KR20150131467A - Composition for protecting skin against UV light and wrinkle improvement comprising the extract of Polygonum viscoferum - Google Patents

Abstract

The present invention relates to a composition for protecting skin against UV lights and improving wrinkles comprising a Polygonum viscoferum extract. The composition comprising the Polygonum viscoferum extract: has an excellent free radical scavenging activity; inhibits apoptosis of dermal fibroblasts caused by ultraviolet rays; inhibits secretion of matrix metalloproteinase-1 (MMP-1) and expression of MMPs; and has an elastase inhibitory activity. Therefore, the composition of the present invention can protect skin against UV lights, and treat or prevent wrinkle, thereby being able to be utilized in biopharmaceutical and cosmetic industries.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing photoprotection and improving skin wrinkles, which comprises an extract of Safflower extract as an active ingredient,

The present invention relates to a composition for anti-aging or wrinkle improvement comprising an extract of Polygonum viscoferum or a pharmaceutically acceptable salt thereof as an active ingredient.

Skin aging is largely divided into intrinsic aging, natural aging, extrinsic aging, and photoaging. Natural aging is due to genetic causes, so it is difficult to control, but photoaging is due to environmental causes. This is easy. Therefore, studies on prevention of photoaging have been continued, and in particular, studies on prevention of wrinkling due to extrinsic aging caused by long-term exposure to ultraviolet light have been attracting attention (Gilchre st BA, J. Am. Acad. Dermatol., 1989: 21: 610-613). Exposure to large amounts of ultraviolet light results in a high concentration of reactive oxygen species in the skin and disruption of the enzymatic and nonenzymatic antioxidant defenses of the skin, resulting in significant collagen, And the expression of matrix metalloproteinase (hereinafter, referred to as 'MMP') is promoted, which has an important influence on collagen reduction, and the skin elasticity is lowered and wrinkles are formed. MMPs are classified into MMP-1, MMP-2, MMP-3 and MMP-9 according to their substrate specificity. Especially, MMP-1 is involved in degradation of extracellular matrix and basement membrane Studies have been reported that MMP-1 plays a very important role in wrinkle formation due to the collagen degradation of the dermis by increasing the activity of MMP-1 in the skin due to the enzyme, It has been reported that MMP-2 and MMP-9 are degraded by gelatinase, a major component of the basement membrane, type collagen (Honda A. et al., J Biotechnol Bioeng., 2005: 20: 40-45). MMP-3 is a stomelysin that degrades proteoglycans, elastin, fibronectin, and laminin in mouse skin, activates various MMPs, and is important for wrinkle formation. (Honda A., et al., J. Dermatol. Sci., 200 8: 49: 63-72). Therefore, there is a demand for development of a substance capable of modulating MMP expression induced in the cell or inhibiting its activity. Until now, the raw materials used as cosmetic materials mostly inhibited only the enzyme activity. Therefore, we tried to control the amount and activity of MMP expression induced by intracellular natural aging and photoaging.

Recently, much attention has been focused on functional cosmetics having functions of antioxidation, wrinkle reduction, whitening, etc., obtained from natural extracts in order to reduce skin irritation caused by various chemical substances and the like. In addition to low adverse effects on the skin, natural materials have recently become increasingly appreciated as a raw material for skin external preparations, as consumers' responses to external preparations using natural materials have increased. For example, U.S. Patent No. 5,972,341 describes the wrinkle-reducing effect of the Commiphora plant, especially the Commiphora mukul extract. Japanese Patent Laid-Open No. 2-245087 describes the antioxidative effect of Sargassum sp. Extract.

The active ingredients of skin wrinkles which are currently being developed are not usable as a cosmetic raw material, are very unstable, are difficult to be delivered to the skin, require a special stabilization system and delivery system, and are not effective in improving skin wrinkles Recently, retinoids have been increasingly attracted attention as a skin protective agent containing retinoids. Currently, retinoids are a means for solving photoaging phenomena such as wrinkles, skin thickening, sagging, and elasticity reduction which are accumulated as sunlight . However, retinoids are very unstable compounds, and they are sensitive to ultraviolet rays, moisture, heat, and oxygen, causing chemical changes easily.

On the other hand, it is a dicotyledonous plant which is native to the mountains and fields. Flowers bloom from July to August, and the height is 40 to 80 cm. There is no known pharmacological action of the known actinomycetes to date.

Accordingly, the inventors of the present invention have found that when investigating natural products having an effect of preventing photoaging or improving wrinkles, they have excellent anti-aging and anti-wrinkle effects on sticky leaves, thus completing the present invention.

Accordingly, an object of the present invention is to provide a composition for improving anti-aging or wrinkle formation, which comprises an extract of glue.

In order to achieve the object of the present invention, the present invention can provide a composition for preventing photoaging and wrinkles, which comprises an extract of glutinosa extract as an active ingredient:

In one embodiment of the present invention, the sticky layer is a protrusion of a sticky layer.

In another embodiment of the present invention, the extract of Safflower extract is an organic solvent extract.

In another embodiment of the present invention, the organic solvent is methanol.

In still another embodiment of the present invention, the extract of Safflower is characterized by scavenging free radicals.

In still another embodiment of the present invention, the extract of Safflower extract is characterized by inhibiting the cell death induced by UVB irradiation.

In still another embodiment of the present invention, the extract of Safflower extract is characterized by inhibiting collagen degradation induced by UVB irradiation.

In still another embodiment of the present invention, the extract of Safflower extract is characterized by inhibiting the activity of elastase.

In addition, the present invention can provide a cosmetic composition comprising the above extract of Safflower extract as an active ingredient.

In addition, the present invention can provide a food composition containing an extract of Safflower extract as an active ingredient.

The present invention relates to a composition for improving skin comprising a extract of glutinosa suppresses the decomposition of collagen induced by irradiation of ultraviolet rays to dermal fibroblasts, has a protective effect against cell death, and has a wrinkle-reducing effect, A composition useful for prevention of aging and improvement of wrinkles can be provided.

FIG. 1 is a graph showing the effect of extracts of the sticky plant on free radicals.
FIG. 2 is a graph showing the protective effect of extracts of the sticky plant on UVB-induced apoptosis.
FIG. 3 is a graph showing the effect of the extract of the present invention on the secretion of MMP-1 which degrades collagen by UVB irradiation in human dermal fibroblast.
FIG. 4 is a graph showing the mRNA expression levels of human dermal fibroblast cultured in UVB irradiated human dermal fibroblast MMP-1 (FIG. 4A), MMP-2 (FIG. 4B), MMP-3 FIG.
FIG. 5 is a graph showing the inhibitory activity of elastase according to the concentration of the extract of the sticky plant extract.

The present inventors have been engaged in research for searching for new natural products having a photoaging and wrinkle-reducing effect on various natural products, and have found that the free radical scavenging effect, the effect of protecting the cell death due to irradiation of ultraviolet rays, Collagen-degrading gene.

That is, it is an object of the present invention to provide a composition for preventing photoaging and improving wrinkles containing an extract of glutinosa extract as an active ingredient. In order to achieve the above object, the present invention provides a method for producing an extract, which comprises using an organic solvent as an outpost, a leaf, a stem, a flower, and a root, will be.

In one embodiment of the present invention, the extract may be extracted using an organic solvent, and the organic solvent may be ethanol or methanol, but most preferably exhibits the best activity when methanol is used.

In another embodiment of the present invention, the extract of the present invention can eliminate free radicals and inhibit cell death or collagen degradation induced by UVB irradiation. In addition, the extract of the present invention can inhibit the activity of elastase. As a result, the extract of the present invention can prevent photo-aging, and thus can have an effect of improving wrinkles.

In addition, the present invention can provide a cosmetic composition comprising an extract of glutinosa extract as an active ingredient.

The cosmetic composition of the present invention can be used in cosmetics such as soft longevity, astringent lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, But the present invention is not limited thereto, and can be manufactured in the form of an essence, a makeup base, a foundation, a hair dye, a shampoo, a rinse, or a body cleanser.

In addition, the composition may be used as a pharmaceutical composition, and may be formulated into ointments, gels, creams, patches, or sprays, but is not limited thereto.

In addition, the present invention can provide a food composition comprising an extract of Safflower extract as an active ingredient.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

The present example is directed to Example 1 for producing sticky beads, Experimental Example 1 for testing free radical scavenging ability using the same, Experimental Example 2 for measuring the protective effect against UVB induced cell death, Experiment for measuring MMP-1 concentration Example 3, Experimental Example 4 for measuring the amount of MMPs expressing UVB-induced collagen degradation, and Experimental Example 5 for measuring elastase inhibitory activity in relation to anti-wrinkles. Through the following examples, it was confirmed that the present invention has anti-aging effect of sticky leaves.

Example  One. It turns into sticky  Preparation of extract

13 kg of outgrowth of polygonum viscoferum was added, and the mixture was immersed in 25 L of methanol for 4 hours and filtered to separate the solid component and the primary liquid component. The separated solids were again dipped in 25 L methanol for 4 hours and filtered to obtain a second liquid component. The obtained primary liquid component and secondary liquid component were mixed and the mixture was concentrated under reduced pressure, and then the residue was lyophilized to obtain 440 g of a sticky extract.

Experimental Example  One. It turns into sticky  Freedom of extract Radical Scatters  Confirm

In this Experimental Example, DPPH radical scavenging activity was measured to confirm the free radical scavenging ability of the extract prepared in Example 1 above.

DPPH Radical  Measurement of scavenging activity

DPPH (1,1-diphenyl-2-picrylhydrazyl) analysis is a method of measuring antioxidative capacity by reducing the amount of purple color by an aromatic compound and an aromatic amine due to the electron donating ability of the antioxidant as an index. Ascobic acid, a well known antioxidant, was used as a positive control. First, 100 μl of the extract of the present invention prepared in Example 1 was added to 100 μl of 5 mM DPPH at each concentration and mixed by vigorous shaking. After 30 minutes, the residual DPPH concentration was measured at 517 nm using a spectrophotometer Were measured. The OD value was calculated as the electron donating ability%.

As a result, as shown in Fig. 1, ascorbic acid, which is well known as a positive control, eradicated 82% of the radicals, whereas extracts of the glutinous extract showed 61% at 100 μg / ml, 77% at 500 μg / And 80% of the radicals were eliminated at 1000 [mu] g / ml. From these results, it was confirmed that the extract of the present invention has a significant contribution to antioxidation. Therefore, it was confirmed that the extract of Safflower extract can be used as a composition for improving skin aging.

Experimental Example  2. UVB  For induction cell death It turns into sticky  Check the efficacy of the extract

Cell culture

Human Hs68 cells, 5% CO ₂ and 37 ℃ in culture medium being kept in a 95% humidity 10% heat-inactivated fetal bovine serum , 100 ㎍ / ㎖ streptomycin and 100 unit / ㎖ DMEM (Dulbeccos containing penicillin modified Eagle's medium) medium.

UVB  The effect of irradiation on cell death

The cultured cells were treated with 10 ㎍ / ㎖ of extracts of glutinosa extract. After 24 hours, the cells were washed once with phosphate-buffered saline and exposed to UVB 100 mJ / cm ² . Cells were further cultured at 37 ° C for 24 hours, and cell viability was measured using MTT assay.

As a result, as shown in Fig. 2, the cell survival rate of human Hs68 fibroblasts was about 70% in the group treated with the extract of the glutinosa extract. In the group treated with 10 ㎍ / ㎖ of the extract of glutinosa extract, no cytotoxicity ). The test results were statistically significant (p <0.0005). The t test of the test group and the control group was performed to verify the significance. From these results, it was confirmed that the treatment of the extract of the present invention produced by the present invention greatly contributes to inhibition of UVB-induced apoptosis.

Experimental Example  3. It turns into sticky  By extract MMP -1 concentration

In the process the sticky water pepper extract samples of various concentrations without FBS in Hs68 cells were cultured for 24 hours exposed to UVB 100 mJ / cm ² and with no FBS medium after 24 hours of incubation, collect the supernatant of the cells used in the MMP-1 Black Respectively. This assay used an MMP-1 ELISA kit (Takara) according to the manufacturer's instructions. The results of the experiments were compared by one way ANOVA of the experimental group and the control group and by the Turkey method (Turkey's multiples comparison test) after the post test, the significance was verified and statistically significant difference was shown *** p &lt; 0.0005).

As a result, as shown in FIG. 3, it was confirmed that MMP-1 secretion was remarkably reduced due to the presence of the extract of the present invention. Therefore, it was confirmed from the above results that the extract of the sticky plant according to the present invention can effectively inhibit collagen degradation by UVB irradiation.

Experimental Example  4. It turns into sticky  Extract UVB  Induced collagen degradation MMP  Confirmation of the effect of inhibition on expression

Studies on the formation of skin wrinkles due to photoaging have shown that matrix metalloproteinase (MMPs) activity, which acts to decompose the elastic collagen and other connective tissue components of the dermis, increases with increasing ultraviolet irradiation dose. Is known to be inhibited by collagen synthesis due to increased activity of MMP-1 (James Varani et al., Journal of Investigative Dermatology, 119: 122-129, 2002).

MMP-2 and MMP-9 play a major role in skin aging by subdividing collagen fragments degraded by MMP-1 (Vanisree Staniforth et al., The Journal of Nutritional Biochemistry, 23: 443-451, 2012) . MMP-3 promotes the secretion of other MMPs from keratinocytes and dermal fibroblasts, and is directly related to the degradation of collagen type I and III in the dermis (Hee-Bong Pyun et al. Preventive Nutrition and food science, 17: 245-253, 2012).

Therefore, when the UVB irradiation increases the expression amount of the MMPs gene, if there is a sample inhibiting the degradation of collagen, the elasticity of the skin tissue can be maintained and wrinkle formation can be prevented. In this experiment, we investigated the effect of UVB irradiation on the expression level of MMPs gene by the treatment with the extracts of Cyprinus arvenosum.

The nucleotide sequences of β-actin, MMP-1, MMP-2, MMP-3 and MMP-9 were as follows.

β-actin forward: GCG AGA AGA TGA CCC AGA T

         reverse: ATC ACG ATG CCA GTG GTA

MMP-1 forward: GCA TAT CGA TGC TGC TCT TTC

      reverse: GAT AAC CTG GAT CCA TAG ATC GTT

MMP-2 forward: GGA GGA GAA GGC TGT GTT

      reverse: TAA AGG CGG CAT CCA CTC

MMP-3 forward: CAA AAC ATA TTT CTT TGT AGA GGA CAA

       reverse: TTC AGC TAT TTG CTT GGG AAA

MMP-9 forward: ATG TAC CCT ATG TAC CGC TTC

reverse: GTG TGG TGG TGG TTG GAG

Cells were seeded at 8 × 10 ⁵ cells / well at 6 × 10 ⁵ cells / well and cultured for 24 hours. Cells were treated with the extracts of Glycyrrhiza glutinosa and cultured for 24 hours. After the medium was removed, the cells were washed 1-2 times with phosphate buffered saline After washing, 1 ml of phosphate buffered saline was added to the cells. UVB irradiation was conducted using UV lamp at a UVB of 100 mJ / cm ² . For the change of MMPs gene by the sample, PBS was removed from the UVB-irradiated cells and cultured for 24 hours in the absence of FBS. Then, RNA was extracted using Trizol and cDNA was prepared using reverse transcriptase polymerase chain reaction Were synthesized. Beta-actin was used as a control. Real-time polymerase chain reaction (PCR) was performed using each primer to examine the expression level of MMPs gene, which has the action effect of decomposing elastic collagen and other connective tissue components of dermis. 95 ° C for 3 minutes, <95 ° C for 10 seconds, 58 ° C for 10 seconds, 72 ° C for 30 seconds> -39 times, 95 ° C for 10 seconds, and 65 ° C for 5 seconds). MMP-1, MMP-2, MMP-3 and MMP-9 were corrected with beta-actin. The results of the experiments were compared by one way ANOVA of the experimental group and the control group and by the Turkey method (Turkey's multiples comparison test) after the post test, the significance was verified and statistically significant difference was shown ** p < 0.005, *** p < 0.0005).

The effect of the extract of the present invention on the expression of collagenase MMPs in UVB-irradiated dermal fibroblasts was investigated by real-time PCR and shown in FIGS. 4a-d. As a result, levels of collagenase MMP gene expression of MMP-1, MMP-2, MMP-3, and MMP-9 were decreased in a concentration-dependent manner due to the treatment of the extract of the present invention.

From the above results, it was judged that the extract of the present invention can effectively inhibit UVB-induced collagen degradation by decreasing expression of all MMPs, that is, MMP-1, MMP-2, MMP-3 and MMP-9.

Experimental Example  5. It turns into sticky  Extract Elastase ( elastase ) Inhibition activity confirmation

Elastase activity was measured by using N-Succinyl- (L-Ala) 3-p-nitroanilide as a substrate and the amount of p-nitroaniline produced at 25 ° C for 20 minutes at 410 nm. Elastase was dissolved in 50 mM Tris-HCl (pH 8.6) to 0.6 units.

N-Succinyl- (L-Ala) 3-p-nitroanilide was dissolved in 50 mM Tris-HCl (pH 8.6) buffer to a concentration of 0.1 mg / μl and 0.1 ml of the sample was added to a 96- 50 μl of 0.4 M Tris-HCl (pH 8.6) was added to the reaction mixture and the mixture was reacted at 37 ° C for 30 minutes in advance. Then 0.1 ml of N-Succinyl- (L-Ala) 3-p-nitroanilide After incubation for a minute, the absorbance was measured at 410 nm and the inhibition rate for elastase was calculated. In the blank test group, all the components except the enzyme were used.

Control experiments show elastase activity in the absence of inhibitor. One unit of activity of elastase is defined as the activity of producing 1 umol of p-nitroanilide per minute. Each group was run in triplicate as shown in Table 1 below, and enzyme inhibition rates were calculated from the following formulas.

% Inhibition = {1- (AB / C)} x 100

Design of Experiments A, B, C, and D to Determine Elastase Inhibitory Activity A B C sample O O X enzyme O O O temperament O X O

The results of the experiments were compared by one way ANOVA of the experimental group and the control group and by the Turkey method (Turkey's multiples comparison test) after the post test, the significance was verified and statistically significant difference was shown *** p < 0.0005).

As a result, as shown in Fig. 5, 10 / / ml of ursolic acid, which is known to have an inhibitory activity of elastase, was used as a positive control, and the extract of Pseudomonas aeruginosa When the inhibitory activity was measured, the inhibitory activity tended to increase with the concentration of the concentration. Thus, it can be seen from the above results that the extract of Safflower extract according to the present invention has a very excellent effect on the inhibition of elastase.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (11)

A composition for preventing photoaging and improving wrinkles, the composition comprising an extract of Safflower extract as an active ingredient.
The method according to claim 1,
Characterized in that the adhesive layer is a protrusion of the adhesive layer.
The method according to claim 1,
The composition of claim 1, wherein the extract is an organic solvent extract.
The method of claim 3,
Wherein the organic solvent is methanol.
The method according to claim 1,
Characterized in that the extracts of sticky leaves scavenge free radicals.
The method according to claim 1,
Wherein said extracts inhibit UVB-induced cell death.
The method according to claim 1,
Characterized in that the extracts of sticky plants inhibit collagen degradation induced by UVB irradiation.
The method according to claim 1,
Characterized in that the extracts of the dragonoids inhibit the activity of elastase.
The method according to claim 1,
Wherein the composition is a cosmetic composition.
The method according to claim 1,
Wherein the composition is a pharmaceutical composition.
The method according to claim 1,
Wherein the composition is a food composition.

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