KR20170105910A - Skin whitening composition comprising extracts of Galla Rhois and use thereof - Google Patents
Skin whitening composition comprising extracts of Galla Rhois and use thereof Download PDFInfo
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- KR20170105910A KR20170105910A KR1020160029272A KR20160029272A KR20170105910A KR 20170105910 A KR20170105910 A KR 20170105910A KR 1020160029272 A KR1020160029272 A KR 1020160029272A KR 20160029272 A KR20160029272 A KR 20160029272A KR 20170105910 A KR20170105910 A KR 20170105910A
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- extract
- skin whitening
- present
- melanin synthesis
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2300/00—Processes
- A23V2300/14—Extraction
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- Chemical & Material Sciences (AREA)
- Nutrition Science (AREA)
- Dermatology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
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- Microbiology (AREA)
- Birds (AREA)
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- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The present invention relates to a skin whitening composition containing an active ingredient extracted from a dermis and a use thereof. More particularly, the present invention relates to a skin whitening composition containing an active ingredient extracted from a dermis, The present invention relates to a composition for skin whitening which is safe and has antioxidative and whitening effect. The composition for skin whitening of the present invention is prepared by dissolving melanin synthesis inhibitor , Thereby inhibiting melanin synthesis. The composition can be usefully used for development of a skin whitening cosmetic material having an antioxidative activity and a whitening effect as a skin whitening composition.
Description
The present invention relates to a composition for skin whitening containing an active ingredient extracted from an obturator and its use, and more particularly to a composition for inhibiting melanin synthesis of a skin whitening composition containing an active ingredient And to develop cosmetic materials having antioxidative and whitening effects.
Melanin in the basal layer of the skin is formed by hydroxylation of tyrosine (amino acid, tyrosine) with tyrosinase (oxidase, tyrosine) followed by making dihydroxy-L-phenylalanine (DOPA) And is oxidized by a catalyst. Then, DOPA quinone (DOPA quinone) is generated and DOPA chrome, 5,6-dihydroxy indole and
Galla Rhois is a species of insect caused by the spawning of aphids (Melaphis chinensis) in the rusks (Ruschinensis) belonging to the Anacardiaceae family. This is an insect (insect that appears to have white fluff) which is an insect with an aphid of aphids and is formed by parasitism on a young leaf of a red tree that is parasitic to a wilderness (an insect is parasitic on a plant, ) Are distributed in various parts of Korea along with Japan and China. In addition, the dwarf is also called anastomosis, white pheasant, and it is also called a fujita, a throat, an ibu, and a rich depending on the appearance. The major components of the larvae are tannin 50-70%, the main component is penta-m-digalloyl-ββ-glucose, and contains gallic acid, fat, resin and starch. Examples of the organic material include malic acid, tartaric acid, citric acid, and flavonoid glucoside. On the other hand, studies on pharmacological effects of dwarf are known to have anticancer effect, intestinal bacterial inhibitory effect, and antidiabetic effect, and the main ingredient of these drugs is gallic methylester. Gallotanins and triterpenoid compounds are known to be the main compounds involved in the pharmacological action of dwarf. It is also used as an external medicine for trauma such as psoriasis, boils and dermatitis. Recently, antimicrobial effects have been intensively studied, and antioxidant, anti-thrombotic and antiviral agents have been reported. A number of studies have been reported on antioxidant and antiinflammatory activity of this dwarf, but studies on the inhibition of melanogenesis in extracted fraction levels have been rarely reported. Therefore, the results of the antioxidant activity and whitening activity of the extract of Opuntia ficus indica according to the present invention are expected to contribute to the development of a skin whitening effect cosmetic containing an active ingredient extracted from Opuntia.
Accordingly, the present inventors have completed the present invention in order to develop a skin whitening composition containing an effective ingredient extracted from an obturator for developing an excellent whitening ingredient from a natural product as a cosmetic material having safety and antioxidative activity and whitening effect on the human body.
It is an object of the present invention to provide a composition for skin whitening which comprises an extract of Galla Rhois as an active ingredient.
Another object of the present invention is to provide a cosmetic composition for whitening skin, which comprises an extract of Galla Rhois as an active ingredient.
It is still another object of the present invention to provide a functional food composition containing an extract of Galla Rhois as an active ingredient.
In order to achieve the above object, the present invention provides a skin whitening composition, which comprises an extract of Galla Rhois as an active ingredient.
The composition according to an embodiment of the present invention provides a composition for skin whitening characterized in that a melanin synthesis inhibitor nucleus is obtained from a solvent-soluble portion obtained by first extracting a dumbbell with methanol and a primary extract with ethyl acetate .
In addition, the composition of the present invention provides a skin whitening composition which inhibits melanin synthesis.
In order to attain the above other objects, the present invention provides a cosmetic composition for whitening skin, which comprises an extract of Galla Rhois as an active ingredient. The composition for skin whitening according to another embodiment of the present invention is characterized in that a melanin synthesis inhibitor nucleus is obtained from a solvent-soluble fraction obtained by first extracting a crude extract with methanol and then with a second extract with ethyl acetate, to provide. In addition, the composition of the present invention provides a skin whitening cosmetic composition characterized by inhibiting melanin synthesis.
According to another aspect of the present invention, there is provided a functional food composition comprising an extract of Galla Rhois as an active ingredient. The composition according to another embodiment of the present invention is characterized in that a melanin synthesis inhibitor nuclear material is obtained from a solvent-soluble portion obtained by firstly extracting the first extract of dumbbell with methanol and then secondly extracting with ethyl acetate do.
Further, the composition of the present invention provides a functional food composition characterized by inhibiting melanin synthesis.
The skin whitening composition of the present invention comprising the Galla Rhois extract as an active ingredient can be prepared by dissolving melanin synthesis inhibitor component (s) from the solvent-soluble portion of the first extract obtained by first extracting dumbbell with methanol, To thereby inhibit melanin synthesis. This composition can be usefully used for development as a skin whitening cosmetic material having an antioxidative activity and a whitening effect as a skin whitening composition.
1 is a flow chart of a fractionation method for Galla Rhois extract according to an embodiment of the present invention,
FIG. 2 shows the measurement results of DPPH radical scavenging ability for the extract of Opuntia sp.
FIG. 3 shows the result of measuring the antioxidant power against the extract of Opuntia sp.
Figure 4 shows the results of the inhibitory effect of tyrosinase on the extract of Opuntia ficus,
Figure 5 is the result of the toxic effect of B16 / F10 melanoma cells in the rhizome extract,
FIG. 6 shows the results of measuring the inhibitory effect of melanin synthesis using rat mouse melanoma cells in the rhizome extract,
FIG. 7 shows the results of expression of tyrosinase, TRP-1, TRP-2 and microphthalmia-associated transcription factor (MITF) protein in horseradish extract using Western blot,
Figure 8 shows the results of tyrosinase, microphthalmia-associated transcription factor (MITF), TRP-1 and TRP-2 transcript RNA (mRNA) expression using real-time PCR.
While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that the invention is not intended to be limited to the particular embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Terms including ordinals such as first, second, etc. may be used to describe various elements, but the elements are not limited by such terms. These terms are used only to distinguish one component from another.
When an element is referred to as being "connected" or "connected" to another element, it may be directly connected or connected to the other element, but other elements may be present in between . On the other hand, when an element is referred to as being "directly connected" or "directly connected" to another element, it should be understood that there are no other elements in between.
The terminology used in this application is used only to describe a specific embodiment and is not intended to limit the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, the terms " comprises ", or " having ", and the like are used interchangeably with a feature, a number, a step, an operation,
One or more other features, integers, steps, operations, elements, components, or combinations thereof, as a matter of principle, without departing from the spirit and scope of the invention.
Hereinafter, a preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings.
The present invention relates to a composition for skin whitening characterized by containing an extract of Galla Rhois as an active ingredient.
In addition, the composition of the present invention may be characterized in that it is a composition for skin whitening characterized in that a melanin synthesis inhibitor nuclear material is obtained from a solvent-soluble portion obtained by firstly extracting the first extract of dumbbell with methanol and then secondly extracted with ethyl acetate have. In the present invention, the first organic solvent may be a polar organic solvent, such as acetone, acetonitrile, dimethylformamide, dioxane, dimethylsulfoxide, ethanol, methanol or the like, It is possible to use the above mixed solvent. The second organic solvent may be a nitrile solvent such as acetonitrile, propionitrile or benzonitrile, an organic halogenated solvent such as carbon tetrachloride, chloroform or dichloromethane, tetrahydrofuran or dioxane, An amide such as N, N-dimethylformamide, an ester such as ethyl acetate, methyl acetate or t-butyl acetate, a ketone such as acetone, methyl ethyl ketone or methyl isobutyl ketone, an aromatic such as benzene or toluene Non-polar organic solvents such as hydrocarbons, or a mixed solvent of at least one of these solvents. In addition, the composition of the present invention may be a skin whitening composition characterized by inhibiting melanin synthesis. In order to confirm the toxicity of melanoma (B16 / F10) cells to methanol when the methanol extract of the composition of the present invention was exposed to the cells, the MTT assay of the cell viability measuring apparatus was carried out. The concentrations were 10, 50, and 100 μg / mL. As shown in FIG. 5 of the present invention, the survival rate was 88.1% at the 10 μg / mL treatment and 60.2% at the 100 μg / mL concentration (see FIG. 5). In order to confirm the whitening effect of the composition, the effect of suppressing melanin synthesis was measured using mouse melanoma cells. Cells were treated with melanocyte stimulating hormone (αα-MSH) to induce the expression of melanin. After that, the extracts were treated at different concentrations and the amount of biosynthesized melanin was measured. As shown in FIG. 6 of the present invention, the amount of melanin generated in cells treated with melanocyte stimulating hormone (αα-MSH) alone was 111.15%, which was increased compared to untreated control. As a control,
In addition, the composition of the present invention may be a skin whitening composition characterized by inhibiting melanin synthesis.
In order to confirm the inhibition characteristics of melanin synthesis of the composition, the present inventors conducted a tyrosinase inhibition assay that inhibits melanin synthesis by participating in the rate determining step of melanin synthesis. The melanin produced by tyrosinase acting as an enzyme was measured and the whitening effect by tyrosinase inhibition of the composition was measured. As shown in FIG. 4, the results of the whitening effect showed that the extract of Opium extract had a dose-dependent inhibition of tyrosinase and that of
The present invention can be characterized as a skin whitening cosmetic composition characterized by containing an extract of Galla Rhois as an active ingredient. In addition, the composition of the present invention may be a composition for skin whitening characterized in that a melanin synthesis inhibitory component is obtained from a solvent-soluble portion obtained by firstly extracting the first extract of dumbbell with methanol and then secondarily extracted with ethyl acetate.
When the composition of the present invention is used as a cosmetic for skin whitening, the cosmetic composition of the present invention can be used as a foundation product cosmetic (cosmetic lotion, cream, essence, cleansing foam, cleansing water, pack), body product cosmetic (body lotion, body oil, body gel) (0.05 to 10.0% by weight) based on the dry weight of cosmetics, such as cosmetics (foundation, lipstick, mascara, make-up base) and cosmetics for hair products (shampoo, rinse, hair conditioner and hair gel).
In addition, the composition of the present invention may be a cosmetic composition for skin whitening, which inhibits melanin synthesis. In order to confirm the inhibition of melanin synthesis of the cosmetic composition, the present inventors evaluated the antioxidative activity through 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. In the present invention, the electron donating ability used as an index of the antioxidant activity was confirmed by measuring DPPH radical scavenging ability. 2, the scavenging ability was increased in a dose-dependent manner, and the scavenging activity was 68.2% at 500 μg / mL (see FIG. 2). In the present invention, ferric tripyridyltriazine (Fe³³-TPTZ) complex is reduced to ferrous tripyridyltriazine (Fe²-TPTZ) by a reducing agent in a method for measuring antioxidant activity. Ferric reducing / antioxidant power (FRAP) assay. In addition, the results of the FRAP assay shown in FIG. 3 showed an increase in the concentration-dependent effect. Vitamin C showed a clearance of 6 mM at a concentration of 1 mg / mL. mL to 5.42 mM (see FIG. 3).
In addition, the composition of the present invention may be characterized as a functional food containing an extract of Galla Rhois as an active ingredient. Examples of the functional food containing the composition include various foods such as confectionery, processed food, combination preservation, milk product, beverage, and the like, and the shape or the shape thereof is not particularly limited, and examples thereof include solid, semi-solid, gel, Or the like. In other words, the whitening effect is exerted by ingesting the composition of the present invention, thereby contributing greatly to the improvement or prevention of glaring skin, spots, freckles, and dark circles, thereby maintaining beautiful skin with a clear feeling.
In addition, the composition of the present invention may be a functional food composition characterized by obtaining a melanin synthesis inhibitor nuclear material from a solvent-soluble portion obtained by firstly extracting the first extract with methanol and then extracting the second extract with ethyl acetate. In addition, the composition of the present invention may be a functional food composition characterized by inhibiting melanin synthesis. &Quot; Functional food "as defined in the present invention means food prepared and processed by using raw materials or ingredients having functionality useful to the human body according to Law No. 6727 on Functional Foods." Functional " And function of the nutrient for the purpose of obtaining a beneficial effect in health use such as controlling the nutrient or physiological action. The functional food of the present invention contains 0.01 to 95% by weight, preferably 1 to 80% by weight, of the above extract relative to the total weight of the composition. In addition, the present invention can be manufactured and processed into functional foods in the form of tablets, capsules, powders, granules, liquids, and rings for skin whitening and antioxidant effects. The functional food containing the extract of the present invention can be used variously for medicines, foods, beverages and the like for the purpose of skin whitening effect and antioxidant effect. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they can be used as powders, granules, tablets, capsules or beverages have.
The present invention may be better understood by the following examples, which are for the purpose of illustrating the invention and are not intended to limit the scope of protection defined by the appended claims.
< Example 1> Extracts
The crude dwarf used in the present invention was milled (produced in China), and then 1 liter of methanol was added to 500 g of the natural product, followed by extraction three times at room temperature for 24 hours, and then the liquid was concentrated. After extracts were suspended in distilled water, the fractions were sequentially extracted with nucleic acid (n-hexane), ethyl acetate and n-butanol to obtain extracts of each solvent. Ethyl acetate (ethyl acetate) Was used for lyophilization. 1 shows a flow chart of a fractionation method for a Galla Rhois extract according to an embodiment of the present invention (see FIG. 1).
< Example 2> Reagent And devices
(DPPH) (Sigma, USA), 2,4,6-tris (2-pyridyl) -s-triazine (TPTZ) (Sigma, USA) were used as reagents for the determination of antioxidant activity. ), 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazoliumbromide (MTT) was used as a starting material. Ascorbic acid, mushroom tyrosinase, (Αα-MSH, Sigma, USA), L-3,4-dihydroxy-β-D-glucosaminidase (DMSO, Sigma, USA), melanocyte stimulating hormone (DMEM, Thermo, USA), fetal bovine serum (FBS, Thermo, USA), tyrosine (trypsin, Thermo, USA), phenylalanine (L-DOPA, Sigma, USA) and cell culture medium Dulbecco's Modified Eagle's Medium Reagents such as 100 U / mL penicillin, 0.1 mg / mL streptomycin (Thermo, USA), and TRIzol (Tri-reagent, Invitrogen, USA) were used. The instrument used in this study was a UV-Vis spectrophotometer (BIO-RAD, USA), PCR (T1, Thermocycler, Biometra, Germany), RT- PCR, CFX connect, Bio-Rad, USA).
< Example 3> 1,1- 피덴 -2- picrylhydrazyl ( DPPH ) Measure( Assay )
1,1-diphenyl-2-picrylhydrazyl (DPPH) affects the initial rate of tyrosinase oxidation by the oxidation of 3,4-dihydroxyphenylalanine (DOPA) in melanogenesis. Therefore, it was estimated by using the reducing power of DPPH that the effect of electron donating ability (EDA) would be effective for the production of melanin. The experimental method was modified by the experimental method of Blios. 100 μL of methanol and 90 μL of 0.25 mM DPPH (in MeOH) were mixed with 10 μL of the sample, reacted in the dark for 30 min, and then measured at 517 nm using a spectrophotometer. Ascorbic acid was used as a positive control and the difference between before and after the addition of the extract was expressed as a percentage.
Inhibition rate (%) = (A-B) / A
A: Absorbance when no sample is added
B: Absorbance when sample is added
< Example 4> Ferric 감 antioxidant power (FRAP) assay
The ferric reducing antioxidant power (FRAP) assay was modified by the method of Benzie and Strain. The FRAP reagent was prepared by heating 25 mL of acetate buffer (300 mM, pH 3.6) at 37 ° C and adding 5 mL of 10 mM TPTZ ((2-pyridyl) -s-triazine) dissolved in 40 mM hydrochloric acid And 2.5 mL of 20 mM ferrous sulfate (FeSO4) were added to prepare an FRAP reagent. 0.03 mL of each concentration of the sample and 0.09 mL of distilled water were added to 0.9 mL of the FRAP reagent, followed by reaction at 37 ° C. for 10 minutes, and the absorbance was measured at 593 nm using a spectrophotometer (Benchmark PLUS Bio-Rad).
< Example 5> Tyrosinase Measurement of inhibitory effect ( Tyrosinase Inhibition Assay )
Melanin is produced by tyrosinase oxidizing tyrosine. Yagi 's method was modified to verify whitening efficacy by competitive inhibition of tyrosinase.
< Example 6> Cell culture
The medium used for culturing melanoma cells (B16 / F10 melanoma cells) was Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin, streptomycin ). Cells were cultured at 37 ° C and 5
< Example 7> Cytotoxicity measurement (cell viability measurement technique, MTT assay )
MTT assay, a cell viability assay, was carried out to examine the toxicity of methanol extract of obtusa on cell exposure. The cells were seeded on a 96-well plate at 5 × 10 4 cells / well and cultured for 24 h. After 24 h, the media was removed and mixed with 9: 1 MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide in
< Example 8> Melanin inhibition measurement ( Melanin Contents Assay )
In order to measure the amount of melanin inhibition, the amount of melanin was determined by treating the extract of Rhus verniciflua with melanocytes. Melanocyte stimulating hormone (α-MSH) was cultured for 24 h at a concentration of 1 × 10 6 cells / mL in a 100-mm cell culture dish (B16 / F10 melanoma cell) Was treated to 10 nM, and then treated with the rhizome extract 4 h later. After 24 h, the cells were washed twice with Dulbecco's Phosphate-Buffered Saline (DPBS) and collected with a cell screaper. 1 mL of lysis buffer (0.1 M sodium phosphate buffer, pH 6.8, 1% (v / v) triton X-100) was added to dissolve the cell wall and allowed to react well at 4 ° C for 30 min. The reaction solution was centrifuged at 13,000 rpm for 10 min. The supernatant was quantitated by BCA (Bicinchoninic acid) protein assay. The precipitate was added to 1 mL of 1 N sodium hydroxide (NaOH) solution, incubated at 90 ° C for 1 h, diluted to the amount of protein, and absorbance measured at 475 nm.
< Example 9> Western blot
The melanoma cells (B16 / F10 melanoma cells) were cultured in a 100 mm cell culture dish at a concentration of 1 × 10 6 cells / mL for 24 h, treated with α-MSH to 10 nM, Cells were harvested by centrifugation at 15,000 rpm for 15 min in a radioimmuno precipitation assay (RIPA) buffer. Protein was quantitated by BCA protein quantitation method. The protein was quantitated by using a 50 μL supernatant and 0.6 mL of x5 SDS sample buffer (0.6 mL of 1 M Tris-HCl (pH 6.8), 5 mL of glycerol, 2 mL of 10% SDS, 0.5 mL of β-mercaptoethanol, H₂O 0.9 mL) was mixed with 10 μL and electrophoresed on 7.5% SDS (sodium dodecyl sulfate) polyacrylamide gel. Then, transfer (transfer of the developed proteins to the membrane) was carried out using a nitrocellulose membrane. After blocking for 1 h with 5% skim milk, the antibody of tyrosinase, actin, TRP-1 and TRP-2 was diluted 1: 1000 and incubated at room temperature For 1 h. After washing five times with PBS-T (10 mM phosphate containing 0.1% tween-20, 2.7 mM potassium chloride, 137 mM sodium chloride, pH 7.4), tyrosinase, actin, TRP- 2 was diluted 1: 1000 with anti-goat Igg (Immunoglobulin G) as a secondary antibody and reacted for 1 h at room temperature. After washing with TBST (Tween 20-containing Tris-Buffered Saline), the solution was color-printed with a western blotting solution.
< Example 10> gene expression analysis
Melanoma cells (B16 / F10 melanoma cells) were subcultured in a 100 mm cell culture dish at a concentration of 1 × 10 6 cells / mL, cultured for 24 h, and treated with α-MSH to 10 nM. After 4 h, the extracts were treated with each concentration. After 24 h, total RNA was isolated by treating with 1 mL TRIzol reagent. 200 μL choloroform: isoamylalcohol (24: 1) was added to the separated total RNA (ribonucleic acid) and centrifuged at 14,000 rpm for 20 min. Respectively. In a centrifuged tube, 500 μL of the supernatant was transferred to a new tube and 500 μL of isopropyl alcohol was added to precipitate the RNA, followed by washing with 70% ethanol. The tube was allowed to dry naturally at room temperature. RNA was dissolved in RNAase-free water, and RNase-free DNase was added and stored at -70 ° C.
< Example 11> complementary DNA ( complementary DNA , cDNA) synthesis
After adding 2 μL of Oligo dT into 1 μg of total RNA isolated from the control and test groups, add 8 μL of DEPC (diethyl pyrocarbonate) water, carefully mix, and incubate at 65 ° C for 5 min. After incubation, add 4 μL of 5X reaction buffer, 1 μL of RiboLock ™ RNase Inhibition (20 U / μL) and 2 μL of 10 mM dNTP mix and 1 μL of RevertAid ™ M-MuLV reverse transcriptase (200 μL) The volume was made up to 20 μL. Thereafter, the reaction was carried out at 42 ° C for 60 minutes, followed by final extraction at 4 ° C. The synthesized cDNA was stored at -20 ° C.
< Example 12> Real-time polymerase chain reaction Real - time RT- PCR )
To determine the whitening efficacy of the extracts, the rate of DNA expression associated with whitening was measured. Taqman Universal Master Mix II (Life technologies) and Taqman gene expression assays were used for quantitative real time PCR. Denaturation was 95 s at 15 ° C and annealing was performed at 68 ° C for 60 s in 40 cycles The final extraction was carried out at 72 ° C for 30 s. Comparison of relative melanogenic protein mRNA expression was compared with GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) mRNA.
Table 1 summarizes the probes used in the PCR of the present invention.
Claims (9)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20200065430A (en) | 2018-11-30 | 2020-06-09 | 이상명 | A cosmetic and a bath composition containing the active ingredient of Buddha's hand |
| KR20200134929A (en) * | 2019-05-24 | 2020-12-02 | 이승호 | Cosmetic composition containing complex medicinal herbs extract for skin whitening and anti-wrinkle effect and manufacturing method thereof |
| KR20210033777A (en) * | 2019-09-19 | 2021-03-29 | 훠리스트 주식회사 | Skin Whitening Composition Containing Active Ingredient Extracted From Callus |
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2016
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20200065430A (en) | 2018-11-30 | 2020-06-09 | 이상명 | A cosmetic and a bath composition containing the active ingredient of Buddha's hand |
| KR20200134929A (en) * | 2019-05-24 | 2020-12-02 | 이승호 | Cosmetic composition containing complex medicinal herbs extract for skin whitening and anti-wrinkle effect and manufacturing method thereof |
| KR20210033777A (en) * | 2019-09-19 | 2021-03-29 | 훠리스트 주식회사 | Skin Whitening Composition Containing Active Ingredient Extracted From Callus |
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