KR20090097232A - Cosmetic compositions containing extract of viburnum dilatatum thunb used for antiwrinkle - Google Patents

Abstract

A mixture extract of leaves and branches of Viburnum dilatatum Thunb. is provided to suppress MMP-1 (matrix metalloproteinase-1) and elastase, promote collagen synthesis and improve anti-wrinkle. A cosmetic composition for improving anti-wrinkle comprises a mixture extract of leaves and branches of Viburnum dilatatum Thunb. The composition a solvent fraction which is obtains by adding hexane, methylene chloride, ethylacetate, and butanol and fractioning. The cosmetic composition is formulated in a general lotion, cream, and massage pack.

Description

Cosmetic composition for improving skin wrinkles including viburnum extract {cosmetic compositions containing extract of Viburnum dilatatum Thunb used for antiwrinkle}

The present invention relates to a viburnum leaf and eggplant mixed extract having an anti-wrinkle effect, and that the viburnum leaf and eggplant mixed extract inhibits MMP-1 and elastase and has an excellent effect of promoting collagen synthesis. It is intended to be used in cosmetic compositions.

Aging is accelerated around 25 years of age in human skin, which is divided into natural and external aging. Natural aging is mainly due to genetic factors, making it difficult to control. External aging is caused by external factors such as pollutants, dust, pollution, and ultraviolet rays, so it can be artificially controlled. Among them, especially when the skin is exposed to ultraviolet rays, reactive oxygen species are generated in fibroblasts, leading to the expression of collagenase (hereinafter referred to as "MMP-1"). (Yoshiharu Kawaguchi et al . , 1996)

MMP-1 is an enzyme that degrades collagen, which is a substrate of cells, and is one of matrix metalloprpteinase (hereinafter referred to as 'MMP').

MMP-1 degrades collagen type I. Therefore, the expression of MMP-1 leads to a decrease in collagen synthesis and wrinkles on the skin (Laure Rittie et. al ., 2002)

In addition, elastin is an important fibrous tissue for wrinkle formation such as collagen, which is involved in skin elasticity. Deficiency and aggregation of elastin fibers, and a dramatic increase in the activity of elastase, are one of the factors in the formation of skin wrinkles. Inhibiting elastase, the only enzyme that can break down elastin, is known to radically reduce skin wrinkles. et al ,. 1999)

Photochemical reaction by ultraviolet light produces highly reactive free radicals such as superoxide radicals, hydroxyl radicals, hydrogen peroxide and mono-oxygen, resulting in non-selective and irreversible destruction of cell components such as lipids, proteins, sugars and DNA Causes skin aging (Halliwell, 1991)

Antioxidants are substances widely distributed in nature, including animals, plants and microorganisms, and can prevent and suppress skin aging by inhibiting free radicals causing oxidative damage.

Retinol, adenosine, and AHA have been developed as anti-wrinkle and skin aging prevention materials reported so far, but they have a problem of poor safety, causing skin irritation, and lack of obvious visible effects. Therefore, there is a need for research on wrinkle improvement and skin aging prevention materials that can supplement the above problems.

The viburnum is a deciduous shrub that grows in the forest below the mountain slopes and grows about 3m. The leaves are college, round and 6 ~ 12cm long. Serrated at the edges, hairy on both sides, occupied at the back, narrow at both ends, without chin leaves. The flower is white and the fruit is red. It is native to south of Gangwon-do, especially Jeju Island, and is often used for ornamental purposes. Viburnum stems and leaves are called stalks and are medicated by harvesting in spring and summer. Fruits and young shoots are used for food, and it is known that the berries of Viburnum have antioxidant effects (Kunihisa Iwai, et. al ,. 2004). However, no physiological activity studies involving viburnum have been conducted.

The present invention is to provide a cosmetic composition for improving skin wrinkles containing natural derived extracts, the purpose of providing a cosmetic composition for improving skin wrinkles found that the viburnum extract is effective in improving skin wrinkles .

The present invention has a viburnum ( Viburnum) having an effect of improving wrinkles dilatatum Thunb.) extract, specifically, viburnum leaf and eggplant mixed extract inhibits elastase and MMP-1 and promotes collagen synthesis. It provides a cosmetic composition for improving wrinkles.

Extraction solvents for producing the viburnum extract of the present invention include methanol, ethanol and the like, most preferably ethanol.

The cosmetic composition for improving skin wrinkles of the present invention contains the viburnum extract (leaf, eggplant mixed extract) 0.005 to 1% by weight of the total weight of the composition.

When it contains 0.005% by weight or less, it is difficult to expect the skin wrinkle improvement effect of the present invention, and when it contains 1% by weight or more, there may be difficulty in formulating.

The cosmetic composition for improving skin wrinkles of the present invention may be provided as a general cosmetic water, cream, massage pack, and the like, and may be formulated according to a conventional formulation method.

The viburnum leaf and eggplant mixed extract of the present invention has excellent antioxidant activity, inhibits elastase and MMP-1, and promotes collagen synthesis, and thus can be usefully used in cosmetic compositions for improving skin wrinkles.

Example 1: Viburnum leaves, eggplant mixed extract (hereinafter referred to as 'VD-LBE') Preparation

Viburnum leaves and branches were collected, the sample was washed with purified water, dried and pulverized. 100 g of a sample of 50 g of dry and crushed viburnum leaves and 50 g of eggplant was extracted twice with ultrasonic wave at 80 ° C in 2 L for 4 hours at room temperature, followed by Advantech NO.4 (manufactured by Toyo Roshi Kaisha Ltd.). ) Filtered through a filter cloth. The filtrate was dried under a reduced pressure concentrator to obtain a dry weight of 19.59 g.

Example 2: Preparation of Solvent Fraction of VD-LBE

10 g of the viburnum leaf and eggplant mixed extract obtained in Example 1 were dissolved in distilled water of sufficient capacity, and then the same volume of hexane, methylene chloride, ethyl acetate, and butanol was sequentially added, followed by solvent fractionation. , 1.621 g of methylene chloride fraction, 1.024 g of ethyl acetate fraction, and 1.981 g of butanol fraction were obtained.

Experimental Example 1: Evaluation of antioxidant efficacy by DPPH radical scavenging activity assay

In this experimental example, the antioxidant activity was confirmed by measuring the DPPH radical scavenging activity of the obtained samples. Antioxidant capacity of extracts was compared with vitamin C, a conventional antioxidant. 2.96 mg of DPPH (1,1-diphenyl-2-picrylhydrazine) was dissolved in 50 ml of ethanol to form a 0.2 mM DPPH solution. 150 μl of the sample to be measured for antioxidant activity was dissolved in ethanol and diluted in a 96-hole flat plate, and then 150 μl of DPPH solution was reacted with the sample solution for 30 minutes, and the absorbance was measured at 517 nm using a microplate reader. Radical scavenging ability was measured using the reaction solution containing ethanol instead of the sample as a control. The calculation formula used is as follows.

Scavenge (%) = {1- (A-B) / C} × 100

A: absorbance of the experimental group in which the sample solution and the DPPH solution were reacted

B: Sample liquid self absorbance

C: absorbance of the experimental group reacted with ethanol and DPPH solution

  Antioxidant Effect Experiment  sample DPPH Scavenge (%) SC ₅₀ (μg / ml) Treatment concentration (µg / ml) One 5 10 30 50 100 VD-LBE 11.10 22.11 35.24 79.77 92.62 94.08 17.03 VD-LBE Hexane Fraction 9.47 11.09 12.41 17.97 23.67 38.55 > 100 VD-LBE Methylenechloride Fraction 8.59 11.13 16.32 28.79 41.68 62.63 61.93 VD-LBE ethyl acetate fraction 15.41 28.20 46.72 87.63 93.93 94.89 13.97 VD-LBE Butanol Fraction 12.37 27.70 49.08 92.06 93.48 94.49 10.30 One 5 10 - - Vitamin c 15.07 58.48 94.82 - 4.64

As shown in Table 1, the free radical scavenging effect of viburnum leaves and branched ethanol extracts was increased in a concentration dependent manner, and the antioxidant activities were increased in the order of ethanol extract, ethyl acetate fraction, butanol fraction. . Therefore, antioxidant activity indicators are expected to exist in the butanol fraction.

Experimental Example 2: activity inhibition measurement experiment of elastase

In order to observe the effect of inhibiting the elastase activity of the samples obtained in this experiment. First, 140 μl of Tris buffer (pH8.00), 20 μl of sample by concentration, and 20 μl of 5 mM N-Succinyl-Ala-Ala-p-nitroanilide (Sigma, S4760) were added. 10 μl / ml of elastase (Sigma, E7885) was mixed well with 20 μl of the reaction at room temperature (25 ° C.) for 15 minutes and the amount of p-nitroaniline produced at 410 nm was measured. As a positive control, betel nut extract (Korean Patent Application: 1997-78817) was used. The inhibitory effect of elastase activity of each sample was expressed as IC ₅₀ , a concentration that eliminates 50% of enzyme activity. At this time, the negative control group was used as the absorbance value of the test solution that did not process the sample.

  elastase inhibitory effect  sample elastase inhibition (%) IC ₅₀ (μg / ml) Treatment concentration (µg / ml) 10 50 100 200 VD-LBE 4.30 39.90 72.20 81.32 63.80 VD-LBE Hexane Fraction 5.51 16.97 20.49 36.54 > 200 VD-LBE Methylenechloride Fraction 17.83 40.94 60.29 67.45 75.03 VD-LBE ethyl acetate fraction 8.39 27.77 42.73 59.86 132.7 VD-LBE Butanol Fraction 20.55 41.59 55.51 72.71 74.53 Betel Nut Extract 4.73 16.47 57.15 - 43

As shown in Table 2, the ethanol extract showed the best inhibitory effect of elastase, and it was confirmed that the elastase inhibitory effect was not improved by the purified fractionation process.

Experimental Example 3: Experiment Inhibition of Activity of Collagenase (MMP-1)

This experiment was performed to observe the inhibitory effect of collagenase (MMP-1) activity of the obtained samples, based on the use of collagen conjugated with purified collagenase and fluorescein. First, 20 µl of 0.25 mg / ml DQ ^TM collagen solution (Molecular probes, USA) in 100 µl of reaction complete solution (0.05 M Tri-HCl, 0.15 M NaCl, 5 mM CaCl ₂ , 0.02 mM NaN ₃ , pH7.3) 40 μl of each sample was added and then mixed well with 40 μl of 5 units of collagenase (GIBCO, USA) and reacted for 20 minutes at dark and room temperature. Thereafter, the fluorescence value was measured at an absorption wavelength of 495 nm and an emission wavelength of 515 nm using a fluorescence spectrophotometer. Green tea extract (S. Pillai, C. Oresajo, and J. Hayward, Int. J. Cosmet. Sci., 27, 17-34, 2005) was used as a positive control. For reference, the inhibitory effect of collagenase enzyme production on green tea extract is disclosed in Korean Patent No. 1999-0056976. The green tea extract inhibits collagenase production by ultraviolet rays and has excellent suppression of erythema and lipid peroxide. It has been said that it can be used as a composition.

The inhibitory effect of collagenase activity of each sample was expressed by IC ₅₀ , a concentration that eliminates 50% of enzyme activity. In this case, the negative control was used as the fluorescence value of the test solution which did not process the sample.

  MMP-1 Inhibitory Effect sample MMP-1 Inhibition (%) IC ₅₀ (μg / ml) Treatment concentration (µg / ml) 10 50 200 VD-LBE 23.12 81.41 99.34 28.50 VD-LBE Hexane Fraction 14.28 25.04 46.61 > 200 VD-LBE Methylenechloride Fraction 16.65 39.46 86.58 83.54 VD-LBE ethyl acetate fraction 31.29 69.97 99.08 22.14 VD-LBE Butanol Fraction 46.19 97.45 99.31 11.27 Green Tea Extract 25.41 35.90 70.90 111.64

As shown in Table 3, it was confirmed that the VD-LBE, methylene chloride fraction, ethyl acetate fraction, butanol fraction showed a higher MMP-1 inhibitory effect than the green tea extract well known as a conventional MMP-1 inhibitor. In particular, butanol fraction showed more than two times better inhibitory activity than ethanol extract. Therefore, it is expected that there is an effective indicator that inhibits MMP-1 activity in butanol fraction.

Experimental Example 4: Collagen synthesis promoting effect measurement experiment

In this Experimental Example, experiments were carried out by treating samples with commercially purchased human fibroblasts in order to measure the collagen synthesis promoting effect of the obtained samples.

Human normal fibroblasts (CCD-985sk) purchased from the American Type Culture Collection (ATCC) with Iscove's Modified Dulbesco's Media (IMD) medium containing 5% fetal bovine serum in a 96-well plate. 5000 cells / well were dispensed and incubated at 37 ° C., 10% CO ₂ conditions for 24 hours. After 24 hours, the samples were diluted by concentration in IMDM medium containing fetal bovine serum, treated for 2 days, and cell cultures were collected. The collected cell culture solution was synthesized using a collagen protein measuring instrument (Procollagen Type IC-peptide EIA Kit (MK101), Takara.Kyoto, Japan) to synthesize Procollagen type IC-peptide (PICP) Measured.

In the measurement method, 100 μl of a POD (perosidase) -enzyme-bound antibody solution and 20 μl of the cell culture solution were added to a 96-hole plate culture in which collagen antibodies were uniformly coated, and reacted at 37 ° C. for 3 hours. After 3 hours, all of the supernatant was removed and each well was washed four times with 400 μl of PBS (phosphate buffered saline). After washing, 100 μl of the substrate solution reacting with POD was added to each well, and left at room temperature for 15 minutes. To stop the reaction, 100 μl of 1N sulfuric acid solution was added and mixed, and then absorbance was measured at 450 nm. PICP amount was calculated by substituting the absorbance of the sample to the standard concentration curve prepared by diluting the standard solution contained in the collagen measurement kit. Moreover, the reaction absorbance of the cell culture which did not process the sample was used as a control.

  Collagen synthesis promoting effect  sample Collagen synthesis rate (%, control) Treatment concentration (µg / ml) 0.5 One 2 Viburnum Leaf, Eggplant Mixed Extract 356.31 358.53 313.27 VD-LBE Hexane Fraction 123.02 169.65 226.00 VD-LBE Chloroform Fraction 251.54 278.83 233.94 VD-LBE ethyl acetate fraction 330.73 340.18 349.40 VD-LBE Butanol Fraction 153.74 203.77 287.63 Control 100

As shown in Table 4, both the VD-LBE and the purified fractions were found to increase collagen synthesis more than two times compared to the control untreated samples.

In the above results, butanol fractions were found to have a common effect on antioxidant and anti-wrinkle effects.

Experimental Example 5: Cytotoxic alleviation effect induced by SLS (Sodiul lauryl sulfate)

Soluul lauryl sulfate (SLS) is a surfactant added to cosmetics and is a representative corrosive irritant that causes contact dermatitis. In this experimental example, an experiment was performed using a cell culture technique to evaluate the SLS stimulating effect of the obtained samples on human normal fibroblasts.

Human normal fibroblasts were seeded in 96-well microplates containing IMDM medium (2 × 10 ⁴ cells / well) and incubated at 37 ° C. for 24 hours at 10% CO ₂ conditions. Normal human fibroblasts were purchased from the American Type Culture Collection (ATCC). First, the preliminary experiment confirmed that the extract of VD-LBE had no cytotoxicity within the concentration of 10µg / ml through experiments under the same conditions. Thereafter, 100 μg / ml SLS and each extract sample diluted by concentration were simultaneously treated, and only the extract sample under the same conditions was compared as a control. After 48 hours of incubation, the medium was removed, and 200 μl of dimethyl sulfoxide (DMSO) solution was added to each well, followed by stirring for 20 minutes to dissolve the formazan. The absorbance was measured at 540 nm using a microplate reader to compare cytotoxicity with SLS.

  Cytotoxicity-inducing effects induced by SLS sample Cell survival rate (%) 0.1 µg / ml 1 µg / ml 5 µg / ml 10 µg / ml Viburnum Leaf, Eggplant Mixed Extract 117.26 119.54 120.57 117.79 Control 100

From the results of Table 5, it was found that the cytotoxic effect was the best at 5 ㎍ / ㎖ in VD-LBE, the extract of viburnum leaf, eggplant of the present invention may be represented by a surfactant when included in the cosmetic composition It was confirmed that the substance can alleviate skin irritation.

Claims (8)

Cosmetic composition for improving skin wrinkles, including viburnum leaves, eggplant mixed extract. Cosmetic composition for improving skin wrinkles comprising one or more solvent fractions obtained by sequentially adding hexane, methylene chloride, ethyl acetate, butanol to the viburnum leaves, eggplant mixed extract. The cosmetic composition for improving skin wrinkles according to claim 2, wherein the solvent fraction is butanol fraction. According to claim 1, Viburnum leaf, eggplant mixed extract is an ethanol extract, the content of the cosmetic composition for skin wrinkle improvement, characterized in that 0.005 to 1% by weight of the total weight of the composition. The cosmetic composition for skin wrinkle improvement according to claim 1 or 2, wherein the skin wrinkle improvement effect is due to the inhibitory effect of elastase. The cosmetic composition for skin wrinkle improvement according to claim 1 or 2, wherein the skin wrinkle improvement effect is due to the inhibitory effect of MMP-1. The cosmetic composition for skin wrinkle improvement according to claim 1 or 2, wherein the skin wrinkle improvement effect is due to collagen synthesis promoting effect. The cosmetic composition for improving skin wrinkles according to claim 1 or 2, wherein the skin cytotoxicity relieving effect is obtained.