KR102560191B1 - Composition for skin whitening comprising Saururus chinensis white leaf extract - Google Patents

Abstract

It relates to a cosmetic composition for skin whitening containing an extract of the white leaf of Sambaekcho as an active ingredient, a pharmaceutical composition for preventing, treating or improving melanin hyperpigmentation disease, and a health functional food composition. Since the white leaf extract of Sambaekcho has excellent melanin production and secretion inhibitory effects and skin pigmentation inhibitory effects compared to the green leaf extract of Sambaekcho, it can be used as a cosmetic composition or pharmaceutical composition for skin whitening.

Description

Composition for skin whitening comprising Saururus chinensis white leaf extract

It relates to a cosmetic composition for skin whitening, and a pharmaceutical composition and health functional food composition for preventing, treating, or improving melanin hyperpigmentation disease.

Pigmentation occurring on the skin, such as spots and freckles, is caused by an increase in melanin in the epidermis, and an important factor determining skin color is also caused by melanin.

Skin pigment formation mechanisms can be largely divided into three types: melanogenesis, melanosome transport, and melanosome transfer. The melanin biosynthesis mechanism refers to the process by which melanin is formed by the action of tyrosinase and TRP-1/2 (tyrosinase related protein-1/2) in melanosomes, which are organelles of melanocytes. The melanosome transport mechanism is a process in which melanosomes are connected to microorganelles and actin and move to the tips of dendrites. Finally, the melanosome transfer mechanism refers to the process in which melanosomes moved to the tip of the dendrites of melanocytes are moved to the surrounding epidermis. Through the above three mechanisms, the synthesis of tyrosinase is promoted in melanocytes activated by ultraviolet rays, and the production of melanin is enhanced to transport and discharge it out of the epidermis (Jody P. Ebanks, Mechanisms Regulating Skin Pigmentation: The Rise and Fall of Complexion Coloration, Int J Mol Sci. Sep; 10(9): 4066-4087, 2009).

Skin color is determined by the content and distribution of melanin, and is related to the number and distribution of melanosomes generated in melanocytes within cells and then released to the outside of the cells. Skin hyperpigmentation (hyperpigmentation) may be caused by various factors such as hormonal abnormalities in the body after an inflammatory reaction of the skin, genetic diseases, ultraviolet irradiation, etc., the main cause is due to abnormal synthesis and distribution of melanin pigments. The main function of melanin is to protect the skin from damage by removing oxygen radicals, and the high amount of melanin means that it has an effective countermeasure system to protect the skin from physical and chemical toxic substances.

However, excessive melanin deposition causes pathological problems such as the skin, but is also recognized as a cosmetic problem such as melasma, freckles, spots, and age spots. Therefore, it is necessary to develop a whitening agent capable of fundamentally inhibiting melanin formation.

According to a recently published fact, developing a substance capable of inhibiting not only the melanin biosynthesis process but also the main factors related to the melanosome transport and delivery process can be a way to prevent skin pigmentation (Jong il Park at al., Inhibitory effect of 2-methyl-naphtho[1,2,3-de]quinolin-8-one on melanosome transport and skin pigmentation, Scientific Reports volume 6, Article number: 29189, 2016).

Therefore, it is necessary to develop a new whitening raw material capable of inhibiting melanin biosynthesis and inhibiting factors related to the melanosome transmission process.

Provided is a composition containing the white leaf extract of Sambaekcho as an active ingredient.

Provides a method for preparing the white leaf extract of Sambaekcho.

Provided is a method for preventing or treating hypermelanin hyperpigmentation disease, comprising administering a composition containing an effective amount of an extract of the white leaf of Sambaekcho to a subject in need thereof.

Provided is the use of a white leaf extract of Sambyeongcho for use in preparing a medicament for preventing or treating hypermelanin hyperpigmentation disease.

One aspect provides a composition comprising the white leaf extract of Sambaekcho as an active ingredient.

“Asian lizard's tail ( Saururus chinensis )” is called “Sambaekcho” because its leaves, roots and flowers are white. The leaves of Sambaekcho are green before blooming, but when it comes to flowering, the leaves turn white to attract insects, and after flowering, they turn green again. It is known that the blooming period of Sambaekcho is from June to August. In other words, Sambaekcho has “white leaf” only at certain times.

Therefore, the "three hundred seconds white leaves" means the white leaves of three hundred seconds harvested at the flowering time of three hundred seconds. The 300 seconds white leaves are different from the 300 seconds green leaves and color, harvest time, etc.

The term "extract" includes all substances obtained by extracting components of natural substances, regardless of the extraction method, extraction solvent, extraction conditions, extracted components or extract form, and also includes substances that can be obtained by extracting components of natural substances and then processing or treating them by other methods. For example, the processing or treatment may include dilution, concentration, drying, purification, fractionation, filtration, fermentation, enzymatic treatment, and the like. Therefore, the extract may include an extract, a dilution or concentrate of the extract, a dried product obtained by drying the extract, or a crude or purified product thereof, or a fraction obtained by fractionating the same.

As the extraction method, known natural product extraction methods such as hot water extraction, ethanol extraction, heat extraction, cold extraction, reflux extraction, reflux cooling extraction, ultrasonic extraction, and subcritical extraction may be used, but are not limited thereto. In one embodiment, the extraction method may be hot water extraction or ethanol extraction.

The extraction solvent may be selected from water, organic solvents, or mixtures thereof. The water may be distilled water or purified water. The organic solvent may include one or more of C1 to C6 lower alcohol, acetone, ether, ethyl acetate, diethyl ether, ethyl methyl ketone, and chloroform, but is not limited thereto.

In one embodiment, the extraction solvent of the extract may be water, C1 to C4 alcohol, or a mixture thereof. In another embodiment, the extraction solvent of the extract may be a mixture of water and C1 to C4 alcohol. In another embodiment, the extraction solvent of the extract may be ethanol. In another embodiment, the extraction solvent of the extract may be 10% (v / v) to 90% (v / v), 20% (v / v) to 80% (v / v), 30% (v / v) to 70% (v / v), 40% (v / v) to 60% (v / v) or 50% (v / v) ethanol. Therefore, the 300 seconds white leaf extract may be a 300 seconds white leaf hot water extract or a 300 seconds white leaf ethanol extract. In one embodiment, it was confirmed that the ethanol extract of Sambaekcho white leaves had superior melanin production inhibitory effect and melanin secretion inhibitory effect compared to the hot water extract of Sambaekcho white leaves. Therefore, the 300 seconds white leaf extract may be 300 seconds white leaf ethanol extract.

The extraction conditions refer to conditions for extracting components of natural substances, such as extraction time and extraction temperature. The extraction time is 30 minutes to 120 hours, 30 minutes to 100 hours, 30 minutes to 80 hours, 30 minutes to 60 hours, 30 minutes to 40 hours, 30 minutes to 20 hours, 30 minutes to 10 hours, 2 hours to 120 hours, 2 hours to 100 hours, 2 hours to 80 hours, 2 hours to 60 hours, 2 hours to 40 hours , 2 hours to 20 hours, or 2 hours to 10 hours, but may be appropriately selected according to other conditions such as extraction solvent and extraction temperature. The extraction temperature may be 10 to 150 ℃, 10 to 120 ℃, 10 to 100 ℃, 10 to 80 ℃, 20 to 150 ℃, 20 to 120 ℃, 20 to 120 ℃, 20 to 100 ℃, 20 to 80 ℃, 20 to 60 ℃, 20 to 40 ℃, or room, but can be selected according to other conditions such as extraction solvents and extraction time.

The "included as an active ingredient" means that the white leaf extract of Sambaekcho is added to the extent that it can exhibit the effects mentioned in this specification, and various ingredients are added as subcomponents for drug delivery and stabilization to form various forms. It means that it is formulated.

The composition containing the white leaf extract of Sambaekcho as an active ingredient may be used for skin whitening, inhibition of melanin production, inhibition of melanin secretion, reduction of melanin, inhibition of melanin hyperpigmentation, or prevention, treatment or improvement of melanin hyperpigmentation disease.

The term "skin whitening" may mean not only brightening the skin tone by inhibiting the synthesis of melanin pigment, but also improving skin hyperpigmentation such as spots or freckles caused by ultraviolet rays, hormones, or heredity.

The term “melanin production inhibition” may mean suppression of new melanin production in cells or the like.

The term “inhibition of melanin secretion” may mean suppression of secretion of melanin from cells or the like.

The term “melanin reduction” may mean reducing the melanin content existing in cells or the like.

The term "melanin hyperpigmentation" is a broad concept that includes all melanin hyperplasia caused by various causes, and means that certain parts of the skin, fingernails, or toenails become darker than other parts due to excessive increase in melanin pigment.

The melanin hyperpigmentation disease is freckles (freckle); melasma; chloasma; liver spots; nevus; solar lentigo; melanosis; peutz-jegher's syndrome; gestational brown spots (chloasma gravidarum); hyperpigmentation after drug use; And it may be any one selected from postinflammatory hyperpigmentation (postinflammatory hyperpigmentation), but is not limited thereto.

The term "prevention" includes inhibiting the occurrence of a disease. The term "treatment" includes the inhibition, alleviation, or elimination of the development of a disease. The term "improvement" can mean any action that at least reduces a parameter associated with alleviation or treatment of a condition, eg, the severity of a symptom.

The white leaf extract of Sambaekcho has a high skin whitening effect, melanin production inhibition, melanin secretion inhibition, melanin reduction, or melanin hyperpigmentation inhibition compared to the green leaf extract of Sambaekcho.

The white leaf extract of Sambaekcho may contain at least one of quercitrin and afzelin. In one embodiment, it was confirmed that the content of quercitrin and afzelin in the white leaf extract of Sambaekcho was significantly higher than that of the green leaf extract of Sambaekcho.

The composition may inhibit melanin production or secretion. In one embodiment, it was confirmed that the white leaf extract of 3 baekcho is excellent in melanin production inhibitory effect and melanin secretion inhibitory effect compared to the green leaf extract of 3 baekcho. In addition, it was confirmed that quercitrin contained in the white leaf extract of Sambaekcho had excellent melanin production inhibitory effects and melanin secretion inhibitory effects. In addition, it was confirmed that the ethanol extract of Sambaekcho leaves had superior melanin production inhibitory effect and melanin secretion inhibitory effect compared to the hot water extract of Sambaekcho leaves.

The composition may inhibit the expression of PAR-2 (protease-activated receptor-2) involved in melanosome transmission. In one embodiment, since it was confirmed that the afgeline contained in the white leaf extract of Sambaekcho inhibits the expression of PAR-2 involved in melanosome transmission, it was confirmed that there is an effect of inhibiting skin pigmentation.

The triticale white leaf extract may be included at a concentration of 0.0001 μg/ml to 0.1 μg/ml, 0.0001 μg/ml to 0.05 μg/ml, or 0.0001 μg/ml to 0.001 μg/ml based on the total composition, but is not limited thereto. The concentration of the white leaf extract of Sambaekcho can be selected in an appropriate range that can exhibit whitening efficacy without exhibiting cytotoxicity.

Quercitrin contained in the triticale white leaf extract is 0.0001 μg / ml to 100 μg / ml, 0.0001 μg / ml to 10 μg / ml, 0.0001 μg / ml to 1 μg / ml, 0.001 μg / ml to 100 μg / ml, 0.001 μg / ml to 10 μg / ml, 0.001 μg / ml to 10 μg / ml, based on the total composition 001 μg/ml to 1 μg/ml, 0.01 μg/ml to 100 μg/ml, 0.01 μg/ml to 10 μg/ml, or 0.01 μg/ml to 1 μg/ml, but is not limited thereto. The concentration of the quercitrin may be selected within an appropriate range capable of exhibiting a whitening effect without exhibiting cytotoxicity.

The triticale white leaf extract is 0.0001% to 20.0% by weight, 0.0001% to 10.0% by weight, 0.0001% to 5.0% by weight, 0.0001% to 1.0% by weight, 0.001% to 20.0% by weight, 0.001% to 10.0% by weight, 0.001% to 10.0% by weight, based on the total composition. 1 wt% to 5.0 wt%, 0.001 wt% to 1.0 wt%, 0.01 wt% to 20.0 wt%, 0.01 wt% to 10.0 wt%, 0.01 wt% to 5.0 wt%, or 0.01 wt% to 1.0 wt%, but is not limited thereto. The content of the white leaf extract of Sambaekcho can be selected in an appropriate range that can exhibit whitening efficacy without exhibiting cytotoxicity.

In one embodiment, the composition may be a cosmetic composition.

The cosmetic composition may be used for skin whitening, inhibition of melanin production, inhibition of melanin secretion, reduction of melanin, or inhibition of excessive melanin pigmentation.

The cosmetic composition may further include ingredients commonly used in cosmetic compositions, functional additives, etc. in addition to the active ingredients disclosed herein, such as antioxidants, stabilizers, solubilizers, surfactants, dispersants, thickeners, preservatives, vitamins, pigments, conventional adjuvants such as fragrances, and cosmetically acceptable carriers.

The cosmetic composition may be prepared in any formulation that is conventionally prepared. For example, the cosmetic composition may be cosmetic formulations such as lotion, cream, essence, cleansing foam, cleansing water, pack, ampoule, body lotion, body oil, body gel, shampoo, rinse, hair conditioner, hair gel, foundation, lipstick, mascara, or makeup base.

In one embodiment, the composition may be an external composition for skin.

The composition for external application for skin may be used for skin whitening, inhibition of melanin production, inhibition of melanin secretion, reduction of melanin, inhibition of melanin hyperpigmentation, or prevention, treatment, or improvement of melanin hyperpigmentation disease.

The external skin preparation may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof. The external skin preparations may be appropriately mixed with ingredients commonly used in external skin preparations such as cosmetics or pharmaceuticals, for example, water-based ingredients, oil-based ingredients, powdered ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, coloring agents, various skin nutrients, or combinations thereof.

In one embodiment, the composition may be a pharmaceutical composition.

The pharmaceutical composition may be used for skin whitening, inhibition of melanin production, inhibition of melanin secretion, reduction of melanin, inhibition of melanin hyperpigmentation, or prevention or treatment of melanin hyperpigmentation diseases.

The pharmaceutical composition may additionally include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and magnesium stearate, talc, or a combination thereof as a lubricant. 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 hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The pharmaceutical composition may be formulated for oral or parenteral administration. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, or combinations thereof. Parenteral dosage forms may be injections.

In one embodiment, the composition may be a health functional food composition.

It may be used for skin whitening, inhibition of melanin production, inhibition of melanin secretion, reduction of melanin, inhibition of melanin hyperpigmentation, or prevention or improvement of melanin hyperpigmentation disease.

The health functional food composition may be used alone with the extract, or in combination with other foods or food ingredients, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). There is no particular limitation on the type of health functional food. Among the types of health functional foods, beverage compositions may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The health functional food composition may also contain nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, or combinations thereof. The health functional food composition may also contain natural fruit juice, fruit juice beverages, fruit flesh for preparing vegetable beverages, or a combination thereof.

Another aspect provides a method for preparing the white leaf extract of Sambaekcho.

The method includes obtaining an extract by incubating the white leaves of Sambaekcho with an extraction solvent.

Details of the three hundred second white leaves, the extraction solvent and the extract are as described above.

The incubation may be performed under conventional extraction conditions. Details of the extraction conditions are as described above.

The method may further include filtering and/or drying the obtained extract. For the filtration and drying, known conventional methods may be used.

Another aspect provides a method for preventing or treating melanin hyperpigmentation disease comprising administering to a subject in need thereof a composition comprising an effective amount of a white leaf extract of triticale.

The term "effective amount" means an amount effective enough to exhibit the above-mentioned effects.

The terms "administering," "introducing," and "implanting" are used interchangeably and can refer to the placement of a composition according to an embodiment into a subject by a method or route that results in at least partial localization of the composition according to an embodiment to a desired site.

Administration may be administered by a method known in the art. Administration can be administered directly to a subject by any means, such as, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. The administration may be administered systemically or locally. The administration may be applied to the skin.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be a subject in need of prevention or treatment of melanin hyperpigmentation disease.

The dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art can adjust the dosage appropriately in consideration of these factors. The number of administrations can be once a day or two or more times within the range of clinically acceptable side effects, and the administration site can be administered to one or two or more sites, and the total number of administration days can be administered from 1 to 30 days at a time of treatment. If necessary, the same treatment can be repeated after a titration period. For animals other than humans, the same dose per kg as that for humans, or an amount obtained by converting the above dose based on, for example, the volume ratio (e.g., average value) of the organ (heart, etc.) between the target animal and the human can be administered.

Another aspect provides the use of a white leaf extract of Sambyeongcho for use in preparing a medicament for the prevention or treatment of melanin hyperpigmentation disease.

Redundant content is omitted in consideration of the complexity of the present specification, and terms not defined otherwise in the present specification have meanings commonly used in the technical field to which the present invention belongs.

The composition according to one aspect may have skin whitening, suppression of melanin production and secretion, and suppression of melanin hyperpigmentation by including the white leaf extract of Sambaekcho. Since the white leaf extract of Sambaekcho has excellent melanin production and secretion inhibitory effects and skin pigmentation inhibitory effects compared to the green leaf extract of Sambaekcho, it can be used as a cosmetic composition or pharmaceutical composition for skin whitening.

Figure 1 shows the results of LC-QTOF-MS analysis of the white leaf extract of Example 1 and the green leaf extract of Sambaekcho of Comparative Example 1.
Figure 2 is the MS fragmentation pattern of quercitrin contained in the white leaves of Sambaekcho.
Figure 3 is the MS fragmentation pattern of afgeline contained in the white leaves of Sambaekcho.
Figure 4 shows the cytotoxicity test results of Example 1, Comparative Example 1, Example 2 and Comparative Example 2.
Figure 5 shows the results of the cytotoxicity test of quercitrin contained in the white leaf extract of Sambaekcho.
Figure 6 shows the results of the cytotoxicity test of afgeline contained in the white leaf extract of Sambaekcho.
Figure 7 shows the melanin biosynthesis results of Example 1, Comparative Example 1, Example 2 and Comparative Example 2.
Figure 8 shows the melanin biosynthesis results of quercitrin contained in the white leaf extract of Sambaekcho.
9 shows the melanin secretion rate results of Example 1 and Comparative Example 1.
10 shows the results of the melanin secretion rate of quercitrin contained in the white leaf extract of Sambaekcho.
Figure 11 shows the PAR-2 expression inhibitory effect of afgeline contained in the white leaf extract of Sambaekcho.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of Sambaekcho white leaf extract

Only the white leaves were separated from Sambaekcho and immersed in 50% ethanol for 3 days at room temperature so that the components were not broken. After filtration, the filtrate was concentrated and vacuum dried.

Comparative Example 1. Preparation of Sambaekcho green leaf extract

Only the green leaves were separated from Sambaekcho and immersed in 50% ethanol for 3 days at room temperature so that the components were not broken. After filtration, the filtrate was concentrated and vacuum dried.

Example 2. Preparation of hot water extract of Sambaekcho white leaves

After separating only the white leaves from 300 seconds, distilled water corresponding to 10 times the weight of the original weight of 300 seconds white leaves was added and mixed, and then extracted at a temperature of 90 ° C. for 4 hours and 30 minutes. The obtained extract was filtered using a filter paper, and then the filtrate was concentrated under reduced pressure using a rotary evaporator. The filtrate subjected to concentration under reduced pressure was lyophilized to remove the remaining solvent to obtain a powdery hot-water extract of Sambaekcho white leaves.

Comparative Example 2. Preparation of hot-water extract of 300 seconds green leaves

After separating only the green leaves from the three hundred seconds, distilled water corresponding to 10 times the weight of the raw material of the three hundred seconds green leaves was added and mixed, and then extracted at a temperature of 90 ° C. for 4 hours and 30 minutes. The obtained extract was filtered using a filter paper, and then the filtrate was concentrated under reduced pressure using a rotary evaporator. The filtrate subjected to concentration under reduced pressure was lyophilized to remove the remaining solvent to obtain a powdery hot-water extract of the green leaves of Sambaekcho.

Experimental Example 1. Identification of secondary metabolites of white leaf and green leaf extracts and comparison of their contents

Secondary metabolites of the white leaf extract of Sambaekcho of Example 1 and the green leaf extract of Sambaekcho of Comparative Example 1 were identified, and experiments were conducted to compare their contents.

First, the white leaf extract and the green leaf extract of Sambaekcho were dissolved in 50% methanol, respectively, to a concentration of 1 mg/mL. Then, after filtering with a 0.45 μm PVDF (Polyamide Fluoride) filter, it was analyzed by LC-QTOF-MS (liquid chromatography-quadrupole-time of flight mass spectrometry). LC-QTOF-MS analysis results are shown in FIG. 1 .

As shown in FIG. 1, four peaks that appeared to be contained more in white leaves than in green leaves of 300 seconds were identified. As a result of analyzing MS fragmentation patterns for the four peaks, all of them could be expected to be flavonoid glycosides.

MS fragmentation patterns for two peaks showing major differences among the four flavonoid glycosides are shown in FIGS. 2 and 3 . Table 1 also shows the MS fragment dereplication results for the two peaks.

RT
(minute) Precursor ion m/z Product ion m/z molecular formula Sympathy 9.76 471.0886
449.0886 303.0499 quercitrin 12.84 455.0952
433.1118 287.0553 afzelin

As shown in Figure 2, Figure 3 and Table 1, the two main peaks that appear to be contained more in white leaves than in the green leaves of 300 seconds were expected to be quercitrin and afzelin. The corresponding components were confirmed by measuring with the standards of quercitrin and afzeline, and it was confirmed that they were contained in a large amount in white leaves rather than green leaves.

Experimental Example 2. Cytotoxicity evaluation on B16 melanoma cells

The cytotoxicity of the white leaf and green leaf extracts of Sambaekcho and the indicator component quercitrin on B16 melanoma cells was evaluated.

Specifically, melanoma cells were inoculated in a 6-well plate and cultured for 24 hours, then replaced with new DMEM medium containing α-MSH and 10% bovine serum, and each sample was treated by concentration and cultured for 72 hours. The cultured cells were reacted by replacing the medium with a 10-fold diluted MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution at 2.5 mg/ml. Thereafter, the supernatant was removed, and 2 ml of dimethyl sulfoxide (DMSO) was added to dissolve the MTT-formazan crystals, and the absorbance was measured at 570 nm using an ELISA reader. What was treated with α-MSH was used as a control. Cytotoxicity was expressed as a percentage of absorbance compared to the control group, and the results are shown in Tables 2 and 3 and FIGS. 4 and 5.

test group Experimental Concentration
(μg/ml) Cell viability (%) standard deviation (Stdev.) P-value untreated group 100.48 2.07 0.856 α-MSH treatment group 100.00 3.73 1.000 α-MSH + arbutin 100 94.31 2.97 0.108 α-MSH + Sambaekcho white leaf extract (Example 1) 0.025 94.29 0.34 0.058 0.05 95.75 0.82 0.127 0.1 93.51 1.98 0.056 0.25 0.70 0.05 0.000 α-MSH + Sambaekcho Green Leaf Extract
(Comparative Example 1) 0.025 93.97 1.30 0.057 0.05 92.24 0.86 0.025 0.1 86.02 0.30 0.003 0.25 0.72 0.03 0.000 α-MSH + Sambaekcho white leaf hot water extract (Example 2) 0.025 93.11 1.10 0.056 0.05 94.31 0.56 0.021 0.1 92.00 0.20 0.012 0.25 0.1 0.01 0.012 α-MSH + hot water extract of Sambaekcho green leaf (Comparative Example 2) 0.025 92.88 1.10 0.055 0.05 91.56 0.76 0.025 0.1 85.01 0.40 0.011 0.25 0.12 0.01 0.010

test group Experimental Concentration
(μg/ml) Cell viability (%) standard deviation (Stdev.) P-value untreated group 107.65 4.02 0.036 α-MSH treatment group 100.00 1.37 1.000 α-MSH + arbutin 100 91.81 0.80 0.001 α-MSH + Quercitrin One 104.10 2.82 0.086 10 102.96 2.01 0.103 25 108.81 4.42 0.030 50 110.19 1.26 0.001 100 104.85 4.67 0.160 250 85.99 0.67 0.000

As shown in Table 2, Table 3, FIGS. 4 and 5, in melanoma cells, the extract of Sambakcho white leaf of Example 1, the green leaf extract of Sambakcho of Comparative Example 1, the hot water extract of Sambakcho white leaf of Example 2, and the green leaf extract of Comparative Example 2 did not show toxicity up to a concentration of 0.1 μg/ml. In addition, quercitrin, a marker component, showed no toxicity up to a concentration of 100 μg/ml.

Experimental Example 3. Evaluation of cytotoxicity to keratinocytes (HaCaT)

The cytotoxicity of the indicator component afgeline on keratinocytes (HaCaT) was evaluated.

Specifically, after inoculating keratinocytes (HaCaT) in a 24-well plate and culturing for 24 hours, the medium of the cultured cells was replaced with a serum-free medium, and the samples were treated by concentration and cultured for 24 hours. The cultured cells were reacted by replacing the 2.5 mg/ml MTT solution with a 10-fold diluted medium. Thereafter, the supernatant was removed and 1 ml of DMSO was added to dissolve the MTT-formazan crystals, and the absorbance was measured at 570 nm using an ELISA reader. The untreated group was used as a control group. Cytotoxicity was expressed as a percentage of absorbance compared to the control group, and the results are shown in Table 4 and FIG. 6.

test group Experimental Concentration
(μg/ml) Cell viability (%) standard deviation (Stdev.) P-value untreated group 107.65 4.02 0.036 Afgeline One 104.10 2.82 0.086 10 102.96 2.01 0.103 25 108.81 4.42 0.030 50 110.19 1.26 0.001 100 104.85 4.67 0.160 250 85.99 0.67 0.000

As shown in Table 4 and FIG. 6, afgeline, a marker component, showed no toxicity to keratinocytes up to a concentration of 100 μg/ml.

Experimental Example 4. Measurement of whitening efficacy of inhibiting melanin biosynthesis per unit cell in melanoma cells

The whitening effect of the white leaf and green leaf extracts of Sambaekcho and the indicator component quercitrin to inhibit melanin biosynthesis per unit cell in melanoma cells was measured.

Specifically, after inoculating melanoma cells in a 6-well plate and culturing for 24 hours, the medium was replaced with new DMEM medium containing α-MSH and 10% bovine serum, and the samples were treated by concentration and cultured for 72 hours. In order to measure the amount of melanin secreted into the medium after 72 hours of culture, the medium was transferred to a 96-well plate, and then the absorbance was measured at 450 nm to obtain the amount of melanin secreted per unit cell. Thereafter, the cells from which the medium was completely removed were washed with 2 ml of phosphate-buffered saline (PBS), treated with 0.25 ml of 1N NaOH, and the cells were collected in a 1.5 ml tube to obtain intracellular melanin. The harvested cell lysate was incubated at 60° C. for 30 minutes, vortexed, and then transferred to a 96-well plate and absorbance was measured at 450 nm to determine the amount of melanin produced per unit cell. α-MSH treatment was used as a control group, and arbutin (manufacturer: Hyundai Bioland) was used as a positive control group. The melanin biosynthesis results per unit cell are shown in Tables 5 and 6 and FIGS. 7 and 8, and the melanin secretion rate results per unit cell are shown in Tables 7 and 8 and FIGS. 9 and 10.

test group Experimental Concentration
(μg/ml) Melanin production rate per unit cell (%) standard deviation (Stdev.) P-value untreated group 58.42 2.71 0.000 α-MSH treatment group 100.00 0.53 1.000 α-MSH + arbutin 100 64.82 2.33 0.000 α-MSH + Sambaekcho white leaf extract (Example 1) 0.025 88.24 1.53 0.000 0.05 73.03 1.26 0.000 0.1 67.55 3.60 0.000 α-MSH + Sambaekcho green leaf extract (Comparative Example 1) 0.025 96.79 1.83 0.043 0.05 76.39 0.71 0.000 0.1 76.19 2.59 0.000 α-MSH + Sambaekcho white leaf hot water extract (Example 2) 0.025 98.12 2.12 0.000 0.05 85.12 1.11 0.000 0.1 79.99 3.12 0.000 α-MSH + hot water extract of Sambaekcho green leaf (Comparative Example 2) 0.025 99.19 1.43 0.043 0.05 88.39 1.51 0.000 0.1 81.19 0.01 0.000

test group Experimental Concentration
(μg/ml) Melanin production rate per unit cell (%) standard deviation (Stdev.) P-value untreated group 45.04 2.47 0.000 α-MSH treatment group 100.00 0.66 1.000 α-MSH + arbutin 100 64.07 1.46 0.000 α-MSH + Quercitrin One 89.58 1.15 0.000 10 92.92 1.72 0.003 25 82.06 2.37 0.000 50 80.00 3.35 0.001 100 84.48 2.02 0.000

test group Experimental Concentration
(μg/ml) Melanin secretion rate per unit cell (%) standard deviation (Stdev.) P-value untreated group 58.03 2.53 0.000 α-MSH treatment group 100.00 2.23 1.000 α-MSH + arbutin 100 73.56 0.76 0.000 α-MSH + Sambaekcho white leaf extract (Example 1) 0.025 91.97 7.18 0.138 0.05 79.30 1.69 0.000 0.1 69.36 1.09 0.000 α-MSH + Sambaekcho green leaf extract (Comparative Example 1) 0.025 100.29 2.40 0.885 0.05 79.65 3.07 0.001 0.1 76.52 0.88 0.000

test group Experimental Concentration
(μg/ml) Melanin secretion rate per unit cell (%) standard deviation (Stdev.) P-value untreated group 47.74 1.84 0.000 α-MSH treatment group 100.00 0.75 1.000 α-MSH + arbutin 100 69.16 1.88 0.000 α-MSH + Quercitrin One 93.96 4.50 0.083 10 96.63 2.46 0.086 25 86.41 2.26 0.001 50 74.12 1.13 0.000 100 82.36 1.49 0.000

As shown in Tables 5 to 8 and FIGS. 7 to 10, the white leaf ethanol extract and green leaf ethanol extract of Sambaekcho decreased the melanin production rate and secretion rate per unit cell increased by α-MSH in a concentration-dependent manner. In particular, it was confirmed that the ethanol extract of the white leaf of Sambaekcho was superior to the ethanol extract of the green leaf in inhibition of melanin production and secretion per unit cell. In addition, it was determined that quercitrin, a marker component, also exhibits a whitening effect by inhibiting melanin biosynthesis and secretion by inhibiting melanin production and secretion per unit cell.

In addition, it was confirmed that the ethanol extract was more effective in concentration-dependently reducing the melanin production rate per unit cell than the hot water extract in both the white and green leaves of Sambaekcho.

Experimental Example 5. Measurement of whitening efficacy to inhibit skin pigmentation by inhibiting melanosomes delivered to the epidermis

The effect of inhibiting skin pigmentation by inhibiting melanosomes of the indicator component afzeline was measured.

Melanosome transfer, the process by which melanosomes are transferred from melanocytes to the epidermis, has various mechanisms, among which phagocytosis by PAR-2 (protease-activated receptor-2), a key mediator, is the most known. By inhibiting the expression of PAR-2, the whitening effect of inhibiting skin pigmentation was confirmed by suppressing melanosomes transmitted to the epidermis (Eun-Jung CHOI at al, Macelignan Inhibits Melanosome Transfer Mediated by Protease-Activated Receptor-2 in Keratinocytes, Biol. Pharm. Bull. 34(5) 748-754, 2011).

Keratinocytes (HaCaT) were inoculated into a 60 mm culture dish (corning), cultured for 24 hours, washed twice with HBSS (Hank's Buffered Salt Solution), and then UVB 30 mJ/cm 2 was irradiated with HBSS. Thereafter, the medium was replaced with a serum-free medium, and each sample was treated for each concentration and cultured for 8 hours. Then, the cells were collected with QIAzol™ Lysis Reagent (Qiagen) and RNA was isolated according to the manufacturer's method. After quantifying the isolated RNA, real-time PCR was performed by synthesizing cDNA with 1 μg of RNA. The PAR-2 and β-Actin primers used in PCR were synthesized and used by Cosmogenetec (Korea), and the primer sequences are shown in Table 9. UVB irradiation was used as a control group, and niacinamide (manufacturer: Sigma) was used as a positive control group. PAR-2 expression results are shown in Table 10 and FIG. 11.

primer order sequence number PAR-2 forward 5'- TGC TAG CAG CCT CTC TCT CC -3' One reverse 5'- CCA GTG AGG ACA GAT GCA GA -3' 2 β-Actin forward 5'-GGC ACC CAG CAC AAT GAA G -3' 3 reverse 5'-CCG ATC CAC ACG GAG TAC TTG -3' 4

test group Experimental Concentration
(μg/ml) PAR-2 mRNA expression rate (%) standard deviation (Stdev.) P-value non-irradiation group 56.11 2.43 0.000 UVB 30 mJ/cm2 100.00 0.00 1.000 UVB + Niacinamide 100 67.90 3.29 0.000 UVB + Afgeline 50 109.05 4.80 0.037 100 81.24 6.64 0.013 250 77.99 4.48 0.001

As shown in Table 10 and FIG. 11, it was confirmed that treatment of keratinocytes with afgeline, which is an indicator component, inhibited the increased PAR-2 expression due to UVB. Through this, it was confirmed that the indicator component afzeline inhibits melanosomes transmitted to the epidermis and exhibits a whitening effect that inhibits skin pigmentation.

SEQUENCE LISTING <110> Cosmax, INC. <120> Composition for skin whitening comprising Saururus chinensis white leaf extract <130> PN139191KR <160> 4 <170> PatentIn version 3.2 <210> 1 <211> 20 <212> DNA <213> artificial <220> <223> PAR-2 forward primer <400> 1 tgctagcagc ctctctctcc 20 <210> 2 <211> 20 <212> DNA <213> artificial <220> <223> PAR-2 reverse primer <400> 2 ccagtgagga cagatgcaga 20 <210> 3 <211> 19 <212> DNA <213> artificial <220> <223> β-Actin forward primer <400> 3 ggcacccagc acaatgaag 19 <210> 4 <211> 21 <212> DNA <213> artificial <220> <223> β-Actin reverse primer <400> 4 ccgatccaca cggagtactt g 21

Claims (10)

A cosmetic composition for skin whitening comprising a white leaf extract of Sambaekcho as an active ingredient,
The 300 seconds white leaf extract is a cosmetic composition that has a higher skin whitening efficacy than the green leaf extract of 300 seconds. The cosmetic composition for skin whitening according to claim 1, wherein the extraction solvent of the extract is water, C1 to C4 alcohol, or a mixture thereof. The cosmetic composition for skin whitening according to claim 1, wherein the extraction solvent of the extract is 10% (v/v) to 90% (v/v) ethanol. delete The cosmetic composition for skin whitening according to claim 1, wherein the extract contains at least one of quercitrin and afzelin. The cosmetic composition for skin whitening according to claim 1, which inhibits melanin production or secretion. The cosmetic composition for skin whitening according to claim 1, which inhibits the expression of PAR-2 (protease-activated receptor-2) involved in melanosome transmission. A pharmaceutical composition for the prevention or treatment of melanin hyperpigmentation disease comprising an extract of white leaves of Sambaekcho as an active ingredient,
The 300 seconds white leaf extract is a pharmaceutical composition that has a high melanin pigment hyper-deposition inhibitory ability compared to the green leaf extract of 300 seconds. The method according to claim 8, wherein the melanin hyperpigmentation disease is freckles (freckle); melasma; chloasma; liver spots; nevus; solar lentigo; melanosis; peutz-jegher's syndrome; gestational brown spots (chloasma gravidarum); hyperpigmentation after drug use; And post-inflammatory hyperpigmentation (postinflammatory hyperpigmentation) of any one selected from, a pharmaceutical composition for the prevention or treatment of melanin hyperpigmentation disease. As a health functional food composition for preventing or improving melanin hyperpigmentation disease comprising an extract of white leaves of Sambaekcho as an active ingredient,
The health functional food composition that the white leaf extract of 300 seconds has a high melanin pigmentation inhibition ability compared to the green leaf extract of 300 seconds.