CN111419743A - Preparation method and application of vitis amurensis extract - Google Patents
Preparation method and application of vitis amurensis extract Download PDFInfo
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- CN111419743A CN111419743A CN202010139014.7A CN202010139014A CN111419743A CN 111419743 A CN111419743 A CN 111419743A CN 202010139014 A CN202010139014 A CN 202010139014A CN 111419743 A CN111419743 A CN 111419743A
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- deionized water
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Images
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—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
- A61K8/9722—Chlorophycota or Chlorophyta [green algae], e.g. Chlorella
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Dermatology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Cosmetics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides a preparation method of a sea grape extract, which is characterized by comprising the following steps: (a) cleaning and crushing the sea grape with deionized water, and then adding deionized water and an antioxidant, heating, stirring and extracting; (b) cooling, coarse filtering and fine filtering; (c) performing suction filtration, and sterilizing and preserving filtrate; (d) precipitating with ethanol, and freeze drying to obtain Vitis heyneana extract. Compared with the prior art, the method for preparing the sea grape extract has the advantages of simple and convenient extraction and separation operation, high extraction rate of effective active ingredients and higher purity of the finally prepared product. The invention directly adopts water as a solvent, and greatly improves the extraction rate of the sea grapes and the dissolution rate of active ingredients under the enzymolysis action of pectinase or cellulase, and improves the content of the active ingredients with the functions of moisturizing and resisting aging.
Description
Technical Field
The invention relates to the field of cosmetics, and particularly relates to a preparation method of a vitis amurensis extract. In addition, the invention also relates to the moisturizing and anti-aging effects of the vitis amurensis extract obtained by the method, and particularly relates to the application of the vitis amurensis extract in cosmetics.
Background
Anti-aging is always the focus of long-term attention in the cosmetic industry, and it is one of the pursuit targets of most people to delay skin aging and make the skin more tender. Environmental factors accelerate the skin aging speed to a certain extent, and simultaneously, with the improvement of economic level and the higher pursuit of people on beauty, cosmetics with the anti-aging effect are developed vigorously. While the basic elements of healthy skin are smooth, full, and elastic, with increasing age, the skin becomes increasingly dry, wrinkled, flaccid, and has an aged appearance. The pathological mechanism of the aging appearance is mainly divided into two types, one is the reduction of the water content of the skin water horny layer, and the other is the skin aging and the occurrence of skin diseases caused by oxidative stress. Therefore, the anti-aging is from the viewpoint of water supplement and oxidation resistance. Therefore, the appearance of the natural plant moisturizing and anti-aging active ingredients fills the gap in the aspect and is favored by the market and consumers.
Acaudina grape science name: caulerpa lentillifera belongs to Chlorophyta, Alopetalae, Caulerpa, and Caulerpa. The grape jelly is full in appearance, is like a string of glittering and translucent grapes, has unique appearance and mouthfeel, can make a radish-like sound when being bitten, has a salty and fresh fragrance of sea flavor given out by beautiful green small particles, has the charm of caviar, is liked to be 'green caviar' in plants, and the extract of the grape jelly can be used as a cosmetic raw material to be widely used in a formula.
The existing extraction process of sea grape usually uses organic solvent such as ethanol, for example, taiwan patent I484966, which is a method of roughly extracting sea grape powder with alcohol, and then sequentially separating effective substances with n-hexane and hexyl acetate, and the extracted effective substances have the effect of inhibiting cancer; korean patent KR101252149 uses an organic solvent to extract the sea grape, and the extract has antioxidant and moisturizing effects. The sea grape is extracted by the organic solvent, and the sea grape is added into the cosmetic formula, so that sensitization is possible, and even if a small amount of organic solvent is remained, the extract can cause harm to skin and even human bodies. Therefore, it is a problem to be solved in the present invention that the extract or active ingredient of the Vitis amurensis which does not use organic solvent and has the effects of moisturizing and delaying skin aging is extracted.
Disclosure of Invention
The invention aims to provide a sea grape extract with the functions of moisturizing and delaying skin aging, which is prepared by the provided method, and the main component of the sea grape extract is sea grape polysaccharide; the extractive of the sea grape can be used as an effective raw material for cosmetics for moisturizing and delaying skin aging.
Accordingly, it is an object of the present invention to provide a method for preparing a vitis amurensis extract, which can be prepared by the following method:
(a) cleaning and crushing the sea grape with deionized water, and then adding deionized water and an antioxidant, heating, stirring and extracting;
(b) cooling, coarse filtering and fine filtering;
(c) performing suction filtration, and sterilizing and preserving filtrate;
(d) precipitating with ethanol, and freeze drying to obtain Vitis heyneana extract.
In one embodiment of the present invention, the step (a) comprises adding pectinase and/or cellulase after grinding, more preferably pectinase and/or cellulase after dilution with deionized water, wherein the pectinase with the model XX L is preferably used as the pectinase.
In one embodiment of the invention, the mass ratio of the sea grapes to the deionized water in the step (a) is 1 (4-20), the mass of the antioxidant is 0.03% of the mass of the sea grapes, the amount of the pectinase and/or cellulase is 0.06% -0.15% of the mass of the sea grapes, and the pectinase and/or cellulase is diluted with the deionized water according to the mass ratio of 1 (50-80); the stirring speed is 50-100r/min, the reaction temperature is 40-60 ℃, and the reaction time is 60-120 min. In a preferred embodiment of the invention, the mass ratio of the sea grape to the deionized water in the step (a) is 1:4, the antioxidant is sodium D-ascorbate, the amount of the pectinase and/or cellulase is 0.1% of the mass of the sea grape, and the pectinase and/or cellulase and the deionized water are diluted according to the mass ratio of 1: 60; the stirring speed is 60r/min, the reaction temperature is 60 ℃, and the reaction time is 120 min.
In one embodiment of the present invention, the step (b) comprises inactivating the enzyme. In a preferred embodiment of the present invention, the enzyme deactivation temperature in the step (b) is 70-100 ℃, and the enzyme deactivation time is 15-30 min; the final temperature of the cooling step is 20-50 ℃; the gauze used for coarse filtration has 60-200 meshes; the aperture of the filter plate used for fine filtration is 1-10 μm. More preferably, the enzyme deactivation temperature in the step (b) is 85 ℃, and the enzyme deactivation time is 20 min; the final temperature of the cooling step is 30 ℃; the gauze used for coarse filtration has 200 meshes; the aperture of the filter plate used for fine filtration is 10 mu m.
In one embodiment of the invention, the aperture of the filter plate used for suction filtration in the step (c) is 0.1-1 μm, the sterilization temperature is 85-95 ℃, the sterilization time is 20-40min, the preservative is selected from phenoxyethanol liquid or PE9010, and the usage amount of the preservative is 0.8% of the mass of the filtrate of the sea grape extract after suction filtration. In a preferred embodiment of the present invention, the final temperature of the cooling step of step (c) is 30 ℃; the aperture of the filter plate used for filtering is 0.2-0.45 μm, the sterilization condition is that the sterilization temperature is 90 ℃, and the sterilization time is 30 min.
In one embodiment of the present invention, the ethanol used in the ethanol precipitation in the step (d) is 70% to 90% by mass; freezing the precipitate after alcohol precipitation at-75 deg.C for 24 hr, and placing in freeze drying machine for freeze drying powder preparation.
The invention also aims to provide the vitis amurensis extract prepared by the preparation method of the vitis amurensis extract. The Vitis heyneana extract contains polysaccharide as effective component, and the Vitis heyneana polysaccharide has effects of keeping moisture, resisting skin aging, stimulating collagen biosynthesis, tightening skin, and reducing wrinkle. The content of polysaccharide in the Vitis amurensis extract is 526-816 mg/g.
The invention also aims to provide application of the vitis amurensis extract prepared by the method, in particular application in cosmetics with moisturizing and anti-aging effects, such as smoothing toner, emulsion, eye cream and the like.
Compared with the prior art, the method for preparing the sea grape extract has the advantages of simple and convenient extraction and separation operation, high extraction rate of effective active ingredients, higher purity of the finally prepared product and greatly improved product quality. The invention abandons the traditional method of extracting by using organic solvent, directly adopts water as solvent, and greatly improves the extraction rate of the sea grape and the dissolution rate of active ingredients under the enzymolysis action of pectinase or cellulase, and improves the content of the active ingredients with moisture retention and anti-aging properties.
Drawings
FIG. 1 is a flow chart of the production process of the sea grape extract provided by the present invention.
FIG. 2 is a diagram showing the results of the short-acting moisturizing test of the sea grape extract provided by the present invention.
FIG. 3 is a graph showing the results of skin elasticity of the extract of Vitis amurensis of the present invention.
FIG. 4 is a graph showing the result of DPPH radical scavenging of the Vitis amurensis extract according to the present invention.
Detailed Description
The invention is further illustrated below with reference to specific examples. It is to be understood, however, that these examples are illustrative only and are not to be construed as limiting the scope of the present invention. The test methods in the following examples, which are not specified under specific conditions, are generally carried out under conventional conditions. All percentages and parts are by weight unless otherwise indicated.
The production place of the sea grapes is Hainan and purchased from an offshore farm in Hainan province;
pectinase or cellulase purchased from Novoxin (China) Biotechnology, Inc.;
PE9010 was purchased from suma, germany.
Example 1 preparation of sea grape extract
(a) Weighing 10kg of cleaned sea grapes, cleaning the surfaces of the sea grapes with deionized water, placing the sea grapes in a colloid mill, slightly adding deionized water into the colloid mill, grinding, and adding deionized water with a mass ratio of 4 to the sea grapes; adding D-sodium ascorbate accounting for 0.03 percent of the weight of the sea grapes, and adding a mixture of cellulase and pectinase accounting for 0.06 percent of the weight of the sea grapes, wherein the mixture of the cellulase and pectinase is diluted with deionized water according to the mass ratio of 1: 50; extracting at 40 deg.C and 50r/min for 120 min;
(b) heating to 70 deg.C, stirring for 30min, and inactivating enzyme; stopping heating, cooling to 20 deg.C with water circulation, and coarse-filtering with 60 mesh gauze; taking the filtrate, and finely filtering the filtrate by using a filter plate with the aperture of 1 mu m to obtain light green liquid;
(c) filtering with a filter plate with the aperture of 0.1 μm to obtain light green liquid; and (3) measuring the pH value, the solid content and the conductivity, wherein the measurement result is as follows: the pH value is 4.7-6.2, the solid content is 0.55-1.1%, and the conductivity is 550-1010 mus/cm2(ii) a Then preserving the heat at 85 ℃ for 20min, and sterilizing; cooling to room temperature in water bath, and adding phenoxyethanol liquid preservative with the mass of 0.8 percent of the sea grape extraction solution;
(d) performing alcohol precipitation by using 90% of ethanol, freezing the precipitate after alcohol precipitation at-75 ℃ for 24h, and drying in a freeze dryer for 24h to obtain the final sea grape extract.
Example 2: preparation method of Vitis amurensis extract
(a) Weighing 10kg of cleaned sea grapes, cleaning the surfaces of the sea grapes with deionized water, placing the sea grapes in a colloid mill, slightly adding deionized water into the colloid mill, grinding, and adding deionized water with a mass ratio of 20 to the sea grapes; adding D-sodium ascorbate accounting for 0.03 percent of the weight of the sea grapes, adding pectinase accounting for 0.15 percent of the weight of the sea grapes, and diluting the pectinase and deionized water according to the mass ratio of 1: 60; extracting at 60 deg.C under stirring at 100r/min for 60 min;
(b) heating to 100 deg.C, stirring for 15min, and inactivating enzyme; stopping heating, cooling to 50 deg.C with water circulation, and coarse-filtering with 150 mesh gauze; taking the filtrate, and finely filtering the filtrate by using a filter plate with the aperture of 8 mu m to obtain light green liquid;
(c) the filter plate having a pore size of 1 μm was usedFiltering to obtain a bright light green liquid; and (3) measuring the pH value, the solid content and the conductivity, wherein the measurement result is as follows: the pH value is 4.6-6.3, the solid content is 0.59-0.98%, and the conductivity is 530-1000 mu s/cm2(ii) a Then keeping the temperature at 95 ℃ for 40min, and sterilizing; cooling to room temperature in water bath, and adding phenoxyethanol liquid preservative with the mass of 0.8 percent of the sea grape extraction solution;
(d) precipitating with 70% ethanol, freezing the precipitate at-75 deg.C for 24 hr, and drying in a freeze-drying machine for 24 hr to obtain final Vitis heyneana extract.
Example 3: preparing Vitis heyneana extract.
(a) Weighing 10kg of cleaned sea grapes, cleaning the surfaces of the sea grapes with deionized water, placing the sea grapes in a colloid mill, slightly adding deionized water into the colloid mill, grinding, and adding deionized water with a mass ratio of 13 to the sea grapes; adding D-sodium ascorbate accounting for 0.03 percent of the weight of the sea grapes and adding cellulase accounting for 0.08 percent of the weight of the sea grapes, wherein the cellulase and deionized water are diluted according to the mass ratio of 1: 70; extracting at 45 deg.C and 80r/min for 80 min;
(b) heating to 80 deg.C, stirring for 25min, and inactivating enzyme; stopping heating, cooling to 40 deg.C with water circulation, and coarse-filtering with 100 mesh gauze; taking the filtrate, and finely filtering the filtrate by using a filter plate with the aperture of 5 mu m to obtain light green liquid;
(c) filtering with a filter plate with the aperture of 0.2 μm to obtain a bright light green liquid; and (3) measuring the pH value, the solid content and the conductivity, wherein the measurement result is as follows: the pH value is 4.5-6.0, the solid content is 0.6-1.05%, and the conductivity is 500-1000 mu s/cm2(ii) a Then preserving the heat for 35min at 88 ℃ and sterilizing; cooling to room temperature in water bath, and adding phenoxyethanol liquid preservative with the mass of 0.8 percent of the sea grape extraction solution;
(d) performing alcohol precipitation by using 80% ethanol, freezing the precipitate after alcohol precipitation at-75 ℃ for 24h, and drying in a freeze dryer for 24h to obtain the final sea grape extract.
Example 4: preparing Vitis heyneana extract.
(a) Weighing 10kg of cleaned sea grapes, cleaning the surfaces of the sea grapes with deionized water, placing the sea grapes in a colloid mill, slightly adding deionized water into the colloid mill, grinding, and adding deionized water with the mass ratio of 10 to the sea grapes; adding D-sodium ascorbate accounting for 0.03 percent of the weight of the sea grapes, and adding pectinase accounting for 0.1 percent of the weight of the sea grapes, wherein the pectinase and deionized water are diluted according to the mass ratio of 1: 80; extracting under stirring at 50 deg.C and 60r/min for 90 min;
(b) heating to 85 deg.C, stirring for 20min, and inactivating enzyme; stopping heating, cooling to 30 deg.C with water circulation, and coarse-filtering with 200 mesh gauze; taking the filtrate, and finely filtering the filtrate by using a filter plate with the aperture of 10 mu m to obtain light green liquid;
(c) filtering with a filter plate with the aperture of 0.45 μm to obtain a bright light green liquid; and (3) measuring the pH value, the solid content and the conductivity, wherein the measurement result is as follows: the pH value is 4.5-5.8, the solid content is 0.7-1.0%, and the conductivity is 500-1000 mu s/cm2(ii) a Then preserving the heat at 90 ℃ for 30min, and sterilizing; cooling to room temperature in water bath, and adding phenoxyethanol liquid preservative with the mass of 0.8 percent of the sea grape extraction solution;
(d) performing alcohol precipitation by using 90% of ethanol, freezing the precipitate after alcohol precipitation at-75 ℃ for 24h, and drying in a freeze dryer for 24h to obtain the final sea grape extract.
Example 5: preparing Vitis heyneana extract.
(a) Weighing 10kg of cleaned sea grapes, cleaning the surfaces of the sea grapes with deionized water, placing the sea grapes in a colloid mill, slightly adding deionized water into the colloid mill, grinding, and adding deionized water with a mass ratio of 17 to the sea grapes; adding D-sodium ascorbate accounting for 0.03 percent of the weight of the sea grapes and adding cellulase accounting for 0.12 percent of the weight of the sea grapes, wherein the cellulase and deionized water are diluted according to the mass ratio of 1: 65; extracting at 55 deg.C and 90r/min for 100 min;
(b) heating to 90 deg.C, stirring for 18min, and inactivating enzyme; stopping heating, cooling to 35 deg.C with water circulation, and coarse-filtering with 180 mesh gauze; taking the filtrate, and finely filtering the filtrate by using a filter plate with the aperture of 7 mu m to obtain light green liquid;
(c) the aperture of the filter plate is 0.35Filtering with a filter plate of a micron meter to obtain a bright light green liquid; and (3) measuring the pH value, the solid content and the conductivity, wherein the measurement result is as follows: the pH value is 5.0-6.5, the solid content is 0.65-0.95%, and the conductivity is 520-1000 mu s/cm2(ii) a Then preserving the heat at 92 ℃ for 25min, and sterilizing; cooling to room temperature in water bath, and adding phenoxyethanol liquid preservative with the mass of 0.8 percent of the sea grape extraction solution;
(d) performing alcohol precipitation by using 90% of ethanol, freezing the precipitate after alcohol precipitation at-75 ℃ for 24h, and drying in a freeze dryer for 24h to obtain the final sea grape extract.
Example 6: preparing Vitis heyneana extract.
(a) Weighing 10kg of cleaned sea grapes, cleaning the surfaces of the sea grapes with deionized water, placing the sea grapes in a colloid mill, slightly adding deionized water into the colloid mill, grinding, and adding deionized water with a mass ratio of 9 to the sea grapes; adding D-sodium ascorbate accounting for 0.03 percent of the weight of the sea grapes, and adding pectinase accounting for 0.12 percent of the weight of the sea grapes, wherein the pectinase and deionized water are diluted according to the mass ratio of 1: 55; extracting at 53 deg.C under stirring at 70r/min for 70 min;
(b) heating to 75 deg.C, stirring for 27min, and inactivating enzyme; stopping heating, cooling to 25 deg.C with water circulation, and coarse-filtering with 80 mesh gauze; taking the filtrate, and finely filtering the filtrate by using a filter plate with the aperture of 4 mu m to obtain light green liquid;
(c) filtering with a filter plate with the aperture of 0.3 μm to obtain a bright light green liquid; and (3) measuring the pH value, the solid content and the conductivity, wherein the measurement result is as follows: the pH value is 4.8-6.4, the solid content is 0.62-0.99%, and the conductivity is 500-1000 mu s/cm2(ii) a Then preserving heat at 87 ℃ for 28min, and sterilizing; cooling to room temperature in water bath, and adding phenoxyethanol liquid preservative with the mass of 0.8 percent of the sea grape extraction solution;
(d) performing alcohol precipitation by using 90% of ethanol, freezing the precipitate after alcohol precipitation at-75 ℃ for 24h, and drying in a freeze dryer for 24h to obtain the final sea grape extract.
Comparative example 1
The extraction procedure and method in this example were the same as in example 1 except that the mixture of cellulase and pectinase was not added in step (a).
Comparative example 2
The extraction procedure and method of this example were the same as those of example 1 except that cellulase was not added in step (a) and pectinase was added.
Comparative example 3
The extraction procedure and method of this example were the same as those of example 1 except that no pectinase was added and only cellulase was added in step (a).
The total sugar content is measured by adopting a phenol-sulfuric acid method, and the method comprises the following steps:
1) drawing a standard curve:
accurately sucking 1mg/m L glucose standard solutions of 1.0, 2.0, 3.0, 4.0 and 5.0m L, placing the solutions in a 50m L volumetric flask for constant volume, accurately sucking 2.0m L of the solutions (taking 2.0m L of deionized water as blank control), respectively placing the solutions in test tubes with plugs for colorimetric reaction, and drawing a standard curve.
2) And (3) colorimetric reaction:
① placing 2.0m L sample (crude polysaccharide dissolved in 50m L volumetric flask) into 15m L graduated test tube (2.0 m L deionized water as blank control), adding 1.0m L5% phenol solution, and shaking to mix well;
② adding 5.0m L concentrated sulfuric acid, mixing for 5min, sealing the tube, and boiling in water bath for 1 h;
③ after removal, the sample was cooled to room temperature and the absorbance was measured at 490 mm.
3) Calculating the formula:
W＝(△A+0.0049)×V/0.0101
w: the content of polysaccharide in the extractive solution (μ g)
△ A light absorption value of reaction liquid
V volume of liquid to be measured (m L)
4) Polysaccharide content of the vitis amurensis extract:
according to a glucose standard curve for measuring the total sugar content by a phenol-sulfuric acid method, the regression equation of the standard curve is as follows:
Y＝0.0101X－0.0049，R2＝0.9999。
the polysaccharide contents of the sea grape extracts prepared in example 1 and comparative examples 1 to 3 were measured according to the above methods as shown in the following table:
TABLE 1 Effect of the Presence or absence of pectinase or cellulase on polysaccharide content of Vitis heyneana extract
Pectin is a high molecular weight polysaccharide present in the primary cell wall and intercellular substance that acts to "bind" cells, and when degraded, results in cell separation. The pectinase can break down the cell wall of the plant cell, so that effective components in the cell, such as the polysaccharide of the grape and the like, can be dissolved out more quickly, and the extraction rate is improved; it also has clarifying effect, and can reduce viscosity to make the extract clear and bright. The cellulase is a general name of a group of enzyme systems for degrading cellulose, the main component of the plant cell wall is cellulose, and the cell wall is easier to damage and the effective components are easier to dissolve out through the enzymolysis of the cellulose. As can be seen from the data in Table 1, the addition of pectinase and/or cellulase for enzymolysis greatly improves the extraction rate of the sea grape and the dissolution rate of the active ingredients.
Comparative example 4 comparison of polysaccharide extraction rates in sea grape extract before and after alcohol precipitation
Taking example 1 as an example, the content of polysaccharides in the vitis amurensis extract before and after alcohol precipitation was determined by the above-described method for measuring total sugar content by phenol-sulfuric acid method, and the results are shown in table 2.
TABLE 2 influence of alcohol precipitation with different concentrations on polysaccharide content of Vitis heyneana extract
As can be seen from the experimental data in Table 2, the alcohol precipitation effect can more effectively improve the content of polysaccharide in the vitis amurensis extract, and further improve the moisture-preserving and anti-aging effects of the vitis amurensis extract in cosmetics.
Example 7 human sensitivity test
Selecting 30 volunteers, half of male and female, before experiment, cleaning the left and right front arms of the volunteers with clear water, sitting still in the environment with temperature of 20-22 deg.C and relative humidity of 50-60%, and performing skin sensitivity experiment without applying any cosmetic to the subject.
The extract of Vitis heyneana prepared in example 1 was applied to the left forearm of the subject once every half hour for 6 times with an area of 3 × 3cm2The right forearm was left without any product as a blank.
The experimental results showed that in 30 subjects, small erythema was not produced, indicating that the extract of Vitis heyneana prepared in the above example has better safety.
The sea grape extract prepared in example 1 was used as a sample for the performance test of sea grape extract.
Example 8 human moisturizing experiments.
The experimental principle is as follows: the human body moisturizing experiment comprises a test group consisting of specific experimental groups, and tests the changes of skin moisture and moisture loss before and after the test subject uses the cosmetics (and the cosmetic active ingredients) so as to determine the moisturizing effect of the cosmetics (or the active ingredients).
The experimental method comprises the following steps: 15 male and female volunteers of 15 years to 60 years of age were selected.
(1) Selecting the left arm and the right arm of the subject to be marked in a circulating mode sequentially: a sample group of a test area and a blank control area, wherein the sample group comprises 2% of a sea grape extract solution, 2% of a sodium hyaluronate solution and 2% of a tremella polysaccharide solution;
(2) the technician uses a skin moisture tester CorneometerCM825, and averages and marks as a blank value.
(3) Smearing the sample to be tested on the experimental part which is cleaned by the test subject with clear water.
(4) Subjects were measured 5 times by a technician using a skin moisture meter corneometer cm825 after one hour, two hours, four hours of continuous application of the cosmetics, and the average was taken.
(5) And counting the measured values, and analyzing the change rule of the skin moisture content.
The average percent moisture content of the skin surface of the subject is shown in figure 2 as a function of time. The result shows that the sea grape extract has good long-acting moisturizing effect. It also shows good properties for enhancing the barrier function of the skin.
Example 9 skin elasticity test
The experimental principle is as follows: skin elasticity test, which comprises a test population consisting of specific experimental population, and tests the skin elasticity change of a subject before and after the subject uses the cosmetics (and the cosmetic functional components), thereby determining the anti-aging effect of the cosmetics (or the functional components).
An experimental instrument: MPA580
The experimental conditions are as follows: room temperature (25 + -1) ° c, humidity (40 + -5)%, subject 90, 45 women, 45 men, and 30-50 years old.
The experimental method comprises the following steps:
a. before testing, 90 subjects are divided into 9 groups, 10 persons in each group have the same gender and age distribution, essence added with the sea grape extract is used in each group, the subjects wash the face with warm water, and after 30 minutes, the testing is started;
b. selecting a position 1cm outside the canthus of the tested person as a test area; detecting and recording initial data.
c. The testing personnel use the MPA580 to test the testing area;
d. the skin of the test subject is smeared at the position of 1cm outside the canthus of the eyes of 2 +/-0.1 mg/cm in the morning and evening every day2The essence of (1);
e. testing the subjects every week for 5 weeks, and recording the test data;
f. the weekly measurements of the test data were averaged R2, Q1 to evaluate the wrinkle-removing effect of the product.
As can be seen from FIG. 3, in the test period, the values of R2 and Q1 both tend to rise, the closer the values of R2 and Q1 are to 1, the better the skin elasticity is, and the essence added with the sea grape extract has better effects of removing wrinkles and delaying skin aging.
EXAMPLE 10DPPH radical scavenging experiment
The experimental principle is as follows: DPPH (1, 1-diphenyl-2-picrylhydrazino radical) is a stable long-life radical in organic solvent, and its lone pair electron has strong absorption near 517nm (dark purple color). When a scavenger is present, the lone pair is paired and absorption is lost or diminished, and the activity of the free radical scavenger can be evaluated by determining the extent of the diminution of absorption. The DPPH method for evaluating the antioxidant activity of the antioxidant is a rapid, simple, sensitive and feasible method.
Reagent preparation, 20 mmol/L DPPH solution preparation method, 20mg DPPH solution is added with absolute ethyl alcohol to make the volume reach 250ml, and 20 mmol/L DPPH solution is obtained.
The experimental steps are that 0.5ml of a sea grape extract solution with the mass fraction of 1%, 2%, 3%, 4% and 5% and 1ml of a DPPH solution with the concentration of 20 mmol/L are put in a colorimetric tube, shaken evenly and reacted for 10min, after absolute ethyl alcohol is used as a blank for zero adjustment, 1ml of the solution is put in a cuvette, an absorbance value A sample with the wavelength of 517nm is measured, 1ml of DPPH solution with the concentration of 20 mmol/L is put in the cuvette, and the absorbance value A with the wavelength of 517nm is measured0。
Free radical clearance calculation formula: clearance (%) ═ a0－ASample (I))/A0×100％
As can be seen from FIG. 4, the DPPH radical scavenging rate increased with the increase of the concentration, demonstrating that the extract has a better radical scavenging ability.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (9)
1. The preparation method of the vitis amurensis extract is characterized by comprising the following steps:
(a) cleaning and crushing the sea grape with deionized water, and then adding deionized water and an antioxidant, heating, stirring and extracting;
(b) cooling, coarse filtering and fine filtering;
(c) performing suction filtration, and sterilizing and preserving filtrate;
(d) precipitating with ethanol, and freeze drying to obtain Vitis heyneana extract.
2. The method of claim 1, wherein in step (a), pectinase and/or cellulase is added after the marine grape is crushed, preferably pectinase and/or cellulase diluted with deionized water.
3. The method of preparing a sea grape extract according to claim 2, wherein the step (b) comprises inactivating the enzyme.
4. The method for preparing the sea grape extract according to claim 2 or 3, wherein the mass ratio of the sea grapes to the deionized water in the step (a) is 1 (4-20), the mass of the antioxidant is 0.03% of the mass of the sea grapes, the amount of the pectinase and/or cellulase is 0.06% -0.15% of the mass of the sea grapes, and the pectinase and/or cellulase is diluted with the deionized water according to the mass ratio of 1 (50-80); the stirring speed is 50-100r/min, the reaction temperature is 40-60 ℃, and the reaction time is 60-120 min.
5. The method of claim 3, wherein the enzyme deactivation temperature in step (b) is 70-100 deg.C, and the enzyme deactivation time is 15-30 min; the final temperature of the cooling step is 20-50 ℃; the gauze used for coarse filtration has 60-200 meshes; the aperture of the filter plate used for fine filtration is 1-10 μm.
6. The method for preparing a Vitis heyneana extract according to any of claims 1-5, wherein the filter plate used in the filtration in step (c) has a pore size of 0.1-1 μm, the sterilization temperature is 85-95 ℃, the sterilization time is 20-40min, the preservative is selected from phenoxyethanol liquid or PE9010, and the usage amount of the preservative is 0.8% of the mass of the Vitis heyneana extract filtrate after filtration.
7. The method for preparing a vitis amurensis extract as claimed in claim 1, wherein the ethanol used in the alcohol precipitation in the step (d) accounts for 70 to 90 percent of the mass fraction; freezing the precipitate at-75 deg.C for 24 hr, and freeze drying in freeze drier.
8. A Vitis heyneana extract prepared by the method of any one of claims 1-7.
9. Use of the extract of Vitis heyneana according to claim 8, in particular in cosmetics with moisturizing, anti-ageing effects.
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