazo [1,2-a ] pyridine compound and pyrazine-oxazole biaryl compound, effectively exert the antioxidation effect, and can endow the cosmetic with remarkable anti-aging effect when being applied to cosmetics, so that the skin is finer, smoother, white and moist. The invention can obviously enhance the antioxidation capability of skin tissues, reduce the content of skin free radicals and delay skin aging by mutually matching the components. However, the composition is only aimed at DPPH free radical, and DPPH experiment only shows that the composition has the oxidation resistance and cannot fundamentally solve the main free radical problem of human body.
Chinese patent CN103228262a discloses a cosmetic composition containing gulfweed extract, sea antler extract and brown seaweed extract, and more particularly, to a cosmetic composition containing one or more extracts selected from the group consisting of gulfweed extract, sea antler extract and brown seaweed extract as an active ingredient, which has a good antioxidant effect, improves skin elasticity, and reduces skin wrinkles. However, the composition only promotes the total amount of Collagen, and does not promote the type I Collagen, so that the overall balance of Collagen I and Collagen III cannot be ensured.
Chinese patent CN110151629B discloses an anti-aging composition for promoting collagen synthesis and a preparation method thereof, wherein the composition comprises 5-15 parts of bergenia linguata root, 3-8 parts of ginseng root, 1-6 parts of astragalus membranaceus root, 2-9 parts of field horsetail, 3-7 parts of Mallotus japonicus bark, 1-4 parts of rhodiola rosea and 1-6 parts of sansevieria glabra, and the composition can effectively stimulate skin to synthesize collagen through synergistic interaction of the components, so as to achieve the effects of endowing skin with elasticity, reducing wrinkles and relieving skin aging. However, the stability of the composition is insufficient, and the requirements of users cannot be met.
Disclosure of Invention
Based on this, it is necessary to provide an anti-aging composition, a preparation method and application thereof, which are different from the prior art, so as to promote the generation of Collagen, in particular to promote the generation of Collagen i, while removing superoxide anion free radicals and hydroxyl free radicals, thereby realizing the anti-wrinkle tightening effect of the composition.
An anti-aging composition comprises, by mass, 0.03% -2.0% of chlorella extract, 0.01% -1.0% of Fucus vesiculosus extract, 0.01% -1.0% of kelp palm extract, 0.5% -1% of tea extract, 0.5% -2% of yeast fermentation product filtrate, 3% -5% of nicotinamide, 0.005% -0.5% of beta-glucan, 1% -6% of butanediol, 0.05% -2% of glycerol, 0.5% -1% of preservative and the balance deionized water, wherein the preservative is one or more selected from 1, 2-hexanediol, 1, 2-pentanediol, octaethylene glycol, sodium benzoate, phenoxyethanol, ethylhexyl glycerol, p-hydroxyacetophenone or octaethylene glycol;
wherein the mass ratio of the chlorella extract, the Fucus vesiculosus extract and the kelp extract is 3:1:1.
In one embodiment, the composition comprises, by mass, 1.5% of chlorella extract, 0.5% of Fucus vesiculosus extract, 0.5% of kelp palmate extract, 1% of tea extract, 2% of yeast fermentation product filtrate, 3% of nicotinamide, 0.5% of beta-glucan, 6% of butanediol, 2% of glycerol, 0.1% of 1, 2-pentanediol, 0.1% of phenoxyethanol, 0.4% of p-hydroxyacetophenone and the balance deionized water.
In one embodiment, the preparation method of the chlorella extract comprises the following steps:
SA 1 : firstly, selecting clean and intact chlorella as a raw material, and cleaning the chlorella for later use;
SA 2 : then adding water and chlorella into a kettle according to the mass fraction of 17:1, heating to 80-85 ℃ for extraction, sampling and detecting after 1-2 hours of reaction, cooling to 38 ℃ by using cooling water when the solid content of a sample is more than or equal to 4%, and filtering by using 800-mesh filter cloth;
SA 3 : finally, for SA 2 Detecting the filtrate obtained in the step, filtering and packaging after the detection is qualified, and obtaining the chlorella extract.
In one embodiment, at step SA 3 In the process, the liquid crystal display device comprises a liquid crystal display device,the total colony count of the chlorella extract is less than or equal to 100CFU/mL, the total count of mould and saccharomycetes is less than or equal to 10CFU/mL, the pH value is 5.0-7.0, the solid content is more than or equal to 4%, the total nitrogen content is more than or equal to 0.2%, and the nucleotide and aromatic amino acid OD260nm is more than or equal to 15.
In one embodiment, the method for preparing the fucus extract comprises the following steps:
SB 1 : firstly, selecting clean and intact Fucus vesiculosus as a raw material, and cleaning the Fucus vesiculosus for later use;
SB 2 : then adding water and Fucus vesiculosus into a kettle according to the mass fraction of 17:1, heating to 80-85 ℃ for extraction, reacting for 1-2 hours, sampling and detecting, cooling to 38 ℃ by cooling water when the polysaccharide content of a sample is more than or equal to 0.3%, and filtering by using 800-mesh filter cloth;
SB 3 : SB is to be the 2 The filtrate obtained in the step and 1,2 hexanediol are mixed according to the mass fraction of 99.5:0.5, uniformly stirring, uniformly mixing, sterilizing, sampling and detecting the finished product, filtering and packaging after the detection is qualified, and obtaining the Fucus vesiculosus extract.
In one embodiment, at step SB 3 Wherein the total number of colonies of the Fucus vesiculosus extract is less than or equal to 100CFU/mL, the total number of moulds and saccharomycetes is less than or equal to 10CFU/mL, the pH value is 4.0-7.0, the refractive index is 1.320-1.350, and the relative density is 0.980-1.100.
In one embodiment, the preparation method of the kelp extract comprises the following steps:
SC 1 : firstly, selecting clean and intact kelp as a raw material, and cleaning the kelp for later use;
SC 2 : then adding water and kelp into a kettle according to the mass fraction of 17:1, heating to 80-85 ℃ for extraction, reacting for 1-2 hours, sampling and detecting, cooling to 38 ℃ by cooling water when the polysaccharide content of a sample is more than or equal to 0.3%, and filtering by using 800-mesh filter cloth;
SC 3 : finally, for SC 2 Detecting the filtrate obtained in the step, filtering and packaging after the detection is qualified to obtain the kelp extract。
In one embodiment, at step SC 3 Wherein the total number of bacterial colonies of the kelp extract is less than or equal to 100CFU/mL, the total number of mould and saccharomycetes is less than or equal to 10CFU/mL, the solid content is more than or equal to 3%, and the pH value is 5.0-8.0.
The anti-aging composition can effectively remove free radicals and promote the formation of collagen through the synergistic effect of the chlorella extract, the fucus extract and the kelp extract, and on the basis, the tea extract is added to further improve the removal rate of the free radicals; in addition, by compounding nicotinamide and beta-glucan, the synthesis of collagen can be further promoted, the skin color is lightened, and the whitening and tightening effects are achieved.
The chlorella extract is used as a skin conditioner, can promote the activity of fibroblasts and the synthesis of Collagen and elastin, can promote the growth of Collagen I and Collagen III, resist the stimulation of ultraviolet rays, and reduce the secretion of metalloproteinase and the degradation of Collagen.
The Fucus vesiculosus extract and the kelp palm extract are used as skin conditioning agents, have good moisturizing capability, can regulate and control the expression of constitutive protein genes of keratinized envelope, improve water locking capability, and simultaneously have antioxidant capability, and can efficiently remove superoxide anion free radicals and hydroxyl free radicals.
The tea extract is used as a skin conditioner, has antioxidation, can effectively remove oxidative free radicals, can reduce aggregation of macrophages and neutrophils, and has anti-inflammatory and soothing effects; the nicotinamide is used as a skin conditioner, has the effects of whitening and controlling oil, and can promote the synthesis of collagen; the beta-glucan is used as a skin conditioner and has the effects of resisting wrinkle and tightening; the yeast fermentation product filtrate is used as a skin conditioner, can balance grease, resist oxidization and repair skin, and simultaneously moisten and moisturize a cuticle, so that the absorption and conversion of nutrient substances are facilitated.
The butanediol and the glycerol are used as moisturizing agents, have good moisturizing effect on skin, can help the skin to lock moisture, and can also construct a protective film on the surface layer of the skin to strengthen the moisturizing effect and keep the skin moist; the 1, 2-hexanediol and the 1, 2-pentanediol are used as moisturizers, play a synergistic effect with preservatives, and can effectively resist fungi and improve the antibacterial effect while changing the permeability of cell membranes; the ethylhexyl glycerol and the octyl glycol are used as moisturizers, so that the moisturizing and skin-moistening effects are achieved, meanwhile, the antibacterial functions are achieved, and the mild and antiseptic effects can be achieved.
The sodium benzoate is used as an acid preservative, the optimal pH value of the preservative is 2.5-4.0, and the sodium benzoate has obvious inhibition effect on various microorganisms when the pH value is 3.5; the phenoxyethanol is used as a preservative, and acts on a cell membrane to increase the passing rate of potassium ions, so that the activity of enzyme is reduced, the growth of cells is inhibited, and the antibacterial effect is further achieved.
A method of preparing an anti-aging composition comprising the steps of:
s1: firstly, adding deionized water, butanediol, glycerol and p-hydroxyacetophenone into an emulsifying pot, heating to 85-90 ℃, and cooling to 45 ℃ for standby after the materials are completely dissolved;
s2: then adding chlorella extract, fucus vesiculosus extract, kelp extract, tea extract, yeast fermentation product filtrate, nicotinamide, beta-glucan, 1, 2-pentanediol and phenoxyethanol into an emulsifying pot one by one at the temperature of 45 ℃, and uniformly stirring at the speed of 500 r/min;
s3: finally, when the temperature of the material body is reduced to 42 ℃, filtering is carried out by using 800-mesh filter cloth, and the anti-aging composition is obtained after discharging.
An anti-aging composition is used for preparing cosmetic or skin care product.
The anti-aging composition can effectively remove free radicals, has an antioxidation effect, has good promotion rate on colla genes, collal genes, colla2 genes and the like, has a remarkable promotion effect on UVA radiation induced fibroblast Collagen I, collagen III and protein concentration, and can be applied to dosage forms such as facial masks, essence, emulsion, cream and the like to lighten skin color, provide moisture for skin, nourish the skin and have good anti-wrinkle tightening effects.
Detailed Description
The following detailed description of the present invention will provide further details in order to make the above-mentioned objects, features and advantages of the present invention more comprehensible. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be embodied in many other forms than described herein and similarly modified by those skilled in the art without departing from the spirit of the invention, whereby the invention is not limited to the specific embodiments disclosed below.
The term "prepared from …" as used herein is synonymous with "comprising". The terms "comprising," "including," "having," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.
When an equivalent, concentration, or other value or parameter is expressed as a range, preferred range, or a range bounded by a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when ranges of "1 to 5" are disclosed, the described ranges should be construed to include ranges of "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a numerical range is described herein, unless otherwise indicated, the range is intended to include its endpoints and all integers and fractions within the range.
Furthermore, the indefinite articles "a" and "an" preceding an element or component of the invention are not limited to the requirements of the number of elements or components (i.e. the number of occurrences). Thus, the use of "a" or "an" should be interpreted as including one or at least one, and the singular reference of an element or component also includes the plural reference unless the amount is obvious to the singular reference.
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. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Example 1
Firstly, adding 6% of butanediol, 2% of glycerol, 0.4% of p-hydroxyacetophenone and deionized water into an emulsifying pot, heating to 85 ℃, and cooling to 45 ℃ after the material is completely dissolved; then, 0.04% of Fucus vesiculosus extract, 0.04% of kelp extract, 0.12% of chlorella extract, 1% of tea extract, 2% of yeast fermentation product filtrate, 3% of nicotinamide, 0.5% of beta-glucan, 0.1% of 1, 2-pentanediol and 0.1% of phenoxyethanol are added into an emulsifying pot at the temperature of 45 ℃ and stirred uniformly at the speed of 500 r/min; finally, after the temperature of the material body is reduced to 42 ℃, filtering by using 800-mesh filter cloth, detecting the filtrate, and discharging after the detection is qualified, thus obtaining the anti-aging composition.
In this process, 3% nicotinamide and deionized water are mixed and dissolved in advance to be transparent, 0.1%1, 2-pentanediol and 0.1% phenoxyethanol are mixed to be transparent, and then added into an emulsifying pot.
Example 2
This example differs from example 1 in that the amounts of the chlorella extract, the fucus extract and the kelp extract are different.
Specifically, the amount of the chlorella extract is 0.24%, the amount of the Fucus vesiculosus extract is 0.08%, and the amount of the kelp extract is 0.08%.
Example 3
This example differs from example 1 in that the amounts of the chlorella extract, the fucus extract and the kelp extract are different.
Specifically, the amount of the chlorella extract is 0.48%, the amount of the Fucus vesiculosus extract is 0.16%, and the amount of the kelp extract is 0.16%.
Example 4
This example differs from example 1 in that the amounts of the chlorella extract, the fucus extract and the kelp extract are different.
Specifically, the amount of the chlorella extract is 0.96%, the amount of the Fucus vesiculosus extract is 0.32%, and the amount of the kelp extract is 0.32%.
Example 5
This example differs from example 1 in that the amounts of the chlorella extract, the fucus extract and the kelp extract are different.
Specifically, the amount of the chlorella extract is 1.5%, the amount of the Fucus vesiculosus extract is 0.5%, and the amount of the kelp extract is 0.5%.
Example 6
This example differs from example 1 in that the amounts of the chlorella extract, the fucus extract and the kelp extract are different.
Specifically, the amount of the chlorella extract was 1.92%, the amount of the fucus extract was 0.64%, and the amount of the kelp extract was 0.64%.
Comparative example 1
This comparative example differs from example 1 in that it does not contain tea leaf extract.
Comparative example 2
Vitamin C (pure substance).
Comparative example 3
100 μg/ml vitamin C and 7 μg/ml vitamin E.
The samples of examples 1 to 6 and comparative examples 1 to 3 were examined respectively, and the specific examination methods and examination results were as follows:
1) Superoxide anion scavenger
4mL of Tris-HCl buffer with the concentration of 0.05mmol/L, pH value of 8.2 is taken, preheated at 25 ℃ for 20min, then the sample liquid of examples 1-6, comparative example 1, 0.5mg/mL comparative example 2, 0.25mg/mL comparative example 2, 0.125mg/mL comparative example 2, 0.0625mg/mL comparative example 2, 0.0312mg/mL comparative example 2 and comparative example 3 is respectively added, 1mL of 0.2mmol/L pyrogallol solution is added, after uniform mixing, the reaction is carried out in a water bath at 25 ℃ for 4min, the reaction is immediately terminated by two drops of concentrated HCl, and the absorbance A sample is measured at 325nm, repeated 3 times per tube and the average value is obtained. The above procedure was repeated, and the sample solution was replaced with distilled water, and the A-source was measured at a wavelength of 325nm, and the results were calculated, and the results are shown in Table 1.
Wherein, the calculation formula is: superoxide anion clearance/% = (pro-a-sample)/(pro-a) ×100.
TABLE 1
| Test sample | Superoxide anion clearance/% |
| Example 1 | 3.68 |
| Example 2 | 16.95 |
| Example 3 | 30.54 |
| Example 4 | 54.22 |
| Example 5 | 75.36 |
| Example 6 | 73.60 |
| Comparative example 1 | 2.37 |
| Comparative example 2 (0.0312 mg/mL) | 56.54 |
| Comparative example 2 (0.0625 mg/mL) | 60.76 |
| Comparative example 2 (0.125 mg/mL) | 66.87 |
| Comparative example 2 (0.25 mg/mL) | 77.84 |
| Comparative example 2 (0.5 mg/mL) | 91.49 |
| Comparative example 3 | 10.68 |
As can be seen from Table 1, the anti-aging composition is effective in scavenging superoxide anion radicals and has antioxidant effect.
2) Radical scavenging rate of hydroxyl groups
Firstly, adding 0.75mmol/L phenanthroline solution into a system containing PBS and distilled water, then adding 0.75mmol/L ferrous sulfate solution, uniformly mixing, then adding 0.01% H 2 O 2 Shaking the solution uniformly and thenIncubation at 37℃for 60min, followed by determination of absorbance A damage at 536 nm; in addition, sample solutions of examples 1-6, comparative example 1, 0.5mg/mL comparative example 2, 0.25mg/mL comparative example 2, 0.125mg/mL comparative example 2, 0.0625mg/mL comparative example 2, 0.0312mg/mL comparative example 2, comparative example 3 were added, respectively, and sample A was tested at 536 nm; repeating the above operation, and substituting distilled water for H 2 O 2 The solution was measured intact at 536nm wavelength. Each tube was repeated 3 times, averaged, and the results were calculated, the results of which are shown in table 2.
The calculation formula is as follows: hydroxyl radical clearance/% = (a-like-a damage)/(a undamaged-a damage) ×100.
TABLE 2
| Test sample | Hydroxyl radical scavenging/% |
| Example 1 | 20.31 |
| Example 2 | 28.49 |
| Example 3 | 45.13 |
| Example 4 | 60.10 |
| Example 5 | 90.26 |
| Example 6 | 87.04 |
| Comparative example 1 | 18.97 |
| Comparative example 2 (0.0312 mg/mL) | 11.41 |
| Comparative example 2 (0.0625 mg/mL) | 21.53 |
| Comparative example 2 (0.125 mg/mL) | 32.05 |
| Comparative example 2 (0.25 mg/mL) | 60.05 |
| Comparative example 2 (0.5 mg/mL) | 100.58 |
| Comparative example 3 | 32.78 |
As can be seen from table 2, the anti-aging composition is capable of effectively scavenging hydroxyl radicals and playing an antioxidant role.
3) DPPH radical scavenging rate
The positive control was diluted with 95% ethanol to: a series of concentration gradients of 0.08mg/ml, 0.04mg/ml, 0.02mg/ml, 0.01mg/ml was used to verify the assay system; the sample to be tested is diluted with water into a multi-stage concentration sample, and the product can be diluted according to actual conditions because the product is liquid.
Specifically, referring to Table 3, a 10ml test tube was used to set up a sample tube (T), a sample background (T 0 ) DPPH tube (C) and solvent background (C) 0 ) The sample tube (T) of each tested concentration of each sample needs to be provided with 3 samplesParallel pipes, and 3 parallel pipes are needed to be arranged in the DPPH pipe (C); in the sample tube (T) and sample background (T 0 ) 1ml of each sample solution of the same concentration was added. In all tubes (T, T 0 、C、C 0 ) Supplementing the solvent, namely supplementing the water-soluble sample with water, supplementing the oil-soluble sample with 95% ethanol, supplementing 3ml of the oil-soluble sample, and uniformly mixing; in addition, 1ml of DPPH ethanol solution was added to the sample tube (T) and the DPPH tube (C), and the sample background (T) 0 ) And solvent background (C) 0 ) The reaction solution was allowed to stand at room temperature for 5 minutes with 95% ethanol instead of the above, and then transferred to a 1cm cuvette, absorbance was measured at 517nm, and the results were calculated after recording the values, as shown in Table 4.
Wherein, the calculation formula of DPPH free radical clearance is:
wherein:
t-absorbance of the sample tube, i.e., absorbance of the solution after reaction of the sample with DPPH;
T 0 -sample background absorbance;
c, the average value of the absorbance of the DPPPH pipe for 3 times, namely the absorbance value of the DPPH solution when no sample is added;
C 0 -solvent background absorbance.
TABLE 3 Table 3
It should be noted that positive control tests were added for each batch of test.
And calculating the free radical clearance of the sample under each tested concentration according to a calculation formula of the DPPH free radical clearance, and simultaneously counting standard deviation (Standard Deviation, SD) among clearance among all groups of parallel pipes, wherein the SD value is less than or equal to 3%, and the test parallelism is considered to be effective.
TABLE 4 Table 4
| Test sample | DPPH radical scavenging/% |
| Example 1 | 41.67 |
| Example 2 | 50.68 |
| Example 3 | 58.83 |
| Example 4 | 63.20 |
| Example 5 | 72.99 |
| Example 6 | 71.56 |
| Comparative example 1 | 39.71 |
| Comparative example 2 | 62.69 |
| Comparative example 3 | 39.47 |
As can be seen from Table 4, the anti-aging composition has a higher clearance rate for DPPH free radicals and has a better antioxidant capacity.
4) Cytotoxicity test:
(1) cell inoculation: cells were seeded at a plating density of 8X 103 cells/well in 96-well plates and in an incubator (37 ℃, 5% CO) 2 ) Incubate overnight.
(2) Experimental grouping: setting a zeroing group, a negative control group, a positive control group and a sample group in an experiment; in the sample group, 5 concentration gradients were set for each sample, and 3 duplicate wells were set for each concentration gradient.
(3) Preparing liquid: sample working solutions with different concentrations are prepared according to a test concentration setting table.
(4) Administration: and when the cell plating rate in the 96-well plate reaches 40% -60%, the administration is carried out. 200. Mu.L of 10% PBS in culture medium was added to each well of the control group; 200. Mu.L of culture medium containing 10% DMSO was added to each well of the positive control group; 200 mu L of culture solution containing samples with corresponding concentrations is added into each hole of the sample group; the zeroed group was inoculated without cells and only 200. Mu.L of cell culture medium was added. After completion of the administration, the 96-well plate was placed in an incubator (37 ℃ C., 5% CO) 2 ) Is cultured.
(5) And (3) detection: after incubation of the cells for 24h, the supernatant was discarded, 0.5mg/mL MTT working solution was added, incubated at 37℃in the absence of light for 2h, after incubation, the supernatant was discarded, and 100. Mu.L DMSO was added to each well and the OD was read at 490 nm.
(6) The relative cell viability was calculated as shown in Table 5, and the calculation formula was:
cell relative viability/% = (sample well OD-zeroed well OD)/(solvent control well OD-zeroed well OD) ×100.
TABLE 5
As can be seen from Table 5, none of examples 1-6 showed fiber cytotoxicity, demonstrating that the anti-aging compositions of the present invention are safe and mild.
5) The detection method for the expression promotion rate of the zebra fish type I collagen gene col1a1a, the expression promotion rate of the zebra fish type I collagen gene col1a1b and the expression promotion rate of the zebra fish type I collagen gene col1a2 comprises the following steps:
(1) healthy 6-day-old zebra fish after fertilization were selected.
(2) Test packets:
a. setting a blank control group (fish embryo culture solution), a positive control group (acetyl hexapeptide-8) and example 5 (test sample); if the organic solvent is used for cosolvent, the concentration of the solvent in each group of solution needs to be the same;
blank group: randomly selecting 36 zebra fish, uniformly distributing the zebra fish into 24 pore plates, and arranging 3 pores, wherein each pore contains 12 zebra fish and 2.5mL fish embryo culture solution;
positive control group: randomly selecting 36 zebra fish, uniformly distributing the zebra fish into 24 pore plates, and arranging 3 pores, wherein each pore contains 12 zebra fish and 2.5mL of acetyl hexapeptide-8 working solution;
sample treatment: randomly selecting 36 zebra fish, uniformly distributing the zebra fish into 24 pore plates, and arranging 3 pores, wherein each pore contains 12 zebra fish and 2.5mL of test object solution;
b. placing the blank control group, the positive control group and the tested sample in an incubator at 28+/-1 ℃ respectively, and culturing 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.
(3) RNA extraction:
a. removing RNAlater solution, washing 3 times with PBS solution, placing in ice bath, removing PBS solution, adding 500 μl TRIzol reagent, homogenizing embryo with particle pestle, and placing in ice bath for 10min;
b. adding 100 mu L of chloroform, swirling for 1min, placing in ice bath for 5min, centrifuging the sample for 20min, transferring the upper layer solution to a 1.5mL centrifuge tube, adding 250 mu L of isopropanol, swirling for 1min, placing in ice bath for 10min, centrifuging the sample for 20min;
c. removing all supernatant, adding 500 μl ethanol, swirling for 1min, centrifuging for 5min, and placing in ice bath for 10min; repeating the steps for 3 times, removing ethanol, centrifuging the sample for 5min, opening a cover in a fume hood, and air-drying the sample for 10min;
d. 10. Mu.L of DNase/RNase-free ultrapure distilled water was added and heated at 55℃for 15min.
(4) cDNA synthesis: RNA samples were diluted to 1000 ng/7. Mu.L with DNase/RNase free ultra-pure distilled water, then RNA was synthesized into cDNA using PrimerScript RT kit containing gDNA Eraser and stored frozen.
(5) Real-time RT-PCR was performed: preparing a primer mixture and a real-time PCR master mix for each primer; the cDNA sample was diluted 10-fold with DNase/RNase-free ultrapure distilled water, 9. Mu.L of the PCR master mix and 1. Mu.L of the diluted cDNA sample were added to each well of the PCR plate, and then the PCR plate was sealed with an optical film, centrifuged for 5min, and real-time PCR amplification was performed.
(6) Data and result calculation: real-time PCR data were collected, ct was used as an amplification result, beta-actin gene amplification amount was used as a housekeeping gene, and the relative expression amounts of each gene (col 1a1a, col1a1b and col1a 2) were calculated as test results, and the results are shown in Table 6.
TABLE 6
As can be seen from Table 6, example 5 has a better promotion rate for the expression of the colla gene, the collal gene, the colla2 gene and the like, and is obviously better than comparative examples 1-3, which demonstrates that the anti-aging composition of the present invention can effectively promote the generation of collagen.
In summary, example 5 is the most preferred example, so further efficacy testing was performed on example 5.
1) Efficacy test (promote the production ability of Collagen I, collagen III, total Collagen):
a. cell inoculation: at a suitable inoculation density (8X 10) 4 Cells were seeded into 24-well plates and incubated in an incubator (37 ℃, 5% CO) 2 ) Incubate overnight.
b. Experimental grouping: the experiment sets a blank control group, a negative control group, a positive control group and a sample group, wherein the sample group is provided with 3 concentration gradients.
c. Preparing liquid: and preparing test object working solutions with different concentrations according to a test concentration setting table.
d. Administration: and (3) when the cell plating rate in the 24-well plate reaches 40% -60%, administration is carried out. Adding 1mL of cell culture solution into each hole of the blank control group and the negative control group; 1mL of a culture solution containing 100pg/mL of vitamin C and 7 mug/mL of vitamin E was added to each well of the positive control group; 1mL of culture medium containing the test substance at the corresponding concentration is added to each hole of the sample group.
e. Radiation: after 24 hours from the completion of the administration, the total dose received by the negative control group, the positive control group and the sample group was 9J/cm 2 Is set in the room; meanwhile, the blank group is placed in the same environment, and the UVA radiation dose is 0J/cm 2 。
f. Cell supernatant was collected: after incubation for 24h, the cell culture supernatant was collected in EP tubes and stored frozen in a refrigerator at-80 ℃.
g. Collecting cells: after incubation for 24h, 0.5mL of lysis solution was added to each well and the lysis was performed by pipetting. The cell sap is collected in an EP tube, and after the cell sap is collected, a sample for detection is placed in a refrigerator at the temperature of-80 ℃ for freezing and preserving.
h. Detecting and analyzing the contents of the CollagenI and the CollagenIII according to the operation instruction of the ELISA detection kit; the BCA protein concentration was assayed according to the instructions of the BCA kit, with the Collagen i assay shown in table 6, the Collagen iii assay shown in table 7, and the BCA protein concentration assay shown in table 8.
TABLE 7
| Test sample | Collagen I content (ng/ml) | SD | p-value |
| Blank control group (BC) | 50.89 | 0.67 | / |
| Negative control group (NC) | 38.85 | 3.67 | 0.0121 |
| Positive control group (comparative example 2) | 56.78 | 3.78 | 0.0005 |
| Positive control group (comparative example 3) | 61.79 | 4.32 | 0.0003 |
| Example 5 | 57.53 | 2.86 | 0.0097 |
As can be seen from Table 7, the total dose received by the fibroblasts in the negative control group was 9J/cm compared with the blank group 2 After UVA irradiation of (c), the levels of Collagen I content were significantly reduced (p<0.05 Indicating that the irradiation stimulus is effective; vitamin C, pair of comparative example 2 compared to the negative control groupThe mixed solution of vitamin C and vitamin E in proportion 3 can obviously improve the content level (p<0.01 And the positive control detection is effective.
TABLE 8
| Test sample | Collagen III content (ng/ml) | SD | p-value |
| Blank control group (BC) | 1178.63 | 3.45 | / |
| Negative control group (NC) | 1156.25 | 4.91 | 0.003 |
| Positive control group (comparative example 2) | 1164.47 | 6.76 | 0.002 |
| Positive control group (comparative example 3) | 1207.04 | 6.89 | 0.003 |
| Example 5 | 1165.39 | 0.76 | 0.035 |
As can be seen from Table 8, the total dose received by the fibroblasts in the negative control group was 9J/cm compared with the blank group 2 After UVA irradiation, the levels of Collagen III content were significantly reduced (p<0.05 Indicating that the irradiation stimulus is effective; the vitamin C of comparative example 2, the vitamin C and vitamin E mixture of comparative example 3 significantly increased the content level (p<0.01 And the positive control detection is effective.
TABLE 9
| Test sample | Average concentration (μg/ml) | SD | p-value |
| Blank control group (BC) | 387.47 | 4.97 | / |
| Negative control group (NC) | 363.75 | 2.93 | 0.017 |
| Positive control group (comparative example 2) | 371.47 | 5.27 | 0.027 |
| Positive control group (comparative example 3) | 386.91 | 7.05 | 0.026 |
| Example 5 | 371.29 | 5.17 | 0.033 |
As can be seen from Table 9, the total dose received by the fibroblasts in the negative control group was 9J/cm compared with the blank group 2 After UVA irradiation of (2) the protein concentration level was significantly reduced (p<0.05 Indicating that the irradiation stimulus is effective; the mixed solution of vitamin C of comparative example 2, vitamin C of comparative example 3 and vitamin E can significantly increase the protein concentration level (p<0.01 The positive control test was shown to be effective.
In summary, this example 5 shows a significant elevation (p < 0.01) of UVA radiation induced fibroblast Collagen I, collagen III and protein concentration under anti-wrinkle compaction-UVA stimulated human fibroblast test method, compared to negative control group, demonstrating anti-wrinkle compaction efficacy of the anti-aging composition of the present invention.
The anti-aging composition can effectively remove free radicals through the synergistic effect of the chlorella extract, the Fucus vesiculosus extract and the kelp extract, promote the synthesis of collagen, and can lighten skin color, provide moisture for skin, nourish skin and achieve the effects of whitening, resisting wrinkles and tightening when being applied to cosmetics or skin care products.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. An anti-aging composition is characterized by comprising, by mass, 0.03% -2.0% of chlorella extract, 0.01% -1.0% of Fucus vesiculosus extract, 0.01% -1.0% of kelp palm extract, 0.5% -1% of tea extract, 0.5% -2% of yeast fermentation product filtrate, 3% -5% of nicotinamide, 0.005% -0.5% of beta-glucan, 1% -6% of butanediol, 0.05% -2% of glycerol, 0.5% -1% of preservative and the balance deionized water, wherein the preservative is selected from one or more of 1, 2-hexanediol, 1, 2-pentanediol, octaethylene glycol, sodium benzoate, phenoxyethanol, ethylhexyl glycerol, p-hydroxyacetophenone or octaethylene glycol;
wherein the mass ratio of the chlorella extract, the Fucus vesiculosus extract and the kelp extract is 3:1:1.
2. The anti-aging composition according to claim 1, comprising, by mass, 1.5% of chlorella extract, 0.5% of fucus extract, 0.5% of kelp extract, 1% of tea extract, 2% of yeast fermentation product filtrate, 3% of nicotinamide, 0.5% of beta-glucan, 6% of butanediol, 2% of glycerol, 0.1% of 1, 2-pentanediol, 0.1% of phenoxyethanol, 0.4% of p-hydroxyacetophenone and the balance deionized water.
3. The anti-aging composition according to claim 1 or 2, wherein the preparation method of the chlorella extract comprises the steps of:
SA 1 : firstly, selecting clean and intact chlorella as a raw material, and cleaning the chlorella for later use;
SA 2 : then adding water and chlorella into a kettle according to the mass fraction of 17:1, heating to 80-85 ℃ for extraction, sampling and detecting after 1-2 hours of reaction, cooling to 38 ℃ by using cooling water when the solid content of a sample is more than or equal to 4%, and filtering by using 800-mesh filter cloth;
SA 3 : finally, for SA 2 Detecting the filtrate obtained in the step, filtering and packaging after the detection is qualified, and obtaining the chlorella extract.
4. An anti-ageing composition according to claim 3, characterised in that in step SA 3 Wherein the total colony count of the chlorella extract is less than or equal to 100CFU/mL, the total count of mould and saccharomycetes is less than or equal to 10CFU/mL, the pH value is 5.0-7.0, the solid content is more than or equal to 4%, the total nitrogen content is more than or equal to 0.2%, and the nucleotide and aromatic amino acid OD260nm is more than or equal to 15.
5. The anti-aging composition according to claim 1 or 2, wherein the method for preparing the fucus extract comprises the steps of:
SB 1 : firstly, selecting clean and intact Fucus vesiculosus as a raw material, and cleaning the Fucus vesiculosus for later use;
SB 2 : then adding water and Fucus vesiculosus into a kettle according to the mass fraction of 17:1, heating to 80-85 ℃ for extraction, reacting for 1-2 hours, sampling and detecting, cooling to 38 ℃ by cooling water when the polysaccharide content of a sample is more than or equal to 0.3%, and filtering by using 800-mesh filter cloth;
SB 3 : SB is to be the 2 The filtrate obtained in the step and 1,2 hexanediol are mixed according to the mass fraction of 99.5:0.5, uniformly stirring, sterilizing after uniformly mixing,and sampling and detecting the finished product, filtering and packaging after the detection is qualified, and obtaining the Fucus vesiculosus extract.
6. An anti-aging composition according to claim 5, wherein in step SB 3 Wherein the total number of colonies of the Fucus vesiculosus extract is less than or equal to 100CFU/mL, the total number of moulds and saccharomycetes is less than or equal to 10CFU/mL, the pH value is 4.0-7.0, the refractive index is 1.320-1.350, and the relative density is 0.980-1.100.
7. An anti-ageing composition according to claim 1 or 2, characterized in that the preparation method of the kelp extract comprises the following steps:
SC 1 : firstly, selecting clean and intact kelp as a raw material, and cleaning the kelp for later use;
SC 2 : then adding water and kelp into a kettle according to the mass fraction of 17:1, heating to 80-85 ℃ for extraction, reacting for 1-2 hours, sampling and detecting, cooling to 38 ℃ by cooling water when the polysaccharide content of a sample is more than or equal to 0.3%, and filtering by using 800-mesh filter cloth;
SC 3 : finally, for SC 2 Detecting the filtrate obtained in the step, filtering and packaging after the detection is qualified, and obtaining the kelp extract.
8. The anti-aging composition of claim 7, wherein at step SC 3 Wherein the total number of bacterial colonies of the kelp extract is less than or equal to 100CFU/mL, the total number of mould and saccharomycetes is less than or equal to 10CFU/mL, the solid content is more than or equal to 3%, and the pH value is 5.0-8.0.
9. A process for the preparation of an anti-ageing composition according to any of claims 1 to 8, comprising the steps of:
s1: firstly, adding deionized water, butanediol, glycerol and p-hydroxyacetophenone into an emulsifying pot, heating to 85-90 ℃, and cooling to 45 ℃ for standby after the materials are completely dissolved;
s2: then adding chlorella extract, fucus vesiculosus extract, kelp extract, tea extract, yeast fermentation product filtrate, nicotinamide, beta-glucan, 1, 2-pentanediol and phenoxyethanol into an emulsifying pot one by one at the temperature of 45 ℃, and uniformly stirring at the speed of 500 r/min;
s3: finally, when the temperature of the material body is reduced to 42 ℃, filtering is carried out by using 800-mesh filter cloth, and the anti-aging composition is obtained after discharging.
10. Use of an anti-ageing composition according to any of claims 1-8 or prepared by the method of preparation of claim 9 in the preparation of a cosmetic or skin care product.
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