KR20070109426A - A cosmetic composition containing an extract of typhae pollen - Google Patents

Abstract

A composition comprising an extract of Typhae pollen is provided to inhibit the activity of tyrosinase and melanogenesis and show anti-oxidative, skin wrinkle improving and irritation alleviating effects. A cosmetic composition comprises 0.001 - 30.0 wt.% of an extract of Typhae pollen as an active ingredient, wherein the extract is prepared by cold-dipping Typhae pollen in at least one solvent selected from the group consisting of purified water, methanol, ethanol, glycerine, ethylacetate, butylene glycol, propylene glycol, dichloromethane and hexane, agitating, heating and filtering it at room temperature to obtain a liquid phase material, and further concentrating the solvent under reduced pressure or freeze-drying it.

Description

Cosmetic composition containing turmeric extract as a main active ingredient {A COSMETIC COMPOSITION CONTAINING AN EXTRACT OF TYPHAE POLLEN}

The present invention relates to a cosmetic composition containing a turmeric extract as a main active ingredient, more specifically 0.001 ~ 30.0% by weight of the turmeric extract, tyrosinase activity inhibitory effect and melanin biosynthesis inhibitory effect, antioxidant effect and skin wrinkle improvement effect The present invention relates to a cosmetic composition having an excellent stimulating alleviation effect.

Factors that determine skin color are basically the differences in race, region, sex, and age, except for blemishes, tanning caused by freckles and UV exposure, or overall pigmentation, as well as the distribution of acne and scars, keratin distribution and blood circulation, Stress, health status. Among the above factors, the most important factor is pigmentation.

The pigments that affect skin color include melanin, carotene, and hemoglobin, the most important of which is melanin. The biggest factor affecting melanin biosynthesis is ultraviolet rays and hormones in the body. The biosynthesis of melanin begins with a series of oxidation processes, starting with the conversion of tyrosine, an amino acid, from the melanocytes of melanocytes to tyrosinase and dihydroxy phenylalanine. It is formed of a polymer of eumelanin. This biosynthesis process is carried out in a special type of brown cell organelle called melanosome. Melanosomes, including melanin granules, move from the periphery of the nucleus to the end of dendritic processes, and into the cytoplasm by the phagocytosis of keratinocytes. Accumulate around the nucleus of the site. The synthesis of melanin, the number of melanosomes, and the movement to surrounding keratinocytes are largely genetically affected, in part by hormones and ultraviolet rays, and in addition to the expression of tyrosinase and the synthesis and transfer of melanin. Intracellular regulators such as cytokines, copper, zinc, iron and other metal ions and interferon, prostaglandins, histamines are known to be involved (Soo-Nam Park, 25: 77-127, 1999). Therefore, the skin whitening effect can be expected by inhibiting the synthesis of melanin, which determines the skin color, and the activity of tyrosinase, an enzyme involved therein.

Next, wrinkles are generated and skin elasticity is reduced due to aging of the skin. As a person ages, his skin is constantly changing due to skin aging and pigmentation. Skin aging is largely divided into natural aging (or endogenous aging) and external aging (EB Doris, Cosmetics & Toiletories, 111, 31-37, 1996), and natural aging (endogenous aging) is artificial because it is influenced by genetic factors. While it is difficult to control, aging is relatively easy because it is influenced by environmental factors. Representative external aging factors include ultraviolet rays, and the most prominent external aging phenomenon is wrinkle formation (HM Daniel, Ann Intern Med, 75, 873-880, 1971, MJ Grove, J Am Acad Dermatol, 21, 631-). 637, 1989, TS Griffiths et al, Hrch Dermatol, 128, 347-351, 1992).

One of the photoaging mechanisms caused by ultraviolet light is via free radical pathways (D. Harman, J. Gerontol, 11, 298-301, 1956). Free radicals are known to cause breakdown of connective tissue formation such as skin collagen, inhibition of cell membrane function, promotion of DNA mutation, modification of protein function, intercellular energy transfer, and metabolism related molecules (AF Kligman and RM Lavker, J. Cutan.Aging Cosmet.Dermatol. 1, 5-12, 1988). The involvement of free radicals in aging means that antioxidants or various chemical scavengers can be used to delay the aging process.

Synthesis and degradation of extracellular matrix such as collagen is regulated in vivo, but its synthesis decreases with aging and expression of matrix metalloproteinase (MMP), an enzyme that breaks down collagen. This is promoted, the elasticity of the skin is reduced and wrinkles are formed. Ultraviolet radiation and free radicals also activate these degrading enzymes.

Matrix metalloproteinases (MMPs) are secreted from various cells in the body, including keratinocytes and fibroblasts in human skin. They act as calcium and zinc-dependent endopeptidase and operate at neutral pH. Various extracellular substrates are used as substrates. MMPs are classified into MMP-1, MMP-2, and MMP-9 according to their substrate specificity. MMP-1 is a fibroblast type collagenase, an enzyme of epilepsy, and collagen type I, II, III, Ⅶ, There are Ⅹ, Ⅹ and gelatin and are cut into peptides of 3/4 to 1/4 length of original collagen length to facilitate the action of nonspecific enzymes (gelatinases). MMP-2 belongs to 72 kD gelatinase (A) and degrades gelatin, collagen types IV, V, VIII, VIII, elastin and fibronectin as substrates. Metallolastase also degrades elastin with MMP-13. Collagen types I and III, elastin, and fibronectin, which occupy most of the dermal layer of human skin, give strength and tension to the skin. Wrinkles and elasticity are degraded when protein is decomposed by abnormally activated MMP by ultraviolet rays and free radicals. Aging such as sagging skin is accelerated. In particular, by inhibiting the activity and biosynthesis of MMP-1, which breaks down the type I collagen, which accounts for the largest proportion of binding proteins, skin binding proteins can be protected to prevent wrinkle formation and elasticity.

In order to prevent aging and pigmentation caused by UV rays, the most common method is to apply a product containing a sunscreen directly to the skin. In 1988, retinoic acid was reported to be effective in reducing skin roughness and fine wrinkles of aged skin. (KS Weiss et al, JAMA, 259, 527-532, 1988), researches are being actively conducted to develop substances that suppress or improve skin aging and have a whitening effect. Among them, derivatives of vitamins such as vitamins A, C, and E and alpha -hydroxy acid (AHA) are representative substances known to improve skin (Hermitte, Cosmetics & Toiletries, 107, 63-67, 1992, DR Rosenthal et al. , J. Invest.Dermatol., 95, 510-515, 1990, TDDitre et al, J. Invest.Dermatol, 34, 187-195, 1996). However, they are very unstable in the natural environment, so there is a problem in use, or a slight lack of skin irritation or a visible effect.

Recently, many cosmetic products using natural products have been developed to reduce skin irritation. Various natural raw materials are extracted by a predetermined method, and the function of the extracts is increasing to develop various functional cosmetics. For example, mung beans have a known function to clean the skin, and other ginseng extracts, mushroom extracts, etc., have been found to have unique anti-aging or whitening effects and are applied to various cosmetic products. These natural materials have less side effects on the skin, and as the consumer's response to cosmetics using natural materials has recently increased, the development value of the natural materials has increased.

Thus, the present inventors studied the possibility of applying as a cosmetic product to various natural products, and selected the turmeric and prepared extracts from them, and measured the antioxidant effect, skin whitening effect, wrinkle improvement effect and stimulating alleviation effect, efficacy as cosmetics. I found that you can expect.

Pohwang (TYPHAE POLLEN,蒲黃), see portions (Typha the portions and (Typhaceae) latifolia L.), Typha orientalis Presl, Typha Angustifolia L.) is the only mature pollen collected and dried. Appearance is a light and fine powder, light yellow to yellow brown, when viewed under the microscope, four pollen grains are combined in a square or ladder shape, the diameter is 35 ~ 40μm (Park Jong Hee, Herbal Encyclopedia, Shinil Corp., 867-868). Dongbogam According to the liquid juice piece and chobu, the ingredients are even, the taste is sweet, there is no poison and the old beef stops bleeding, It is to treat the subdivision and morning pain, bleeding, and gestation. It emerges from the water of the pond and is located in several places, that is, it is yellow sulfur in the fortune, and it is written that it is received at the time when the minute occurs (Ku Bon-hong, the Korean-American Hang-cho Pan-dong, Korean Educational Culture History, 1411).

Studies on the parts and plants are disclosed in the Republic of Korea Patent Publication No. 10-2003-4499 relates to the skin whitening of "Hyangpo", which refers to the outpost of the parts and plants, "Hyangpo" in the same parts and plants Although it is a natural product obtained, it is clearly distinguished from "sulfur" which means only pollen of wealth. Even in the agreement, the huangpo and hyangpo are distinguished. The yangpo, which is called the outpost of the wealth, is the shoot of the huang, which heals the scent of five intestines and the rotten odor of the mouth, and firms the teeth and brightens the eyes and ears. It can be seen that Hyangpo and Pohang were classified into different medicines (Ku Bon-hong, Korean-American Pancho-dong, Korean Educational Culture History, 1411; Jang Il-mu, et al., Oriental Medical Science Institute, Seoul National University, 440 ~ 441). , 456). Therefore, the published patent is not about the research and discovery of the cosmetic application using the improvement effect on the skin of the pollen of plants and plants.

The present inventors selected the turmeric among a variety of herbal medicines, to confirm its efficacy as a cosmetic raw material and to develop a method of using the same, to increase the range of selection of raw materials of various cosmetics. In another aspect, the present invention was to provide a method for producing a cosmetic having a predetermined functional effect without the skin irritation by using the extract of the sulfur as a raw material.

That is, an object of the present invention is to provide a natural extract that is applicable to skin cosmetics and exhibits excellent whitening effect by inhibiting the production of melanin and the activity of enzymes related thereto when contained in cosmetics.

In addition, an object of the present invention is to provide a natural extract that can be applied to skin cosmetics and exhibits excellent anti-wrinkle and preventive effects such as anti-wrinkle effect by acting on the antioxidant effect, collagen synthesis effect, substrate metalloproteinase, when contained in cosmetics will be.

In addition, the present invention was to provide a method for producing a cosmetic using this material as a cosmetic raw material and the cosmetics from the point that the target efficacy was more greatly selected when selecting a specific extraction solvent in the preparation of the extract of turmeric.

Thus, the present inventors have studied the possibility of applying as a cosmetic in the natural materials that have not been known so far, confirmed that the turmeric extract has an effect such as whitening, wrinkle improvement, stimulation-reducing effect, etc. to prepare a cosmetic composition containing the same.

The present invention relates to a cosmetic composition containing the turmeric extract.

In addition, the present invention is characterized in that it is contained in 0.001 to 30.0% by weight based on the total weight of the total composition of the turmeric extract. When the content of the extract is less than 0.001% by weight, the efficacy is weak, and when the content of the extract exceeds 30.0% by weight, it is not economical because there is no apparent increase in the effect of the content increase.

In addition, the present invention is a liquid product obtained by the cold extract at room temperature using the at least one solvent selected from the sulfur extract is purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane and hexane In addition, the solvent may be obtained by concentrating under reduced pressure or lyophilization.

In addition to the extraction method, the turmeric extract of the present invention includes an extraction method using a supercritical fluid such as carbon dioxide.

Furthermore, the present invention, the cosmetic composition is a lotion, gel, water-soluble liquid, cream, essence, other oil-in-water (O / W) and water-in-oil (W / O) type base cosmetic formulation and oil-in-water or water-in-oil makeup base , Foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, cheek color and eyebrow pencil is characterized in that it is selected.

In addition, the present invention provides a cosmetic composition, characterized in that the extraction by using 40 to 95% by weight of ethanol as the extraction solvent.

The cosmetic composition may provide an anti-aging effect and a skin irritant effect.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are merely illustrative of the present invention and the scope of the present invention is not limited to these examples.

Example 1 Preparation of Sulfur Extract

The turmeric purchased from the medicinal herb was refluxed with a hot 95% (V / V) ethanol aqueous solution for 3 hours, cooled, and filtered through Whatman # 10 filter paper. The filtered extract was concentrated under reduced pressure and lyophilized at 50 ° C or lower. Sulfur extract was prepared using one or more solvents selected from purified water, ethanol, butylene glycol, and propylene glycol so that the vacuum concentrate and the lyophilisate contained 0.001 to 30.0% by weight.

Example 2 Antioxidant Effect Measurement Experiment Using NBT Method

In this example, to determine the antioxidant effect of the sulfur extract obtained in Example 1, the antioxidant sample, green tea extract and vitamin E as a comparative sample under laboratory conditions, and the antioxidant activity was measured using the NBT method.

In humans, molecular oxygen is essential to sustain life, but the consumption of oxygen produces a small amount of free radicals in vivo, and its strong oxidation destroys cell membranes and cellular components. Since these active oxygen has a great effect on the damage and aging of skin cells, antioxidant active substances having an active oxygen scavenging action will have an important effect on the living body.

In order to measure the antioxidant effect, the active oxygen produced by xanthine and xanthine oxidase is measured by the NBT method, and the effect of the test substance removing the active oxygen, that is, the active oxygen scavenging effect, is evaluated. Free radicals are produced by xanthine and xanthine oxidase. This active oxygen reacts with Nitro Blue Tetrazolium (NBT) to measure the active oxygen scavenging rate by measuring the blue color produced by this at a wavelength of 560 nm.

As the reagent used

1) 0.05M Na ₂ CO ₃ buffer (MW = 105.99): A solution of 5.25 g (50 mM) of sodium carbonate (pure photochemical) in purified water and a 50 mM NaHCO ₃ (MW = 84.01) solution are mixed to pH 10.2.

2) 3mM xanthine solution: 45.6 mg of xanthine (MW = 152.11) is dissolved in water to make 10 ml.

3) 3mM EDTA solution: Sodium ethylenediamine tetraacetate (MW = 60.1) is dissolved in distilled water to make 3mM.

4) 0.15% BSA solution: Dissolve 15mg of BSA (Fraction V, powder, Sigma) in distilled water to make 10ml.

5) 0.75 mM NBT solution: 61.32 mg of nitroblue tetrazolium (MW = 817.65, Tokyo University) is dissolved in distilled water to make 100 ml.

6) xanthine oxidase solution: Dilute the xanthine oxidase (Boehringer) with distilled water about 100 times and make the absorbance of the blank test within the range of 0.2 ~ 0.23 in the measurement operation. In other words, dilute with purified water so that the change in absorbance is A ₅₆₀ = 0.3 / 20 minutes.

7) 6mM CuCl ₂ solution: Dissolve 102.29mg of copper chloride (CuCl ₂ -2H ₂ O, MW = 134.45) in distilled water to make 100ml.

As a measuring method

0.05M Na ₂ CO ₃ ---------- ------------------------ 2.4ml

2.3mM xanthine solution -------------------------------- 0.1ml

3.3mM EDTA Solution ---------------------------------- 0.1ml

4.BSA solution --------------------------------------- 0.1ml

5.0.72mM NBT Solution -------------------------------- 0.1ml

6.Xanthine Oxidase Solution --------------------------- 0.1ml

7.6 mM CuCl ₂ solution ---------------------------------- 0.1ml

① Add 1, 2, 3, 4, 5 to the Bayer bottle, and add 0.1 ml of sample solution to it and leave at 25 ° C for 10 minutes.

② Add 6 solutions, stir rapidly, and start incubation for 20 minutes at 25 ℃.

(3) Then, add the 7 solutions to stop the reaction and measure the absorbance St at 560 nm.

④ For the blank test, measure the absorbance Bt by using distilled water instead of the sample solution as above.

⑤ Also, the blank of the sample solution was operated in the same manner using distilled water instead of the above 6 solution to measure the absorbance Bo.

The result of the effect was calculated by Equation 1, the results are shown in Table 1.

% Inhibition = [1- (St-So) / (Bt-Bo)] X 100

St: absorbance at 560 nm after enzyme reaction of sample solution

Bt: absorbance at 560 nm after enzymatic reaction of blank test solution

So: absorbance at 560 nm before reaction without enzyme in sample solution

Bo: absorbance at 560 nm before reaction without enzyme in blank test solution

The results showed excellent antioxidant effects in all samples tested as shown in Table 1 (active oxygen scavenging effect using NBT method). The turmeric extract was similar to green tea extract, which is widely used as an antioxidant, and had better antioxidant power than vitamin E. It was confirmed.

Sample Name Antioxidative effect(%) Turmeric extract 94 Green Tea Extract 95 Vitamin E 90

Example 3 Collagen Synthesis Effect Measurement Experiment

In order to observe the collagen synthesis effect of the turmeric extract obtained in Example 1, this Example was tested by treating human fibroblasts obtained directly from humans or purchased commercially.

In a 96-well plate, add DMEM (Dulbecco's Modified Eagle's Media) containing 2.5% fetal bovine serum and human fibroblasts (5000 cells / well), and incubate until 70-80% growth. It was. Then, the cultures were collected after 1 day treatment with the concentration of 0.01% and 0.001%, respectively. The collected cell culture solution was measured for collagen synthesis using a collagen protein measuring instrument (Catalog N .: MK 101, Takara Shuzo Co. Ltd, Japan).

In the measurement method, first, a cell culture solution collected in a 96-hole plate incubator uniformly coated with primary collagen antibody was subjected to antigen-antibody reaction for 3 hours. After 3 hours, the chromophore-bound secondary collagen antibody was placed in a 96-hole plate incubator and allowed to react for 15 minutes. After 15 minutes, the color development material was added and developed at room temperature, and 1M sulfuric acid was added to stop the color development. The color of the reaction color is yellow, depending on the intensity of the reaction. A yellowish 96-hole plate incubator was measured at 450 nm using an absorbance spectrometer, and the degree of collagen synthesis was calculated by Equation 5 below. At this time, the reaction absorbance of the cell culture solution without the extract was used as a control.

Collagen synthesis effect was calculated by the equation (2), the results are shown in Table 2.

Collagen Synthesis Effect (%) = (Responsive Absorbance of Extract Cell Cultures / Responsive Absorbance of Control) × 100

The results show that the turmeric extract of the present invention has a collagen production promoting effect as shown in Table 2.

sample Collagen Synthesis Effect (%) Turmeric extract 0.05% 74 0.005% 46 Control 0

Example 4 Inhibitory Activity Measurement of Substrate Metalloproteinase (MMP-1)

Substrate metalloproteinase (MMP-1) inhibitory activity was measured in vitro in a screening type biochemical model based on the use of purified collagenase and collagen conjugated to its substrate, fluorescein ( EnzChek ™ collagenase kit, molculaprobe). Collagenase purified from Clostridium histocomum was fed into the EnzChek collagenase kit. This enzyme possesses dual functionality against collagen types I and IV. The reaction buffer consisting of DQ-collagen purified from pig skin and conjugated to fluorescein and 0.05M Tris-HCl, 0.15M, NaCl, 5mM CaCl ₂ , and 0.02mM sodium azide (pH 7.6) was found in the EnzChek collagenase kit. (Molcula Probes) was used. The sulfur extract prepared in Example 1 was dissolved in the reaction buffer. It was tested at 4; 2; 0.4; 0.2; 0.04% (v / v). Dilutions of the samples were incubated with 25 μg / ml of DQ-collagen and 0.1 U / ml of collagenase for 15 minutes, 45 minutes, and 120 minutes at room temperature.

In each experimental condition, the control corresponding to the collagenase and DQ-collagen mixture was also incubated in the same manner. Blanks, hereinafter called 'enzyme-free blanks', were also incubated in the presence of DQ-collagen and in the absence of collagenase in each experimental condition. After 15, 45 and 120 minutes, the signal corresponding to the degradation of DQ-collagen was measured with a fluorometer (excitation; 485 nm, emission; 505 nm). The fluorescence value of each sample was measured based on the 'enzyme free blank' fluorescence value. Results are expressed as percent variance for fluorescence units per sample and control. The results are shown in fluorescein units / sample. All tests used 1,10-phenanthroline, a nonspecific metal chelator inhibitor, as a control inhibitor. At 0.1 U / ml collagenase the inhibitor is suitably at a concentration of 0.2 mM. In conclusion, the turmeric extract tested at 0.04 to 4% (v / v) under selected experimental conditions retained dose dependent anticollagenase activity. The results are shown in Table 3. The turmeric extract inhibited about 80% of the collagen degradation activity of Clostridium collagenase at 0.5% treatment, which was superior to the inhibitory effect of 2 mM 1,10-phenanthroline.

0.1U collagenase 2 mM 1,10-phenanthroline Sulfur Extract (%) 1.0 0.5 0.1 0.05 0.01 Fluorescence 232.6 57.5 15.8 46.8 66.5 97.7 169.1 Enzyme activity 100 24.7 6.8 20.1 28.6 42.0 72.7 Inhibition rate - 75.3 93.2 79.9 71.4 58.0 27.3

Example 5 Evaluation of Inhibition of MMP-1 Expression by Sulfur Extract after UV Irradiation

In this example, an ELISA (enzyme linked immunosorbent assay) was performed to measure the concentration of MMP-1 after UV irradiation and sample addition of the turmeric extract obtained in Example 1.

Human dermal fibroblasts are irradiated with UVA at an energy of 5 J / cm ² using a UV chamber. UV irradiation dose and culture time were established through preliminary experiments to maximize the expression of MMP in fibroblasts. Negative controls were wrapped in tinfoil and maintained at the same time in the environment of UVA. UVA emission was measured using a UV radiometer. The cells during the UVA irradiation are intact with the previously dispensed medium and irradiated with UVA and then exchanged with the medium containing the sample for 24 hours of incubation, and then the medium is recovered and coated in 96-well. The primary antibody (MMP-1 (Ab-5) monoclonal antibody) is treated and reacted at 37 ° C. for 60 minutes. After reacting the secondary antibody, anta mouse IgG (Whole mouse, alkaline phosphatase conjugated) for about 60 minutes, the alkaline phosphatase substrate solution (1 mg / ml p-nitrophenyl phosphate in diethanolamine buffer solution) was reacted at room temperature for 30 minutes and then Measure absorbance at 405 nm with a plate reader. As a control, the one without addition of the sample is used.

In case of MMP-1 expression induced by UV irradiation, the turmeric extract showed 92% inhibition rate compared to the control group without treatment, which is similar to the inhibition rate of retinol used as a control. Table 4 shows the inhibitory effect of MMP-1 expression of turmeric extract.

Test group % Inhibition of MMP-1 expression Control - Turmeric Extract 0.1% 92 Retinol 93

Example 6 Experiment of Measurement of Tyrosinase Inhibitory Effect Using Mushroom Tyrosinase

This Example is to determine the whitening effect by looking at the degree of inhibition of the function of the enzyme tyrosinase (tyrosinase) to confirm the whitening effect of the sulfur extract obtained in Example 1.

Tyrosinase is an enzyme that accelerates the oxidation process of tyrosine in vivo and helps melanin production. In this example, the method of inhibiting the function of this enzyme to inhibit the oxidation of tyrosine to form a black polymer called melanin (Pomerantz SH: J. Biochem . , 24: 161-168, 1996) is applied. To determine the whitening effect.

The inhibitory activity against tyrosinase of each sample was determined by placing 15 μl of the sample into a 96-well plate, adding 150 μl of 50 mM phosphate buffer (pH 6.5), 25 μl of 1.5 mM L-tyrosine solution, and then using mushroom tyrosinase (1,500 units / ml, Sigma). 10 μl was added and reacted at 37 ° C. for 20 minutes, and then the absorbance was measured at 490 nm using a microplate reader (ELx800, USA) to measure the inhibition rate against tyrosinase. % Inhibition of tyrosinase was calculated by Equation 3, IC ₅₀ The value is the concentration of the substance that inhibits tyrosinase enzyme activity by 50%.

% Inhibition = [(D-C)-(B-A)] / (D-C) × 100

A: Absorbance before reaction of the well in which the sample is placed

B: Absorbance after reaction of the well in which the sample was placed

C: Absorbance before reaction of the well without sample

D: absorbance after reaction of the well without sample

As a result of testing the inhibitory effect of tyrosinase activity, the IC ₅₀ value of the turmeric extract was 0.52%, which was lower than that of koji acid and oil-soluble licorice extract, which is known to be known previously, but showed an effect similar to that of arbutin (Table 5).

sample Mushroom tyrosinase inhibitory effect (IC ₅₀ ) Turmeric extract 0.52% Kojic acid 0.04% Arbutin 0.47% Lettuce extract 10.20% Soluble Licorice Extract 0.08%

Example 7 Measurement of Inhibitory Activity of Tyrosinase in Cells Using B16F1 Melanocyte

This Example was determined by measuring the degree of inhibition of tyrosinase activity in B16F1 melanocytes in order to confirm the whitening effect of the sulfur extract obtained in Example 1.

The B16F1 melanocytes used in this example are cell strains derived from mice and cells that synthesize an enzyme called tyrosinase. The degree of inhibition of tyrosinase activity was compared by evaluating the activity of the sample by treating the sample during the artificial culture of the cells and by separating the tyrosinase from the cells.

The B16F1 melanocytes used in this example were used after being sold from the American Type Culture Collection (Accession No .: 6323). The activity inhibitory effect of tyrosinase of B16F1 melanocytes was measured as follows.

B16F1 melanocytes were dispensed in 6 well plates at a concentration of 2 × 10 ⁶ per well, cells were attached, and the samples were incubated for 72 hours by treating the samples at a concentration that does not cause toxicity. After 72 hours of incubation, the cells were detached with Trypsin-EDTA, and the cells were counted and centrifuged to recover the cells. Wash the cell pellet once with PBS, add 1 ml of homogenization buffer (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF), vortex for 5 minutes, disrupt the cells, and centrifuge (3,000 rpm, 10 Min) to recover the supernatant. Put 1.5mM L-tyrosine, 0.06mM L-DOPA, and cell supernatant into 50mM sodium phosphate buffer (pH 6.5), incubate at 37 ℃ for 30 minutes, and measure the absorbance at 490nm with microplate reader to inhibit tyrosinase enzyme activity. Was measured. The inhibition rate of tyrosinase activity (%) of B16F1 melanocytes was calculated by the equation (4), IC ₅₀ value is the concentration of a substance that inhibits the activity of tyrosinase enzyme 50%.

% Inhibition = [(A-B) / A] × 100

A: Tyrosinase enzyme activity in wells without sample

B: Tyrosinase Enzyme Activity of Well Added Samples

As a result of measuring the inhibitory effect of tyrosinase enzyme of B16F1 melanocytes, IC ₅₀ value of turmeric extract was 0.84%, which was lower than conventional whitening agent Koji acid and oil-soluble licorice extract, but showed similar effect compared to lettuce extract and arbutin. (Table 6).

sample Inhibitory Effect of Melanosite on Tyrosinase Activity (IC ₅₀ ) Turmeric extract 0.84% Kojisan 0.05% Arbutin 0.82% Lettuce extract 2.65% Soluble Licorice Extract 0.19%

Example 8 Measurement of Melanin Inhibition Effect Using B16F1 Melanosite

This Example is to determine the whitening effect by looking at the degree of inhibition of melanin production for B16F1 melanocytes in order to confirm the whitening effect of the sulfur extract obtained in Example 1.

B16F1 melanocytes used in this example are cell strains derived from mice, and cells that secrete a black pigment called melanin. Samples were treated during the artificial culture of these cells to evaluate the degree of melanin reduction. The B16F1 melanocytes used in this example were used after being sold from the American Type Culture Collection (Accession No .: 6323).

The melanin biosynthesis inhibitory effect of B16F1 melanocytes was measured as follows. B16F1 melanocytes were dispensed in 6 well plates at a concentration of 2 × 10 ⁶ per well, cells were attached, and the samples were incubated for 72 hours by treating the samples at a concentration that does not cause toxicity. After 72 hours of incubation, the cells were detached with trypsin-EDTA, the cell number was measured, and the cells were recovered by centrifugation. Determination of intracellular melanin was performed by Rotan: Cancer Res . , 40: 3345-3350, 1980). The cell pellet was washed once with PBS, and then 1 ml of homogenization buffer solution (50 mM sodium phosphate, pH 6.8, 1% Triton X-100, 2 mM PMSF) was added to vortex for 5 minutes to disrupt the cells. Melt the extracted melanin by adding 1N NaOH (10% DMSO) to the cell filtrate obtained by centrifugation (3,000 rpm, 10 minutes), and measure the absorbance of melanin at 405 nm with a microplate reader. Percent inhibition of production was measured. Melanin inhibition rate (%) of B16F1 melanocytes was calculated by the equation (5), IC ₅₀ value is the concentration of the substance inhibiting 50% melanogenesis.

% Inhibition = [(A-B) / A] × 100

A: Melanin amount in wells without sample

B: Melanin amount in the wells to which the sample was added

The results of testing the melanin production inhibitory effect of B16F1 melanocytes showed that the IC ₅₀ value of the turmeric extract was 0.52%, which was weaker than the existing whitening agents hydroquinone and oil-soluble licorice extract, but showed a similar effect to arbutin (Table 7).

sample Melanin synthesis inhibitory effect (IC ₅₀ ) Turmeric extract 0.52% Hydroquinone 0.03% Arbutin 0.46% Lettuce extract 5.32% Soluble Licorice Extract 0.04%

Example 9 Inhibitory Effect of Inflammation-Related Enzyme (hyaluronidase) Activity

This example is to determine the anti-inflammatory effect of the turmeric extract obtained in Example 1 by measuring the enzyme activity of the inflammation-inducing enzyme hyaluronidiase.

Hyaluronidase is an enzyme that hydrolyzes hyaluronic acid and causes inflammation. In this Example, the method of inhibiting the activity of this enzyme to measure the anti-inflammatory effect (Kakegawa Y., Japanese J. of Inflammantion , 4: 437-438, 1984) was used to determine the anti-inflammatory effect.

The inhibitory effect of hyaluronidiase activity was investigated using comfrey, seesaw, triticale, oil-soluble licorice extract, and turmeric extract as samples.

Inhibitory activity of hyaluronidiases of each sample was performed as follows.

100 μl of the sample and 50 μl of a hyaluronidase solution (type IV-S, Sigma, 400 U / ml) were added and reacted at 37 ° C. for 20 minutes, followed by enzyme activation solution (compound 48/80 CaCl _{2 ·} 2H ₂ O, Sigma). , 0.1mg / ml) 100μl was added and reacted again at 37 ° C for 20 minutes. Add 250 μl of a hyaluronic acid solution (0.4 mg / ml) and react for 40 minutes at 37 ° C., and add 100 μl of 0.4N NaOH to terminate the reaction. 100 μl of potassium borate solution was added and reacted at 95 ° C. for 3 minutes and cooled. Then, 3 ml of ρ-dimethylaminobenzaldehyde solution was added and reacted at 37 ° C. for 20 minutes for color development. Absorbance was measured at 585 nm to determine the inhibition rate for hyaluronidiases. Inhibition rate (%) for hyaluronidiases was calculated by Equation 6, IC ₅₀ value is the concentration of a substance that inhibits the hyaluronidiase enzyme activity by 50%.

% Inhibition = [(A-B) / A] x 100

A: Enzyme activity of wells without sample

B: Enzyme Activity of Well Added Samples

As a result of testing the inhibitory effect of hyaluronidase enzyme activity, the IC ₅₀ value of the turmeric extract was 0.36%, which is a useful anti-inflammatory licorice extract, comfrey extract, tritical herb extract, and seesaw extract which are known to be effective in inhibiting hyaluronidase. It showed superior or similar effects compared to the back (Table 8).

sample Inhibitory effect of hyaluronidiases (IC ₅₀ ) Comfrey Extract 0.22% Seesaw extract 0.58% Soluble Licorice Extract 0.18% Triticale extract 0.43% Turmeric extract 0.36%

Example 10 Evaluation of Stimulation Relaxation Effect Using Cell Culture Technology

In this example, the cell death by Sodium Lauryl Sulfate (SLS), which is a substance causing skin irritation, was evaluated using a cell culture technique to evaluate the stimulating relaxation effect of the sulfur extract obtained in Example 1. The degree of stimulation alleviation was evaluated to the extent of inhibition.

SLS is a substance that is used as a criterion for evaluation of skin irritation based on cosmetics. It is bound to cell membranes to inhibit cell metabolism and destroys cell membranes to cause skin irritation.

This example evaluates the effect of reducing cell death by SLS when SLS and sulfur extract were added simultaneously to human fibroblasts.

The Hs68 fibroblasts used in this example were used as a dermal dermal cell in humans, sold at ATCC (American Type Culture Collection, Accession No. 1635). Stimulation relaxation evaluation experiment using the cell culture technology was carried out as follows. Human-derived fibroblasts were dispensed in 96 well plates at a concentration of 1 × 10 ⁵ per well, incubated for 24 hours, treated with 0.01% SLS alone, 0.01% SLS and turmeric extract (0.05%) simultaneously, and further cultured for 24 hours. It was. After incubation, MTT (Sigma) reagent was added and incubated for 4 hours, the culture solution was removed, 1N NaOH / isopropanol solution was added and stirred for 20 minutes, and the absorbance was measured at 565 nm using a microplate reader. The effect of inhibiting apoptosis was measured.

Experimental results have been found that the turmeric extract is an irritant that reduces skin irritation that may occur when applied with a cosmetic substrate by inhibiting apoptosis caused by SLS (Table 9).

sample Fibroblast survival rate (%) 0.01% SLS 21.8 0.01% SLS + 0.05% Sulfur Extract 54.2

<Examples 11 to 13 and Comparative Example 1>

In this example, a cosmetic containing the turmeric extract obtained in Example 1 was prepared. Cosmetics were prepared in the form of a cream, the composition is shown in Table 10. First, the phase b) recorded in Table 10 is heated and stored at 70 ° C. To this, a) phase is added, and after pre-emulsification, it is emulsified uniformly with a homomixer, and then cooled slowly to prepare a cream (Examples 11 to 13, Comparative Example 1).

 Raw material Example  Comparative Example 1 11 12 13 end Stearyl alcohol 8 8 8 8 Stearic acid 2 2 2 2 Stearic Acid Cholesterol 2 2 2 2 Squalane 4 4 4 4 2-octyldodecyl alcohol 6 6 6 6 Polyoxyethylene (25 mol addition) alcohol ester 3 3 3 3 Glyceryl monostearic acid ester 2 2 2 2 I Turmeric extract 10 3 0.5 - Propylene glycol 5 5 5 5 Purified water Quantity Quantity Quantity Quantity

Note) unit; weight%

Experimental Example 1

In this experimental example, the cream form cosmetics prepared in Examples 11-13 and Comparative Example 1 were applied to humans to evaluate skin wrinkle improvement effects.

Forty experimenters (women aged 20-35 years) were applied to the right face of the cream prepared in Examples 8 to 10 and the left face to the cream prepared in Comparative Example 1 twice a day for 2 consecutive months. It was.

After completion of the experiment, the skin wrinkle relief effect is obtained by using a silicone resin copy (replica) before and after using the product for 2 months, and comparing the state of the eye wrinkles with a skin fine wrinkle device and a skin image analyzer.

Table 11 compares the average eye wrinkle depth and wrinkle reduction rate of the experimenter using the creams prepared in Examples 11 to 13 with the experimenter using the cream of Comparative Example 1. As shown in Table 11, it can be seen that the effect of improving skin wrinkles is excellent in the skin around the face of the experimenter who applied the cream containing the turmeric extract.

Example 12 Comparative Example 1 Before use 2 months later Before use 2 months later Average fine wrinkle depth 0.218 0.192 0.214 0.206 Wrinkle Reduction (%) 11.93% 3.74%

n = 40, p <0.01

Experimental Example 2

In this experimental example, the stimulating alleviation effect of the cosmetic containing the turmeric extract obtained in Example 1 was evaluated by human patch experiment.

This evaluation is a result of evaluation obtained by directly applying the result obtained in Example 10 to the human body. Stimulation index can be obtained by mixing SLS (sodium lauryl sulfate) which causes irritation in general cosmetic prescriptions (cream, lotion, skin, essence) and the product prepared in Examples 11 to 13 for 24 hours, 48 hours and 72 hours. The stimulation-relaxation effect was evaluated based on this.

FINN CHAMBER (FINLAND) was applied to each arm of 50 healthy men and women aged 50 to 50 with 0.3 mg of each product, and the acute irritation index was evaluated after 24 hours. After evaluation, the same amount of product was applied to the same site again, and the delayed stimulation index after 48 and 72 hours was evaluated.

The test resulted in no skin seizures after 24 hours, 48 hours, 72 hours after patching the product containing the turmeric extract.

The results of this evaluation indicate that there is a significant effect that can reduce skin irritation by substrates (surfactants, fragrances, alcohols) that cause irritation when turmeric extract is mixed in cosmetics.

Other examples are shown below. That is, the turmeric extract of the present invention and the composition containing the same showed an excellent effect on skin improvement such as antioxidant effect, wrinkle improvement effect, stimulation alleviation effect as described above.

<Example 14> Preparation of the lotion containing the turmeric extract obtained in Example 1

Into 8 g of 95% ethanol, 0.05 g of polypyrrolidone, 0.1 g of oleic alcohol, 0.2 g of polyoxyethylene monooleate, 0.2 g of fragrance, 0.1 g of paraoxybenzoic acid methyl ester, a small amount of antioxidant, and a small amount of pigment are dissolved. . 0.1 g of the turmeric extract obtained in Example 1 and 5 g of glycerin were dissolved in 85.33 g of purified water, and the mixed solution was added thereto and stirred to obtain a lotion having a skin improvement effect.

<Example 15> Preparation of the milk liquid containing the turmeric extract obtained in Example 1

1.2 g of cetyl alcohol, 10 g of squalane, 2 g of petroleum jelly, 0.2 g of paraoxybenzoic acid ethyl ester, 1 g of glycerin monoesteraryide, 1 g of polyoxyethylene (20 mole addition) monooleate and 0.1 g of fragrance at 70 ° C The mixture was dissolved by heating, and 0.5 g of the turmeric extract obtained in Example 2, 5 g of dipropylene glycol, 2 g of polyethylene glycol 1500, 0.2 g of triethanolamine, and 76.2 g of purified water were heated to 75 ° C to dissolve it. Both mixtures were emulsified and then cooled to obtain an emulsion having a water / oil-based skin improvement effect.

<Example 16> Preparation of the cosmetic liquid containing the turmeric extract obtained in Example 1

5 g of 95% ethyl alcohol, 1.2 g of polyoxyethylene sorbitan monooleate, 0.3 g of kitulose, 0.2 g of sodium hyaluronate, 0.2 g of vitamin E-acetate, 0.2 g of sodium licoate, 0.1 g of paraoxybenzoic acid ester, Each 1 g of the turmeric extract obtained in Example 1 and an appropriate amount of pigments were mixed to obtain a cosmetic liquid having a skin improvement effect.

As described above, the turmeric extract according to the present invention was found to be excellent in free radical scavenging effect and wrinkle reducing effect such as inhibiting the activity and expression of matrix metalloproteinase (MMP), in particular collagenase (MMP-1). . It has also been found to be excellent in whitening effects such as inhibition of tyrosinase activity and inhibition of melanin biosynthesis. In addition, it was found that the skin stimulating effect of reducing skin irritation caused by cosmetic substrates, skin stimulation-reducing effects such as inflammation-related enzyme activity inhibitory effect is excellent.

Therefore, it can be seen that the cosmetic composition such as a lotion, cream, milky lotion, and pack containing such a turmeric extract has a skin wrinkle improvement effect, a whitening effect, and a stimulating alleviation effect.

Claims (5)

Cosmetic composition containing turmeric extract as the main active ingredient The method according to claim 1, wherein the extract is extracted using one or more solvents selected from purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane and hexane as the extraction solvent Cosmetic composition containing as a component According to claim 1 or 2, wherein the extract is a liquid obtained by cooling, stirring, heating, filtration at room temperature, further comprising a sulfur extract as a main active ingredient, characterized in that obtained by concentrated under reduced pressure or lyophilized solvent Cosmetic composition The cosmetic composition according to any one of claims 1 to 3, wherein the content of the turmeric extract is 0.001 to 30.0% by weight based on the total cosmetic composition. The cosmetic composition according to any one of claims 1 to 3, wherein the cosmetic composition is a lotion, a gel, a water-soluble liquid, a cream, an essence, an oil-in-water (O / W) type and a water-in-oil (W / O) type. , Cosmetic base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, cheek color and eyebrow pencil formulation