CN106074263B

CN106074263B - Herbal extract and application thereof in cosmetics

Info

Publication number: CN106074263B
Application number: CN201610520127.5A
Authority: CN
Inventors: 洪民华; 刘丹; 赵兆; 洪奇; 吕智; 卢艳花; 何浩
Original assignee: Shanghai Inoherb Cosmetic Co ltd; East China University of Science and Technology
Current assignee: Shanghai Inoherb Cosmetic Co ltd; East China University of Science and Technology
Priority date: 2016-07-05
Filing date: 2016-07-05
Publication date: 2020-08-07
Anticipated expiration: 2036-07-05
Also published as: CN106074263A

Abstract

The invention discloses a snow tea extract, which comprises a depside compound, such as any one or more of squamatic acid, capric acid, snow tea essence and kojic acid. The invention also discloses a preparation method of the snow tea extract and application of the snow tea extract in the field of cosmetics. The snow tea extract adopts polar solvent extraction method, has simple extraction process, and is easy for industrialization. The snow tea extract provided by the invention has excellent anti-aging performance, the capability of repairing damaged cells and excellent moisturizing performance, and can be added into cosmetics as a compound additive.

Description

Herbal extract and application thereof in cosmetics

Technical Field

The invention belongs to the field of biochemistry, relates to a herbal extract, a preparation method thereof and cosmetics containing the extract, and particularly relates to a snow tea extract, a preparation method thereof and application thereof in cosmetics.

Background

Thamnolia vermicularia, also known as Thamnolia japonica, Thalassia alba and Thamnolia asiatica, is a plant of the family Thamnaceae. The ground clothes are in a branch shape, grey white or white, 3-7cm high, multi-branched, long round bar shape or flat bar shape, and the branches of the common committee 2-3 or single branch are in a small thorn shape.

Snow tea has been a famous and precious traditional Chinese medicine and health-care drink since ancient times. There are the following reports in compendium Shigao (supplement to compendium): xue cha, in Yunnan, is white in color and slightly yellow in color after long-term use, so as to be rich in Yu and stand out with faint scent. Turen adopts stir-baked tea, so it is named Xue tea. The tea has the strong function of warming stomach, and people suffering from tuberculosis and loss of blood must have cold stomach and are most prohibited to eat tea, but the tea is not prohibited, and people who cook Yu and eat the tea have warm abdomen. The snow tea has the effects of clearing heat, promoting fluid production, restoring consciousness, soothing nerves, reducing blood pressure and reducing blood fat.

The snow tea grows on a mountain with the altitude of thousands of meters and is divided into red snow tea and white snow tea. The snow tea contains various components beneficial to human bodies, and can reduce blood fat and blood pressure, lose weight, refresh the brain, moisten the lung, clear away summer heat, promote the production of body fluid, relieve cough, clear away the liver, improve eyesight, nourish yin, moisten the lung and the like, and can clear away garbage in the body and discharge toxins, so the snow tea has been widely applied to the fields of beverages, medicines and the like for a long time. Patent application No. 201210190931.1 discloses a composite snow tea and its preparation method, wherein the composite snow tea has effects of clearing away heat and toxic materials, lowering hypertension, hyperlipemia, hyperglycemia, and increasing coronary artery and cerebral blood flow. The patent with the application number of 201210042650.3 discloses a black snow tea extract, which is prepared by taking dry red snow tea coarse powder as a raw material and carrying out the steps of water extraction, alcohol precipitation, trypsin and papain hydrolysis, dialysis, drying and the like, and the extract can be used for preventing and treating diseases such as fatty liver and the like. However, no reports on the application of snow tea in cosmetics are seen at present.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a snow tea extract by taking snow tea as a raw material, and researches the application of the extract in the field of cosmetics.

The snow tea extract provided by the invention comprises depside compounds.

As a preferable embodiment of the invention, the phenolic acid compound is any one or a combination of several of crystalline flake-shaped chlamydia acid, capric acid, snowtea extract and digaleic acid.

As a preferred embodiment of the present invention, the content of the phenolic acid compound is 5% to 50%, such as 8%, 45%, preferably 10% to 35%, such as 15%, 30%, more preferably 18% to 25%, such as 20%, etc. by mass.

As a preferred embodiment of the invention, the snow tea extract further comprises α -cedrol, and the content of α -cedrol is 0-5%, such as 1%, 3% and the like.

As a preferred embodiment of the present invention, the snow tea extract may be an aqueous extract, an alcohol extract, an oil extract, supercritical CO₂Any one or combination of several of the extracts.

As a preferred embodiment of the present invention, the alcohol extract is preferably an ethanol extract.

The second aspect of the present invention provides a method for preparing a snow tea extract, comprising the steps of:

step 1, taking the crushed snow tea raw material, adding a solvent with the volume 10-20 times that of the snow tea raw material into the snow tea raw material, extracting for multiple times at 40-80 ℃, and combining extracting solutions;

and 2, filtering the combined extracting solution obtained in the step 1 under reduced pressure to obtain a filtrate, and concentrating the filtrate under reduced pressure to obtain the snow tea extract.

As a preferred embodiment of the present invention, the solvent in step 1 is any one or a combination of polar solvent and non-polar solvent, such as methanol, aqueous methanol solution, methanol, aqueous ethanol solution, diethyl ether, etc., preferably a polar solvent, such as aqueous methanol solution and aqueous ethanol solution.

As a preferred embodiment of the present invention, the snowtea extract is physically pulverized powder and/or extract.

As a preferred embodiment of the present invention, the preparation method comprises the steps of:

step 1, taking the crushed snow tea raw material, extracting at least 2 times at the temperature of 50-120 ℃ for 0.5-10h each time according to the proportion of the snow tea raw material to a solvent of 1g (5-20) m L, and mixing the extracting solutions each time after the extraction is finished to obtain a mixed extracting solution;

and 2, concentrating the mixed extracting solution to prepare the snow tea extract.

As a preferred embodiment of the invention, the solvent for extracting the snow tea in the step 1 is any one or a combination of water, methanol water solution, ethanol water solution, diethyl ether and the like.

As a preferred embodiment of the present invention, the volume concentration of the methanol aqueous solution is 30% to 100%, such as 40%, 95%; preferably 50% -90%, such as 60%, 70%, 80%, etc., wherein, when the volume concentration is 100%, the solvent is the anhydrous methanol.

As a preferred embodiment of the present invention, the volume concentration of the ethanol aqueous solution is 30% to 100%, such as 40%, 95%; preferably 50% -90%, such as 60%, 70%, 80%, etc., wherein, when the volume concentration is 100%, the solvent is absolute ethyl alcohol.

As a preferred embodiment of the present invention, the extraction conditions of the snow tea are 0.2-0.8MPa, such as 0.3MPa, 0.6MPa, preferably 0.4-0.5MPa, such as 0.45 MPa.

As a preferred embodiment of the present invention, the extraction conditions of the snow tea are 1.2-1.8MPa, such as 1.3MPa, 1.6MPa, preferably 1.4-1.5MPa, such as 1.45 MPa.

In a preferred embodiment of the present invention, in the step 2, the mixed extract is first concentrated to obtain a first concentrated solution, and the first concentrated solution is purified by a chromatography column for a plurality of times and then concentrated for a second time to obtain the olive leaf powder.

As a preferred embodiment of the invention, the purification process is any one or a combination of macroporous adsorption resin purification and silica gel column adsorption purification.

As a preferred embodiment of the present invention, the conditions of the macroporous adsorption resin purification process are as follows: the temperature is 25-50 ℃, preferably 30-40 ℃, and the type of the macroporous adsorption resin is any one or more of AB-8 type, D-101 and ADS-5.

As a preferred embodiment of the present invention, after the macroporous adsorbent resin is adsorbed, gradient elution is performed by using a mixed solution of ethanol and/or water, wherein the volume ratio of the mixed solution is ethanol: water 1: (0-10), preferably ethanol: water 1: (1-5).

As a preferred embodiment of the invention, in the elution process, the flow rate of the mixed solution is 2-4BV/h, and the elution temperature is 30-50 ℃.

As a preferred embodiment of the present invention, the conditions of the silica gel column adsorption purification process are as follows: 25-50 ℃, chloroform: methanol: water ═ 1-3: (1-5): (1-2) eluting with eluent at the flow rate of 2-4BV/h and the elution temperature of 30-50 ℃.

As a preferred embodiment of the present invention, the step 2 may further comprise a drying step after concentration under reduced pressure.

As a preferred embodiment of the present invention, the drying step is spray drying and/or hot air drying.

As a preferred embodiment of the invention, in the spray drying process, the high-pressure pump passes the filtrate through the atomizer under the pressure of 80-120MPa to form particles of 100-200 μm, the temperature of hot air contacting with the particles is 60-100 ℃, and the contact time is 5-50 s.

The third aspect of the invention is to provide application of any one of the snow tea extracts.

The snow tea extract is preferably applied to the outer surface of the skin, and more preferably applied to the preparation of an article to be applied to the outer surface of the skin.

The snow tea extract is applied to preparing daily chemical products, preferably cosmetics and/or skin care products.

The fourth aspect of the invention provides a daily chemical product containing any one of the snow tea extracts, preferably a cosmetic and/or a skin care product containing any one of the snow tea extracts, more preferably the cosmetic and/or the skin care product, and also can comprise nutritional additives.

As a preferred embodiment of the present invention, the content of the snowtea extract in the cosmetic and/or skin care product is 0.001% -50%, such as 0.003%, 40%, preferably 0.005% -30%, such as 1%, 3%, 5%, 10%, 20%, etc.

The preparation process of the snow tea extract provided by the invention is simple, and the obtained snow tea extract has excellent moisturizing performance and anti-aging capability, can be added into cosmetics as a compound additive, and is an ideal cosmetic additive.

Drawings

FIG. 1 is a graph showing the effect of a snow tea extract on type I collagen secretion in HDF cells in a natural state;

FIG. 2 is a graph of the effect of hydrogen peroxide on cell viability;

FIG. 3 is a graph of the effect of snow tea extract on collagen type I secretion from HDF cells damaged by hydrogen peroxide;

FIG. 4 is a graph of the effect of snow tea extract on MMP-1 secretion from hydrogen peroxide damaged HDF cells;

FIG. 5 is a graph of the effect of snow tea extract on malondialdehyde content in hydrogen peroxide damaged HDF cells;

FIG. 6 is a graph showing the effect of snow tea extract on DPPH radical removal rate;

FIG. 7 effect of snow tea extract on HAS3 content in HDF cells damaged by hydrogen peroxide;

FIG. 8 is a graph of the effect of snow tea extract on HA content of hydrogen peroxide damaged HDF cells;

FIG. 9 is a graph of the effect of snow tea extract on the content of hydrogen peroxide damaged HaCat cells L OR.

Detailed Description

Preparation of snow tea extract

Example one

Taking 10kg of dried snow tea as a raw material, washing the raw material with deionized water for 2 times, removing impurities, crushing the raw material, sieving the raw material with a sieve of 80 meshes, adding 70% ethanol solution with the volume ratio of 14 times into the snow tea raw material, extracting the raw material for 2 hours in a water bath at 75 ℃ under normal pressure, adding 50% ethanol solution with the amount of 10 times of the raw material after the extraction is finished, extracting the raw material for 2 hours in the water bath at 75 ℃ under normal pressure, combining the extracting solutions for 2 times after the extraction is finished, concentrating the obtained extracting solution to 4L under the condition of 5kPa and slight boiling to obtain an extract, and keeping the extract at 4 ℃, wherein the extract is the snow tea extract.

Analyzing the extract of Thamnolia vermicularis (snowtea extract dissolved in anhydrous methanol in an amount 10 times the volume of the extract to prepare a methanol solution) with a liquid chromatography-mass spectrometer using a C18 column, wherein the ratio of methanol: 0.5% formic acid solution 75: 25 as mobile phase, 250nm wavelength, column temperature 25 ℃.

Analysis shows that the snow tea extract contains 10% of depside compounds, 2% of squamatic acid, 2% of capric acid and 5% of snow tea essence by mass.

Example two

Taking 10kg of dried snow tea as a raw material, washing the raw material with deionized water for 2 times, removing impurities, crushing the raw material, sieving the raw material with an 80-mesh sieve, adding 70% ethanol solution with the volume ratio of 10 times into the snow tea raw material, extracting the mixture for 2 hours in a 75 ℃ water bath at normal pressure, adding 50% ethanol solution with the volume ratio of 10 times into the mixture after the extraction is finished, extracting the mixture for 2 hours in a 65 ℃ water bath at normal pressure, adding deionized water with the volume ratio of 10 times into the mixture after the extraction is finished, extracting the mixture for 2 hours in a 80 ℃ water bath at normal pressure, combining the extracting solutions for 3 times after the extraction is finished, and concentrating the obtained extracting solution to 5L under the condition of.

Cooling, placing two layers of filter paper on the decompression funnel, decompressing and filtering the concentrated solution, and spray drying the filter cake to obtain the snow tea extract. Wherein the conditions in the spray dryer are: making the extract into 150 μm microparticles under 100MPa, and drying the microparticles with 70 deg.C hot air for 15 s.

Analysis shows that the snow tea extract contains 20% of depside compounds, 10% of scale coating acid, 5% of sheep-horn coating acid and 3% of snow tea essence by mass.

EXAMPLE III

Taking 10kg of dried snow tea as a raw material, washing the raw material with deionized water for 2 times, removing impurities, crushing the raw material, sieving the raw material with an 80-mesh sieve, adding a 70% methanol solution and a 10 times of 70% ethanol solution in a volume ratio into the snow tea raw material, extracting the mixture for 2 hours in a 70 ℃ water bath at normal pressure, after the extraction is finished, adding a 10 times of 50% ethanol solution in an amount of the raw material, extracting the mixture for 2 hours in a 65 ℃ water bath at normal pressure, after the extraction is finished, adding 10 times of deionized water in an amount of the raw material, extracting the mixture for 2 hours in a 80 ℃ water bath at normal pressure, combining 3 times of extracting solutions after the extraction is finished, and concentrating the obtained extracting solution to 5L under the condition.

Cooling, placing two layers of filter paper on the decompression funnel, decompressing and filtering the concentrated solution, and spray drying the filter cake to obtain the snow tea extract. Wherein the conditions in the spray dryer are: making the extract into 100 μm microparticles under 120MPa, and drying the microparticles with 70 deg.C hot air for 30 s.

Analysis shows that the snow tea extract contains 40% of depside compounds, 25% of scale coating acid, 10% of sheep-horn coating acid and 3% of snow tea essence by mass.

Example four

Taking 10kg of dried snow tea as a raw material, washing the raw material with deionized water for 2 times, removing impurities, crushing the raw material, sieving the raw material with a 30-mesh sieve, adding 15 times of 70% methanol solution and 10 times of 70% ether solution in volume ratio into the snow tea raw material, extracting the mixture for 2 hours in a 70 ℃ water bath under 1.2MPa, after the extraction is finished, adding 10 times of 50% ethanol solution, extracting the mixture for 2 hours in a 85 ℃ water bath under 1.2MPa, after the extraction is finished, adding 10 times of deionized water, extracting the mixture for 2 hours in a 80 ℃ water bath under 0.8MPa, combining 3 times of extracting solutions after the extraction is finished, and concentrating the obtained extracting solution to 5L under the condition of 5kPa and slight boiling.

After cooling, the concentrate was purified using a 200 mesh silica gel column, in volume ratio chloroform: methanol: water 3: 5: 2 as a mobile phase, the flow rate of the mobile phase is 3BV/h, elution is carried out at 25 ℃, the eluent is concentrated, and secondary purification is carried out by using a 200-mesh silica gel column, wherein the volume ratio of chloroform: methanol: water-4: 2: 4 as a mobile phase, eluting at 25 deg.C at a flow rate of 2BV/h, evaporating the secondary eluent at 5kPa and 30 deg.C to remove chloroform and methanol, standing for 12h, placing two layers of filter paper on a decompression funnel, filtering the concentrated solution under reduced pressure, and spray drying the filter cake to obtain the Thamnolia vermicularis (Ach.) Asa-hina extract. Wherein the conditions in the spray dryer are: making the extract into 100 μm microparticles under 120MPa, and drying the microparticles with 70 deg.C hot air for 30 s.

Analysis shows that the snow tea extract contains 50% of depside compounds, 30% of scale coating acid, 10% of sheep-horn coating acid and 6% of snow tea essence by mass.

EXAMPLE five

Taking 10kg of dried snow tea raw material, washing for 2 times by deionized water, removing impurities, crushing, and sieving by a 30-mesh sieve. Adding 15 volume times of diethyl ether into the raw material of the white snow tea, and extracting for 2 times in water bath at 60 deg.C under 1.1MPa for 2 hr each time. Adding 20 times of 80% methanol solution into the white snow tea, extracting at 70 deg.C under 1.1MPa for 2 times (2 hr each time), and combining the extractive solutions 4 times after extraction.

Placing two layers of filter paper on a decompression funnel, decompressing and filtering the combined extracting solution to obtain filtrate, concentrating the filtrate to 4L under the pressure of 5kPa and saturated steam, and keeping the temperature of the extract at 4 ℃, wherein the extract is the snow tea extract.

Taking 10kg of dried snow tea as a raw material, washing the raw material with deionized water for 2 times, removing impurities, crushing the raw material, sieving the raw material with a 30-mesh sieve, adding a 50% ethanol solution and a 70% diethyl ether solution which are 15 times in volume ratio into the snow tea raw material, extracting the mixture for 2 hours in a 70 ℃ water bath under 1.2MPa, after the extraction is finished, adding a 60% methanol solution which is 10 times in amount of the raw material into the mixture, extracting the mixture for 2 hours in a 85 ℃ water bath under 1.2MPa, combining the extracting solutions for 2 times after the extraction is finished, and concentrating the obtained extracting solution to 5L under the condition of 5kPa and slight boiling.

Analysis shows that the snow tea extract contains 30% of depside compounds, 15% of squamatic acid, 10% of capric acid and 5% of snow tea essence by mass.

Control test

Weighing 10kg of snow tea, putting the snow tea into an extraction pool of 200L, placing the snow tea on a rotary disc, adding dichloromethane serving as an extraction solvent, preheating for 1min at the temperature of 60 ℃ and the pressure of 10.34MPa, statically extracting for 2h, purging with nitrogen for 90s, collecting extract liquor, adding 1 volume times of anhydrous sodium sulfate into the extract liquor, standing the mixture at 0 ℃ for overnight, evaporating and concentrating the overnight extract liquor at 30 ℃ and 0.7MPa to remove most of dichloromethane, and performing spray drying on the concentrated solution at 40 ℃ to obtain the snow tea extract.

Analysis shows that the snow tea extract contains 30% of depside compounds, 22% of crystalline flake coating acid, 6% of capric acid and 1% of snow tea essence by mass.

Effect of Thamnolia vermicularis extract on survival rates of HDF cells and HaCat cells

Taking the logarithmic growth phase with the concentration of 10⁵Each m L of human HDF cells were seeded in 96-well cell culture plates at 100 μ L per well of cell broth, the number of wells plated finally depending on the number of samples, 3 wells per well.

The 96-well plate is placed in a cell culture box at 37 ℃ for adherent culture for 6h.

Adding snow tea extract with final concentration of 1mg/m L, 0.1mg/m L and 0.01mg/m L into 96-well cell culture plate, adding equal volume of cell culture solution to control group, and adding 5% CO at 37 deg.C₂The cell culture box of (1) was incubated for 48 h.

Then 0.5mg/m L MTT 100. mu. L containing 5% CO at 37 ℃ was added to each well₂The cell culture box is incubated for 4h under the dark condition, the supernatant is removed, 150 mu L DMSO is added, the oscillation is carried out, the 570nm is taken as the experimental wavelength, the 630nm is taken as the reference wavelength, and the absorbance is detected.

Cell viability was calculated and the results are shown in table 1:

TABLE 1 influence of snow tea extract on the viability of HDF cells and HaCat cells

Taking the cell survival rate higher than 80% as the standard of no toxicity to human HDF cells and HaCat cells, as can be seen from Table 1, the highest concentration of the snow tea extract which is nontoxic to human HDF cells is 0.01mg/m L, and as can be seen from Table 1, the highest concentration of the snow tea extract which is nontoxic to human HaCat cells is 0.01mg/m L, so that the concentration of the snow tea extract is determined to be 0.01mg/m L, which is the subsequent test concentration.

Anti-aging property of snow tea extract

Influence of snow tea extract on HDF collagen secretion in natural state

In order to examine the effect of the snow tea extract provided by the present invention on HDF collagen secretion, the present example divided the test into a blank group, an experimental group 1, an experimental group 2, an experimental control group, and a standard group.

Taking the logarithmic growth phase with the concentration of 10⁵Cells/m L, 100 μ L per well, CO at 37 ℃₂After 24 hours, the supernatant was removed and centrifuged at 3000rpm for 10min to obtain the supernatant.

To the standard group, 50 μ L of the standard solution was added, to the experimental group 1, 10 μ L of the supernatant and 40 μ L of the dilution of the snow tea extract provided in the third example, to the experimental group 2, 10 μ L of the supernatant and 40 μ L of the dilution of the snow tea extract provided in the fourth example, to the experimental control group, 10 μ L of the supernatant and 40 μ L of the dilution of the snow tea extract prepared by the method described in the comparative example were added, to the blank group, no sample was added, the plates were incubated at 37 ℃ for 1 hour, respectively, and washed 5 times with 3 wells per well.

To each of the five groups of samples, 50. mu. L of biotin-labeled anti-lgG antibody was added, incubated at 37 ℃ for 30min, and washed 5 times.

To five groups of samples, 50. mu. L of streptomycin and element-HRP were added, mixed by gentle shaking, incubated at 37 ℃ for 30min, and washed 5 times.

And adding 50 mu L of the color-developing agent A and the color-developing agent B into the five groups of samples respectively, shaking gently and mixing uniformly, incubating for 30min at 37 ℃ in the dark, and adding 50 mu L of stop solution into the five groups of samples.

The absorbance of each group was measured at a wavelength of 450nm within 15min after termination of the reaction by zeroing with a blank group, and the results are shown in Table 2 and FIG. 1.

TABLE 2 influence of Thamnolia vermicularis extract on type I collagen secretion of HDF cells

Group of	Type I collagen content
		Experimental group 1	104
Experimental group 2	118.88
		Control experiment group	102
Blank group	100

As shown in Table 2, after the snow tea extract with the concentration of 0.01mg/m L provided by the invention is added into HDF cells, the content of the type I collagen in the skin is respectively 1.04 times and 1.18 times of that of a blank group, and the difference between the content of the type I collagen in the snow tea extract prepared by adopting the prior art and the content of the type I collagen in the blank group without the snow tea extract is almost zero, so that the snow tea extract provided by the invention can obviously promote the expression of the type I collagen and is a potential anti-aging additive.

Effect of Hydrogen peroxide on survival of HDF cells

Selecting hydrogen peroxide with different concentrations, such as 50 μ M, 100 μ M, 200 μ M, 400 μ M, 600 μ M, 800 μ M and 1000 μ M, incubating with HDF cells for different times, such as 3h, 6h and 9h, and detecting the influence of hydrogen peroxide with different concentrations on the survival rate of HDF cells by adopting an MTT method.

Culturing to a concentration of 10⁵HDF cells of L/m were seeded in 96-well cell culture plates at 100. mu. L/well containing CO at 37 ℃₂The culture box is used for adherent culture for 6 hours, and each hole is provided with 3 repeated holes.

Incubating the samples for 3h, 6h and 9h with hydrogen peroxide of different concentrations, adding MTT100 μ L of 0.5mg/m L to each sample well, and adding CO at 37 deg.C₂The incubator (2) was incubated for 4 hours in the dark.

Removing supernatant, adding 150 μ L DMSO to the sample, shaking, measuring absorbance of each sample with 570nm as experimental wavelength and 630nm as reference wavelength, and calculating cell survival rate.

As shown in FIG. 2, the survival rate of human HDF cells after 3-9h incubation in 50-800 μ M hydrogen peroxide is significantly reduced, and when the incubation concentration exceeds 400 μ M, the cell survival rate is lower than 50%. After the action of 200 mu M hydrogen peroxide for 6 hours, the cell survival rate is reduced to 70 percent.

Effect of Thamnolia vermicularis extract on survival rate of HDF cells damaged by hydrogen peroxide

The test is divided into a blank group, a negative control group, a positive control group, an experimental group 1, an experimental group 2 and a control experimental group.

Culturing until the density is 10⁵HDF cells at a density of L/m were seeded in 96-well plates at 100. mu. L/well in 37 ℃ with CO₂The culture box is used for adherent culture for 6 hours.

Tretinoin was added to the positive control group, the snow tea extract provided in example three of the present invention was added to the experimental group 1, the snow tea extract provided in example four of the present invention was added to the experimental group 2, the snow tea extract provided in comparative example was added to the control experimental group, and no sample was added to the blank group and the negative control group.

Using hydrogen peroxide damage modeling, 0.5mg/m L MTT 100. mu. L was added to each well at 37 ℃ with 5% CO₂Removing the supernatant, adding DMSO 150 mu L, shaking to detect absorbance, and calculating the cell survival rate, wherein the results are shown in Table 3.

TABLE 3 Effect of snow tea extract on Hydrogen peroxide damaged HDF cell survival

Group of	Cell survival rate (%)
		Negative control group	70
Positive control group	88
		Experimental group 1	80.01
Experimental group 2	77.19
		Control experiment group	73
Blank group	100

As can be seen from table 3, the survival rate of HDF cells after hydrogen peroxide incubation was reduced to 70%, as shown by the negative control group in table 3. After the snow tea extract provided by the third embodiment of the invention is added into HDF cells damaged by hydrogen oxide, the survival rate of human HDF cells reaches 80.01%; after the snow tea extract provided by the fourth embodiment of the invention is added into HDF cells damaged by hydrogen oxide, the survival rates of human HDF cells reach 77.19 percent, which are both higher than 73 percent in a control experiment group, and 70 percent in a negative control group, which shows that compared with the prior art, the snow tea extract provided by the invention can more effectively repair the damage of hydrogen peroxide to the HDF cells.

Effect of Thamnolia vermicularis (Ach.) Asa-hina extract on collagen type I secretion of HDF cells damaged by hydrogen peroxide

The test is divided into a blank group, an experimental group 1, an experimental group 2, a control experimental group, a negative control group and a positive control group.

Culturing until the density is 10⁵HDF cells at a density of L/m were seeded in 24-well cell culture plates at 1m L/well with 3 multiple wells per well at 37 ℃ in the presence of CO₂The culture box is used for adherent culture for 6 hours.

The blank group and the negative control group are not added with samples, the snow tea extract provided by the third embodiment of the invention is added into the experiment group 1, the snow tea extract provided by the fourth embodiment of the invention is added into the experiment group 2, the snow tea extract prepared by the comparative example is added into the control experiment group, and the retinoic acid is added into the positive control group.

The experimental group 1, the experimental group 2, the positive control group and the control experimental group were modeled using hydrogen peroxide damage.

0.5mg/m L MTT 100. mu. L was added to each well at 37 ℃ with 5% CO₂Removing the supernatant, adding DMSO 150 mu L, shaking to detect absorbance, and calculating the collagen content, wherein the results are shown in Table 4 and FIG. 3:

TABLE 4 Effect of snow tea extract on collagen type I secretion from HDF cells damaged by Hydrogen peroxide

As is clear from table 4 and fig. 3, the amount of type I collagen secretion in HDF cells after hydrogen peroxide damage was 72% of that when intact, as shown in the negative control group in table 4. Whereas, after addition of retinoic acid to HDF cells that had been damaged by hydrogen oxide, the amount of type I collagen secreted from HDF cells increased to 94%, as shown in the positive control group in table 4. After the snowtea extract provided by the invention is added into HDF cells damaged by hydrogen peroxide, the content of type I collagen in the human HDF cells reaches 85% and 89%. After the snowtea extract prepared by the prior art is added into HDF cells damaged by hydrogen oxide, the content of type I collagen in the HDF cells of a human body is 78%, which is obviously lower than the content of type I collagen in the HDF cells of the human body after the snowtea extract is added, namely, compared with the snowtea extract prepared by the prior art, the snowtea extract provided by the invention has excellent capability of repairing cell damage, can obviously repair the damage of hydrogen peroxide to the HDF cells, and is an excellent anti-aging cosmetic additive.

Effect of Thamnolia vermicularis extract on hydrogen peroxide-damaged HDF cell matrix metalloproteinase (MMP-1)

Materials and reagents

Cell culture solution 24-hole cell culture plate

Hydrogen peroxide in cell culture box

Centrifuge E L ISA kit

Test procedure

The test was divided into a standard group, a blank group and an experimental group.

Taking the concentration of logarithmic growth phase as 10⁵HDF cells at a density of L/m were seeded in 24-well plates at 1m L per well and at 37 ℃ in the presence of CO₂The cell culture box is used for culturing for 6 hours.

The snow tea extract provided by the third embodiment of the present invention was added to the experimental group 1, the snow tea extract provided by the fourth embodiment of the present invention was added to the experimental group 2, the standard was added to the standard group, and no sample was added to the blank group.

And (3) inducing to establish a hydrogen peroxide damage model, carefully absorbing a supernatant culture medium, centrifuging, taking 10 mu L cell suspension for experiment according to the instruction of the kit, adjusting zero by using a blank hole, measuring the absorbance of each hole by using a wavelength of 450nm within 15min after the reaction is ended, calculating a linear regression equation of a standard curve according to the concentration of MMP-1 corresponding to the standard product and the corresponding absorbance value, and calculating the concentration of MMP-1 in the experimental group on the regression equation according to the absorbance value of the sample.

TABLE 5 Effect of snow tea extract on MMP-1 content in Hydrogen peroxide damaged HDF cells

Group of	MMP-1 concentration (%)
		Negative control group	167.21
Positive control group	125
		Experimental group 1	103
Experimental group 2	91
		Control experiment group	130
Blank group	100

As can be seen from table 5 and fig. 4, MMP-1 levels in HDF cells significantly increased to 167% of the original levels after the stimulation with hydrogen peroxide in human HDF cells, as shown in the negative control group in table 5. MMP-1 values were reduced after retinoic acid addition, as shown in the positive control in Table 5. After the snow tea extract provided by the invention is added into HDF cells stimulated by hydrogen peroxide, the MMP-1 content is reduced to about 100 percent, even below 100 percent, for example, after the snow tea extract provided by the fourth embodiment of the invention is added, the MMP-1 content is 91 percent. After the snowtea extract prepared by the prior art is added into the HDF cells stimulated by the hydrogen peroxide, the MMP-1 content in the HDF cells is 130%, which shows that the snowtea extract provided by the invention has more obvious MMP-1 inhibition capability compared with the snowtea extract prepared by the prior art. Namely, the snow tea extract provided by the invention has excellent capacity of inhibiting MMP-1 expression under oxidative stress conditions.

Effect of Thamnolia vermicularis (Ach.) Asa-hina extract on Malondialdehyde (MDA) in HDF cells damaged by Hydrogen peroxide

When heated under acidic conditions, MDA can be condensed with thiobarbituric acid (TBA) to form a red product, 3,5, 5-trimethyloxazole-2, 4-dione, having a maximum absorption peak at 532 nm. The substrate is thiobarbituric acid (TBA), so the method is called TBA method.

Taking the logarithmic growth phase with the concentration of 10⁵HDF cells of L/m were seeded in 6-well cell culture plates at 2m L per well and cultured for 6h.

The snowtea extract prepared in the third example of the invention diluted with fresh medium was added to the experimental group 1, the snowtea extract prepared in the fourth example of the invention diluted with fresh medium was added to the experimental group 2, the snowtea extract prepared in the control example diluted with fresh medium was added to the control group, retinoic acid was added to the positive control group, and no sample was added to the blank group and the negative control group.

Inducing and establishing a hydrogen peroxide damage model, centrifuging at 1000rpm for 10min, collecting cells and adherent cells in culture medium supernatant, adding 500 mu L PBS into cell sediment, slightly reversing and uniformly mixing, centrifuging at 2000rpm for 10min at 4 ℃, removing supernatant and reserving bottom sediment, adding 500 mu L PBS into sediment, ultrasonically crushing the cells (400A, 5 s/time, and repeating for 3-5 times in a gap of 10 s), and preparing cell homogenate;

experiments were performed according to the kit instructions. Mixing the reagents uniformly, fastening the opening of the test tube by using a preservative film, puncturing the test tube by using a needle for ventilation, boiling the test tube for 40min by using a pot cover, taking out the test tube, cooling the test tube by flowing water, and centrifuging the test tube at 4000rpm for 10min to ensure that the precipitation is complete. Taking the supernatant, and measuring the absorbance at the wavelength of 532nm by adopting a spectrophotometry; the snowtea extract prepared by the invention is diluted by proper times by using a BCA protein detection kit according to the instruction method, and the protein content in the cells is determined.

TABLE 6 influence of snow tea extract on MDA content of HDF cells damaged by Hydrogen peroxide

As can be seen from table 6 and fig. 5, when the snow tea extract was not added, the corresponding MDA content in HDF cells was recorded as 100, and after hydrogen peroxide damage, the MDA concentration rises rapidly, the MDA content increases 3.5 times to 326.34 in 6 hours, and the increase of MDA content is inhibited after the retinoic acid is added, and is reduced to 2.6 times of that of the blank group, after the snow tea extract provided by the third embodiment of the invention is added into HDF cells damaged by hydrogen peroxide, the MDA content is obviously reduced to about 200, after the snow tea extract prepared in the fourth embodiment is adopted, the MDA content is reduced to 185, after the snow tea extract prepared by the prior art is added, the MDA content in HDF cells damaged by hydrogen oxide is 262, compared with the snow tea extract provided by the prior art, the snow tea extract provided by the invention has more obvious capacity of inhibiting MDA secretion in the HDF cells, namely, the snow tea extract provided by the invention has a remarkable repairing effect on human HDF cell damage.

Free radical removing performance of snow tea extract

1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) is a very stable free radical with a nitrogen center, and the stability of the DPPH mainly comes from the resonance stabilization effect and the steric hindrance of 3 benzene rings, so that an unpaired electron on a nitrogen atom sandwiched in the middle cannot play the due electron pairing effect. As a stable free radical, it can scavenge other free radicals. The method is widely used for quantitatively measuring the oxidation resistance of biological samples and food at present. The method is based on the characteristic that DPPH free radical has single electron, strong absorption is provided at 517nm, and alcoholic solution is purple. In the presence of a free radical scavenger, its absorption gradually disappears by pairing with its single electron, and its degree of discoloration is quantitatively related to the number of electrons it receives, so that a rapid quantitative analysis can be performed with a spectrophotometer.

Materials and reagents

DPPH Anhydrous ethanol

ddd water 200 mu L suction head

8 passageway application of sample rifle of 96 orifice plates

Enzyme-linked immunosorbent assay (ELISA) instrument

Test procedure

Preparing 0.2 mmol/L DPPH solution with anhydrous ethanol, storing in dark, and preparing 0.01mg/m L epigallocatechin gallate (EGCG) solution.

Adding 20u L of snow tea extract solution with the concentration of 0.01mg/m L and 0.2 mmol/L of DPPH ethanol solution 180u L into a 96-well plate in an experimental group 1, adding 180u L of absolute ethanol and 20u L of snow tea extract solution provided by the invention into a blank group 1, adding 180u L of DPPH solution and 20u L of distilled water into a standard group, setting at least 3 multiple wells in each well, shaking up respectively, standing in the dark at room temperature for 30min, and measuring the absorbance of each group.

20u L of snow tea extract solution of 0.01mg/m L and 0.2 mmol/L of DPPH ethanol solution 180u L are added to the experimental group 2 to a 96-well plate, 180u L of absolute ethanol and 20u L of snow tea extract solution of the invention are added to the blank group 2, 180u L DPPH solution and 20u L of distilled water are added to the standard group, at least 3 multiple wells are arranged in each well, the wells are shaken up respectively, the mixture is left to stand in the dark at room temperature for 30min, and the absorbance of each group is measured.

Adding 20u L snow tea extract solution with concentration of 0.01mg/m L and 0.2 mmol/L DPPH ethanol solution 180u L to 96-well plate, adding 180u L absolute ethanol and 20u L snow tea extract solution with concentration of at least 3 wells to blank group 3, shaking up, standing in dark at room temperature for 30min, and measuring absorbance of each group.

Adding 20u L solution with concentration of 0.01mg/m L EGCG and 0.2 mmol/L DPPH ethanol solution 180u L to a 96-well plate to a positive control group, adding 180u L absolute ethanol and 20u L EGCG solution to a blank group 4, setting at least 3 multiple wells in each well, shaking up respectively, standing in the dark at room temperature for 30min, and measuring the absorbance of each group.

The DPPH radical scavenging ability is expressed as follows:

when the DPPH clearance rate of the experimental group is calculated, the absorbance of the test sample is the absorbance of the experimental group 1 and the absorbance of the experimental group 2 respectively, and the absorbance of the blank group is the absorbance of the blank group 1 and the blank group 2 respectively;

when the DPPH clearance rate of the control experiment group is calculated, the absorbance of the test sample is the absorbance of the control experiment group, and the absorbance of the blank group is the absorbance of the blank group 3;

when the DPPH clearance rate of the positive control group is calculated, the absorbance of the test sample is the absorbance of the positive control group, and the absorbance of the blank group is the absorbance of the blank group 4;

the higher the clearance rate, the stronger the antioxidant capacity, and the results are shown in fig. 6, when no DPPH free radical exists in the solution, the DPPH clearance rate of the blank group is 0, the DPPH free radical removal rate of the snow tea extract prepared in the third embodiment of the present invention is 33.62%, the DPPH free radical removal rate of the snow tea extract prepared in the fourth embodiment of the present invention is 28.12%, and the DPPH free radical removal rate of the snow tea extract prepared by the prior art is less than 20%, which indicates that the snow tea extract prepared by the present invention has the superior DPPH clearance rate compared with the prior art.

Moisturizing performance of snow tea extract

The amount of moisture in the skin directly affects the elasticity, luster, etc. of the skin, and the epidermis, dermis, and subcutaneous tissues of the skin all play different roles in maintaining moisture in the skin. During the process of skin moisturization, there are two main mechanisms that affect the water maintenance of the skin:

1) the skin is used as a natural barrier to avoid water loss;

the epidermis of the skin is the natural barrier of humans, where the stratum lucidum, stratum granulosum and stratum corneum play an important role in the water-holding capacity of the skin. The transparent layer contains phospholipids and keratin, and can prevent moisture and electrolyte from penetrating skin. The cells in the granular layer are arranged compactly, and have important functions of storing moisture and preventing moisture from permeating. The stratum corneum is a thin, tough, elastic lamellar structure formed by the keratin, and the cells are filled with keratin.

2) Many moisturizing factors are present in the skin, absorbing and locking up moisture.

Effect of Thamnolia vermicularis extract on expression level of hyaluronic acid synthase (HAs3) in HDF cells

In normal skin, HA is mainly synthesized by HAs, the HAs mainly comprise three types, namely HAS1, HAs2 and HAs3, wherein HAs3 HAs the largest catalytic activity and catalyzes the generation of HA with small molecular weight (200 and 300ku), and the HA is a known substance with the strongest moisturizing capability, so that the content of HAs3 in the skin is increased to indirectly promote the moisturizing capability of the skin.

The test was divided into blank, experimental 1, experimental 2, control and standard groups.

Taking the logarithmic growth phase with the concentration of 10⁵HDF cells/m L, the cultured cells were seeded in 24-well plates at a cell sap of 1m L per well and contained CO at 37 ℃₂The cell culture box is used for culturing for 6 hours.

Adding a certain amount of the snow tea extract provided by the third embodiment of the invention into the cell sap of the experiment group 1, adding a certain amount of the snow tea extract provided by the fourth embodiment of the invention into the cell sap of the experiment group 2, adding the snow tea extract provided by the comparative example into the control experiment group, adding the standard solution into the standard group, and adding no sample into the blank group.

Placing at 37 deg.C with 5% CO₂And culturing in a cell culture box with saturated humidity for 24h, respectively, removing supernatant, washing the cells twice with precooled PBS, adding 100 mu L lysate into the culture hole, lysing the cells on ice for 10min, collecting cell lysate, centrifuging at 13000rpm for 10min, and taking the supernatant for later use.

Taking cell lysate for testing according to the instruction of an E L ISA kit;

the BCA kit is used for detecting the content of different groups of proteins, and the expression level of HAs3 in each group is corrected by protein amount, and the results are shown in Table 7 and FIG. 7.

TABLE 7 influence of snow tea extract on expression of HAs3

Group of	Content of HAs3 (%)
		Experimental group 1	126.04
Experimental group 2	124.22
		Control experiment group	112.35
Blank group	100

As can be seen from table 7 and fig. 7, the content of HAs3 in human HDF cells was increased by 27% after adding the snowtea extract provided by the present invention, compared to the content without adding the snowtea extract, which indicates that the snowtea extract provided by the present invention HAs better moisture retention than the snowtea extract prepared by the prior art.

Influence of Thamnolia vermicularis extract on hyaluronic acid secretion

In the dermis layer of the skin, Hyaluronic Acid (HA) is a high-content aminopolysaccharide, HAs extremely high viscosity and can be combined with water molecules in a high proportion, and the content of HA directly influences the content of moisture in the skin. As an important moisture retention component in skin, the skin moisturizing cream has obvious effects on proliferation and differentiation of skin cells and is important for maintaining normal metabolism of the skin cells.

Taking the concentration of logarithmic growth phase as 10⁵Culturing human HDF cells at a concentration of L/m, inoculating the cultured cells into 24-well plates at a cell sap concentration of 1m L/well at 37 ℃,Containing CO₂The cell culture box is used for culturing for 6 hours.

Adding the extract of Thamnolia vermicularis (Ach.) Asa-hina provided in example III of the present invention to the cell sap of experiment group 1, adding the extract of Thamnolia vermicularis (Ach.) Asa-hina provided in example IV of the present invention to the cell sap of experiment group 2, adding the extract of Thamnolia vermicularis (Ach.) Asa provided in comparative example to the cell sap of control experiment group, and standing at 37 deg.C and 5% CO₂And culturing in a cell culture box with saturated humidity for 24h respectively;

the supernatant medium was carefully collected and centrifuged to obtain the supernatant for use.

Adding 50 mu L standard solution into the standard group, adding 10 mu L supernatant and 40 mu L diluent of the snow tea extract provided by the third embodiment of the invention into the experimental group 1, adding 10 mu L supernatant and 40 mu L diluent of the snow tea extract provided by the fourth embodiment of the invention into the experimental group 2, adding 10 mu L supernatant and 40 mu L diluent of the snow tea extract provided by the comparative embodiment into the control experimental group, adding no sample into the blank group, respectively incubating for 1h at 37 ℃, and washing the plate for 5 times.

Color reagent A and B50 μ L each were added to the five groups of samples, incubated for 30min at 37 ℃ in the dark, and stop solution 50 μ L was added to the five groups of samples.

The blank was set to zero, and absorbance values of each group were measured at a wavelength of 450nm within 15min after termination of the reaction, and the HA content in the cell sap was calculated from the absorbance values, with the results shown in Table 8 and FIG. 8.

TABLE 8 influence of snow tea extract on HA secretion

Group of	HA content (%)
		Experimental group 1	112.99
Experimental group 2	113.75
		Control experiment group	105.32
Blank group	100

As can be seen from table 8 and fig. 8, the HA content in human HDF cells increased by about 13% after the addition of the snow tea extract provided by the present invention, compared to that without the addition of the snow tea extract. And after the snow tea extract provided by the prior art is added, the HA content in the HDF cells is improved by 5%, which shows that compared with the snow tea extract prepared by the prior art, the snow tea extract provided by the invention can obviously promote the HDF cells to secrete HA, effectively keep the water in the cells and further play a role in moisturizing.

Influence of Thamnolia vermicularis extract on the expression of loricrine (L OR)

The stratum corneum is a tough, elastic lamellar structure composed of keratinocytes, which are filled with keratin. Keratin is water-insoluble scleroprotein and has the function of preventing the internal permeation of chemical substances and the external permeation of body fluid, wherein the keratin with the highest content is called paphiopediin, which accounts for about 80 percent of the keratin, and the content of the paphiopediin directly influences the loss condition of skin moisture.

The test examines the expression of Hacat cell pangolin (L OR) to examine the moisturizing performance of the snow tea extract provided by the invention, and the test is divided into a blank group, an experiment group 1, an experiment group 2, a control experiment group and a standard group.

Taking the concentration of logarithmic growth phase as 10⁵Hacat cells at a cell density of L/m, cultured cells were seeded in 24-well plates at a cell sap of 1m L/well and containing CO at 37 deg.C₂The cell culture box is used for culturing for 6 hours.

Adding the extract of Thamnolia vermicularis (Ach.) Asa-hina provided in the third embodiment of the present invention to the cell sap of experiment group 1, and adding the extract of Thamnolia vermicularis (Ach.) Asa-hina to the cell sap of experiment group 2Adding the extract of Thamnolia vermicularis (Ach.) Asa-hina of the fourth embodiment of the present invention into the solution, adding the extract of Thamnolia vermicularis (Ach.) Asa of the comparative embodiment into the cell fluid of the control experiment group, and standing at 37 deg.C with 5% CO₂And culturing in a cell culture box with saturated humidity for 24h respectively;

discarding the supernatant, washing the cells twice with precooled PBS, adding 100 mu L lysate into the culture well, lysing the cells on ice for 10min, collecting the cell lysate, centrifuging at 13000rpm for 10min, and taking the supernatant for later use.

Taking cell lysate for testing according to the instruction of an E L ISA kit;

the BCA kit is used for detecting the protein content of different groups, the expression level of L OR of each group is corrected by protein amount, and the results are shown in Table 9 and FIG. 9.

TABLE 9 influence of snow tea extract on L OR expression

Group of	Experimental group 1	Experimental group 2	Blank group	Control experiment group
					L OR content (%)	86.75	131.85	100	112.95

As can be seen from table 9 and fig. 9, the L OR content in human HaCat cells was increased by 31% compared to the blank group after adding the snowtea extract provided in example four of the present invention, while the L OR content was increased by only 12% compared to the blank group after adding the snowtea extract prepared by the prior art, which indicates that the snowtea extract provided by the present invention can significantly promote the expression of L OR.

In conclusion, the snow tea extract provided by the invention has remarkable moisturizing performance and excellent anti-aging and anti-oxidation capacity, and can be added into cosmetics as a dual-function additive.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. The snow tea extract is characterized by comprising depside compounds, wherein the depside compounds are scale-coating acid, sheep-horn-coating acid and snow tea essence,

wherein the snow tea extract is prepared by a method comprising the steps of:

taking 10kg of dried snow tea as a raw material, washing the raw material with deionized water for 2 times, removing impurities, crushing the raw material, sieving the raw material with an 80-mesh sieve, adding a 70% methanol solution and a 70% ethanol solution which are 10 times of the volume ratio of the raw material into the raw material, extracting the mixture for 2 hours in a 70 ℃ water bath at normal pressure, adding a 50% ethanol solution which is 10 times of the amount of the raw material into the raw material after the extraction is finished, extracting the mixture for 2 hours in a 65 ℃ water bath at normal pressure, adding deionized water which is 10 times of the amount of the raw material into the mixture after the extraction is finished, extracting the mixture for 2 hours in a 80 ℃ water bath at normal pressure, combining the extracted solutions for 3 times after the extraction is finished, concentrating the obtained extracted solution to 5L under the condition of 5kPa and slightly boiling, cooling the obtained solution, placing two layers of filter paper on a decompression funnel, filtering the concentrated solution under reduced pressure, and spray-drying a filter cake to obtain the snow tea extract, wherein the extract is prepared into particles with.

2. The snow tea extract according to claim 1, wherein the depside compound is contained in an amount of 40% by mass.

3. A method for preparing a snow tea extract according to any one of claims 1 to 2, comprising the steps of:

4. Use of the extract of Thamnolia vermicularis as claimed in any one of claims 1-2 for preparing cosmetics.

5. Use of a snow tea extract according to claim 4, characterized in that the cosmetic is for moisturizing and/or anti-aging.