KR20090003144A - Cosmetic composition containing allithiamine - Google Patents

Abstract

A cosmetic composition is provided to improve the antiaging effect, the tyrosinase inhibiting effect, the melanin synthesis inhibiting effect and the wrinkle improving effect. A cosmetic composition contains allithiamine. Preferably the content of the allithiamine is 0.01-70 wt%. Also the cosmetic composition contains the mixture which comprises an extract of a plant containing vitamin B1 and an extract of a plant containing allithiamine, or the dried powder of the mixture.

Description

Cosmetic composition containing alithiamine {Cosmetic composition containing allithiamine}

The present invention relates to a cosmetic composition containing alliamine (allithiamine, active type vitamin B ₁ ), and more particularly, anti-wrinkle effect, atopic skin improvement effect, anti-inflammatory effect, stimulation alleviating excellent alithiamine (allithiamine, activity It relates to a cosmetic composition containing type vitamin B ₁ ).

Human skin is accompanied by physical and chemical changes such as loss of skin elasticity, formation of skin wrinkles, change of skin color, loss of skin barrier function, loss of skin immune function as aging progresses. In particular, stimulants from the external environment such as heavy metals, yellow sand, ozone, ultraviolet rays, cosmetics substrates, etc. promote the deactivation of skin cells, necrosis of skin cells, and cell degeneration, thereby promoting wrinkle formation, pigmentation, loss of immune function, and increased inflammation. To promote the back.

On the other hand, the most important factors for determining the skin color are melanin, carotene, hemoglobin and the most important of these is melanin. 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, to dihydroxy phenylalanine. It is formed of a polymer of eumelanin. This biosynthesis process is carried out in a special type of brown intracellular organelles called melanosomes. Melanosomes, including melanin granules, move from the periphery of the nucleus to the dendritic end, and then 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 migration to surrounding keratinocytes are primarily genetically influenced by the external environment, such as heavy metals, yellow dust, ozone, and ultraviolet rays, and by the internal environment such as abnormal hormone secretion. Other expression and the synthesis of melanin in the tyrosinase, is within the regulator cells which participate in the transmission cytokine (cytokine), also metal ions and interferon, prostaglandins, histamine, such as copper, zinc and iron are known to affect (for baksunam, Society of Cosmetic Scientists, 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.

Meanwhile, ascorbic acid, kojic acid, arbutin, hydroquinone, glutathione, or the like in order to treat or alleviate excessive melanin pigmentation caused by skin pigmentation abnormalities such as blemishes, freckles, pigmentation, and ultraviolet exposure, etc. These derivatives, substances having tyrosinase inhibitory activity, have been used in cosmetics and pharmaceuticals. However, they cause insufficient whitening effects, safety problems on the skin, formulation and stability problems when formulated in cosmetics, and their use is limited.

Therefore, there is a need for the development of alternative materials for this, at present, the research to find the whitening active ingredient in natural products continues to be made, among them baekbaekpi (Japanese Patent Publication No. 55-44375, So 64-26507, So 64-83009, Hei 1-25687, Hei 5-139950, Korean Laid-Open Patent Nos. 1992-002109, 1997-021273), Licorice (Japanese Laid-Open Patent Nos. 60-214721, Small 63-23809, Small 64-63506, Flat 1-49706 , Korean Laid-Open Patent No. 1992-002109, 1997-025601) have been found to inhibit melanin production by inhibiting the action of tyrosinase in plant extracts. However, these also have a lot of problems in the use of more than the effective concentration in cosmetics or pharmaceuticals in terms of safety, discoloration and the like does not have a satisfactory effect.

On the other hand, the human skin is constantly changing due to skin aging and pigmentation with age. Skin aging is largely divided into natural aging (or endogenous aging) and external aging (EB Doris, Cosmetics & Toiletories , 111 : 31-37, 1996). While natural aging is influenced by genetic factors, it is difficult to control artificially. However, external aging is relatively easy to control because it is influenced by environmental factors. Representative external aging factors include heavy metals, yellow sand, ozone, ultraviolet rays, and chemicals. The most prominent external aging phenomenon is wrinkle formation (HW Daniel, Ann). Intern Med , 75 (6): 873-880, 1971; GL Grove, et al ., J Am Acad Dermatol , 21 : 631-637, 1989; CE Griffiths, et al ., Arch Dermatol , 128 : 347-351, 1992).

These external stimulators are via free radical pathways (D Harman, J Gerontol, 11 (3): 298-301, 1956). Free radicals inhibit the activity of skin cells and induce the necrosis and degeneration of skin cells to destroy connective tissue formation such as skin collagen, impair cell membrane function, promote DNA mutation, modify protein action, intercellular energy transfer, metabolism It is known to cause modification of 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.

In vivo, the synthesis and degradation of extracellular matrix such as collagen is properly regulated, but as aging progresses, its synthesis decreases and matrix metalloproteinase (MMP), an enzyme that breaks down collagen, In particular, the expression of collagenase (hereinafter referred to as 'MMP-1') and gelatinase (gelatinase (hereinafter referred to as 'MMP-2')) is promoted, thereby reducing the elasticity of the skin and forming wrinkles. Ultraviolet irradiation and free radicals also activate these degrading enzymes.

MMP is classified into MMP-1, MMP-2, and MMP-9 according to its substrate specificity. MMP-1 is a fibroblast type collagenase, an enzyme of epilepsy, and collagen type I, II, III, Ⅶ, Ⅷ, Ⅹ and gelatin, originally cut into 3/4 or 1/4 length peptides to facilitate the action of nonspecific enzymes (gelatinase). MMP-2 belongs to 72 KD of gelatinase (A) and degrades substrates such as gelatin, collagen types IV, V, VIII, VIII, elastin and fibronectin. MMPs of neutrophils and polymorphonuclear leukocytes are primarily activated by oxidation, which first generates hydrogen peroxide in the cell and is converted to hypochlorous acid by myeloperoxidase in the presence of chlorine ions. Activate it.

In order to prevent aging due to external stimulation, the method of applying sunscreen products directly to the skin is the most common method.However, 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), and there are active studies around the world to develop substances that inhibit or improve skin aging. Among them, vitamins A, C, E, derivatives of vitamins, and AHA (alpha-hydroxy acid) are representative substances for improving skin aging known to date (Hermitte, Cosmetics & Toiletries , 107 : 63-67, 1992; DR Rosenthal, et al ., J Invest Dermatol , 95 : 510-515, 1990; TD Ditre, 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 somewhat insufficient to show a visible effect.

Accordingly, the present inventors searched for various natural materials for the purpose of developing new materials. Among them, the inventors focused on alithiamine and focused on the possibility of development into cosmetic compositions.

Alithiamine (active vitamin B ₁ ) is a derivative of vitamin B ₁ , a compound of Latin allium (garlic) and thiamine (vitamin B ₁ ), and has the same effect as vitamin B ₁ . Alithiamine is a derivative that is hardly susceptible to degradation of the vitamin B ₁ degrading enzyme and is easily absorbed in the digestive tract, and is highly likely to be developed as a cosmetic composition.

Accordingly, an object of the present invention is to develop and provide a cosmetic composition using alithiamine as a raw material.

In order to achieve the above object, the present invention is a cosmetic composition characterized in that it contains alithiamine; And it provides a cosmetic composition, characterized in that it contains a mixture of the extract of the plant containing vitamin B ₁ and the extract of the plant containing allicin or a dry powder of the mixture.

Hereinafter, the problem solving means of the present invention will be described in more detail.

The present invention relates to a cosmetic composition containing alliamine (active vitamin B ₁ ). The alithiamine used in the present invention may be a purified synthetic compound, or may be a mixed liquid obtained by mixing an extract of a plant containing vitamin B ₁ with an extract of a plant containing allicin or a dry powder of the mixed liquid. Mixing the extracts of plants which contain plant extracts and allicin that contain vitamin B _1, it is reported that the synthesis is possible without any special conditions at room temperature by the combination of vitamin B ₁ and allicin (allicin) (M Taizo et al, Yakugaku Zasshi, 72: 502-506, 1952; IH Kim, et al., Korean J. Food Sci. Technol., 30: 293-298, 1998). Therefore, it is also used to inform thiamine as a single compound, but also that the mixture may be used a mixture of extracts of plants that contain extracts of the plant and allicin which contain vitamin B _1.

On the other hand, it is preferable that the content of the alithiamine in the total cosmetic composition is preferably 0.01 to 70% by weight, and the content of the mixed liquid in the cosmetic composition or the dry powder content of the mixed liquid is an alithiamine content in the total cosmetic composition of 0.01 to 70 It is good that it is quantity to become weight%. At this time, the cosmetic composition according to the present invention, in addition to the above extracts, and other additives and the like that can give a synergistic effect to various supplements and main effects within the range that does not impair the effect of whitening, etc. described below for the purpose of the present invention It is okay to do it. Examples of adjuvants may include conventional adjuvants such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, blockers, pigments, odorants and dyes, etc. Amounts of the various adjuvants may be added by amounts conventionally used in the art. Such adjuvants may be incorporated into and added to the fatty phase, aqueous phase, lipid vesicles and / or nanoparticles. However, the type and ratio of the adjuvant should be selected and added so as not to adversely affect the desirable properties of the cosmetic composition according to the present invention.

On the other hand, to describe the meaning of the 'content of the mixed solution in the cosmetic composition or the dry powder content of the mixture of the amount of the alithiamine content in the total cosmetic composition of 0.01 to 70% by weight' in more detail, in the total cosmetic composition It means that the content of the mixed liquid to be added or the dry powder content of the mixed liquid is added by weighing the amount of alithiamine contained in the mixed liquid or the dry powder of the mixed liquid as a reference, and the content of the alithiamine is the total cosmetic composition. It is preferable that it is 0.01 to 70% by weight.

On the other hand, the extract of the plant can be extracted using a variety of solvents, preferably obtained easily purified water commonly used in the art, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, It is preferable to use at least one selected from dichloromethane and hexane.

On the other hand, the vitamin B ₁ is a plant extract that contains the vitamin B ₁ is if the plants are contained, and can be extracted from any plant without limitation particular to, a plant that allicin is contained extract is allicin containing Plants that are present are not limited to any particular plant and can be extracted from any plant. However, preferably, the extract of the plant containing vitamin B ₁ may be any one or more plant extracts selected from eggplant, beer yeast, wheat germ, rice bran, alfalfa and malt, to which potatoes belong, and the plant containing allicin The extract of is preferably at least one plant extract selected from the genus Lilium and garlic (allium) belonging to.

On the other hand, the present invention is a cosmetic composition includes all forms of formulations that can be used as a cosmetic, not necessarily limited to a specific formulation, for example, lotion, gel, water-soluble liquid, cream, essence, oil-in-water ( Basic cosmetic formulation consisting of O / W) and water-in-oil (W / O) type, oil-in-water or water-in-oil makeup base, foundation, skin cover, lipstick, lip gloss, face powder, two-way cake, eye shadow, cheek Tincture and cosmetic formulations of color and eyebrow pencils.

On the other hand, the cosmetic composition of the present invention, the following experiment results, the whitening ability is confirmed, from which the present invention preferably provides a cosmetic composition, characterized in that the whitening ability.

In addition, the cosmetic composition of the present invention, the following experimental results, the wrinkle improvement ability is confirmed, from which the present invention preferably provides a cosmetic composition, characterized in that the wrinkle improvement ability.

In addition, the cosmetic composition of the present invention is confirmed by the following experiments, the skin irritation relief is confirmed, from which the present invention provides a cosmetic composition characterized in that it has a skin irritation relief ability.

As described above, the cosmetic composition containing the alithiamine of the present invention having the above means has a tyrosinase activity inhibitory effect, melanin synthesis inhibitory effect, which prevents melanin production, which is a substance causing blemishes, freckles and skin pigmentation. Excellent anti-wrinkle effect such as whitening effect, free radical scavenging effect, collagenase (MMP-1) activity and expression suppression, and inhibits skin cell death which reduces skin irritation caused by cosmetic substrate It is excellent in alleviating skin irritation such as related enzyme activity inhibitory effect.

Therefore, the present invention can prepare a cosmetic composition, such as a lotion, cream, emulsion, pack containing alithiamine having the above-described efficacy can provide a functional cosmetic composition having a skin whitening effect wrinkle improvement effect, irritation relief effect.

Hereinafter, the content and effect of 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  One: Alithiamine  Preparation of Mixed Extracts Containing>

Potatoes and garlic were each chopped and dried, refluxed three times for 5 hours with 70% (V / V) ethanol aqueous solution, and then cooled and filtered with Whatman # 10 filter paper. The filtered extract was concentrated under reduced pressure and lyophilized at 50 ° C or lower. After diluting the purified water to a concentration of 0.001 to 70.0% by weight of the vacuum concentrate and freeze-dried, these two were mixed to prepare a mixed extract.

< Example  2: Alithiamine  Solution Preparation>

After purchasing the commercially available alithiamine, it diluted with purified water and manufactured the alithiamine liquid.

< Example  3: Mushroom Tyrosinase  Used Tyrosinase  Activity Inhibition Effect Measurement>

Inhibition of the function of tyrosinase (tyrosinase) was investigated to confirm the whitening effect of the mixed extract and alithiamine solution obtained in Examples 1-2.

Tyrosinase is an enzyme that accelerates the oxidation process of tyrosine in vivo and helps melanin production. In this example, the degree of inhibiting the function of this enzyme to oxidize tyrosine to inhibit the formation of a black polymer called melanin is measured (SH Pomerantz, J Biol chem , 241 : 161-168, 1966) to determine the whitening effect.

Inhibition of tyrosinase activity was investigated using kojic acid, arbutin, lettuce extract, oil-soluble licorice extract, mixed extract obtained in Example 1 and alithiamine solution obtained in Example 2 as samples.

Inhibitory activity of tyrosinase of each sample was placed in a 96-well plate, 15 µl of a sample, 150 µl of 50 mM phosphate buffer (pH 6.5) and 25 µl of 1.5 mM L-tyrosine solution, followed by mushroom tyrosinase. 10 μl of (1,500 units / ml, Sigma) was added and reacted at 37 ° C. for 20 minutes, and then irradiated at 490 nm using an absorbance photometer (microplate reader, ELx800, USA).

The activity inhibition rate (%) for tyrosinase was calculated by Equation 1, and the IC ₅₀ value is the concentration of a substance that inhibits tyrosinase enzyme activity by 50%.

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

A: Absorbance before reaction of the well containing the sample

B: Absorbance after reaction of the well containing the sample

As a result of testing the inhibitory effect of tyrosinase activity, the IC ₅₀ values of the mixed extract and the alithiamine solution were 0.011% and 0.009%, respectively, which were superior to the conventional whitening agents koji acid, arbutin, and lettuce extract, which are known to be excellent in inhibiting tyrosinase. (Table 1).

 Sample Name Inhibitory effect of tyrosinase (IC ₅₀ )  Mixed Extract  0.011%  Alithiamine liquid  0.009%  Arbutin  0.382%  Lettuce extract  0.900%  Soluble Licorice Extract  0.028%

< Example  4: B16F1 Melanosite  Intracellular Use Tyrosinase  Determination of Enzyme Inhibitory Effect>

In order to confirm the whitening effect of the mixed extract and alithiamine solution obtained in Examples 1 and 2, the tyrosinase enzyme activity in the B16F1 melanocytes was examined for the inhibition of tyrosinase enzyme activity in cells.

The B16F1 melanocytes used in this example are cell lines derived from mice, and cells that synthesize an enzyme called tyrosinase. The degree of inhibition of tyrosinase enzyme activity was evaluated by comparing the degree of inhibition of tyrosinase activity by treating a sample during the artificial culture of these cells and by separating the tyrosinase from the cells and measuring the activity of the enzyme. The B16F1 melanosite used in this example was sold from the American Type Culture Collection (Accession No .: 6323). The tyrosinase enzyme activity inhibitory effect of B16F1 melanocytes was measured as follows.

B16F1 melanocytes were dispensed at a concentration of 2 × 10 ⁶ per well in a 6-hole plate incubator, and the cells were attached and incubated for 72 hours by treating the sample 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. 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 minutes) to recover the supernatant. Put 1.5mM L-tyrosine, 0.06mM L-dopa, and cell supernatant into 50mM sodium phosphate buffer (pH 6.5), and incubate at 37 ℃ for 30 minutes and measure the absorbance at 490nm with an absorbance photometer to inhibit tyrosinase enzyme activity. Was measured.

The inhibition rate of tyrosinase enzyme activity (%) of B16F1 melanocytes was calculated by the equation (2), IC ₅₀ value is the concentration of a substance that inhibits the activity of the intracellular tyrosinase enzyme 50%.

% Inhibition = [(A-B) / A] × 100

A: Absorbance of Wells Without Samples

B: Absorbance of Wells Added Samples

As a result of measuring the inhibitory effect of tyrosinase enzyme activity of B16F1 melanocytes, IC ₅₀ values of natural extracts and alithiamine solution were 0.013% and 0.010%, respectively, and the conventional whitening agents, koji acid, arbutin, oil-soluble licorice extract and lettuce extract Even at low concentrations, it effectively inhibited the activity of tyrosinase (Table 2). As a result, it can be seen that the mixed extract of the present invention and the alithiamine solution exhibited a whitening effect by inhibiting the activity of the tyrosinase enzyme in the cells.

 Sample Name Inhibitory effect of intracellular tyrosinase activity (IC ₅₀ )  Mixed Extract  0.013%  Alithiamine liquid  0.010%  Arbutin  0.320%  Lettuce extract  0.950%  Soluble Licorice Extract  0.030%

< Example  5: B16F1 Melanosite  Measurement of melanin production inhibitory effect>

In this example, to determine the whitening effect of the natural product mixed extract and alithiamine solution obtained in Examples 1 and 2, the degree of inhibition of melanin production on B16F1 melanosite was investigated.

The B16F1 melanocytes used in this example are cell lines 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 black pigment reduction. The B16F1 melanosite used in this example was 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 at a concentration of 2 × 10 ⁶ per well in a 6-hole plate incubator, and the cells were attached and incubated for 72 hours by treating the sample 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 then centrifuged to recover the cells. Quantification of intracellular melanin was performed by slightly modifying the method of Rotan (R Lotan and D Lotan, 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 an absorbance spectrometer. Melanin production inhibition rate (%) was measured.

Melanin inhibition rate (%) of B16F1 melanocytes was calculated by the equation (3), IC ₅₀ value is the concentration of the substance inhibits 50% melanin production.

% 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 IC ₅₀ values of natural extracts and alithiamine were 0.003% and 0.001%, respectively, as a result of testing the melanin inhibition effect of B16F1 melanocytes, which is superior to the existing whitening agents hydroquinone, arbutin, useful licorice extract, and lettuce extract. Is shown (Table 3).

 Sample Name Melanin synthesis inhibitory effect (IC ₅₀ )  Mixed Extract  0.003%  Alithiamine liquid  0.001%  Arbutin  0.400%  Lettuce extract  0.450%  Soluble Licorice Extract  0.035%

< Example  6: NBT ( Nitro Blue Tetrazolium Antioxidant Effect Measurement Using the

In this example, the active oxygen radical scavenging effect was investigated to confirm the antioxidant effect of the natural product mixture extract and alithiamine solution obtained in Examples 1 and 2.

Free radical radical scavenging is described by Furuno et al. (K Furuno, et. al ., Biol Pharm Bull , 25 (1): 19-23, 2002), the effect of scavenging the active oxygen produced by the xanthine-xanthine oxidase system of each sample was measured. ₃ mM xanthine, 3 mM EDTA, 0.72 mM nitroblue tetrazolium (NBT), 0.15% bovine serum albumin (BSA) solution and sample were added to 0.05 M Na ₂ CO ₃ buffer, and then mixed at 25 ° C. The reaction was carried out for 10 minutes. Then, xanthine oxidase (0.25 U / ml) solution was added to each test tube, and it reacted at 25 degreeC for 30 minutes, and the absorbance was measured at 565 nm.

The result of the active oxygen scavenging effect was calculated by Equation 4, and the results are shown in Table 4.

Free radical scavenging effect (%) = [1- (St-So) / (Bt-Bo)] × 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 enzyme reaction of enzyme-free sample solution

Bo: absorbance at 560 nm before enzyme reaction of enzyme-free blank test solution

As shown in Table 4, all the samples tested showed excellent antioxidant effects, and natural extracts and alithiamine were similar to or superior to retinol and BHT.

 Sample Name  Antioxidative effect (%)  Mixed Extract  96.0%  Alithiamine liquid  98.0%  Retinol  94.0%  BHT  90.0%

< Example  7: DPPH  ( Nitro Blue Tetrazolium Antioxidant Effect Measurement Using the

In this example, the antioxidant activity was measured by using the DPPH method to confirm the antioxidant effect of the natural product mixed extract and alithiamine solution obtained in Examples 1 and 2.

DPPH method is to measure the antioxidant activity by reducing power using a free group called DPPH (2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl free radical). The free radical scavenging rate is measured at a wavelength of 560 nm by comparing the degree to which the DPPH is reduced by the test substance to decrease the absorbance.

Reagents used were evaluated by dissolving 61.88 mg in methanol as a 0.1 mM solution of DPPH (Aldrich, chem. Co ,. MW = 618.76) to 100 ml.

As an experimental method, 0.15 ml of 0.15 ml of 0.1 mM DPPH solution was added to 96-well plate, stirred quickly and reacted at 25 ° C. for 10 minutes, and the absorbance St was measured at 560 nm.

In the blank test, the absorbance Bt was measured using distilled water instead of the sample solution in the same manner as described above, and the blank of the sample solution was measured in the same manner using methanol instead of the 0.1 mM DPPH solution to measure the absorbance Bo.

Inhibition rate was calculated by the equation (5), the results are shown in Table 5.

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

St: absorbance at 560 nm after free radical scavenging of sample solution

Bt: absorbance at 560 nm after free radical scavenging of blank test solution

So: absorbance at 560 nm before reaction when no free radical of sample solution is added

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

 Sample Name  Antioxidative effect (%)  0.05% mixed extract  95.0%  Alithiamine liquid  96.0%  Retinol  88.0%  BHT  91.0%

< Example  8: Experiment of promoting collagen biosynthesis>

This Example evaluated the collagen biosynthesis promoting effect of the mixed extract and alithiamine solution obtained in Examples 1 and 2.

For the test, first human skin fibroblasts (Human skin fibroblast) was cultured in a 24-well plate until 3% 10 ⁴ per well (Seeding) to grow to about 90%. This was washed once with PBS, and then treated with the mixed extract and alithiamine solution obtained in Examples 1 and 2 and incubated in a 48 hour CO ₂ incubator. The supernatant was removed and the amount of procollagen (procollagen) type (1) C-peptide (PICP) was tested using ELISA kits to promote procollagen biosynthesis. Synthetic capacity is prepared by using a control group (Biriri group) as 100.

Collagen biosynthesis effect was calculated by the formula 6, the results were as shown in Table 6, the collagen biosynthesis promoting effect of the mixed extract and alithiamine solution obtained in Examples 1 and 2 was very excellent.

Collagen biosynthesis effect (%) = (reaction absorbance of extract cell culture / response absorbance of control) × 100

 Sample Name  Collagen Biosynthesis (%)  Mixed Extract 0.1%  144.0%  Alithiamine liquid  145.0%  Control (untreated)  100.0%

< Example  9: substrate metalloproteinase ( MMP -1) Determination of Activity Inhibition Effect>

The inhibitory effect of the mixed extract obtained in Examples 1 and 2 and alithiamine solution on the substrate metalloproteinase (MMP-1) activity was measured. Wang et al. (Y Wang, et. al ., J Biol Chem , 274 : 33043-33049, 1999). That is, 20 μl of DQ-collagen and 40 μl of sample dissolved in the reaction buffer at 0.25 mg / ml were added to 100 μl of the reaction buffer, and 40 μl of collagenase diluted to 0.5 units was added thereto. After 20 minutes at room temperature, the fluorescence value was measured by using a fluorescence spectrophotometer (LS55, PERKIN ELMER, USA) at an absorption wavelength of 495 nm and an emission wavelength of 515 nm. The fluorescence value was measured. The fluorescence value of the sample itself was also measured and corrected in enzymatic activity calculation. As a positive control, 1,10-phenanthroline (phenanthroline), which is known to have an inhibitory effect on MMP-1 activity, was used.

As shown in Table 7, the collagen degrading activity of Clostridium collagenase was decreased when the mixed extracts of two or more natural products obtained in Examples 1 and 2 and the alithiamine solution were treated by 0.1%, respectively. The inhibition was 90% and 90.5%, respectively, which was superior to the inhibitory effect of green tea extract.

 Sample Name  Collagen Biosynthesis (%)  Mixed Extract 0.1%  90.0%  Alithiamine liquid  90.5%  Green Tea Extract  83.5%

< Example  10: after ultraviolet irradiation MMP -1 expression inhibition evaluation>

In this Example, ELISA (Enzyme Linked ImmunoSorbent Assay) was performed to measure the expression level of MMP-1 after UV irradiation and sample addition of the mixed extract obtained in Examples 1 and 2 and alithiamine solution (SE Dunsmore, et. al ., J Biol Chem , 271 : 24576-24582, 1996).

Human fibroblasts were irradiated with UVA at an energy of 6.3 J / cm 2 using a UV chamber. Ultraviolet radiation dose and incubation time were established under the 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 were intact with the previously dispensed medium, and then irradiated with UVA and exchanged with the medium containing the sample, followed by culture for 24 hours, and then the medium was recovered and coated in a 96-hole plate incubator. The primary antibody (monoclonal antibody) was treated and reacted at 37 ° C. for 90 minutes. The secondary antibody, anti-mouse immunoglobulin (alkaline phosphatase conjugated anti-mouse IgG) was reacted for another 90 minutes, washed with buffer solution, and then washed with alkaline phosphatase substrate solution (1 mg / ml p -nitrophenyl phosphate in). diethanolamine buffer solution) was reacted at room temperature for 30 minutes, and the absorbance was measured at 405 nm using an absorbance spectrophotometer. At this time, the reaction absorbance of the cell culture solution without the extract was used as a control. MMP-1 expression inhibitory effect was calculated by the equation (7), the results are shown in Table 8.

% Inhibition of MMP-1 expression = [(A-B) / A] × 100

A: Absorbance of Wells Without Samples

B: Absorbance of Wells Added Samples

As shown in Table 8, the mixed extract and alithiamine solution obtained in Examples 1 and 2 according to the present invention exhibited excellent MMP-1 expression inhibitory effect by 0.1% to 84% and 86%, respectively. Could know.

 Sample Name  MMP-1 expression inhibition rate (%)  Control  -  Mixed Extract  84.0%  Alithiamine liquid  86.0%  Retinol  45.0%

< Example  11: Effect of inhibiting prostaglandin biosynthesis by ultraviolet irradiation>

In this example, the effect of inhibiting prostaglandin biosynthesis of the mixed extracts obtained in Examples 1 and 2 and alithiamine solution was evaluated.

First, 5 × 10 ⁴ keratinocytes isolated from human epidermal tissue were placed in 24-well test plates, attached for 24 hours, exchanged with medium containing no FBS, and cultured for 18 hours. Treatment of aspirin (aspirin) to a final concentration of 50uM keratinocytes of the keratinocytes to remove the activity of prostaglandin biosynthesis (prostaglandin E2 synthetase, or cyclooxygenase, COX). After 21 hours of aspirin treatment, each well containing keratinocytes was washed twice with PBS and 100 ul of PBS was added to each well. The keratinocytes were irradiated with ultraviolet rays 30mJ / cm ² using an ultraviolet B (UVB) lamp (Model: F15T8, UV B 15W, Sankyo Dennki, Japan), and then PBSfmf was removed and keratinocyte culture medium (Keratinocyte) was added to each well. growth media, Clonetics, Biowhittacker, MD, USA) 250ul was added. After treating the mixed extract to be evaluated therein, it was incubated for 16 hours. An appropriate amount of the culture supernatant was taken to quantify PGE2 (prostaglandin E2) synthesized for 16 hours to determine the effect of prastaglandin inhibition. The amount of PGE2 was quantified using enzyme immunoassay, and the experimental results showed that the natural extract and alithiamine solution effectively inhibited the production of prostaglandins by UV light. Particularly, the produced PGE2 is known to be mainly produced by the COX-2 enzyme, and thus, it was found that the above-described experiment inhibits the COX-2 induction or activity of the mixed extract (Table 9).

 Sample Name PGE ₂ Inhibition Rate (%)  (-) UV B  -  (+) UV B  -  (+) UV B + Mixed Extract  88.0%  (+) UV B + Alithiamine solution  87.0%

< Example  12: inflammation-associated enzyme ( hyaluronidase ) Activity Inhibition Effect Measurement>

This example measured the enzymatic activity of hyaluronidiase, an inflammation-related enzyme, in order to confirm the anti-inflammatory effects of the mixed extract and alithiamine solution obtained in Examples 1 and 2.

Hyaluronidase is an enzyme that hydrolyzes hyaluronic acid and has an inflammation-inducing function. In this example, the method for measuring the anti-inflammatory effect (Y Kakegawa, et al . , Japanese J of Inflammation , 4 : 437-438, 1984) was applied to determine the anti-inflammatory effect by inhibiting the activity of this enzyme.

The inhibitory effect of hyaluroniadiasis activity was investigated using a mixed extract, alithiamine solution, comfrey, seesaw, and three hundred sec extract as a sample. Inhibitory activity of hyaluronidiases of each sample was performed as follows.

100 μl of sample and 50 μl of hyaluronidiase solution (type IV-S, Sigma, 400U / mL) were added and reacted at 37 ° C. for 20 minutes, followed by enzyme activation solution (compound 48/80 CaCl ₂ ㆍ 2H ₂ O, Sigma). , 0.1 mg / ml) was added to 100 µl and reacted again at 37 ° C. for 20 minutes. 250 µl of a hyaluronic acid solution (0.4 mg / ml) was added thereto and reacted at 37 ° C. for 40 minutes, and 100 µl of 0.4N NaOH was added to terminate the reaction. 100 μl of potassium borate solution was added thereto, reacted at 95 ° C. for 3 minutes, cooled, and then, 3 ml of ρ -dimethylaminobenzaldehyde solution was added and reacted at 37 ° C. for 20 minutes, followed by color development. Absorbance was measured at 565 nm to determine the inhibition rate for hyaluronidiases. Inhibition rate (%) for hyaluronidiases was calculated by the equation (7), IC ₅₀ value is the concentration of a substance that inhibits the hyaluronidiase enzyme activity by 50%.

% Inhibition = [(A-B) / A] × 100

A: Enzyme activity of wells without sample

B: Enzyme Activity of Well Added Samples

The IC ₅₀ values of the mixed extract and alithiamine were 0.03% and 0.02%, respectively. It was possible to show an excellent effect compared to the seesaw extract and the like (Table 10).

 sample Inhibitory effect of hyaluronidiases (IC ₅₀ )  Mixed extract  0.03%  Alithiamine liquid  0.02%  Comfrey Extract  0.3%  Seesaw extract  0.5%  Triticale extract  0.2%

< Example  13: Evaluation of Stimulation Relaxation Effect Using Cell Culture Technology>

In this embodiment, sodium lauryl sulfate (SLS), which is a substance causing skin irritation by using cell culture technology, in order to evaluate the stimulating relaxation effect of the mixed extract and alithiamine solution obtained in Examples 1 and 2 Stimulation-releasing effect was evaluated to the extent of inhibiting apoptosis by.

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

This example evaluates the effect of reducing the cell death by SLS when SLS and mixed extract or alithiamine solution are added to human fibroblasts at the same time.

Hs68 fibroblasts used in this example were used as human dermal dermal cells obtained from the ATCC (American Type Culture Collection, Accession No .: 1635).

Dispensing human-derived fibroblasts at a concentration of 1 × 10 ⁵ per well in a 96-well plate incubator and incubating for 24 hours, followed by '0.01% SLS alone', '0.01% SLS and mixed extract (0.01%)' or '0.01 % SLS and alithiamine solution (0.01%) were simultaneously treated and further incubated for 24 hours. After incubation, MTT (Sigma) reagent was added and incubated for 4 hours, and then the culture solution was removed, stirred for 20 minutes by adding 1N NaOH / isopropanol solution, and the absorbance was measured at 565 nm with an absorbance spectrophotometer. It was determined whether the solution inhibited apoptosis by SLS.

As a result of the experiment, it was found that the mixed extract and the alithiamine solution are irritant substances that reduce skin irritation that may occur when the cosmetic base is prescribed with the cosmetic base by inhibiting apoptosis caused by SLS (Table 11).

 Sample Name  Fibroblast survival rate (%)  0.002% SLS  50  0.002% SLS + 0.01% Mixed Extract  87  0.002% SLS + 0.01% alithiamine solution  85

< Experimental Example  1> Skin whitening effect evaluation

In this Experimental Example, the cosmetics containing the alithiamine solution obtained in Example 2 were prepared, and the skin whitening effect of humans was compared with Comparative Example 1.

The cosmetics used in the comparative experiments were in the form of a cream, the composition of which is shown in Table 12. First, the phase b) recorded in Table 12 was heated and stored at 70 ° C. To this, a) phase is added and pre-emulsified, then it is emulsified uniformly with a homomixer, and then cooled slowly to prepare a cream (Examples 14 to 16, Comparative Example 1).

 Raw material  Example  Comparative Example 1 14 15 16    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 Alithiamine 10 One 0.1 - Propylene glycol 5 5 5 5 Purified water Quantity Quantity Quantity Quantity

Note) Unit: Weight%

For 20 experimenters (20-35 year old female), the cream prepared in Examples 14 to 16 on the right side of the face and the cream prepared in Comparative Example 1 on the left side of the face for 2 consecutive days Applied.

After completion of the test, the skins of the application areas on both sides of the face were compared by using an image analyzer and a color difference meter, and the darkest color was set to 5, the middle color to 3, and the brightest color to 1, and the intermediate level was estimated. Table 13 compares the facial skin color of the experimenter using the cream prepared in Example 16 with the facial skin color of the experimenter using the cream made in Comparative Example 1.

As shown in Table 13, it can be seen that the whitening effect is excellent in the facial skin of the experimenter who applied the cream containing alithiamine solution.

Product used Example 16 Comparative Example 1 Experiment number Right skin color Left skin color One 2 2.5 2 2.5 3.5 3 1.5 2.5 4 2 2.5 5 3 3 6 1.5 2 7 2.5 3.5 8 2 2.5 9 2 3.5 10 1.5 2.5 11 2 3.5 12 1.5 2.5 13 1.5 2 14 2 2 15 2.5 3.5 16 1.5 2.5 17 2 3 18 2 3.5 19 1.5 2.5 20 1.5 2.5

< Experimental Example  2> Evaluation of skin fine wrinkle improvement effect

In the present experimental example, the cream form cosmetics prepared in Examples 14 to 16 and Comparative Example 1 were applied to humans to evaluate the effect of improving skin wrinkles.

For 20 experimenters (20-35 year old female), the cream prepared in Examples 14 to 16 was applied to the right side of the face, and the cream prepared in Comparative Example 1 was applied twice a day for 2 consecutive months to the left side of the face. It was.

After the experiment was completed, wrinkles around the face of the face were collected with a silicone resin copy (replica) before and after using the product for 2 months, and the skin fine wrinkles device and skin image analyzer were analyzed to compare the condition of the wrinkles around the eyes. .

Table 14 compares the average eye wrinkle depth and wrinkle reduction rate of the experimenter using the cream prepared in Example 15 with the experimenter using the cream of Comparative Example 1. As shown in Table 14, it can be seen that the effect of improving skin wrinkles is excellent in the skin around the face of the experimenter to which the cream containing alithiamine solution is applied.

Example 15 Comparative Example 1 Before use 2 months later Before use 2 months later Average fine wrinkle depth (AU) 0.201 0.165 0.199 0.194 Wrinkle Reduction (%) 17.9% 2.5%

n = 20, p <0.01

< Experimental Example  3> Evaluation of stimulus relief effect

This experimental example evaluated the stimulating alleviation effect of the cosmetics containing the alithiamine liquid obtained in Example 2 by the human patch test. This evaluation is a result of evaluation obtained by directly applying the result obtained in Example 14 to the human body.

SLS that causes irritation in general cosmetic prescriptions (creams, lotions, skins, essences) and the products prepared in Examples 14 to 16 are mixed and patched for 24 hours, 48 hours, and 72 hours to stimulate stimulation based on the stimulation index. The effect was evaluated.

FINN CHAMBER (FINLAND) was used to coat 0.2 mg of each product on the upper arm of 50 healthy men and women aged 20 to 50 years, 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.

As a result of the test, it was confirmed that no skin redness was caused even after 24 hours, 48 hours, 72 hours after patching the product containing alithiamine solution.

The results of this evaluation indicate that there is a significant effect of reducing skin irritation by the substrate (surfactant, fragrance, alcohol) that causes irritation when the alithiamine solution is mixed in cosmetics.

In the following, other examples are shown.

< Example  17: Alithiamine  Make-Up Containing Lotion>

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 were dissolved. .

Subsequently, the mixed solution was added to 0.05 g of the alithiamine solution obtained in Example 2 and 5 g of glycerin in 85.33 g of purified water, followed by stirring to prepare a lotion having an effect of improving skin.

< Example  18: Alithiamine  Preparation of Containing Latex>

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 Heat mixing dissolved.

Subsequently, 0.05 g of the alithiamine solution 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.

Thereafter, both of the above were emulsified by mixing, followed by cooling to prepare an emulsion having a water / oil-based skin improvement effect.

< Example  19: Alithiamine  Containing Essence  Manufacture

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 licorice, 0.1 g of paraoxybenzoic acid ester, Each 1 g of the alithiamine liquid obtained in Example 1 and an appropriate amount of pigments were mixed to obtain a cosmetic liquid having a skin improvement effect.

Claims (10)

A cosmetic composition comprising alithiamine. The method of claim 1, The alithiamine, Cosmetic content, characterized in that the content of 0.01 to 70% by weight. A cosmetic composition comprising a mixture of a plant extract containing vitamin B ₁ and an extract of a plant containing allicin or a dry powder of the mixture. The method of claim 3, The content of the mixed solution or the dry powder content of the mixed solution in the cosmetic composition, Cosmetic composition, characterized in that the amount of the alithiamine content in the total cosmetic composition is 0.01 to 70% by weight. The method of claim 3, Extract of the plant, Cosmetic composition, characterized in that any one or more selected from purified water, methanol, ethanol, glycerin, ethyl acetate, butylene glycol, propylene glycol, dichloromethane and hexane. The method of claim 3, Extract of the plant containing the vitamin B ₁ , A plant extract of any one or more selected from eggplant, beer yeast, wheat germ, rice bran, alfalfa and malt, to which the potato belongs, The extract of the plant containing allicin is a cosmetic composition, characterized in that any one or more plant extracts selected from the genus Lilium and garlic (allium) belongs to garlic. The method according to any one of claims 1 to 6, Formulation of the cosmetic composition, Basic cosmetic formulation consisting of lotion, gel, water soluble liquid, cream, essence, oil-in-water (O / W) and water-in-oil (W / O) type, makeup base in oil-in-water or water-in-oil type, foundation, skin cover, lipstick, Cosmetic composition, characterized in that any one selected from lip gloss, face powder, two-way cake, eye shadow, cheek color and eyebrow pencil formulation. The method according to claim 1 or 3, The cosmetic composition, Cosmetic composition, characterized in that there is a whitening ability. The method according to claim 1 or 3, The cosmetic composition, Cosmetic composition, characterized in that the wrinkle improvement. The method according to claim 1 or 3, The cosmetic composition, Cosmetic composition, characterized in that there is a skin irritation relieving ability.