KR101820085B1 - Composition for skin whitening comprising Wisteria floribunda extract and use thereof - Google Patents

Abstract

A waxy stem extract, or a fraction thereof as an active ingredient, and a use thereof.

Description

A composition for whitening comprising a wisteria stem extract, or a fraction thereof as an active ingredient, and a use thereof. (Composition for skin whitening comprising Wisteria floribunda extract and use thereof)

The present invention relates to a skin whitening composition containing a rattan extract and a fraction. More particularly, the present invention relates to a skin whitening composition containing a wisteria extract and a fraction, The present invention relates to a composition for whitening skin.

Several factors are involved in determining the skin color of a person. Among them, the activity of melanocyte which makes melanin pigment, distribution of blood vessels, thickness of skin, carotenoid, bilirubin and other factors related to pigment are important. In particular, the most important factor is melanin, which is produced by the action of various enzymes such as tyrosinase in melanocytes in the human body. Skin pigmentation is caused by stimuli such as ultraviolet rays or inflammation, and it is also known to be caused by factors such as genetic factors, hormones, foods, and chemicals.

Melanin is converted from tyrosine to dopa and dopaquinone by the action of tyrosinase present in the pigment cells, and is produced through dopachrome. At this time, three major enzymes involved in melanin synthesis are tyrosinase, tyrosinase related protein 1 (TRP1), and tyrosinase related protein 1 (TRP2). This melanin is present in the skin and has an important function to protect the body from ultraviolet rays and the like. However, excessive production of melanin is known to form spots and freckles, accelerate skin aging, and play an important role in skin cancer induction. Recently, development of drugs to prevent melanin excess production is actively under way. P-methoxyphenol, hydroquinone, kojic acid, and arbutin have been used as the whitening effect agents, but they are weakly active or cause denaturation or lethality of pigment cells Or damage the original function of the cell. On the other hand, vitamin C and its derivatives and the like have been used for the purpose of inhibiting melanin production, but these also have a disadvantage of low inhibitory activity. Therefore, it is required to develop a substance exhibiting melanin biosynthesis inhibitory activity even in a small amount.

Wisteria (Wisteria floribunda) is a tree with bean and soybean. A deciduous, fallen leaf of a tree. A vine is 10 meters or longer. It extends to the right and covers other objects. Leaves are alternate phyllotaxis and have 4-6 pairs of small leaves. Small leaves have short petiole with pointed oval shape. In the spring, many bluish - purple butterfly flowers run on axillary axillaries with lengths of several tens of centimeters. The fruit is long, about 15cm long, hanging down, and when it is ripe, the seed pops out.

Rattan extracts are known to have anticancer activity. However, there is no known skin whitening effect of rattan stems.

Published Patent Application No. 10-2011-0001538 (2011.01.06.)

One aspect provides a whitening composition comprising a wisteria extract, or a fraction thereof, as an active ingredient.

Another aspect provides a composition for suppressing or reducing melanogenesis comprising wisteria stem extract, or a fraction thereof, as an active ingredient.

Another aspect provides a method of making a rattan stem extract comprising contacting a rattan stem with a C1-C6 alcohol, water, or a mixture thereof under microwave irradiation.

Another aspect provides a method of improving the whitening state of an individual comprising administering to the subject an amount of the composition described above that is effective to ameliorate the whitening condition.

The first aspect provides a whitening composition comprising a wisteria extract, or a fraction thereof, as an active ingredient.

The second aspect provides a composition for inhibiting or reducing melanogenesis comprising wisteria stem extract, or a fraction thereof, as an active ingredient.

The wisteria stalk may be a part of the stem of the wisteria without leaves and flowers. The wisteria stem used for the extraction may be whole, part thereof, or a material derived therefrom, may be ground or chopped or suitably dried. The portion may be a bark.

The wisteria extract may be an organic solvent, water or an extract of a mixture thereof. The extraction may be such that the wisteria stalk is contacted with an organic solvent, water or a mixture thereof so that the components of the wisteria stalk are distributed to the organic solvent, water or a mixture thereof. The organic solvent may be a C1-C6 alcohol, for example, a C1-4 alcohol. The alcohol may be methanol, ethanol, propanol, isopropanol, 1,3-propanediol, butanol, pentanol, hexanol and the like. The solvent may be, for example, a mixture of water and an alcohol, that is, an aqueous solution of an alcohol. The alcohol concentration of the alcohol aqueous solution may be 1 to 99.5 (v / v)%, for example, 10 to 99.5 (v / v)%, 1 to 70 (v / 5 to 25 (v / v)%, 7 to 20 (v / v)%, 5 to 25 (v / v)%, or 10 to 20 (v / v)%. The alcohol aqueous solution may be an aqueous ethanol solution.

The extraction may be carried out in an amount of 3 to 10 (volumes / weight) times, for example 3 to 7 (volumes / weight) times, 3 to 10 times 5 (volume / weight) times, 5 to 10 (volume / weight) times, or 4 to 10 (volume / weight) times. For example, 3 to 10 L of the extraction solvent may be added to 1 kg of the whole waxy stem, a portion thereof, or materials derived therefrom.

The extraction may be performed by warmed liquid extraction, pressurized liquid extraction (PLE), microwave assisted extraction (MAE), subcritical extraction (SE), or a combination thereof . The subcritical extraction may be subcritical water extraction (SWE). Sub-critical water extraction is also referred to as superheated water extraction or pressurized hot water extraction (PHWE). The warmed liquid extraction may be a reflux extraction. The rattan extract may be extracted under microwave irradiation. The microwave irradiation may be performed at room temperature, 10 to 25 캜, 10 to 30 캜, 10 to 40 캜, 10 to 45 캜, 10 to 50 캜, 10 to 55 캜, 10 to 60 캜, Lt; RTI ID = 0.0 > 70 C. < / RTI > The microwave frequency may be 1,000 to 3,000 MHz, 1,500 to 3,000 MHz, 2,000 to 3,000 MHz, or 2,450 MHz. The output may be 100 to 300 W, 100 to 250 W, or 120 to 240 W. The irradiation time is 10 to 30 minutes. 10 minutes to 1 hour, 10 minutes to 2 hours, 10 minutes to 3 hours, 10 minutes to 4 hours, 10 minutes to 5 hours, 10 minutes to 6 hours, 10 minutes to 9 hours, 10 minutes to 12 hours, 10 minutes To < / RTI > 24 hours. The microwave irradiation may be performed in an airtight container.

The extraction may be carried out at a temperature of 4 ° C to 70 ° C, for example 4 ° C to 50 ° C, 4 ° C to 40 ° C, 4 ° C to 30 ° C, 10 ° C to 70 ° C, 15 ° C to 70 ° C, 10 ° C to 50 ° C, 10 ° C to 50 ° C, 4 ° C to 40 ° C, 4 ° C to 30 ° C, 10 ° C to 40 ° C, 10 ° C to 35 ° C, or 10 ° C to 30 ° C. The extraction time may vary depending on the temperature selected and may range from 1 hour to 2 months, such as 1 hour to 1 month, 1 hour to 15 days, 1 hour to 10 days, 1 hour to 5 days, 1 hour to 3 days 5 hours to 5 days, 5 hours to 3 days, 5 hours to 2 days, 5 hours to 2 days, 1 hour to 1 day, 5 hours to 1 month, 5 hours to 15 days, 5 hours to 10 days, Hour to 1 day, 10 hours to 1 month, 10 hours to 15 days, 10 hours to 10 days, 10 hours to 5 days, 10 hours to 3 days, or 10 hours to 2 days. Said extraction may comprise mixing and leaving for a period of time the entire wicker stem, a portion thereof, or a material derived therefrom, in said solvent. The setting may include moderate agitation. The extraction may be repeated one or more times, for example, 1 to 5 times.

The above extraction can be performed by separating the plant residue and the extract by a known method such as filtration. The extraction can also include removing the solvent from the resulting extract by known methods such as concentration under reduced pressure. The extraction may also comprise preparing the dried extract by drying, such as lyophilization, of the resulting extract.

In the above composition, the term "fraction" refers to a substance in which the wisteria extract is divided into a part of its components, i.e., a fractioned substance. The fraction may be obtained by solvent fractionation. The solvent fractionation may be a step of mixing the wisteria extract with a solvent and separating the substance present in the solvent. The fraction may be a methylene chloride fraction, an ethyl acetate fraction, a butanol fraction, or a water fraction obtained by suspending the wisteria extract in water and sequentially fractionating it with methylene chloride, ethyl acetate, butanol, and water.

Specifically, the methylene chloride fraction is prepared by mixing the wisteria extract with water, mixing the mixture with methylene chloride, allowing the mixture to stand for a certain period of time, separating the methylene chloride layer, separating the separated methylene chloride layer And separating the fractions. Separation of the fractions may include removing methylene chloride from the methylene chloride layer. The ethyl acetate fraction is obtained by mixing the methylene chloride fraction with water, mixing the mixture with ethyl acetate, allowing to stand for a predetermined time, separating the ethyl acetate layer, separating the separated fraction from the ethyl acetate layer . ≪ / RTI > Separation of the fractions can include removal of the ethyl acetate from the ethyl acetate layer. The butanol fraction may be prepared by mixing the ethylacetate fraction with water, mixing the mixture again with butanol, allowing to stand for a certain period of time, separating the butanol layer, and separating the fraction from the separated butanol layer . Separation of the fractions may include removing butanol from the butanol layer. Conditions for such fractionation, such as temperature conditions, pressure conditions, time, amount or concentration of solvent used, stirring, and the like, may be as described for the extracts used to prepare the rattan stem extract described above. The fractionation may be repeated one or more times, for example, 1 to 5 times.

Separation of the fractions may be accomplished by known methods such as filtration. The fractionation may also include removing the solvent from the fraction obtained by known methods such as concentration under reduced pressure. The fractionation may also include concentration and / or drying of the obtained fractions. The concentration may be reduced pressure concentrated. The drying may include vacuum drying, boiling drying, spray drying, room temperature drying or freeze drying.

In the present specification, "melanin reduction" may mean reduction of melanin content existing in cells and the like, and "melanin production inhibition" may mean inhibiting new production of melanin in cells and the like.

The composition may be one that inhibits tyrosinase activity. The composition may be for the treatment, improvement, or prevention of pigmentation.

The composition may be present in an amount of from 0.001% to 80%, such as from 0.01% to 60%, from 0.01% to 40%, from 0.01% to 30%, from 0.01% to 20% %, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20% From 0.05% to 10%, from 0.05% to 5%, from 0.1% to 60%, from 0.1% to 40%, from 0.1% to 30%, from 0.1% to 20% % To 10 wt%, or 0.1 wt% to 5 wt% of the wisteria extract or fraction thereof.

The composition may comprise a pharmaceutically, nutraceutically or cosmetically acceptable diluent or carrier. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low substituted hydroxy cellulose, or a combination thereof. The disintegrant may be sodium starch glycolate, calcium monohydrogen phosphate anhydrous, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or combinations thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The composition may be formulated into a parenteral dosage form. The parenteral dosage form may be an injection or an external preparation for skin. The external skin preparation may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, a lotion, or a combination thereof.

The external preparation for skin is usually used as a component used in external skin preparations such as cosmetics or medicines such as an aqueous component, an oily component, a powder component, an alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, an antiseptic, , Coloring agents, various skin nutrients, and the like can be appropriately blended as needed.

The external preparation for skin may be a metal blocker such as sodium edetate, sodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate or gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridine, Vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, fructose, fructose and other herbal medicines, various herbal medicines, tocopherol acetate, glycyrrhizic acid, Sugars such as trehalose and the like can also be appropriately compounded.

The composition may be a pharmaceutical, food or cosmetic composition.

Another aspect provides a method of improving the whitening state of an individual comprising administering to said individual an amount of said composition effective to ameliorate whitening. The composition is the same as described above.

Administration can be by any method known in the art. Administration may be by any means directly administered to a subject, such as by intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration, . The administration can be systemically or locally administered. The administration may be topical administration to the site where the wound is present.

The subject may be a mammal, such as a person, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat. The entity may be an entity that requires improved whitening.

The administration may be carried out in an amount of from 0.1 mg to 1,000 mg, for example 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg.

Another aspect provides a method of making a rattan stem extract comprising contacting a rattan stem with a C1-C6 alcohol, water, or a mixture thereof under microwave irradiation.

The microwave irradiation may be performed at room temperature, 10 to 25 캜, 10 to 30 캜, 10 to 40 캜, 10 to 45 캜, 10 to 50 캜, 10 to 55 캜, 10 to 60 캜, Lt; RTI ID = 0.0 > 70 C. < / RTI > The microwave frequency may be 1,000 to 3,000 MHz, 1,500 to 3,000 MHz, 2,000 to 3,000 MHz, or 2,450 MHz. The output may be 100 to 300 W, 100 to 250 W, or 120 to 240 W. The irradiation time is 10 to 30 minutes. 10 minutes to 1 hour, 10 minutes to 2 hours, 10 minutes to 3 hours, 10 minutes to 4 hours, 10 minutes to 5 hours, 10 minutes to 6 hours, 10 minutes to 9 hours, 10 minutes to 12 hours, 10 minutes To < / RTI > 24 hours. The microwave irradiation may be performed in an airtight container.

Waxy stem extract, or a fraction thereof as an active ingredient, can be used to improve whitening of an individual.

Can be used for inhibiting or reducing melanin production of an individual according to a composition for suppressing or reducing melanin production comprising wisteria stem extract, or fractions thereof as an active ingredient.

According to the method for producing the rattan stem extract comprising the step of contacting the rattan stem with the C1-C6 alcohol, water or a mixture thereof under microwave irradiation, the rattan stem extract can be efficiently produced.

The method for improving the whitening state of an individual, comprising the step of administering to the individual an amount of the above composition effective for improving the whitening state, can effectively improve the whitening state of the individual.

Figure 1 shows the amount of melanin production versus cell viability.
Figure 2 shows the rate of inhibition of tyrosinase activity.
FIG. 3 shows the effect of Wisteria crassi extract and its fractions on mRNA expression of MITF, tyrosinase, TRP1, and TRP2 genes in the untreated group as 100, inhibiting the expression of MITF, tyrosinase, TRP1, and TRP2 genes .
FIG. 4 shows Western blot analysis of the effect of the wisteria extract and its fractions on the protein expression of MITF, tyrosinase, TRP1, and TRP2 genes, .

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

Example  1: Preparation of wisteria stem extract

In this embodiment, wisteria floribunda (Willd.) DC.)), And the effect of the extract on skin whitening treatment was confirmed.

1. Wisteria stem extract and his Fraction  Produce

(1) Preparation of wisteria stem extract

The wisteria stems used in this example were directly collected from Gyeongbu-dong, Gangneung-gu, Korea. The used wisteria stem had a diameter of about 0.5 cm. Collection was done on November 17, 2015.

Rattan stalks Cutting scissors are used to cut 610 g of stem with a diameter of 0.5 cm or less in BANDELIN electronic GmbH & Co. KG. The ultrasonic wave was irradiated for 2 hours by immersing in 6.1 L of 99% methanol in water contained in a bath of BANDELIN SONOREX Ultrasonic baths, an ultrasonic device manufactured by KG. At this time, the reaction vessel was sealed with the lid closed, and the ultrasonic wave was irradiated at about 100 W (frequency 2455 MHz), and the temperature of the reactant during the reaction was 25 ° C. The extract obtained by repeating this three times was dried under reduced pressure and concentrated to obtain 44.4 g (7.3% based on the dry weight) of the concentrate.

(2) Wisteria stem extract Fraction  Produce

(44.4 g) of the wisteria-methanol extract prepared in (1) above was suspended in distilled water and extracted three times with the same amount of methylene chloride, ethyl acetate (EtOAc) and butanol ( n- BuOH) And then concentrated under reduced pressure to obtain 10.24 g (1.7% of dry weight) of methylene chloride layer, 11.34 g (1.9% of dry weight) of EtOAc layer and 12.66 g (2.1% of dry weight) of butanol layer, EA fraction and Bu fraction.

2. Wisteria stem extract and its The fraction  Influence on melanogenesis inhibition

The mouse melanocytes (melanocytes) used in this experiment were donated by Bennett, University of London, UK. Melanin production and tyrosinase activity were measured according to the following method.

Melan-a cells were inoculated in 96-well plates at 2 x ¹⁰ cells / well and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, (TPA, 12-O-Tetradecanoylphorbol 13-acetate) in an incubator at 37 ° C and 10% CO ₂ for 24 hours, and the sample was added to a concentration of 10 μg / ml and cultured for 72 hours . The sample is the rattan stem extract prepared in (1) or a fraction thereof. The amount of melanin was determined by adding 100 μl of 1N NaOH solution to the wells. Melanin in the cells was dissolved in the solution at 70 ° C for 1 hour, and the amount of melanin dissolved therein was measured. The amount of melanin was determined by measuring the absorbance at 475 nm using a spectrophotometer according to the degree of the change in the amount of melted in 1 N NaOH. The amount of melanin production was calculated as (measured absorbance / absorbance of untreated group) x 100.

The cell viability of the rattan stem extracts and fractions was determined by inoculating melan-a cells in a 96 well plate at 2 x ¹⁰ cells / well and culturing with 10% fetal bovine serum (FBS) and 1 % penicillin / streptomycin myth, and 200 nM UTP a (TPA, 12-O-Tetradecanoylphorbol 13-acetate) is contained in a medium of RPMI 1640 37 ℃, 10㎍ a 24-hour incubation and the sample in the incubator of 10% CO ₂ condition / Ml < / RTI > and incubated for 72 hours. After that, 100 μl of MTT solution (Thiazolyl blue tetrazolium bromide 5 mg / ml in PBS) was dispensed into each well. After 1 hour and 30 minutes, the formazan formed was dissolved in DMSO. The concentration of formazan dissolved in DMSO was measured at 595 nm using a spectrophotometer. Cell viability was calculated as (measured absorbance / absorbance of untreated group) x 100. Control experiments were the same except that the arbutin was added to the wells at 30 占 퐂 / ml. Table 1 shows the amount of melanin production versus cell viability.

sample The amount of melanin production relative to the survival rate Untreated group 100 Arbutin 57.75 Methanol extract 69.42 Methylene chloride fraction 36.28 Ethyl acetate fraction 66.55 Butanol fraction 93.02

Figure 1 shows the amount of melanin production versus cell viability. In FIG. 1, CON represents a non-treated group, and M, D, E, and B represent fractions of methanol, methylene glycolide, ethyl acetate and butanol. In FIG. 1, the amount of melanin is a percentage (%) of the control and the viability is the percent survival (%) of the control. Therefore, there is no unit of melanin production (%) / survival rate (%) compared to the control group.

As shown in Table 1 and Fig. 1, rattan stem extract and fraction inhibited melanin biosynthesis. In particular, the methylene chloride fraction had remarkably excellent melanin biosynthesis inhibiting activity as compared with arbutin, which is a whitening component.

3. Confirmation of tyrosinase activity inhibitory effect of wisteria stem extract and its fractions

This experiment was carried out by using intracellular tyrosinase method to confirm the inhibitory effect of tyrosinase activity on the extract of Rattan Stem.

Melan-a cells were inoculated in 96 wells at 4 x ¹⁰ cells / well and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, (TPA, 12-O-Tetradecanoylphorbol 13-acetate) in an incubator at 37 ° C and 10% CO ₂ for 24 hours. The sample was added to a concentration of 10 μg / ml and cultured for 72 hours.

Cells cultured in the wells of 6-well plates were washed once with PBS, added with 100ul of lysis buffer (1% Triton X-100) and stirred for 1 minute to obtain a total 100ul cell containing medium. The resulting cell-containing medium was centrifuged at 15,000 rpm for 15 minutes, and then the supernatant was separated from the precipitate.

Add 2 ul of the separated supernatant to the wells of the 96-well plate and add 23 ul of distilled water to a final volume of 25 ul. Then, absorbance was measured at 562 nm using BCA Protein Assay Kit (Thermo scientific, USA), and the amount of protein was measured. Based on the measured amount of protein, the cell supernatant containing 40 μg of the protein contained in the supernatant was added to 0.1 M potassium phosphate buffer (pH 6.8) to make a final volume of 100 μl. Then, L-DOPA (L-3,4-dihydroxyphenylalanine) was diluted to 20 mM in 0.1 M potassium phosphate buffer, and then 100 μl of the solution was further added. The amount of dopachrome produced by the action of tyrosinase from L-DOPA was measured using absorbance. Absorbance was measured at 475 nm on a spectrophotometer after incubation at 37 [deg.] C after L-DOPA addition and after 30 minutes. The rate of inhibition of tyrosinase activity was calculated according to (measured absorbance / no-treatment group absorbance) x 100. Table 2 and Fig. 2 show inhibition rates of tyrosinase activity.

sample Inhibition rate of tyrosinase activity (%) Untreated group 100 Methanol extract 97.12 Methylene chloride fraction 50.46 Ethyl acetate fraction 69.67 Butanol fraction 62.09

As shown in Table 2 and Fig. 2, tyrosinase activity was inhibited by the rattan stem extract and its fractions.

4. Rattan stem extract and its fractions were treated with tyrosinase, TRP1 , TRP2 , MITF  Gene mRNA  Effect on expression

Melan-a cells were inoculated in a 6 well-plate well at 4 × 10 ⁵ / well and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, (TPA, 12-O-Tetradecanoylphorbol 13-acetate) in an incubator at 37 ° C and 10% CO ₂ for 24 hours. The sample was added to a concentration of 10 μg / ml and cultured for 72 hours.

RNA was extracted from the cultured cells using RNeasy mini kit (Qiagen). The extracted RNA was checked for integrity using an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, Calif., USA). The cDNA was synthesized from cDNA of 1 의 of RNA using ImProm-II Reverse Transcription System (Promega, Madison, Wis., USA). PCR was performed using GeneAmp PCR System 9700 (Applied Biosystems, Foster City, Calif., USA).

The PCR reaction was carried out by adding 0.02 μl Ex Taq polymerase (TAKARA, Otsu, Shiga, Japan), 2 μl Ex Taq polymerase buffer, 1.6 μl dNTP (10 mM), 2 μl forward primer (20 μM), 2 μl reverse primer 11.38 μl of nuclease free water and 1 μl of the synthesized first-strand cDNA were mixed well and PCR was performed on a reaction mixture of 20 μl in total. The PCR conditions for amplification of cDNA from MITF, tyrosinase, TRP1, and TRP2 genes were 94 ° C for 4 minutes, 94 ° C for 50 seconds, 63 ° C for 50 seconds, and 72 ° C for 35 seconds. , 72 ° C for 5 minutes (1 cycle), GAPDH conditions were 94 ° C for 4 minutes, 94 ° C for 50 seconds, 63 ° C for 50 seconds, 72 ° C for 50 seconds (35 cycles) 5 minutes (1 cycle). The RT-PCR result was electrophoresed on 2% agarose gel and the intensity of the band was measured using ImageJ. The efficacy was determined by comparison with the ratio of untreated group to 100. The primers used for RT-PCR are shown in Table 3.

Target gene Forward primer Reverse primer MITF SEQ ID NO: 1 SEQ ID NO: 2 Tyrosinase SEQ ID NO: 3 SEQ ID NO: 4 TRP1 SEQ ID NO: 5 SEQ ID NO: 6 TRP2 SEQ ID NO: 7 SEQ ID NO: 8 GAPDH SEQ ID NO: 9 SEQ ID NO: 10

Table 4 and FIG. 3 show the effect of the wisteria stem extract and its fractions on mRNA expression of MITF, tyrosinase, TRP1 and TRP2 genes in the untreated group as 100 and expression of MITF, tyrosinase, TRP1 and TRP2 genes The degree of inhibition is shown. Methanol extract inhibited the gene expression of tyrosinase and TRP1. The methylene chloride fraction inhibited the gene expression of MITF, tyrosinase, and TRP1. The ethyl acetate fraction inhibited gene expression of TRP1. The butanol fraction inhibited tyrosinase and TRP1 Lt; / RTI >

sample MITF Tyrosinase TRP1 TRP2 Untreated group 100 100 100 100 Methanol extract 124.13 96.20 83.91 107.94 Methylene chloride fraction 92.55 56.17 56.01 109.82 Ethyl acetate fraction 134.73 106.40 83.28 118.58 Butanol fraction 140.44 56.40 96.61 116.41

5. Wisteria stem extract Tyrosinase , TRP1 , TRP2 , And MITF  Effect of gene expression on protein expression

Melanin production and tyrosinase activity of melan-a cell, a mouse melanocyte, were measured in the following manner.

Melan-a cells were inoculated into wells of a 6 well plate at 4 x ¹⁰ cells / well and incubated with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, Was cultured in RPMI 1640 medium containing TPA (12-O-Tetradecanoylphorbol 13-acetate) for 24 hours in an incubator at 37 ° C and 10% CO 2, and the sample was added to 10 μg / ml and cultured for 72 hours.

Cells cultured in wells of 6-well plates were washed once with PBS, and 100ul of lysis buffer (1X RIPA Buffer, 25X Protease Inhibitor, 100X Phosphatase inhibitor) was added and cells were collected using a cell lifter, 100 ul of total medium was obtained and the cells were lysed using an ultrasound cell crusher. After centrifugation at 15,000 rpm for 15 minutes, the supernatant and the precipitate were separated.

2 ul of the separated supernatant was added to the wells of a 96-well plate and 23 ul of distilled water was added to make a final volume of 25 ul. Then, absorbance was measured at 562 nm using BCA Protein Assay Kit (Thermo scientific, USA), and the amount of protein was measured. Based on the measured results, a cell supernatant containing 20 μg of protein was Western blotted to confirm the protein expression pattern.

Table 5 and FIG. 4 show the effect of the wisteria extract and its fractions on protein expression of MITF, tyrosinase, TRP1, and TRP2 gene by Western blot analysis, As shown in FIG. As a result, the methanol extract inhibited the protein expression of the tyrosinase gene, the methylene chloride fraction inhibited the protein expression of the genes of MITF and TRP2, and the ethyl acetate fraction inhibited the protein expression of the genes of MITF, tyrosinase, and TRP2 And the butanol fraction inhibited the protein expression of the gene in the MITF and tyrosinase fractions.

sample MITF Tyrosinase TRP1 TRP2 Untreated group 100 100 100 100 Methanol extract 101.73 83.51 253.98 177.72 Methylene chloride fraction 45.16 110.49 264.15 26.16 Ethyl acetate fraction 29.43 52.52 185.95 28.81 Butanol fraction 56.42 39.42 307.01 112.94

<110> Korea Institute of Science and Technology <120> Composition for skin whitening comprising Wisteria floribunda          extract and use thereof <130> PN114837KR <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for MITF <400> 1 tccgrctctc actggattgg tg 22 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for MITF <400> 2 cgtgaatgtg tgttcatgcc tgg 23 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for tyrosinase <400> 3 gtggatgacc gtgagtcctg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for tyrosinase <400> 4 tctgtgccaa ggcagaaacc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for TRP1 <400> 5 atggaacggg aggacaaacc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for TRP1 <400> 6 aaccccgaaa atggcagcta 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for TRP2 <400> 7 ctttgcaacc gggaagaacg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for TRP2 <400> 8 aggcattggt cccattcagg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for GAPDH <400> 9 tccaccaccc tgttgctgta 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for GAPDH <400> 10 accacagtcc atgccatcac 20

Claims (13)

A composition for whitening comprising a Wisteria floribunda stem extract or a fraction thereof as an active ingredient, wherein the wisteria stem extract is an extract of C1-C6 alcohol, water, or a mixture thereof. A composition for inhibiting or reducing melanin production comprising Wisteria floribunda stem extract or a fraction thereof as an active ingredient, wherein the wisteria stem extract is an extract of C1-C6 alcohol, water, or a mixture thereof. delete The composition according to claim 1 or 2, wherein the wisteria extract is an extract of ethanol, methanol, water, or a mixture thereof. The method according to claim 1 or 2, wherein the fraction is a methylene chloride fraction, an ethyl acetate fraction, a butanol fraction or a water extract obtained by sequentially suspending the wisteria extract in water and then fractionating the extract into methylene chloride, ethyl acetate, Lt; / RTI &gt; fraction. The composition according to claim 1 or 2, wherein the wisteria extract, or fraction thereof, is from 0.001% to 10% by weight, based on the total weight of the composition. The composition according to claim 1 or 2, wherein the composition inhibits tyrosinase activity. The composition according to claim 1 or 2, wherein the wisteria extract is extracted under microwave irradiation. The composition of claim 1 or 2, further comprising a cosmetically, pharmaceutically, or pharmaceutically acceptable excipient or carrier. The composition according to claim 1 or 2, wherein the composition is a pharmaceutical, food or cosmetic composition. delete delete WHAT IS CLAIMED IS: 1. A method for improving the whitening state of an individual comprising administering to a subject an amount of the composition of claim 1 or 2 effective to improve whitening, wherein said individual is a non-human mammal.

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