KR101398392B1 - cosmetic compositions containing extract of Viburnum dilatatum Thunb used for antiwrinkle - Google Patents

Abstract

The present invention relates to a method for producing wrinkle- dilatatum Thunb.) Leaf and Branch Mixture Extracts, Viburnum dilatatum Thunb.) leaves and leaves have an excellent effect of inhibiting MMP-1 and elastase, and are intended to be used in cosmetic compositions for improving skin wrinkles.

Giblets, cosmetics, skin wrinkles, elastase, MMP-1

Description

[0001] The present invention relates to a cosmetic composition containing an extract of Cinnamomum cassia var. Viburnum dilatatum Thunb used for antiwrinkle,

The present invention relates to a mixed extract of cinnamon leaf and eggplant having a wrinkle-reducing effect, and it has been found that an extract of cinnamon leaf and gangliosate inhibits MMP-1 and elastase and has an excellent effect of promoting collagen synthesis. Which is intended to be used in a cosmetic composition for use in cosmetics.

Skin aging is promoted around the age of 25, and is divided into natural aging and external aging. Natural aging is difficult to control because it is mainly caused by genetic factors. External aging is caused by external factors such as pollutants, dust, pollution, and ultraviolet rays, so that man-made control is possible. In particular, when skin is exposed to ultraviolet rays, reactive oxygen species are generated in fibroblasts and induce the expression of collagenase (hereinafter referred to as "MMP-1") (Yoshiharu Kawaguchi et al . , 1996)

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

MMP-1 degrades collagen type I. Therefore, when MMP-1 expression is induced, collagen synthesis is reduced and wrinkles occur on the skin. (Laure Rittie et al ., 2002)

In addition, elastin is a fiber structure important for the generation of wrinkles, which is involved in skin elasticity like collagen. Deficiency and aggregation of elastin fibers, and a marked increase in the activity of elastase is one of the causes of skin wrinkles. It is known that inhibiting elastase, the only enzyme capable of degrading elastin, can fundamentally reduce skin wrinkles (Lee meat al . 1999)

Ultraviolet light-induced photochemical reactions produce highly reactive reactive oxygen species such as superoxide radicals, hydroxyl radicals, hydrogen peroxide, and mono-polymerized oxygen, which causes non-selective, irreversible destruction of cellular components such as lipids, proteins, , Which causes skin aging (Halliwell, 1991).

Antioxidants are widely distributed in the natural world, ranging from plants, plants and microorganisms, and can prevent and inhibit skin aging by inhibiting free radicals that cause oxidative damage.

Although retinol, adenosine, and AHA have been developed as anti-wrinkle agents and anti-aging agents reported in the previous researches, they are less safe, cause skin irritation, and have poor visibility. Therefore, it is necessary to study wrinkle improvement and skin aging prevention materials that can solve the above problems.

It is a deciduous shrub that grows in the forest below the middle of the mountain and grows about 3m in length. Its leaves are round, round and 6 ~ 12cm long. There are serrations on the edge, hairs on both sides, there is a prefix on the back, both ends are narrow, and there is no stipple. The flower is white and the fruit is red. It is native to Gangwon-do in Korea and especially in Jeju-do, and is widely used for ornamental purposes. It is called spring coat and leaves, and it is collected in spring and summer and used medicinally. Fruit and lysoon are also used for edible purposes, and it is known that fenugreek fruit has antioxidant effects (Kunihisa Iwai, et al . 2004). However, studies on the physiological activity related to giblets have not been conducted yet.

The present invention aims to provide a cosmetic composition for improving skin wrinkles containing a naturally derived extract, and it is an object of the present invention to provide a cosmetic composition for improving skin wrinkles containing the extract of Cinnamomum cassia bark extract to improve skin wrinkles .

The present invention relates to a method for producing wrinkle- dilatatum Thunb.) extracts. Specifically, it has been found that the extracts of gabbrook leaves and leaves have an effect of inhibiting elastase and MMP-1 and promoting collagen synthesis. A cosmetic composition for improving wrinkles is provided.

The extraction solvent for preparing the cinnamon extract of the present invention includes methanol, ethanol and the like, and ethanol is most preferable.

The cosmetic composition for improving skin wrinkles of the present invention contains from 0.005 to 1% by weight of the total amount of the cinnamon bark extract (leaf, branch mixture).

When the content is 0.005% by weight or less, the effect of improving the skin wrinkles of the present invention is hardly expected, and when the content is 1% by weight or more, it may be difficult to formulate the composition.

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

The mixed extract of V. falcatum and leaves of the present invention has an excellent antioxidative activity, and has an effect of inhibiting elastase and MMP-1 and promoting collagen synthesis, so that it can be usefully used in a cosmetic composition for improving skin wrinkles.

Example 1: Production of a mixed extract of cinnamon leaf and garlic (hereinafter referred to as "VD-LBE")

The leaves and branches of V. chinensis were collected and the samples were washed with purified water, dried and pulverized. 100 g of a sample obtained by mixing 50 g of the dried pulp leaves with 50 g of the branch was repeatedly extracted with 2 L of 80% by weight ethanol at room temperature for 4 hours twice by using ultrasonic waves, followed by addition of Advanan NO.4 (manufactured by Toyo Roshi Kaisha Ltd.). ) Filter cake. The filtrate was dried to a weight of 19.59 g using a rotary evaporator under reduced pressure.

Example 2: Preparation of solvent fractions of VD-LBE

10 g of the mixed extract of cinnamomum flavia and leaves obtained in Example 1 was dissolved in a sufficient volume of distilled water and then hexane, methylene chloride, ethyl acetate and butanol were added in the same volume as that of the same, followed by solvent fractionation to obtain 0.930 g 1.621 g of methylene chloride fraction, 1.024 g of ethyl acetate fraction and 1.981 g of butanol fraction.

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

In this experimental example, DPPH radical scavenging activity of the obtained samples was measured to confirm antioxidant activity. The antioxidant capacity of the 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 prepare a 0.2 mM DPPH solution. The samples to be tested for antioxidant activity were dissolved in ethanol and diluted to a volume of 150 μl in a 96-well plate plate. 150 μl of the DPPH solution was reacted with the sample solution for 30 minutes and absorbance was measured at 517 nm using a microplate reader. The reaction solution containing ethanol instead of the sample was used as a control to measure the radical scavenging ability. The formula used at this time is as follows.

Scavenge (%) = {1- (A-B) / C} 100

A: Absorbance of the experimental group reacted with the sample solution and the DPPH solution

B: Sample liquid self-absorbance

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

  Experiment of antioxidant activity
sample DPPH Scavenge (%) SC ₅₀
(占 퐂 / ml) Treatment concentration ([mu] g / 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
Methylene chloride 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 the ethanol extract of V. fructus was increased in a concentration-dependent manner, and ethanol extract, ethyl acetate fraction and butanol fraction showed an increase in antioxidant activity . Therefore, antioxidant activity indicators are expected to exist in the butanol fraction

Experimental Example 2: Measurement of activity inhibition of elastase

In order to observe the elastase activity inhibitory effect of the samples obtained in this Experimental Example, First, 140 μl of Tris buffer (0.2 M Tris buffer, pH 8.00), 20 μl of each concentration sample, and 20 μl of 5 mM N-Succinyl-Ala-Ala-p-nitroanilide (Sigma, S4760) After mixing well with 20 μl of 10 μl / ml of elastase (Sigma, E7885) and reacting at room temperature (25 ° C) for 15 minutes, the amount of p-nitroaniline produced at 410 nm was measured. As a positive control, bingo extract (Korea patent application: 1997-78817) was used. The inhibitory effect of each sample on the elastase activity was expressed as IC _{50, which} is a concentration for eliminating the enzyme activity by 50%. As a negative control, the absorbance of the untreated sample was used.

  elastase inhibitory effect
sample elastase inhibition (%) IC ₅₀
(占 퐂 / ml) Treatment concentration ([mu] g / 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
Methylene chloride 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 Bingo extract 4.73 16.47 57.15 - 43

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

Experimental Example 3: Measurement of inhibitory activity of collagenase (MMP-1)

In this Experimental Example, the effect of the collagenase (MMP-1) on the activity of the obtained samples was examined, and the use of the collagenase conjugated with purified collagenase and fluorescein was performed. First, 20 μl of 0.25 mg / ml DQ ^TM collagen solution (Molecular Probes, USA) was added to 100 μl of the reaction complete layer solution (0.05 M Tri-HCl, 0.15 M NaCl, 5 mM CaCl ₂ , 0.02 mM NaN ₃ , pH 7.3) And 40 μl of each sample were added to each well and mixed well with 40 μl of 5 units of collagenase (GIBCO, USA). The mixture was reacted in a dark place at room temperature for 20 minutes. Thereafter, fluorescence values were 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 action of collagenase production on green tea extract is disclosed in Korean Patent Publication No. 1999-0056976. The green tea extract inhibits collagenase production by ultraviolet rays, and the degree of suppression of erythema and lipid peroxidation is excellent, It is possible to use it as a composition.

The inhibitory effect of collagenase on the activity of each sample was expressed by IC _{50, which} is a concentration for eliminating the enzyme activity by 50%. As a negative control, the fluorescence value of the untreated test solution was used.

  MMP-1 inhibitory effect sample MMP-1 Inhibition (%) IC ₅₀
(占 퐂 / ml) Treatment concentration ([mu] g / 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
Methylene chloride 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 VD-LBE, methylene chloride fraction, ethylacetate fraction, and butanol fraction exhibited higher MMP-1 inhibitory effect than green tea extract, which is well-known as a conventional MMP-1 inhibitor. Particularly, the butanol fraction showed more than two times more inhibitory activity than the ethanol extract. Therefore, it is expected that there will be an effective indicator substance which inhibits MMP-1 activity in the butanol fraction.

EXPERIMENTAL EXAMPLE 4: Experiment for Measuring Effect of Promoting Collagen Synthesis

In this Experimental Example, in order to measure the promoting effect of the collagen synthesis of the obtained samples, a commercially available human fibroblast was treated with a sample.

Human normal fibroblasts (CCD-985sk) purchased from the American Type Culture Collection (ATCC) with IMD (Iscove's Modified Dulbesco's Media) medium containing 5% fetal bovine serum were inoculated into 96-well 96- 5000 cells / well, and cultured at 37 ° C and 10% CO ₂ for 24 hours. After 24 hours, the samples were diluted with IMDM medium containing no fetal bovine serum for 2 days, and cell culture medium was collected. The amount of pro-collagen type IC-peptide (Type IC-peptide: PICP) synthesized by using the collagen protein assay system (Procollagen Type IC-peptide EIA Kit (MK101) Respectively.

For the measurement, 100 μl of the antibody solution in which the POD (perosidase) enzyme was bound and 20 μl of the collected cell culture were added to a 96-well plate incubator uniformly coated with the collagen antibody, and reacted at 37 ° C. for 3 hours. After 3 hours, the supernatant was removed and each well was washed 4 times with 400 μl of PBS (phosphate buffered saline). After washing, 100 μl of substrate solution reacting with POD was added to each well and left at room temperature for 15 minutes. To stop the reaction, 100 1 of 1 N sulfuric acid solution was added and mixed, and the absorbance at 450 nm was measured. The PICP amount was calculated by substituting the absorbance of the sample into the standard concentration curve prepared by diluting the standard solution contained in the collagen measurement kit. The reaction absorbance of the cell culture without sample treatment was used as a control.

  Promoting collagen synthesis
sample Collagen synthesis rate (%, control) Treatment concentration ([mu] g / ml) 0.5 One 2 Extract of gabbro leaves, eggplant mixture 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 group 100

As shown in Table 4, both VD-LBE and purified fractions increased collagen synthesis more than twice as much as the control group not treated with the sample.

From the above results, it was found that the butanol fraction had common efficacy in antioxidative and wrinkle - reducing effects.

Experimental Example 5: Cytotoxic relaxation effect induced by SLS (Sodium lauryl sulfate)

SLS (Sodiulylauryl sulfate) is a surfactant added to cosmetics. It is a typical corrosive irritant causing contact dermatitis. It causes loss of skin barrier function and increases water loss. In this Experimental Example, experiments were carried out using cell culture technology to evaluate the SLS stimulation relaxation effect on the human normal fibroblasts of the obtained samples.

Human normal fibroblasts were inoculated (2 x 10 ⁴ cells / well) into 96-well microplates containing IMDM medium and cultured at 37 ° C for 24 hours at 10% CO ₂ . The normal human fibroblasts were purchased from the American Type Culture Collection (ATCC). First, preliminary experiments confirmed that the extract of VD-LBE was not cytotoxic within 10 μg / ml concentration. Thereafter, 100 μg / ml SLS and diluted extract extracts were simultaneously treated and, in the case of no extract, only control samples were used as a control. After 48 hours of culture, the medium was removed, and 200 μl of dimethyl sulfoxide (DMSO) solution was added to each well. The resulting solution was stirred for 20 minutes to dissolve the generated formazan. Absorbance was measured at 540 nm using a microplate reader, and the cytotoxic relaxation effect of SLS by the sample was confirmed through comparison with the control group.

  SLS-induced cytotoxicity mitigation effect sample Cell survival rate (%) 0.1 mu g / ml 1 mu g / ml 5 / / ml 10 mu g / ml Extract of gabbro leaves, eggplant mixture 117.26 119.54 120.57 117.79 Control group 100

From the results shown in Table 5, it was found that the cytotoxic relaxation effect of VD-LBE was the best at 5 μg / ml, and that the mixed extract of V. falcatum and leaves of the present invention can be exhibited by a surfactant It was confirmed that it is a substance that can alleviate skin irritation.

Claims (8)

A cosmetic composition for improving skin wrinkles, comprising a mixture of the leaves of Vincennesiaceae and the mixture of plant extracts. Wherein at least one solvent fraction obtained by sequentially adding hexane, methylene chloride, ethyl acetate, and butanol to a mixture of the leaves of cinnamomum oil and the mixture of branches is fractionated and fractionated. The cosmetic composition for improving skin wrinkles according to claim 2, wherein the solvent fraction is butanol fraction. [Claim 2] The cosmetic composition for improving skin wrinkles according to claim 1, wherein the extract of V. fructus is ethanol extract and the content thereof is 0.005 to 1% by weight of the total weight of the composition. The cosmetic composition for improving skin wrinkles according to claim 1 or 2, wherein the wrinkle-reducing effect of the skin is caused by an activity inhibiting effect of elastase. The cosmetic composition for improving skin wrinkles according to claim 1 or 2, wherein the wrinkle improving effect is due to the inhibitory effect of MMP-1. The cosmetic composition for improving skin wrinkles according to any one of claims 1 to 3, wherein the effect of improving skin wrinkles is due to the effect of promoting collagen synthesis. The cosmetic composition for improving skin wrinkles according to claim 1 or 2, which has a skin cell toxicity-reducing effect.