KR20200107880A - Composition for protecting skin containing oenothera biennis extract as active ingredient - Google Patents

Abstract

The present invention relates to a composition for protecting skin, containing an Oenothera tetraptera extract as an active ingredient. The Oenothera tetraptera uses an aerial part thereof excluding a root part. The composition according to the present invention has no cytotoxicity, has excellent antioxidant activity due to high reducing power and DPPH radical scavenging ability, and increases the survival rate of cells by inhibiting the production of reactive oxygen species, DNA damage, apoptosis and mitochondrial membrane damage, thereby protecting the skin.

Description

Skin protection composition containing evening primrose extract as an active ingredient {COMPOSITION FOR PROTECTING SKIN CONTAINING OENOTHERA BIENNIS EXTRACT AS ACTIVE INGREDIENT}

The present invention relates to a composition for skin protection containing an evening primrose extract as an active ingredient.

Skin aging can be classified into endogenous aging that occurs naturally and exogenous aging that is caused by an external environment.

Endogenous aging is caused by genetic factors and cell metabolism imbalance, and exogenous aging is mainly caused by external stimuli such as UV stimulation.

When the skin is irritated by exposure to UV rays, reactive oxygen species (ROS) are generated, which induces damage due to oxidative stress in the skin and promotes skin aging.

When skin aging is promoted due to oxidative stress, DNA damage is caused. If such damage is severe enough to be irreparable, apoptosis is induced, destroying the epithelial structure of the skin and causing skin diseases.

Accordingly, research and development of new antioxidant substances capable of treating skin aging and skin diseases caused by oxidative stress are required.

Antioxidants that have been used up to now include butyrate hydroxytoluene (BHT), but it has been reported to be involved in the occurrence of malformations or cancer in recent animal experiments, and thus there is a problem in the use of BHT.

Therefore, there is a need for research to develop antioxidants that are safe to be absorbed by the human body due to improved side effects, and have excellent antioxidant effects.

Meanwhile, the present inventor intends to develop a composition for skin protection with low cytotoxicity and excellent antioxidant activity by using an evening primrose extract. Looking at the evening primroses as follows.

Evening primrose ( Oenothera biennis ) is a biennial herb of the needle flower family. It is also called Yaraehyang or Wolhahyang, and can be found all over Korea.

In oriental medicine, the root is called Wolgyeoncho, and when you have a cold and sore throat, put it in water and take it after decoction.

Looking at the prior art related to evening primrose, Korean Patent Publication No. 10-1892626 describes "a composition for preventing or improving allergic diseases comprising an evening primrose extract as an active ingredient".

However, when comparing the prior art with the present invention, it is similar in that an evening primrose is used, but the effect of the present invention is different in that the evening primrose is used to protect the skin from oxidative stress.

Accordingly, the present invention has completed the present invention by finding that the extract of the arial part of the evening primrose is effective in suppressing the damage caused by oxidative stress in human keratinocytes.

Republic of Korea Patent Publication No. 10-1892626 (2018.08.22)

It is an object of the present invention to provide a composition for skin protection containing an evening primrose extract as an active ingredient.

More specifically, it has no cytotoxicity, high reducing power and DPPH radical scavenging ability, so it has excellent antioxidant activity, and has the effect of increasing the survival rate of cells by inhibiting the production of reactive oxygen species, DNA damage, apoptosis and mitochondrial membrane damage. It is intended to provide a composition for skin protection containing as an active ingredient.

The present invention is to solve the technical problem by providing a composition for skin protection, characterized in that it contains an evening primrose extract as an active ingredient.

The evening primrose is characterized in that the above-ground portion excluding the root portion.

The treatment concentration of the evening primrose extract is characterized in that 50 to 70 ug/ml in 3 x 10 ⁵ cells/ml of human keratinocytes (HaCaT).

The composition has no cytotoxicity, high reducing power and DPPH radical scavenging ability, so it has excellent antioxidant activity, and inhibits the production of reactive oxygen species, DNA damage, apoptosis, and mitochondrial membrane damage, thereby increasing the survival rate of cells.

In addition, the present invention provides a cosmetic composition or a health functional food composition, characterized in that it contains evening primrose as an active ingredient.

The composition for skin protection containing the evening primrose extract according to the present invention has no cytotoxicity, has high reducing power and DPPH radical scavenging ability, has excellent antioxidant activity, and inhibits the production of reactive oxygen species, DNA damage, apoptosis and mitochondrial membrane damage. Thus, it has the effect of increasing the survival rate of cells.

Figure 1 is a diagram showing the effect of the evening primrose extract on the survival rate of HaCaT cells, (a) is a graph showing the effect on the survival rate of HaCaT cells when the evening primrose extract is treated at various concentrations, (b) is an evening primrose extract It is a graph showing the effect of hydrogen peroxide treatment on the survival rate of HaCaT cells induced oxidative stress.
Figure 2 is a diagram showing the reducing power and DPPH radical scavenging ability of the evening primrose extract, (a) is a graph showing the reducing power of the evening primrose extract, (b) is a graph showing the DPPH radical scavenging ability of the evening primrose extract.
Figure 3 is a view showing the effect of the evening primrose extract on the production of reactive oxygen species in HaCaT cells induced oxidative stress by treatment with hydrogen peroxide, (a) is a photomicrograph showing the level of reactive oxygen species production in HaCaT cells (200 Magnification), (b) is a graph showing the result of measuring the production level of reactive oxygen species with a flow cytometer, (c) is a graph showing the degree of fluorescence expression measured by a fluorescence microscope, and (d) is the production of reactive oxygen species It is a graph showing the degree.
Figure 4 is a diagram showing the effect of the evening primrose extract on the comet generation of HaCaT cells induced oxidative stress by treatment with hydrogen peroxide, (a) is a fluorescence micrograph showing the degree of comet generation, (b) is the tail of the comet It is a graph measuring motion (tail moment, TM), and (c) is a graph measuring the length of the comet tail (tail length, TL).
5 is a view showing the effect of the evening primrose extract on the protein expression of HaCaT cells induced oxidative stress by treatment with hydrogen peroxide, (a) is an electrophoresis picture showing the expression level of γH2AX and phosphorylated γH2AX, (b) Is a graph showing the change in the ratio of phosphorylated γH2AX protein according to the level of γH2AX expression, (c) is an electrophoresis picture showing the degree of activation of caspase-3 and PARP, and (d) is keap1 according to the treatment time of evening primrose extract , Is an electrophoresis picture showing the expression level of Nrf2 and HO-1.
Figure 6 is a view showing the effect of the evening primrose extract on the mitochondrial membrane potential of HaCaT cells induced oxidative stress by treatment with hydrogen peroxide.(a) is a flow cytometer measuring the degree to which JC-1 aggregates change into JC-1 monomers And (b) is a graph showing the red fluorescence value that changes as the JC-1 aggregate changes to the JC-1 monomer.
7 is a diagram showing the effect of the evening primrose extract on the death of HaCaT cells induced by oxidative stress by treating hydrogen peroxide, (a) is 4',6'-diamino-2-phenylindole (4',6 '-diamidino-2-phenylindol, DAPI) stained cell nucleus is a fluorescence micrograph, (b) is a graph showing the degree of apoptosis in the experimental group and the control group, and (c) apoptosis induced cells This is a picture showing the degree of DNA cleavage by extracting and electrophoresis of DNA.

Terms and words used in this specification and claims should not be construed as being limited to their usual or dictionary meanings, and that the inventor can appropriately define the concept of terms in order to describe his own invention in the best way. Based on the principle, it should be interpreted as a meaning and concept consistent with the technical idea of the present invention.

Therefore, the embodiments and experimental examples described in the present specification are only the most preferred embodiments of the present invention and do not represent all of the technical spirit of the present invention, and various equivalents and modifications that can replace them at the time of application It should be understood that there may be examples.

Experimental Example 1. Preparation of evening primrose extract

Evening primroses collected in Sangju, Gyeongsangbuk-do, were washed with water and air dried at room temperature.

After selecting only the above-ground part excluding the root part of the evening primrose, it was pulverized using a grinder to obtain powder.

100 g of the powder was added to 2 L of ethanol and mixed with shaking at 100 rpm for 24 hours at 25°C.

The obtained evening primrose extract was filtered using a filter, and a solvent removed evening primrose powder was obtained using a vacuum rotary concentrator (N-1000S).

Evening primrose powder was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 100 mg/ml and then stored at 4°C.

The stock solution was diluted to a desired concentration using physiological saline, and the experiment was conducted.

The results of all experiments conducted below were expressed as ±S.D. (standard deviation) based on the average value, and if the P-valus of the result was less than 0.05, it was determined to be significant.

Experimental Example 2. Cell culture and cell viability analysis

1. Cell culture

The human skin keratinocyte (HaCaT) cell line used in this experiment was purchased from the American Type Culture Collection (ATCC).

HaCaT cells were plated with Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (heated to inactivate), streptomycin (100 μg/mL), and penicillin (100 unit/mL). Using, it was cultured while maintaining 5% CO2 and 37 ℃ in a carbon dioxide incubator.

2. Analysis of viability of cells treated with evening primrose extract

To analyze the cell viability, HaCaT cells were inoculated into a 6-well plate at 3 x 10 ⁵ cells/ml, incubated for 24 hours, treated with evening primrose extracts of various concentrations, and cultured for 24 hours. .

1 (a) is a graph showing the survival rate of HaCaT cells when the evening primrose extract was treated at various concentrations.

As a result, the evening primrose extract did not significantly affect cell viability at concentrations up to 60 ug/ml, but gradually decreased cell viability at concentrations of 80 ug/ml or more.

Therefore, in order to examine the effect of the evening primrose extract on the hydrogen peroxide-treated HaCaT cells, the experiment was conducted at a concentration of 60 ug/ml.

To investigate the effect of evening primrose extract on HaCaT cells induced oxidative stress by treating hydrogen peroxide for 30 minutes, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) analysis was performed. I did.

After the cells were treated with 0.5 mg/ml of MTT, they were incubated at 37°C for 3 hours.

After the culture was completed, the medium was removed and the formazan precipitate was dissolved in dimethyl sulfoxide.

The absorbance of the formazan product was measured at a wavelength of 540 nm using a Cytation-3 microplate reader.

Cells that were not treated with anything were treated with normal group, cells treated with 0.5 mM hydrogen peroxide as control 1, antioxidant N-acetyl-L-cysteine (NAC) 10 mM, and 0.5 mM hydrogen peroxide. The treated cells were treated with the control group 2 and the evening primrose extract, and then the cells treated with the evening primrose extract were designated as the experimental group.

1(b) is a graph showing the effect of the evening primrose extract on the survival rate of HaCaT cells induced oxidative stress by treatment with hydrogen peroxide.

As a result, the viability of the control group 1 treated with hydrogen peroxide was significantly reduced.

However, the experimental group pretreated with the evening primrose extract increased the survival rate in a concentration-dependent manner of the evening primrose extract, as in the control group 2 pretreated with NAC.

Experimental Example 3. Analysis of reducing power and DPPH radical scavenging ability of evening primrose extract

1) Analysis of reducing power of evening primrose extract

In order to analyze the reducing power of the evening primrose extract, the degree of reduction of the Fe ³⁺ / ferric cyanide complex to the Fe2 ⁺ form was analyzed.

Evening primrose extract (0-100 ug/ml) was dissolved in 50 ml of 0.1M phosphate buffer (pH 6.6), and 0.5 g of potassium ferricyanide was added and mixed.

The mixture was reacted at 50° C. for 30 minutes, and 10% trichloroacetic acid (trichloroacetic acid, 50 ml, 5 g) was added and mixed.

100 ul of distilled water and 0.1% ferric chloride (10 ml, 0.01 g) were added to 100 ul of the mixed solution, and the absorbance was measured at a wavelength of 700 nm using a Cytation-3 microplate reader.

100 ug/ml of ascorbic acid, a representative antioxidant, was used as a positive control, and evening primrose extract was used as an experimental group.

The reducing power of the evening primrose extract was analyzed by setting the degree of reduction of ascorbic acid to Fe ³⁺ / ferric cyanide complex to Fe 2 ⁺ form at 100%.

Figure 2 (a) is a graph showing the reducing power of the evening primrose extract.

As a result, the reducing power of the evening primrose extract increased in a concentration-dependent manner, and showed the highest reducing power at a concentration of 60 ug/ml.

2) DPPH radical scavenging activity analysis of evening primrose extract

To analyze the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of evening primrose extract, DHHP in 100 ul of evening primrose extract (0-100 ug/ml) 100 ul of a methanol solution containing radicals (final concentration 0.2 mM) was added and mixed.

After reacting the mixed solution at 25° C. for 30 minutes, the absorbance of the mixed solution was measured at a wavelength of 517 nm using a spectrophotometer.

100 ug/ml of ascorbic acid, a representative antioxidant, was used as a positive control, and evening primrose extract was used as an experimental group.

2(b) is a graph showing the DPPH radical scavenging activity of the evening primrose extract.

As a result, DPPH radical scavenging activity of evening primrose extract increased in a concentration dependent manner, and the highest scavenging activity was shown at a concentration of 60 ug/ml.

Experimental Example 4. Measurement of ROS production level of cells treated with evening primrose extract

To measure the level of production of reactive oxygen species (ROS) in cells, 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA), an oxidation-sensitive agent, was used.

After washing the cultured HaCaT cells twice with phosphate-buffered saline (PBS), 10 uM DCF-DA was added and reacted at 37° C. for 30 minutes in the dark.

After washing the reaction solution with PBS, FL-1 fluorescence was measured with a flow cytometer (FACS Calibur).

Control 1 for cells treated with only 60 ug/ml of evening primrose extract, control 2 for cells treated with only 10 mM NAC, positive control for cells treated with 0.5 mM hydrogen peroxide, and 10 mM NAC After that, the cells treated with 0.5 mM hydrogen peroxide were treated with a negative control and 60 ug/ml of evening primrose extract, and then the cells treated with 0.5 mM hydrogen peroxide were designated as the experimental group.

Figure 3 is a view showing the effect of the evening primrose extract on the production of reactive oxygen species in HaCaT cells induced oxidative stress by treatment with hydrogen peroxide, (a) is a photomicrograph showing the level of reactive oxygen species production in HaCaT cells (200 Magnification), (b) is a graph showing the result of measuring the production level of reactive oxygen species with a flow cytometer, (c) is a graph showing the degree of fluorescence expression measured by a fluorescence microscope, and (d) is the production of reactive oxygen species It is a graph showing the degree.

As a result, the positive control treated with hydrogen peroxide for 30 minutes showed a significant increase in the production of intracellular reactive oxygen species, whereas the negative control pretreated with NAC and the experimental group pretreated with evening primrose extract suppressed the production of reactive oxygen species.

Experimental Example 5. Measurement of DNA damage in cells treated with evening primrose extract using comet assay

The comet assay is an experimental method that measures the degree of DNA damage to cells by using a single cell gel electrophoresis assay.

When the cell is damaged, the double strands of DNA are cut to form fragments, and the traces of the fragments resemble the shape of a comet passing by.

The smaller the molecular weight of the DNA is, the farther it is from the nucleus, so the more damaged and cut DNA strands, the longer a comet tail is formed.

In order to analyze the effect of the evening primrose extract on the DNA damage of HaCaT cells induced oxidative stress by treating hydrogen peroxide for 30 minutes, a Comet assay was performed.

HaCaT cells treated with evening primrose extract were washed with PBS, and then mixed with 0.5% agarose gel.

The mixture was put on a slide glass coated with 1% agarose gel on a slide glass, covered with a cover glass, and stored at 4°C for 10 minutes.

After the gel was hardened, the cover glass was removed, and 0.5% agarose gel was dropped on the slide, and then stored at 4° C. for 40 minutes.

After confirming that the gel is solid, mix the solution buffer solution (2.5 M NaCl and 500 mM Na-ethylene diamine tetra acetic acid (EDTA)) and 1% triton X-100, and immerse the slide glass with the cover glass removed. It was immersed in for 1 hour.

Then, the slide glass was immersed in an unwinding buffer solution (300 mM NaOH, 10 mM Na ₂ EDTA, pH 13) to unwind the double strands of DNA, and then the slide glass was transferred to an electrophoresis tank.

Then, electrophoresis was performed for 20 minutes at a voltage of 25 V/300 mA.

After electrophoresis was completed, the slide was washed 3 times with 0.4 M Tris (pH 7.5) for 5 minutes at 25°C, stained with 20 ug/ml propidium iodide (PI), and a fluorescence microscope (Carl Zeiss) Was observed.

The cells treated with nothing were normal, cells treated with only 60 ug/ml of evening primrose extract were treated as control 1, cells treated with 0.5 mM hydrogen peroxide were positive control, and cells treated with 10 mM NAC and then 0.5 mM hydrogen peroxide were negative. A control group, treated with 60 ug/ml of evening primrose extract, and then treated with 0.5 mM hydrogen peroxide, was designated as an experimental group.

* Figure 4 is a diagram showing the effect of the evening primrose extract on the comet generation of HaCaT cells that induce oxidative stress by treating hydrogen peroxide, (a) is a fluorescence micrograph showing the degree of comet generation, (b) is a comet tail It is a graph measuring the motion (tail moment, TM), and (c) is a graph measuring the length of the comet tail (tail length, TL).

As a result, the tail length and tail motion values of the positive control treated with hydrogen peroxide increased, but the tail length and tail motion values of the negative control pretreated with NAC and the experimental group pretreated with evening primrose extract decreased.

Accordingly, it was confirmed that the evening primrose extract inhibits DNA damage.

Experimental Example 6. Western blot analysis after protein extraction of cells treated with evening primrose extract

Hydrogen peroxide-treated cells are subjected to oxidative stress, intracellular reactive oxygen species are generated, and the generated reactive oxygen species cleave DNA double strands.

At this time, the histone protein γH2AX, a protein that maintains the double strands of DNA, is phosphorylated, and the degree of phosphorylation of the γH2AX protein can be analyzed to determine the degree of DNA damage.

In addition, when the cells are subjected to oxidative stress to generate reactive oxygen species, damage to the cells is caused, and if the damage caused is severe enough to be difficult to repair, apoptosis occurs.

When apoptosis progresses, when apoptosis factor is activated, proteolytic enzymes caspase-3 and poly(ADP-ribose) polymerase are used to degrade unnecessary cell proteins. , PARP) is activated.

The degree of apoptosis can be determined according to the degree of caspase-3 and PARP activation.

In addition, when cells are subjected to oxidative stress, the expression of Kelch-like ECH-associated protein 1 (Keap1) is increased, and nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase (HO-1) are released. It is activated and acts as an antioxidant against oxidative stress.

Nrf2 and HO-1 play an important role in antioxidant activity that inhibits oxidative stress in cells, and antioxidant activity against oxidative stress in cells can be judged according to the expression level of Nrf2 and HO-1.

To analyze the effect of evening primrose extract on the DNA damage, apoptosis and antioxidant activity of HaCaT cells induced by oxidative stress by treating hydrogen peroxide for 30 minutes, γH2AX, caspase-3, PARP, Keap1, NrF2 and HO-1 proteins The expression level of was measured.

Lysis buffer solution (20 mM sucrose, 1 mM EDTA, 20 μM Tris-Cl (pH 7.2), 1 mM dithiothreitol, DTT), 10 mM KCl, 1.5 mM MgCl ₂ and 5 ug/ml for HaCaT cells Aprotinin) was added and reacted for 30 minutes, and then centrifuged at 12,000 rpm for 20 minutes to obtain a supernatant from which cell membrane components were removed.

Protein present in the supernatant was quantified using a protein extraction kit (Bio-Rad Protein Assay Kit).

30-50 ug of lysate was separated by polyacrylamide gel electrophoresis (SDS-PAGE), and this was transferred to a polyvinylidene fluoride (PVDF) membrane.

After treatment with the primary antibody and secondary antibody binding to the protein for which the expression level is to be checked, enhanced chemiluminescence (ECL, Amersham Corp) solution was treated, and then photosensitized on an X-ray film.

* The control group and the experimental group were set in the same manner as in Experimental Example 6.

5 is a view showing the effect of the evening primrose extract on the protein expression of HaCaT cells induced oxidative stress by treatment with hydrogen peroxide, (a) is an electrophoresis picture showing the expression level of γH2AX and phosphorylated γH2AX, (b) Is a graph showing the change in the ratio of phosphorylated γH2AX protein according to the level of γH2AX expression, (c) is an electrophoresis picture showing the degree of activation of caspase-3 and PARP, and (d) is keap1 according to the treatment time of the evening primrose extract. , Is an electrophoresis picture showing the expression level of Nrf2 and HO-1.

As a result, the positive control treated with hydrogen peroxide increased phosphorylation of γH2AX, indicating that DNA was damaged, and that caspase-3 and PARP were activated, leading to apoptosis.

However, in the negative control pretreated with NAC and the experimental group pretreated with evening primrose extract, it was found that the phosphorylation of γH2AX was reduced, so DNA damage was suppressed, and apoptosis was suppressed because caspase-3 and PARP were not activated. there was.

In addition, since the expression of Nrf2 and HO-1 was increased according to the treatment time of the evening primrose extract, it was found that antioxidant activity was active.

Experimental Example 7. Mitochondrial membrane potential analysis of cells treated with evening primrose extract

Reactive oxygen species attack the mitochondria in cells and cause damage.

When damage to the mitochondria is caused, the mitochondrial membrane potential (MMT) changes to a depolarization state, and the degree of damage to the mitochondria can be measured by analyzing the degree of depolarization.

To determine the mitochondrial membrane potential, a fluorescent dye called JC-1 can be used.

In the case of JC-1, in cells where apoptosis has not occurred, JC-1 is bound in the form of an aggregate and exhibits red fluorescence, whereas in cells where apoptosis occurs, JC-1 is bound as a monomer, reducing red fluorescence and green color. Fluorescence increases.

Through the change in expression of red fluorescence and green fluorescence, the degree of apoptosis can be analyzed by measuring the change in mitochondrial membrane potential.

After culturing the HaCaT cells, only the cells were collected and suspended in phosphate buffered saline containing JC-1 (10 μg/ml).

After reacting at 37° C. for 15 minutes, only cells were collected, suspended in phosphate buffered saline, and measured using a flow cytometer.

The control group and the experimental group were set in the same manner as in Experimental Example 6.

6 is a view showing the effect of the evening primrose extract on the mitochondrial membrane potential of HaCaT cells induced oxidative stress by treating hydrogen peroxide.(a) is a flow cytometer measuring the degree to which the JC-1 aggregate changes to the JC-1 monomer. And (b) is a graph showing the red fluorescence value that changes as the JC-1 aggregate changes to the JC-1 monomer.

As a result, in the positive control treated with hydrogen peroxide, the mitochondrial membrane was damaged, resulting in a change in potential (depolarization), the red fluorescence value of JC-1 decreased, and the green fluorescence value increased.

However, no change in mitochondrial membrane potential was observed in the negative control group pretreated with NAC and the experimental group pretreated with evening primrose extract, and the red fluorescence value of JC-1 was similar to that of the normal group.

Experimental Example 8. Analysis of the degree of death of cells treated with evening primrose extract

Reactive oxygen species cause damage to cells and induce apoptosis.

In the cell membrane of normal cells, a protein called phosphatidylserine exists, and in cells in which apoptosis is induced, the inside and outside of the cell membrane are turned over, and the phosphatidylserine, which should be in the cell membrane, exists outside the cell membrane.

At this time, the presence or absence of apoptosis can be measured by analyzing the degree of fluorescence expressed by treatment with annexin V, a fluorescent dye that binds to phosphatidylserine.

In order to investigate the effect of evening primrose extract on the death of HaCaT cells induced oxidative stress by treating hydrogen peroxide for 30 minutes, an annexin V-FITC apoptosis detection kit (BD Biosciences) was used.

After treating HaCaT cells with evening primrose extract for 24 hours, wash with phosphate buffered saline, and use 5 ul of binding buffer mixed with annexin V and propidium iodide (PI) in the dark. After staining for 15 minutes at room temperature, the stained cells were measured for annexin V positive cells indicating dead cells using a flow cytometer (FACS calibur).

The control group and the experimental group were set in the same manner as in Experimental Example 6.

7 is a view showing the effect of the evening primrose extract on the death of HaCaT cells induced oxidative stress by treating hydrogen peroxide, (a) is 4',6'-diamino-2-phenylindole (4',6 '-diamidino-2-phenylindol, DAPI) stained cell nucleus is a fluorescence micrograph, (b) is a graph showing the degree of apoptosis in the experimental group and the control group, and (c) apoptosis induced cells This is a picture showing the degree of DNA cleavage by extracting and electrophoresis of DNA.

As a result, in the positive control treated with hydrogen peroxide, apoptosis was induced and cell nuclei were destroyed, and annexin V-positive cells and DNA cleavage were increased.

However, the cell nuclei of the negative control group pretreated with NAC and the experimental group pretreated with evening primrose extract were similar to those of the normal group, and annexin V-positive cells and DNA cleavage were decreased.

Therefore, when the results of the experimental example are summarized, the evening primrose extract has no cytotoxicity, has excellent antioxidant activity due to high reducing power and DPPH radical scavenging ability, and inhibits the production of reactive oxygen species, DNA damage, apoptosis, and mitochondrial membrane damage. Since it increases the survival rate, it was confirmed that it is an effective composition for skin protection.

Example 1. A cosmetic composition for skin protection containing evening primrose extract as an active ingredient.

The composition according to the present embodiment is a cosmetic composition for skin protection containing an evening primrose extract as an active ingredient, based on the experimental results of the experimental example.

The concentration of the active ingredient is preferably 50 to 70 ug/ml in 3 x 10 ⁵ cells/ml of human keratinocytes (HaCaT) based on the above experimental examples.

The cosmetic composition is skin, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, eye cream, moisture cream, hand cream, essence, nutrition essence, pack, cleansing foam , Cleansing water, cleansing lotion, cleansing cream, body lotion, body cleanser, soap, and powder can be prepared in any one formulation selected from the group consisting of.

When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fibers, plant fibers, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide as carrier components Etc. can be used.

When the formulation of the cosmetic composition of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydro Propellants such as carbon, propane-butane or dimethylether may be included.

When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, a solvating agent or an emulsifying agent may be used as a carrier component, and water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan may be used.

When the formulation of the cosmetic composition of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like can be used.

When the formulation of the cosmetic composition of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate , Fatty acid amide ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty diethanolamides, vegetable oils, linoline derivatives or ethoxylated glycerol fatty acid esters, and the like may be used.

In addition, the cosmetic composition is a fatty substance, an organic solvent, a solubilizing agent, a thickening and gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, ionic or nonionic Emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic activators or lipid vesicles, and the like may be used.

Example 2. Skin protection health food composition containing evening primrose extract as an active ingredient

The composition according to the present embodiment is a health functional food composition for skin protection containing an evening primrose extract as an active ingredient, based on the experimental results of the experimental example.

The concentration of the active ingredient is preferably 50 to 70 ug/ml in 3 x 10 ⁵ cells/ml of human keratinocytes (HaCaT) based on the above experimental examples.

The health functional food composition of the present invention can prepare a health functional food such as foods, beverages, gum, tea or vitamin complex by adding food supplements.

The health functional food composition of the present invention is not limited in the use of other ingredients, and may contain flavoring agents or natural carbohydrates as additional ingredients.

Taumatin, stevia extract, rebaudioside A, glycyrrhizin, saccharin or aspartame may be advantageously used as the flavoring agent.

As the natural carbohydrate, glucose, fructose, maltose, sucrose, dextrin, cyclodextrin, xylitol, sorbitol, or erythritol may be used.

In addition to the above, the health functional food composition of the present invention includes nutrients, vitamins, minerals (electrolytes), synthetic flavors, natural flavors, colorants, heavy agents, pectic acids and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers. , Preservatives, glycerin, alcohols or carbonation agents.

Claims (1)

Pulverizing the aerial part of the evening primrose to obtain a powder;
Shaking and mixing the powder at 100 rpm for 24 hours after putting the powder in ethanol; And
In the method for producing a cosmetic composition for preventing skin cell death comprising the step of removing the solvent after filtering the mixture using a filter,
The treatment concentration of the cosmetic composition is 50 to 70 ug/ml in 3 x 10 ⁵ cells/ml of human keratinocytes (HaCaT),
The prevention of apoptosis is a method for producing a cosmetic composition for preventing skin apoptosis, characterized in that preventing apoptosis by preventing DNA damage, inactivating caspase-3 and PARP, and inhibiting a change in mitochondrial membrane potential.

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