KR101923916B1 - Composition for skin whitening comprising Impatiens textori extract - Google Patents

Abstract

The present invention relates to an antiseptic composition comprising an extract of Bordeaux vulgaris as an active ingredient, a cosmetic composition for prevention or improvement of skin aging, a cosmetic composition for health functional food or skin whitening, and a health functional food, wherein said Bordeaux methanol extract is Escherichia coli, And Pseudomonas aeruginosa, and exhibits anti-wrinkle effect by increasing collagenase and elastase inhibitory activity and type I procollagen synthesis, decreasing MMP-1 mRNA and protein expression, exhibiting tyrosinase activity and tyrosinase activity Since it exhibits a whitening effect by decreasing the expression of azine mRNA and protein, it can be usefully used as a preservative in foods, cosmetics or medicines, and can be usefully used for improving skin aging and whitening.

Description

Composition for skin whitening comprising Impatiens textori extract as an active ingredient

The present invention relates to a preservative, a cosmetic composition for preventing or improving skin aging, and a health functional food or a cosmetic composition for skin whitening, and a health functional food comprising a natural substance, Impatiens textori, as an active ingredient.

Antiseptic (防腐劑) refers to a drug that prevents spoilage of substances. Preservative is to prevent decay of animal and vegetable organic matter by the action of microorganisms, and preservatives are drugs added to preservative for the purpose of preservation. Microorganisms that cause decay include fungi belonging to fungi, yeast and bacteria, which are lower microbes.

These preservatives are generally added to prevent deterioration of foods, cosmetics, or pharmaceuticals, and to maintain their purity while using or preserving them. Therefore, it is essential that there is no harm to the human body, and the quality must not be impaired by the addition.

Food preservatives, also known as food preservatives, must prevent food from being oxidized when exposed to the air, or microbes such as mold and bacteria from spoiling the food. Conventionally, as food preservatives, chemical preservatives such as calcium propionate, sodium benzoate, sodium nitrite and sorbic acid have been used. Propionic acid prevents mold formation and is used in bread, cheese, and chocolate. Sodium benzoate prevents microbial growth when added to orange or grapefruit juice. Sodium nitrite prevents the growth of microorganisms in meat. Sorbic acid and its salts inhibit the growth of mold and yeast, especially in cheese. However, when the amount of these chemical preservatives is exceeded, it is harmful to both humans and livestock, so its use is regulated by law (Pharmaceutical Affairs Act and Food Sanitation Act).

Cosmetic preservatives allow cosmetics to be stored at room temperature for a long time. Since cosmetic products have to be used for a long time after opening, preservatives (preservatives) are added to protect consumers so that they do not deteriorate easily. Preservatives mainly used in cosmetics are methyl paraben, ethyl paraben, propyl paraben, butyl paraben, quaternium-15, imidazolidinyl urea. (Imidazolidinyl Urea) and others. However, since cosmetics are used directly on the human body for a lifetime, even a small amount of preservatives can have a significant effect on the human body. In fact, dermatologists say that 70% of patients who visit the hospital for symptoms of skin troubles are the cause of cosmetic toxicity (preservatives). Preservatives in cosmetics can cause an allergic reaction or irritate the skin. Recently, it has been discovered that parabens, a type of chemical substance contained in various cosmetics and perfumes, can accumulate inside the human body and cause breast cancer.

As pharmaceutical preservatives, sodium salts of benzoic acid are added to eye drops, homemade preparations, injections, etc. However, these chemical preservatives can cause fatal harm to humans if the amount is exceeded. And usage are strictly controlled.

Since the above-described chemical preservatives cause many problems with respect to human safety, it is preferable to use natural preservatives obtained by extracting from natural organisms in consideration of human safety aspects. Until now, as natural preservatives that inhibit microbial contamination and growth, hinokitiol, a cypress tree extract, megnonol, a magnolia extract, and DF-100, a grapefruit seed extract, have been developed, but the scale is 17 for synthetic preservatives. It is only a percentage, so it is not meeting the demand.

One of the most obvious phenomena due to skin aging is the decrease in the extracellular matrix of the dermal layer, resulting in wrinkles. The extracellular matrix of the dermal layer contains various proteins such as collagen, elastin, and glycoprotein as a major component. More than 90% of these proteins are collagen, of which about 90% is type 1 collagen. It was confirmed that the amount of type 1 collagen in the extracellular matrix of the dermal layer decreased by about 70% due to aging.

Hyaluronic acid is a type of polysaccharide consisting of amino sugar and uronic acid, and is distributed in all tissues of a living body, and about 50% of the total amount in the body is distributed in the skin. Hyaluronic acid can bind up to 1000 times the mass of water and fills the extracellular matrix of dermal tissue. In addition, hyaluronic acid plays a role in storing or preserving various growth factors by forming an aggregate between various glycoproteins and proteoglycans. According to the results of clinical studies, hyaluronic acid increases the proliferation of dermal fibroblasts, collagen production, and secretion of growth factors, and is known to decrease by more than 40% of the skin with increasing age.

Skin aging is largely divided into two types: intrinsic aging and extrinsic aging. Exogenous skin aging is mainly caused by external environmental factors such as ultraviolet rays and smoking, and endogenous skin aging is skin aging that accompanies aging. Currently, there are various methods such as cosmetics with UV blocking function to prevent exogenous skin aging, but there is no way to prevent endogenous skin aging.

In addition, there are various causes of blackening of a person's skin in general, but the main cause in particular is exposure of the skin to ultraviolet rays. When the skin is exposed to ultraviolet rays, melanin is synthesized and released in melanocytes, a kind of skin cells, resulting in black skin.

Looking at the process of synthesizing melanin in melanocytes, an enzyme called tyrosinase acts using tyrosine in the cell as a substrate to produce dopaquinone, and spontaneous reactions and enzyme reactions from dopaquinone are performed. Through this, melanin, which is a copolymer black pigment, is produced.

Therefore, in order to prevent the skin from becoming black, a method of reducing the synthesis of melanin by inhibiting some reactions during the melanin production process is generally easily performed.

Conventionally, various plant extracts such as ascorbic acid, kojic acid, arbutin, hydroquinone, and the like have been used for this purpose. Among these, kojic acid acts to inhibit enzymatic activity by chelating copper ions present in the active site of tyrosinase. Although it has good performance, it has a safety problem when it is incorporated in cosmetics, so its use is limited. Because of high irritation, this raw material is also not currently applied to cosmetics due to safety issues.

Therefore, it is necessary to develop a composition that exhibits excellent antibacterial, anti-wrinkle or skin whitening effect.

1. Korean Patent Registration No. 10-1430643.

Accordingly, an object of the present invention is to provide a preservative composition containing a natural substance having few side effects.

In addition, another object of the present invention is to provide a cosmetic composition for preventing or improving skin aging, including a natural substance having less side effects.

In addition, another object of the present invention is to provide a health functional food for preventing or improving skin aging, including natural substances with less side effects.

In addition, the present invention has another object to provide a cosmetic composition for skin whitening comprising a natural substance with less side effects.

In addition, the present invention has another object to provide a health functional food for skin whitening containing a natural substance with less side effects.

In order to achieve the above object, the present invention provides a preservative composition comprising a water balsamic extract as an active ingredient.

In order to achieve the above another object, the present invention provides a cosmetic composition for preventing or improving skin aging, comprising an extract of water balsamic lump as an active ingredient.

In order to achieve the above another object, the present invention provides a health functional food for preventing or improving skin aging, comprising the extract of water balsamic lump as an active ingredient.

In order to achieve the above another object, the present invention provides a cosmetic composition for skin whitening comprising a water balsamic extract as an active ingredient.

In order to achieve the above another object, the present invention provides a health functional food for skin whitening comprising the extract of water balsamic lump as an active ingredient.

According to the present invention, the methanol extract of water balsamic acid shows excellent antibacterial effect against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, and increases collagenase and elastase inhibitory activity, type I procollagen synthesis, and decreases MMP-1 mRNA and protein expression. It shows anti-wrinkle effect, reduces tyrosinase activity and tyrosinase mRNA and protein expression to show whitening effect, so antiseptic composition, cosmetic composition for preventing or improving skin aging, and health functional food or cosmetic composition for skin whitening and health function It can be usefully used as a food.

1 is a result of confirming the antioxidant ability of the methanol extract (I. textori methanol extract, ITME) according to the present invention,
2 is a result of confirming the electronic donation ability of ITME,
3 is a result of confirming the antibacterial activity of ITME,
Figure 4 is a result of confirming the antimicrobial properties of 1,2-hexanediol (1,2-hexanediol),
Figure 5 is a result of confirming the antimicrobial properties of methyl paraben (methyl paraben),
Figure 6 is a result of confirming the collagenase (Collagenase) inhibitory activity of ITME,
Figure 7 is a result of confirming the inhibitory activity of the elastase (Elastase) of ITME,
8 is a result of confirming the maximum permissible level (MPL) of ITME,
9 is a result of observing morphological changes after treating human skin fibroblasts with various concentrations of ITME,
10 is a result of confirming the effect of synthesizing Type I procollagen after treating human skin fibroblasts with various concentrations of ITME,
11 is a result of confirming the change in expression of matrix metalloproteinase-1 (MMP-1) mRNA after treating human skin fibroblasts with various concentrations of ITME,
12 is a result of confirming the change in the expression of matrix metalloproteinase-1 (MMP-1) protein after treating human skin fibroblasts with various concentrations of ITME,
13 is a result of confirming the cell growth rate after treatment of various concentrations of ITME on melan-a cells, which are pigment synthesis cells,
14 is a result of observing the morphological changes of cells after treatment with various concentrations of ITME on melan-a cells,
15 is a result of observing the melanin content of cells after treatment with various concentrations of ITME on melan-a cells,
16 is a result of observing intracellular tyrosinase activity after treatment with various concentrations of ITME on melan-a cells,
17 is a result of confirming the change in intracellular tyrosinase mRNA expression after treatment with various concentrations of ITME in melan-a cells,
18 is a result of confirming the change in the expression of the intracellular tyrosinase protein after treatment with various concentrations of ITME in melan-a cells.

Hereinafter, the present invention will be described in detail.

While trying inventors of the present invention have studied the efficacy of natural product extracts, mulbongseonhwa (Impatiens textori) the methanol extract showed an excellent antibacterial effect on Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa collagenase and elastase inhibitory activity and the type I pro The present invention was completed by confirming that it showed an anti-wrinkle effect by increasing collagen synthesis and reducing MMP-1 mRNA and protein expression, and showing a skin whitening effect by reducing tyrosinase activity and tyrosinase mRNA and protein expression.

The Impatiens textori grows in groups in the waterside or wetland of the mountain valley. The stem stands straight, many branches are split, and the height is 40~80cm. Leaves are alternate, 6~15cm long, broad lancet shape, sharp end, serrated edge, fruit is capsule, 1~2cm long lancet shape, when ripe, it bursts and seeds sprout. Distributed in Korea, Japan, and northeastern China, the dark purple flowers are called Gayamulbongseon, and the white flowers blooming are called whitemulbongseons.

Because leaves and stems act as detoxification and small bells, they are used to treat boils or when being bitten by snakes, and the roots are known to have a tonic effect and relieve bruised blood.

However, no antibacterial effect, anti-wrinkle effect, or whitening effect of the water balsam extract has been reported.

Therefore, the present invention provides a preservative composition comprising the water balsamic extract as an active ingredient.

The extract is extracted with one or more solvents selected from the group consisting of methanol, ethanol, distilled water, and mixtures thereof, and it is preferable to use methanol, but is not limited thereto.

According to an embodiment of the present invention, the methanol extract (ITME) according to the present invention showed antibacterial properties similar to 1,2-hexanediol or methyl paraben, which are currently used as preservatives, against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa.

Therefore, the preservative composition can be used in food, cosmetics or pharmaceuticals.

According to another embodiment of the present invention, the methanol extract of water balsamic acid according to the present invention exhibits excellent collagenase and elastase inhibitory activity, and increases the synthesis of type I procollagen in human skin fibroblasts and genes and proteins of MMP-1. Decreased expression. In addition, it was confirmed that the MPL of the ITME concentration for human skin fibroblasts was 200 μg/mL or more.

Accordingly, the present invention provides a cosmetic composition for preventing or improving skin aging, comprising an extract of water balsamic lump as an active ingredient.

The skin aging may be a wrinkle caused by a decrease in the extracellular matrix of the dermal layer of the skin.

The extract is extracted with one or more solvents selected from the group consisting of methanol, ethanol, distilled water, and mixtures thereof, and it is preferable to use methanol, but is not limited thereto.

In addition, according to another embodiment of the present invention, the methanol extract of water balsamic acid according to the present invention reduced tyrosinase activity and expression of tyrosinase mRNA and protein in melan-a cells, which are pigment-synthetic cells.

Accordingly, the present invention provides a cosmetic composition for skin whitening comprising the water balsamic extract as an active ingredient.

The extract is extracted with one or more solvents selected from the group consisting of methanol, ethanol, distilled water, and mixtures thereof, and it is preferable to use methanol, but is not limited thereto.

The cosmetic composition may include a stabilizer, a solubilizing agent, vitamins, pigments and flavors, and a carrier in addition to the water balsamic acid extract, which is an active ingredient.

The cosmetic composition may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, oil, powder foundation, emulsion foundation, It may be formulated as a wax foundation or spray, but is not limited thereto. In more detail, it may be prepared in the form of a sun cream, a flexible lotion, a converging lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a pack, a spray, or a powder.

When the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. .

When the formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propane/butane Or a propellant such as dimethyl ether.

When the formulation is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -Butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the above formulation is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose , Aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.

In addition, the present invention provides a health functional food for preventing or improving skin aging, comprising the extract of water balsamic plant as an active ingredient.

The extract is extracted with one or more solvents selected from the group consisting of methanol, ethanol, distilled water, and mixtures thereof, and it is preferable to use methanol, but is not limited thereto.

In addition, the present invention provides a health functional food for skin whitening comprising the extract of water balsamic plant as an active ingredient.

The extract is extracted with one or more solvents selected from the group consisting of methanol, ethanol, distilled water, and mixtures thereof, and it is preferable to use methanol, but is not limited thereto.

The health functional food may be provided in the form of powder, granule, tablet, capsule, syrup, or beverage, and the health functional food is used with other foods or food additives in addition to the water balsam extract, which is an active ingredient, and is appropriate according to a conventional method. Can be used. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use, for example, prevention, health or therapeutic treatment.

There are no particular restrictions on the types of health functional foods, examples of which include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea. , Drinks, alcoholic beverages, and vitamin complexes.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.

< Reference example 1> Reagents and laboratory equipment

Dimethyl sulfoxide (DMSO), 2,6-di-tert-butylate hydroxytoluene (BHT), 3,4-dihydroxy-L-phenyl -Alanine (3,4-dihydroxy-L-phenyl-alanine, L-DOPA), 1,1-diphenyl-2-picryl-hydrazyl (1,1-diphenyl-2-picryl hydrazyl, DPPH), arbutin (arbutin), tannic acid, L-tyrosine, Foline-Ciocalteu's phenol reagent, 3-(4,5-dimethyl-thiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, MTT) and 12- o -tetradecanoyl-porbol- 13-acetate (12- o- tetradecanoyl-phorbol-13-acetate, TPA) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

DMEM (Dulbecco's modified Eagles medium), RPMI-1640 medium (Roswell Park Memorial Institute (RPMI)-1640 medium), fetal bovine serum (FBS) and penicillin/streptomycin mixture (P/S) ) Was purchased from Lonza (Cascade, MD, USA).

The morphology of the cells was observed using an inverted microscope (CKX41, Olympus, Tokyo, Japan), and each cell was cultured in a CO ₂ incubator (MCO-17AC, Sanyo electric, Osaka, Japan).

<Reference Example 2> Cell culture

Human dermal fibroblasts were purchased from Amore Pacific Company (Yongin, Korea) and supplemented with 10% FBS and 1% P/S in a humidified incubator of _{5% CO 2 and 37°C for 72 hours.} Was cultured in DMEM medium.

The pigment-synthetic cells, melan-a cells, were obtained from Dr. Dorothy Bennett (St. George's Hospital, UK), and persistent cells with many pigments were isolated from C57BL/6 mice. Each cell was cultured in RPMI-1640 medium, supplemented with 10% FBS, 1% P/S and 200 nM TPA, and cultured for 72 hours in an incubator at _{37° C. and 10% CO 2.}

<Reference Example 3> Culture of Microorganisms

Mulbongseonhwa methanol extract to determine the antimicrobial activity of (I. textori methanol extract, ITME) , E. coli (Escherichia coli, ATCC 8739), Staphylococcus aureus (Staphylococcus aureus, ATCC 6538) and Pseudomonas aeruginosa (Pseudomonas aeruginosa, ATCC 9027) microorganism in Korea They were pre-sold at the Korean Culture Center of Microorganisms (KCCM) and cultured at 37° C. for 24 hours in bacteria nutrient agar medium.

<Example 1> Preparation of water balsamic methanol extract

Waterbongseonhwa (Impatiens textori) was purchased at a floricultural pot in Goyang, Gyeonggi-do, South Korea, located in Goyang-si, Gyeonggi-do, Korea in May 2014, and crushed and used for experiments.

50 g of a powdery sample was put into a flask and extracted three times with 500 mL of 80% methanol at 25°C every 24 hours.

This was filtered with filter paper, concentrated using a rotary vacuum evaporator (BUR-205, BULabortechnik AG, Switzerland), and then freeze-dried.

< Example 2> Antioxidant ability measurement

2.1 total Polyphenol and Total flavonoids ( flavonoid ) Content measurement

In order to measure the total polyphenol content of the ITME prepared in Example 1, 0.2 mg of the extract was first dissolved in 1 mL of distilled water and placed in a test tube, and then a foline-reagent (1 mL) was added and left for 3 minutes. After 3 minutes, 1 mL of 10% Na ₂ CO ₃ was added, shaken vigorously, and left for 60 minutes. Then, the polyphenol concentration was measured at 725 nm and quantified by a standard curve using tannic acid.

Next, to measure the flavonoid content, 0.2 mg of the extract was dissolved in 1 mL DMSO, and 100 μL of this solution was mixed with 1 mL of di-ethylene glycol reagent and 100 μL of 1N NaOH in a test tube. Mixed. Then, after vigorously shaking, the reaction was performed at 37°C for 60 minutes, and the absorbance was measured at 420 nm, and this was quantified by a standard curve using rutin.

As a result, as shown in FIG. 1, the contents of polyphenols and flavonoids in ITME were 193.8 and 146.8 mg/g, respectively.

2.2 Electron-donating ability

In order to measure the electron donating ability of the ITME prepared in Example 1, ITME was dissolved in distilled water to a final concentration (125, 250, 500 or 1000 μg/mL).

Then, 1 mL of ITME was added to the test tube, and 4 mL of 0.4 mM DPPH was added thereto.

The mixture was shaken vigorously and reacted at 60° C. for 10 seconds, and the absorbance was measured at 525 nm. At this time, ascorbic acid was used as a positive control.

The DPPH radical-scavenging activity of each solution was calculated as the inhibition rate according to Equation 1 below.

[Equation 1]

% Electron donation capacity = [1-(A _sample / A _control )] × 100

The A _control is the absorbance of the control (reagent excluding the test compound).

It was shown that the electron donating ability of ITME of various concentrations increased to 50.4% in the case of 1000 μg/mL, in a dose-dependent manner, as shown in FIG. 2.

< Example 3> Anti-microbial activity

3.1 Antimicrobial activity assay

Antimicrobial activity of ITME was measured using gram-negative bacteria, E. coli, Pseudomonas aeruginosa, and gram-positive bacteria, Staphylococcus aureus.

At this time, the already known literature ( Bioorg Med Chem Lett 14: 5831-5833, 2004), the minimum inhibitory concentrations (minimal inhibitory concentrations, MICs) determined through the agar streak dilution method based on the radial diffusion described in (2004) were measured.

The same 0.5 Mycobacterium avium complex (Mac) Farland suspension was diluted in Retin broth and dispensed into a 24-well plate.

The final inocula determined by the colony count of the growth control wells was approximately 1.5×10 ⁶ cells/mL per well.

Accordingly, ITME (4%, 3%, 2%, 1.5% or 1%), 1,2-hexanediol (4%, 2%, 1%, 0.5% or 0.25%) and methyl paraben (2%, 1%) , 0.5%, 0.25% or 0.125%) was added to each well and then incubated at 37°C for 48 hours.

MIC was determined as the minimum inhibitory concentration of ITME that inhibits the growth of microorganisms.

As a result, as shown in FIG. 3, the MIC values of ITME for Escherichia coli (A), Staphylococcus aureus (B) and Pseudomonas aeruginosa (C) were measured as 3%, 1.5% and 1.5%, respectively.

4 and 5, the MIC values of 1,2-hexanediol used as a preservative were measured as 1%, 2% and 1%, respectively, and the MIC values of methyl paraben were 0.25%, 0.5% and 0.25%, respectively. It was measured as.

Therefore, the ITME according to the present invention showed antimicrobial activity similar to the preservatives currently used.

<Example 4> Anti-winkle effect

4.1 Collagenase activity assay

Calcium chloride (4 mM) was dissolved in 0.1M Tris-HCL buffer (pH 7.5), and then used as a final buffer and substrate. In this buffer 4-phenylazo-benzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg(4-phenylazo-benzyloxy-carbonyl-Pro-Leu-Gly-Pro-D-Arg(0.15 mg/mL) ) Was dissolved.

Then, 250 μL of substrate soultion and 100 μL of ITME were injected into the tube.

After dissolving water-encapsulated collagenase in the final buffer to 0.2 mg/mL, 150 μL of this solution was added to the tube.

After reacting this at 37° C. for 30 minutes, citric acid (6%) was added to stop the reaction, and ethyl acetate was added to separate the reaction mixture, and the absorbance of the supernatant was measured at 324 nm. .

At this time, ascorbic acid was used as a positive control, and collagenase activity was calculated through Equation 2 below, and was calculated as a relative inhibitory activity with respect to the control group.

[Equation 2]

Inhibition (%) = - a (sample OD ₃₂₄ OD ₃₂₄ of control) / control OD _324] × 100

As shown in FIG. 6, the collagenase inhibitory activity of ITME at various concentrations was 50.4% at a concentration of 10 mg/mL, and it was shown to increase in a concentration-dependent manner.

4.2 Elastase activity assay

In order to evaluate the inhibitory effect of ITME on elastase activity, absorbance was measured at 410 nm, and the substrate released by elastase, N-succinyl-(L-Ala)) ₃ -p -nitroanilide (N- was confirmed that the amount of fluoride is not a nitro (p -nitroanilide) - succinyl- (L -Ala) 3 - p -nitroanilide) is an exploded p singer.

First, 2.9 mM N-succinyl-(L-Ala)) ₃ -p -nitroanilide was prepared in 100 mM Tris-HCl buffer (pH 8.0), and then added to the lyophilized ITME, and each sample solution was finalized. It was diluted to a concentration of 1.25, 2.5, 5 or 10 mg/mL.

This was thoroughly mixed by tapping, and a stock solution of elastase (0.2 unit/mL) was added, followed by reaction at 37° C. for 10 minutes, and absorbance was measured at 410 nm.

The elastase inhibition rate was calculated by substituting the absorbance value measured at 410 nm in Equation 2, and was calculated as the relative inhibitory activity with respect to the control group.

As a result, as shown in FIG. 7, ITME showed an excellent inhibitory effect on elastase activity even at a low concentration of 1.25 mg/mL, and this inhibitory activity slightly increased depending on the concentration.

4.3 MTT analysis

The cell growth rate was confirmed through MTT analysis to determine the maximum permissible level (MPL) of ITME in human skin fibroblasts.

^{First, human skin fibroblasts were dispensed by 1×10 4} cells/well per well of a 96-well plate, and cultured in an incubator at 37° C., 5% CO _{2 and humidified for 24 hours.}

ITME (200 μL) diluted in red-free DMEM (PRF-DMEM) at a concentration of 25, 50, 100 or 200 μg/mL was added to each well, followed by further incubation for 48 hours.

After the cultivation is complete, MTT (0.5 μg/mL) was added to each well, incubated for 3 hours, centrifuged for 10 minutes at 1,000 rpm to remove the supernatant, and then DMSO (200 μL) was added to each well. gave.

After resuspending the cells in a plate-shaker for 15 minutes, the absorbance was measured at 540 nm with a microplate reader, and the cell growth rate was calculated as a percentage by dividing the measured value by the absorbance of the control reagent.

As a result, as shown in A in FIG. 8, when ascorbic acid was treated at a concentration of 100 or 200 μg/mL, growth inhibition rates of 4.5% and 29.9% were shown, respectively. Therefore, it was confirmed that the MPL of ascorbic acid for human skin fibroblasts was 100 μg/mL.

On the other hand, as shown in B in FIG. 8, when the ITEM was treated at a concentration of 100 or 200 μg/mL, growth rates of 21.1% and 27.4% were shown, respectively. Therefore, it was confirmed that the MPL of ITME for human skin fibroblasts was 200 μg/mL or more.

4.4 Morphological observation of human skin fibroblasts

Human skin fibroblasts were treated with ITME at 25, 50, 100 or 200 μg/mL and cultured for 48 hours. After that, the medium was replaced and the cells were observed under a microscope, and the results are shown in FIG. 9.

In FIG. 9, A is a control group, B is an experimental group treated with 25 μg/mL ITME, C is an experimental group treated with 50 μg/mL ITME, D is an experimental group treated with 100 μg/mL ITME, and E is 200 μg/mL It is an experimental group that treated ITME of

The human skin fibroblasts treated with ITME showed a flat and spindle-like shape as in the control, even with a high concentration of 200 μg/mL.

4.5 Type I procollagen synthesis assay

Human skin fibroblasts were treated with 25, 50, 100 or 200 μg/mL of ITME, and then the supernatant was removed and analyzed with an enzyme immunoassay kit (EIA kit, Takara, Japan).

The production of type I procollagen protein is already known in the literature ( Biol Pharm Bull 30: 1395-1399, 2007), and 5 ng/mL of transforming growth factor-β1 (TGF-β1) was used as a positive control.

Human skin fibroblasts were stabilized in DMEM containing 10% FBS and 1% P/S, and cultured in _{an incubator at 37° C., 5% CO 2} and humidified for 24 hours.

These cells were collected and dispensed in a 500-μL volume so as to enter ^{0.7×10 5} cells/well for each well of a 6-well plate, and cultured for 24 hours.

After removing the medium and washing the cells with phosphate-buffered saline (PBS), 1 mL of PRF-DMEM and 1% P/S were added and cultured for 24 hours.

After the culture was completed, the cells were washed with PBS, 3 mL of PRF-DMEM containing 25, 50, 100, or 200 μg/mL of ITME was added, followed by incubation for 24 hours. And centrifuged at a speed of 12,000 rpm for 10 minutes to separate the supernatant, which was used as a sample for quantitative analysis of procollagen protein.

100 μL of antibody-peroxidase-conjugate solution was added to each well of the 96-well EIA kit, and 20 μL of the sample diluted in 4 steps was added. And it was wrapped in foil (foil) and incubated for 3 hours at 37 ℃.

After washing the cells 4 times with PBS, adding 100 μL of a substrate solution, the kit wrapped with foil was incubated at room temperature for 15 minutes.

To this, 100 μL of 1N H ₂ SO ₄ was added as a reaction stop solution, and the absorbance was measured three times at 450 nm with a microplate reader.

The production of procollagen protein is previously known in the literature ( J Biol Chem 193: 265-275, 1951) and quantified using bovine serum albumin (BSA).

In addition, cell lysis buffer (80 μL; CelLytic ^TM B Cell Lysis Reagent, Sigma-Aldrich) was added to the plate, and then frozen at -20°C, thawed three times before removing the mixture from the plate, and diluted to 1/10. Then, the protein content was quantified using the BSA standard curve.

As a result, as shown in FIG. 10, when the ITME of 25, 50, 100 or 200 μg/mL was treated, the effect of increasing type I procollagen production of 16.7%, 22%, 28.1% and 36.7% (p<0.05), respectively, was obtained. Was observed, which increased in a concentration dependent manner.

4.6 Reverse transcription-polymerase chain reaction (RT-PCR)

After treating human skin fibroblasts with ITME at a concentration of 25, 50, 100 or 200 μg/mL, the total RNA thereof was prepared by the manufacturer using Trizol reagent (Trizol-reagent, Invitrogen, New York, NY, USA). Extracted according to the instructions.

In addition, after UVA treatment on human skin fibroblasts, its mRNA was extracted and used as a control. After culturing the cells for 80% of the culture dish area at 200 nm, the medium was removed and washed with PBS. The sample was treated with 1.5×10 ⁵ cells/mL cells ^{cultured in DMEM not containing FBS, irradiated with 6.3 J/cm 2} UVA, and cultured for 24 hours, and then its mRNA was extracted.

Total RNA (5 μg) was added to 8 μL of Moloney murine leukemia virus Reverse Transcriptase (M-MLV RT) 5× buffer, 3 μL of 10 mM eoxyribonucleotide triphosphates (dNTPs), 0.45 μL of 40 U/μL ribonuclease inhibitor (RNasein inhibitor), 0.3 μL of 200 U/μL M-MLV RT (Promega, Madison, WI, USA) and 1.5 μL of 50 μM oligo dT (oligo dT, Bioneer). , Daejeon, Korea).

Single-stranded cDNA was amplified using PCR, in which case 4 μL of 5× Green Go Taq flexi buffer, 0.4 μL of 10 mM dNTPs, 0.1 μL of 5 U/μL tagged polymerase (Taq polymerase) ), 1.2 μL of 25 mM MgCl ₂ (Promega) was used, and 0.4 μL of primers for matrix metalloproteinase-1 (MMP-1) or β-actin in Table 1 below (20 μM) was used to amplify.

Gene name direction order Sequence number MMP-1 Forward direction CGA CTC TAG AAA CAC AAG AGC AAG A One Reverse AAG GTT AGC TTA CTG TCA CAC GCT T 2 β-actin Forward direction ACC GTG AAA AGA TGA CCC AG 3 Reverse TAC GGA TGT CAA CGT CAC AC 4

The expected sizes of the PCR products of MMP-1 or β-actin were 237 and 248 base fairs, respectively.

PCR was performed as follows. MMP-1 was subjected to 28 cycles of denaturation at 94°C for 60 seconds, annealing at 50°C for 60 seconds, and extension at 72°C for 60 seconds.

β-actin was denatured at 94°C for 30 seconds, annealing at 51°C for 30 seconds, and extended 30 times at 72°C for 60 seconds.

It was evaluated for the relative expression of MMP-1 using the - (β actin) control group - this a PCR product generated by 1.2% agarose gel was analyzed using the β-actin.

As a result, as shown in FIG. 11, in the case of treatment with 25, 50, 100 or 200 μg/mL of ITME, 15.9% (p<0.05), 17.4% (p<0.05), 23.1% (p<0.01), and 36.8, respectively. In% (p<0.01), the expression of MMP-1 mRNA was significantly reduced in a concentration-dependent manner.

4.7 Western Blot

As in Example 4 of 6, human skin fibroblasts irradiated with UVA and human skin fibroblasts treated with 25, 50, 100 and 200 μg/mL ITME were 1% nonidet P-40 (NP-40), It was sonicated in 0.1 M Tris-HCl (pH 7.2) buffer containing 0.01% sodium dodecyl sulfate (SDS) and a protease inhibitor cocktail (Roche, Mannheim, Germany).

The protein concentration of the cell lysate was measured using a Pierce Protein Assay Kit (Pierce Biotechnology, Inc., Rockford, IL, USA), and bovine serum albumin (BSA) was used as a standard. .

Equal amounts of protein (10 μg) were loaded onto each lane and separated by electrophoresis on a 10% polyacrylamide gel.

This was transferred to a nitrocellulose membrane, and incubated with an antibody ab137332 (1:1,000 dilution, anti-MMP-1, Abcam, Cambridge, UK).

Then, it was washed with TBST (Tween 20-containing Tris-Buffered Saline) and incubated with a secondary antibody, anti-rabbit IgG (anti-rabbit IgG, 1:1,000 dilution, Santa Cruz Biotechnology, Inc.).

Bands showing an immune response were detected using an enhanced chemiluminescence (ECL, Amersham, Bucks, UK) system.

The intensity of the band was measured using an Image J program (Image J program, NIH, Bethesda, MD, USA), and at this time, β - actin was designated as an internal control to evaluate the band intensity.

As a result, as shown in FIG. 12, when the ITME of 25, 50, 100 or 200 μg/mL was treated, the MMP-1 protein was significantly expressed in a concentration-dependent manner at 34%, 34.9%, 35.3% and 37.2%, respectively ( p<0.01) decreased.

<Example 5> Whitening effect

5.1 MTT analysis

The growth rate of melan-a cells treated with ITME was confirmed through MTT analysis. melan-a cells were dispensed into a 96-well plate by 0.5×10 ⁴ cells/well and cultured in an incubator at 37° C. and 10% CO _{2 for 24 hours.}

200 μL of ITME was diluted in RPMI-1640 medium to a concentration of 25, 50, 100, or 200 μg/mL, and then added to each well and cultured in an incubator for 48 hours.

After 48 hours, the cells were placed in a medium containing 0.5 μg/mL MTT, cultured for 3 hours, and centrifuged for 10 minutes at a rate of 200×g to fix the cells and remove all the medium.

Accordingly, 200 μL of DMSO was added to each well, and the cells were resuspended for 15 minutes in a plate-shaker, and then absorbance was measured at 540 nm using a plate reader (680, Bio-Rad, Tokyo, Japan). The cell growth rate was calculated as a percentage by dividing the measured value by the absorbance of the control reagent.

As a result, as shown in FIG. 13, it was confirmed that the growth rate of the cells was not decreased when the ITME was treated at a concentration of 25 to 100 μg/mL, but the growth rate of the cells was slightly decreased when the ITME at a concentration of 200 μg/mL was treated.

5.2 Morphological observation of melan-a cells

melan-a cells were treated with 25, 50, 100, or 200 μg/mL of ITME, respectively, and _{cultured in an incubator at 37°C and 10% CO 2} for 48 hours, and then the medium was replaced and the shape was changed with an inverted microscope. Observed.

In FIG. 14, A is a control group, B in FIG. 14 is an experimental group treated with 25 μg/mL of ITME, C in FIG. 14 is an experimental group treated with 50 μg/mL of ITME, and D in FIG. 14 is treated with 100 μg/mL of ITME. One experimental group, and E in FIG. 14 is an experimental group treated with 200 μg/mL of ITME.

As a result, as shown in FIG. 14, it was confirmed that melanin accumulation and dendrites in the experimental group treated with ITME were significantly reduced when compared to the control group.

5.3 Melanin synthesis assay

Melan-a cells were dispensed into a 48-well plate by 2×10 ⁴ cells/well for each well, and cultured for 24 hours in a 37°C and 10% CO _{2 incubator.} To this, 100 μg/mL of arbutin (PC) and 500 μL of ITME diluted in RPMI-1640 medium at various concentrations (25, 50, 100 or 200 μg/mL) were added each, and then in an incubator at _{37°C and 10% CO 2} Incubated for 72 hours and washed. Then, this process was repeated once more.

Next, melanin was dissolved in 1N NaOH, and the absorbance was measured at a wavelength of 490 nm using a plate reader, and the change in melanin content was calculated as a percentage by dividing the measured value by the absorbance of the control reagent.

As a result, as shown in FIG. 15, when compared to the control group (V) without any treatment, the concentration of melanin in the experimental group treated with ITME at a concentration of 25, 50, 100 or 200 μg/mL was 15.6%, 18.2%, and 19.1, respectively. % Or 20.5% significantly (p<0.05).

5.4 Intracellular tyrosinase activity assay

Melan-a cells were dispensed into a 60 mm cell culture dish ^{as much as 4×10 5} cells/well, incubated for 24 hours, and then 100 μg/mL arbutin (PC) and various concentrations (25, 50, 100 or 200 μg). /mL) was added to each of the diluted ITME in RPMI-1640 medium and incubated for an additional 48 hours.

After washing each cell with phosphate-buffered saline (PBS), remove the cells from the bottom of the dish with 200 μL of 1% Triton X-100, transfer them to an Eppendorf tube, and place them on ice for 60 minutes. I put it. The mixture was stirred 6 times at 10 minute intervals for a total of 60 minutes.

This was centrifuged for 20 minutes at a rate of 14,000×g at 4° C., and 100 μL of 0.2% L-DOPA was added and _{incubated in a 37° C. and 10% CO 2} incubator for 1 hour, and then at 490 nm. The absorbance was measured.

The tyrosinase activity was calculated as a percentage by dividing the measured value by the absorbance of the control reagent, and the result is shown in FIG. 16.

When compared with the control group treated with the medium, it was confirmed that the tyrosinase activity of the experimental group treated with ITME at a concentration of 100 μg/mL was significantly reduced by 8.4% (p<0.05).

5.5 Reverse transcription-polymerase chain reaction (RT-PCR)

Tyrosinase and β in melan-a cells treated with 100 μg/mL arbutin (PC) and ITME diluted in RPMI-1640 medium at various concentrations (25, 50, 100 or 200 μg/mL), respectively. -Actin mRNA was determined using RT-PCR in the same manner as 4.6 in Example 4.

The tyrosinase primer sequence used at this time is shown in Table 2 below, and the primer sequence of β-actin is as shown in Table 1 above.

Gene name direction order Sequence number Tyrosinase
Forward direction CAT TTT TGA TTT GAG TGT CT 5 Reverse TGT GGT AGT CGT CTT TGT CC 6

The expected size of the PCR product of tyrosinase or β-actin was 1192 or 528 base fairs, respectively.

PCR was performed as follows. Tyrosinase was subjected to 28 cycles of denaturation at 94°C for 60 seconds, annealing at 56°C for 60 seconds, and extension for 60 seconds at 72°C.

β - actin was performed 30 times with denaturation at 94°C for 30 seconds, annealing at 51°C for 30 seconds, and extension at 72°C for 30 seconds.

The resulting PCR product was analyzed using a 1.2% agarose gel. β - actin (β - actin) was used as a control to evaluate the relative expression of tyrosinase.

As a result, as shown in FIG. 17, the expression of tyrosinase mRNA in the experimental group treated with ITME at a concentration of 25, 50, 100 or 200 μg/mL was 8.1% and 13.1%, respectively, when compared to the control group (V) without any treatment. , 17.2% (p<0.05) or 26.3% (p<0.01) was found to decrease in a concentration-dependent manner.

5.6 Western Blot

Melan-a cells were treated with 100 μg/mL arbutin (PC) and ITME diluted in RPMI-1640 medium at various concentrations (25, 50, 100 or 200 μg/mL), 1% NP-40, 0.01% Sonication was performed in 0.1 M Tris-HCl (pH 7.2) buffer containing SDS and protease inhibitor mixture.

The protein concentration of the cell lysate was measured using a Bio-Rad protein assay kit (Bio-Rad Laboratories USA, Inc., Hercules, CA, USA), and bovine serum as a standard. albumin, BSA) was used.

Equal amounts of protein (10 μg) were loaded onto each lane and separated by electrophoresis on a 10% polyacrylamide gel.

This was transferred to a nitrocellulose membrane and incubated with an anti-tyrosinase (SC 15341, 1:1,000 dilution) antibody.

Then, it was washed with TBST (Tween 20-containing Tris-Buffered Saline) and incubated with a secondary antibody, anti-rabbit IgG (anti-rabbit IgG, 1:1,000 dilution, Santa Cruz Biotechnology, Inc.).

Bands showing an immune response were detected using an enhanced chemiluminescence (ECL, Amersham, Bucks, UK) system.

The intensity of the band was measured using an Image J program (Image J program, NIH, Bethesda, MD, USA), and at this time, β - actin was designated as an internal control to evaluate the band intensity.

As a result, as shown in FIG. 18, the expression of tyrosinase protein in the experimental group treated with ITME at a concentration of 25, 50, 100 or 200 μg/mL was 1.9% and 16.5%, respectively, when compared to the control group (V) without any treatment. (p<0.05), 22.0% (p<0.01), or 31.2% (p<0.01) was found to decrease in a concentration-dependent manner.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it will be apparent that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> INDUSTRY ACADEMIC COOPERATION FOUNDATION KEIMYUNG UNIVERSITY <120> Composition for skin whitening comprising Impatiens textori extract <130> ADP-2018-0180 <160> 6 <170> KopatentIn 2.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> MMP-1 forward primer <400> 1 cgactctaga aacacaagag caaga 25 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> MMP-1 reverse primer <400> 2 aaggttagct tactgtcaca cgctt 25 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin forward primer <400> 3 accgtgaaaa gatgacccag 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta-actin reverse primer <400> 4 tacggatgtc aacgtcacac 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> tyrosinase forward primer <400> 5 catttttgat ttgagtgtct 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> tyrosinase reverse primer <400> 6 tgtggtagtc gtctttgtcc 20

Claims (4)

A cosmetic composition for skin whitening comprising an extract of Wollastonite extracted with an aqueous methanol solution as an active ingredient. delete A health functional food for skin whitening containing an extract of Wollastonite extracted with an aqueous methanol solution as an active ingredient. delete

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