KR20220135121A - Composition for skin whitening comprising Sophora tonkinensis root extract or its fractions and uses thereof - Google Patents

Abstract

The present invention relates to: a composition which comprises a Sophora tonkinensis Gagnep. root extract or a fraction thereof, and is for skin whitening or preventing, alleviating or treating skin hyperpigmentation. The Sophora tonkinensis Gagnep. root extract or the fraction thereof of the present invention can inhibit the activity of tyrosinase and melanin synthesis in a concentration-dependent manner, and thus can be effectively used as a composition for skin whitening or preventing, alleviating or treating skin hyperpigmentation.

Description

Composition for skin whitening comprising Sophora tonkinensis root extract or its fractions and uses thereof

The present invention relates to a composition for skin whitening or preventing, improving or treating skin hyperpigmentation, comprising an extract or a fraction thereof.

Humans naturally produce melanin for photo-protection. Melanin biosynthesis is a step of catalyzing the hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) and the oxidation of L-DOPA to dopaquinone. It is initiated by the rate-limiting enzyme tyrosinase. After a series of redox processes, the reaction produces intermediate dihydroxyindole (DHI) and DHI carboxylic acid (DHICA) to form eumelanin by polymerization.

Melanogenesis (melanogenesis) occurs in the melanosomes of melanocytes, stem cell factor (SCF), adrenaline (adrenaline), noradrenaline (noradrenaline), α- melanocyte stimulating hormone (α-MSH), including a variety of hormones including Wnt hormone It may be induced by paracrine cytokines. These factors interact with their respective receptors and activate various signaling pathways, eventually upregulating microphthalmia-associated transcription factor (MITF) in the nucleus. Up-regulated MITF is tyrosine hydroxylase isoform I, phenylalanine hydroxylase, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein- Activate 2 (TRP-2). Thus, upregulation of tyrosinase is associated with increased melanogenesis. In contrast, phosphorylation of MEK/ERK and P13K/AKT during extracellular signaling downregulates MITF, triggering an anti-melanogenic effect.

Most of the existing anti-melanin production agents are tyrosinase inhibitors obtained from synthetic raw materials. These tyrosinase inhibitors have been attracting attention in the cosmetic industry as a skin bleaching agent and in the food industry as a food additive, and act as various types of inhibitory mechanisms.

In order to provide an anti-melanin agent derived from a natural product, many plants have been reported internationally. Plant extracts show an effect on melanin production through inhibition of melanin biosynthesis in melanocytes or melanoma cells. Some plants are known to reduce the production of melanin by reducing the expression of MITF, tyrosinase, and tyrosinase-related proteins, and have been proposed as promising candidates for the treatment of hyperpigmentation disorders or whitening (non-patent literature). 01).

Sophora tonkinensis root, as the root and rhizome of Tojibisari belonging to the legume family, has been used as a traditional medicinal plant with analgesic or blood pressure lowering effects in the oriental region since ancient times. Neurodegenerative disease or cognitive function improvement (Patent Document 01), anti-tumor activity (Patent Document 02), or antiviral activity against hepatitis B virus (Patent Document 03), etc. with respect to the physiological and pathological activity of the biceps extract it is known

However, there has been no study or description of the effects of dry acid muscles on skin aging and melanin production.

Patent Document 01: KR 10-2010-0002432 (2010-01-11) Patent Document 02: CN 201811101728.8 (2018-09-20) Patent Document 03: CN 201510874926.8 (2015-12-03)

Non-Patent Document 01: Jeong, M.-H., Yang, K.-M., Kim, J.-K., Nam, B.-H., Kim, G.-Y., Lee, S.- W., Seo, S.-Y., Jo, W.-S., 2013. Inhibitory effects of Asterina pectinifera extracts on melanin biosynthesis through tyrosinase activity. Int. J. Mol. Med. 31, 205212.

Accordingly, the present inventors have made an earnest effort to provide a natural product-derived anti-melanin generating agent, and as a result, it was confirmed that Sophora tonkinensis root extract or a fraction thereof inhibits tyrosinase activity and melanin synthesis, and completed the present invention.

Accordingly, it is an object of the present invention to provide a composition for skin whitening or preventing, improving or treating skin hyperpigmentation, comprising an acid muscle extract or a fraction thereof.

The present invention provides a cosmetic composition for skin whitening comprising an Sophora tonkinensis root extract or a fraction thereof.

According to a preferred embodiment of the present invention, the acid root extract may be extracted using water, an organic solvent, or a mixture thereof as a solvent.

According to a preferred embodiment of the present invention, the fraction of the biceps root extract may be fractionated using at least one selected from the group consisting of nucleic acid, chloroform, ethyl acetate, butanol and water as a solvent. .

The present invention also provides a pharmaceutical composition for preventing or treating skin hyperpigmentation-related diseases, including a Sophora tonkinensis root extract or a fraction thereof.

According to a preferred embodiment of the present invention, the hyperpigmentation-related diseases include freckles, senile spots, liver spots, melasma, brown or dark spots, solar pigmentation, cyanic melasma, hyperpigmentation after drug use, pregnancy It may be at least one selected from the group consisting of gravidic chloasma and hyperpigmentation after injury or inflammation.

The present invention also provides a health functional food composition for preventing or improving skin hyperpigmentation-related diseases, including a Sophora tonkinensis root extract or a fraction thereof.

Since the acid muscle extract or a fraction thereof of the present invention can inhibit tyrosinase activity and melanin synthesis in a concentration-dependent manner, it can be effectively used as a composition for skin whitening or preventing, improving or treating skin hyperpigmentation.

Figure 1 is a mushroom tyrosina by ethanol extract (STR-E) and chloroform (STR-EC), ethyl acetate (STR-E-EA), butanol (STR-EB) and water (STR-EW) fractions of sanguin A graph is shown of the relative inhibition (%) of the enzyme activity. Statistically significant differences from the negative control group were expressed as ***P < 0.001, **P < 0.01 and *P < 0.05.
Figure 2 shows the cytotoxicity test results of STR ethanol extract (STR-E), chloroform (STR-EC) and ethyl acetate (STR-E-EA) fractions in B16F10 melanoma cells by MTT analysis. Data are presented as mean±SEM of 3 replicates. Statistically significant differences from the negative control group were expressed as ***P < 0.001 and **P < 0.01.
Figure 3 shows the effect of chloroform (STR-EC) and ethyl acetate (STR-E-EA) fractions of ethanol extract (STR-E) extracted from acid muscle on the tyrosinase activity of cells. A statistically significant difference from the control group of B16F10 melanoma cells stimulated with α-MSH was indicated as ###P < 0.001.
Figure 4 shows the effect of the chloroform (STR-EC) and ethyl acetate (STR-E-EA) fractions of the ethanol extract (STR-E) extracted from the biceps muscle on melanogenesis. Statistically significant differences from the control B16F10 melanoma cells stimulated with α-MSH were indicated by ###P < 0.001 and ##P < 0.05.
5 shows the results of Western blot analysis of the effect of STR-EC at various concentrations (10, 20 and 30 μg/ml) on MITF and tyrosinase expression in B16F10 melanoma cells stimulated with α-MSH. β-actin was used as a loading control.
Figure 6 shows the band intensities after western blot analysis of the effect of STR-EC at various concentrations (10, 20 and 30 μg/ml) on MITF and tyrosinase expression in B16F10 melanoma cells stimulated with α-MSH. The results measured by the Chemi-doc imaging system are shown.

The ethanol extract (STR-E) and the four fractions of the present invention showed concentration-dependent inhibitory activity on mushroom tyrosinase, respectively (Example 3), and α-MSH In stimulated B16F10 melanoma cells, STR-E and two selected fractions inhibited cellular tyrosinase activity and melanin synthesis (Examples 5 and 6). In addition, the inhibitory effect of the chloroform fraction of STR-E (STR-E-C) on melanogenesis in α-MSH-stimulated B16F10 melanoma cells was shown in relation to the MITF pathway (Example 7).

Accordingly, the present invention can provide a cosmetic composition for skin whitening comprising an Sophora tonkinensis root extract or a fraction thereof.

'Whitening' of the present invention may refer to any action that inhibits, prevents, or improves skin pigmentation by inhibiting the production of melanin.

According to a preferred embodiment of the present invention, the acid root extract may be extracted using water, an organic solvent, or a mixture thereof as a solvent. The solvent is preferably water or C ₁ to C ₄ lower alcohol, more preferably water, methanol, ethanol or propanol, and most preferably ethanol as the solvent. The ethanol may be 1% to 100% ethanol. The acid muscle extract may be included at a concentration of less than 10 μg/ml, but may exhibit toxicity when included at a concentration of 10 μg/ml or more.

According to a preferred embodiment of the present invention, the fraction of the biceps root extract is prepared by using the biceps extract as a solvent at least one selected from the group consisting of nucleic acid, chloroform, ethyl acetate, butanol, and water. It may be fractionated. Preferably, the fraction of the biceps root extract may be fractionated using any one or more selected from the group consisting of chloroform, ethyl acetate, butanol and water as a solvent.

The chloroform fraction of the biceps root extract may be included at a concentration of less than 20 μg/ml, and may exhibit toxicity when included at a concentration of 20 μg/ml or more. Preferably, the chloroform fraction of the acid muscle extract may be included at a concentration of 5 or more and less than 20 μg/ml, and more preferably, it may be included at a concentration of 10 μg/ml.

The ethylacetate fraction of the sanguin extract may be included at a concentration of less than 2 μg/ml, but may exhibit toxicity when included at a concentration of 2 μg/ml or more. Preferably, the ethylacetate fraction of the acid muscle extract may be included at a concentration of 0.1 or more and less than 2 μg/ml, and more preferably, it may be included at a concentration of 1 μg/ml.

The cosmetic composition of the present invention contains an extract of lichen serrata or a fraction thereof as an active ingredient, and a basic cosmetic composition (lotion, skin protectant, cream, essence, face wash such as cleansing foam and cleansing water, pack, Body oil, whole body cleanser), color cosmetic composition (foundation, lipstick, mascara, makeup base), hair product composition (shampoo, conditioner, scalp cleaner, hair conditioner, hair gel), and soap.

The excipient is not limited thereto, but may include, for example, an emollient, a skin penetration enhancer, a colorant, a fragrance, an emulsifier, a thickening agent, and a solvent. In addition, fragrances, dyes, bactericides, antioxidants, preservatives and moisturizing agents may be additionally included, and thickeners, inorganic salts, synthetic polymer materials, etc. may be included for the purpose of improving physical properties. For example, in the case of preparing a face wash and a soap with the cosmetic composition of the present invention, it can be easily prepared by adding the acid root extract or a fraction thereof to a conventional face wash and soap base. In the case of preparing a cream, it can be prepared by adding an extract or a fraction thereof to a general oil-in-water type (O/W) cream base. Here, synthetic or natural materials such as proteins, minerals, vitamins, etc. for the purpose of improving physical properties such as fragrances, chelating agents, pigments, antioxidants, and preservatives may be additionally added.

Although not limited to this, the content of the algae extract or a fraction thereof contained in the cosmetic composition of the present invention is preferably 0.001 to 10% by weight, more preferably 0.01 to 5% by weight, based on the total weight of the composition. If the content is less than 0.001% by weight, the desired skin whitening effect cannot be expected, and if it exceeds 10% by weight, there may be difficulties in safety or formulation.

The present invention also provides a pharmaceutical composition for preventing or treating skin hyperpigmentation-related diseases, including a Sophora tonkinensis root extract or a fraction thereof.

Since the acid muscle extract or its fractions are the same as those included in the cosmetic composition, the description is replaced with the description thereof.

The term 'prevention' of the present invention refers to any action that suppresses or delays the onset of hyperpigmentation-related diseases due to the biceps muscle or its fractions of the present invention.

'Improvement' or 'treatment' of the present invention means any action that improves or benefits the parameters related to hyperpigmentation-related diseases, for example, the degree of symptoms due to the acid muscle or a fraction thereof of the present invention.

According to a preferred embodiment of the present invention, the hyperpigmentation-related diseases include freckles, senile spots, liver spots, melasma, brown or dark spots, solar pigmentation, cyanic melasma, hyperpigmentation after drug use, pregnancy It may be at least one selected from the group consisting of gravidic chloasma and hyperpigmentation after injury or inflammation.

The pharmaceutical composition of the present invention may be in various oral or parenteral formulations. When formulating the composition, one or more buffers (eg, saline or PBS), antioxidants, bacteriostatic agents, chelating agents (eg, EDTA or glutathione), fillers, bulking agents, binders, adjuvants (eg, aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrating agents or surfactants, diluents or excipients.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in one or more compounds, for example, starch (corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl - It is prepared by mixing cellulose or gelatin. For example, tablets or dragees can be obtained by blending the active ingredient with a solid excipient, grinding it, adding suitable adjuvants, and processing it into a granular mixture.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, solutions, emulsions, or syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, for example, wetting agents, sweeteners, fragrances or preservatives may be included. can In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant, and an anti-aggregating agent, flavoring, emulsifying agent, solubilizing agent, dispersing agent, flavoring agent, antioxidant, packaging agent, etc. , and may further include pigments and preservatives.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspension solutions, emulsions, lyophilized formulations, or suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerol, gelatin, etc. may be used.

The composition of the present invention may be administered orally or parenterally, and when administered parenterally, for external use; intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection; or transdermal administration; It can be formulated according to methods known in the art in the form of.

In the case of the injection, it must be sterilized and protected from contamination of microorganisms such as bacteria and fungi. For injection, examples of suitable carriers include, but are not limited to, water, ethanol, polyols (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and/or a solvent or dispersion medium containing vegetable oil. can More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) with triethanolamine or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. etc. can be used. In order to protect the injection from microbial contamination, it may further include various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In addition, in most cases, the injection may further contain an isotonic agent such as sugar or sodium chloride.

In the case of transdermal administration, forms such as ointment, cream, lotion, gel, external solution, pasta, liniment, and air are included. In the above, transdermal administration means that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin by topically administering the pharmaceutical composition to the skin.

The composition of the present invention is administered in a pharmaceutically effective amount. A pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level refers to the patient's disease type, severity, drug activity, drug sensitivity, and administration time. , route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. That is, the total effective amount of the composition of the present invention may be administered to a patient as a single dose, and may be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time. . In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the pharmaceutical composition of the present invention varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of disease. The daily dosage is preferably 0.01 to 50 mg, more preferably 0.1 to 30 mg per 1 kg of body weight per day based on the acid muscle extract or a fraction thereof when administered parenterally, and when administered orally may be administered in 1 to several divided doses so as to be administered in an amount of preferably 0.01 to 100 mg, more preferably 0.01 to 10 mg per 1 kg of body weight per day based on the acid muscle extract of the present invention or a fraction thereof. However, since it may increase or decrease depending on the route of administration, the severity of obesity, sex, weight, age, etc., the dosage does not limit the scope of the present invention in any way.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

The pharmaceutical composition of the present invention may also be provided in the form of an external application containing the sandsicum extract or a fraction thereof as an active ingredient. When the pharmaceutical composition for preventing or treating skin hyperpigmentation disease of the present invention is used as an external preparation for skin, additionally, a fatty substance, an organic solvent, a solubilizer, a thickening agent and a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, and a foaming agent (foaming agent), fragrance, surfactant, water, ionic emulsifier, nonionic emulsifier, filler, sequestering agent, chelating agent, preservative, vitamin, blocker, wetting agent, essential oil, dye, pigment, hydrophilic active agent, It may contain adjuvants commonly used in the field of dermatology, such as lipophilic active agents or any other ingredients commonly used in external preparations for skin, such as lipid vesicles. In addition, the above ingredients may be introduced in an amount generally used in the field of dermatology.

When the pharmaceutical composition for preventing or treating skin hyperpigmentation disease of the present invention is provided as an external preparation for the skin, the present invention is not limited thereto, but may be in the form of an ointment, a patch, a gel, a cream, or a spray.

The present invention also provides a health functional food composition for preventing or improving skin hyperpigmentation-related diseases, including a Sophora tonkinensis root extract or a fraction thereof.

Since the acid muscle extract or its fractions are the same as those included in the cosmetic composition, the description is replaced with the description thereof.

Since the skin hyperpigmentation-related disease is the same as the disease targeted in the pharmaceutical composition, the description is replaced with the description thereof.

The health functional food composition according to the present invention can be prepared in various forms according to conventional methods known in the art. Common foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods (eg, canned fruit, bottled, jam, marmalade, etc.), fish, meat and their processed foods (eg, ham, sausages) Corn beef, etc.), breads and noodles (eg udon noodles, soba noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (eg butter, cheese, etc.), edible vegetable oils and fats, margarine , vegetable protein, retort food, frozen food, various seasonings (eg, soybean paste, soy sauce, sauce, etc.) can be prepared by adding the extract of the present invention or a fraction thereof. In addition, the nutritional supplement is not limited thereto, but it can be prepared by adding the sanguin extract of the present invention or a fraction thereof to capsules, tablets, pills, and the like. In addition, the health functional food is not limited thereto, but for example, liquefied, granulated, and encapsulated so that it can be consumed (health drink) by preparing the extract or a fraction thereof of the present invention in the form of tea, juice, or drink. And it can be ingested by powdering. In addition, in order to use the acid root extract or a fraction thereof of the present invention in the form of a food additive, it may be prepared and used in the form of a powder or a concentrate. In addition, it can be prepared in the form of a composition by mixing the acid muscle extract of the present invention or a fraction thereof and a known active ingredient known to be effective in preventing or improving skin hyperpigmentation disease.

In the case of using the extract of the present invention or a fraction thereof as a health drink, the health drink composition may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional drink. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as taumatine, stevia extract; A synthetic sweetener such as saccharin or aspartame may be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition, the acid muscle extract or a fraction thereof of the present invention may be contained as an active ingredient in a health functional food composition for preventing or improving skin hyperpigmentation disease, and the amount is effective to achieve the skin hyperpigmentation disease prevention or improvement action. The amount is not particularly limited, but is preferably 0.01 to 100% by weight based on the total weight of the total composition. The health functional food composition of the present invention may be prepared by mixing the acid muscle extract or a fraction thereof with other active ingredients known to be effective in skin hyperpigmentation diseases.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives , glycerin, alcohol, or a carbonation agent. In addition, the health functional food of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it is obvious to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Preparation of Ethanol Extracts and Fractions from Biceps

STR ( Sophora tonkinensis root; 50 g) was chopped into small pieces, ground into a fine powder using a blender, and extracted with 500 ml of ethanol in a shaking incubator for 24 hours (STR-E). Aliquots of STR-E were subjected to Whatman No. 1 filtered using filter paper, reduced to 50 ml using a vacuum rotary evaporator at 60° C., and freeze-dried for 4 days to obtain a dried powder. Then, the dried powder was dissolved in 50 ml of methanol/distilled water (1:1, v/v), and the same volume of chloroform (fraction 1: STR-EC), ethyl acetate (fraction 2: STR-E-EA), n -Butanol (fraction 3: STR-EB) and distilled water (fraction 4: STR-EW) were re-extracted in that order. Each fraction was evaporated under reduced pressure at low temperature, lyophilized and pulverized for sample use.

Characterization of Ethanol Extracts and Fractions of Biceps

<2-1> Determination of total phenol content

To determine the total phenolic content of STR-E and the four fractions, 50 μl aliquots of methanolic gallic acid (0.024, 0.075, 0.105 and 0.3 mg/ml) solutions were dissolved in water for standard curve preparation. 500 μl of 10% Folin-Ciocalteau reagent and 400 μl of 1M sodium bicarbonate were mixed. After treatment in dark conditions for 30 minutes, absorption was read at 765 nm at 20°C and a standard curve (y = 140.3187 x + 3.4873, r2 = 0.9998) was determined. 50 μl of each sample (1 mg/ml in methanol) was mixed with the same reagent described above, and a reagent blank was prepared using methanol. After 30 minutes, absorbance was measured for the determination of plant phenolic, and the result was expressed as gallic acid equivalent (GAE) mg/g dry powder. All measurements were performed in triplicate.

As a result, the highest value of total phenol content was observed in STR-E-C (395.12 mg GAE/g), and the lowest value was observed in STR-E-W (97.22 mg GAE/g) (Table 1).

<2-2> Total flavonoid measurement

For determination of the total flavonoid content of STR-E and the four fractions, 20 μl of each sample (1 mg/ml in methanol) was mixed with 20 μl of 10% (w/v) aluminum nitrate and 4 μl of 1M potassium acetate. , mixed with 60 μl of methanol and 96 μl of distilled water. The mixture was kept at room temperature for 30 min and then the absorption at 415 nm was read using a UV-VIS spectrophotometer Libra S22 (Biochrom Ltd., Cambridge, UK). A standard curve (y = 32.5862 - 1.6915, r2 = 0.9999) was prepared by measuring the absorption of a quercetin solution in methanol (0-100 μg/ml) under the same conditions. Results were expressed as quercetin equivalent (QE) mg/g dry powder, and all measurements were repeated at least three times.

As a result, the highest value of total flavonoid content was observed in STR-E (51.67 mg QE/g), and the lowest value was observed in STR-E-W (6.49 mg QE/g) (Table 1).

<2-3> Total terpenoid measurement

The total terpenoid content of STR-E and 4 fractions was determined using linalool as a standard reagent. To 200 µl of each sample (1 mg/ml in methanol) was added 1.2 ml of chloroform and 20 µl of aluminum nitrate (10%). The mixture was thoroughly vortexed and incubated at room temperature for 3 minutes before the addition of 100 μl of concentrated sulfuric acid. The microcentrifuge tube containing the reaction mixture was incubated at room temperature for 30 minutes in the dark. When a reddish-brown precipitate formed, the supernatant reaction mixture was harvested. After adding 1.5 ml of methanol (95%, v/v) to a microcentrifuge tube, the precipitate was completely dissolved by vortexing, and the resulting mixture was transferred to a cuvette and absorbance at 538 nm for a methanol blank was measured. Results were calculated as linalool equivalents (mg/g) using the regression equation of the linalool standard curve (y = 80.5429 x + 18.3385, r2 = 0.9999). All measurements were repeated at least three times.

As a result, the highest value of total terpenoid content was observed in STR-E (31.82 mg LE/g), and the lowest value was observed in STR-E-W (24.16 mg LE/g) (Table 1).

extract/fraction Total phenol content
(mg GAE/g ± SD) Total flavonoid content
(mg QE/g ± SD) Total terpenoid content
(mg LE/g ± SD) EtOH 282.16±9.87 51.67±0.84 31.82±0.038 Chloroform 364.81±2.46 15.24±1.25 27.65±0.012 Ethyl acatate 395.12±1.87 8.64±1.26 26.15±0.008 Butanol 163.73±1.38 14.38±1.65 27.23±0.018 Water 97.22±2.70 6.49±0.82 24.16±0.012

Mushroom Tyrosinase Activity Inhibition Assay

The purpose of this study was to determine the potential to inhibit the mushroom tyrosinase activity of STR-E and the four fractions.

Inhibition of mushroom tyrosinase activity was achieved by diluting STR-E, STR-E-C, STR-E-EA, STR-E-B and STR-E-W in sodium phosphate buffer (0.1M, pH 6.8) and adding 5-1,000 in a 96-well plate. Concentrations in the μg/ml range were tested. First, 100 μl of the diluted sample was mixed with 20 μl of sodium phosphate and 50 μl of mushroom tyrosinase (110 U/ml). After that, 30 μl of L-DOPA (10 mM) dissolved in sodium phosphate buffer was added to each mixture and incubated at 37° C. for 20 minutes. Absorbance was measured at 490 nm with a time zero reading on a microplate reader (Bio-Rad Inc., Hercules, Calif.) at the start of the reaction. The inhibition rate of tyrosinase was calculated by the following [Equation 1] (A = absorbance of the enzyme lacking the sample; B = absorbance of the sample and enzyme; C = absorbance of the sample lacking the enzyme; D = the absorbance of the sample and the enzyme lack of absorbance). Dose-dependent inhibition experiments were performed in triplicate to determine the IC50 of each sample.

[Equation 1]

Inhibition rate = {1-(B-C)/(A-D)}×100

As a result, as shown in [Fig. 1], each sample inhibited mushroom tyrosinase in a concentration-dependent manner, and at a concentration of 1,000 μg/ml, inhibited the enzyme activity much more strongly than arbutin at the same concentration. This indicated that STR-E and four other fractions strongly bind to mushroom tyrosinase and effectively inhibit the enzyme activity. Among the four fractions, STR-E-B showed the highest inhibition of mushroom tyrosinase activity. The IC50s of STR-E, STR-E-C, STR-E-EA, STR-E-B and STR-E-W were 65.81, 63.10, 32.48, 30.81 and 64.36 μg/ml, respectively, when L-DOPA was used as a substrate. This suggests that STR has an inhibitory effect on mushroom tyrosinase activity.

Cytotoxicity assay

Rodent B16F10 melanoma cell line in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (10,000 U/100 μg/ml) at 37° C. in 5% CO ₂ cultured under conditions. After 5 min incubation, B16F10 cells were centrifuged, washed with Dulbecco's phosphate buffered solution (DPBS), and the sediment was resuspended in 1 ml DMEM containing 10% FBS. For continuous culture, 10 ml of DMEM containing 10% fetal bovine serum was transferred to 50 μl of the cell suspension into a new culture dish. When the B16F10 melanoma cell line reached 80-90% confluency, trypsinization and seedling were performed at a density of 1 × 10 ⁵ cells/well in a 96-well microtiter plate. After culturing for one day, MTT {3-(4,5 dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} analysis was performed to evaluate the cytotoxic effect of each sample, and PBS buffer was compared as a negative control. . Concentrations of STR-E (5 and 10 μg/ml), STR-EC (10, 20 and 30 μg/ml) and STR-E-EA (1,2 and 3 μg/ml) were found in B16F10 melanoma cells 24 processed over time. After washing the cells with DPBS, 90 μl of fresh DMEM medium containing 10 μl MTT (1 mg/ml) was added to each well. After incubation at 37° C. for 4 hours, the solution was replaced with 100 μl DMSO to dissolve intracellular formazan, and absorbance was measured at 595 nm. The relative viability was calculated by the following [Equation 2] for the PBS-treated control cells.

[Equation 2]

% Cytotoxicity = (1- Absorbance of target cells / Absorbance of control cells) × 100

Various concentrations of STR-E (5 and 10 μg/ml), STR-E-C (10, 20 and 30 μg/ml) and STR-E-EA (1, 2 and 3 μg/ml) at various concentrations by the above method for 48 hours As a result of treatment with B16F10 melanoma cells during the experiment, STR-E, STR-E-C and STR-E-EA did not show a cytotoxic effect (non-toxic) at concentrations of 5, 10 and 1 μg/ml, respectively (Fig. 2) . However, all concentrations of STR-E, STR-E-C and STR-E-EA were included for further experiments.

Cellular Tyrosinase Inhibitory Activity Assay

To observe tyrosinase inhibitory activity in B16F10 melanoma cells (2 x 10 ⁵ cells/well), cells were pretreated with 500 nM of α-MSH followed by various concentrations of STR-E (5 and 10 μg/ml). , STR-EC (10, 20 and 30 μg/ml) and STR-E-EA (1,2 and 3 μg/ml) or arbutin (30 μg/ml). After incubating the plates in a CO2 incubator at 37°C for 72 hours, the cells were harvested and washed twice with 1x PBS. To observe tyrosinase activity, cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.5, 159 mM NaCl, 1% Nonidet P-30, 0.5% sodium deoxycholate, 0.1% SDS) and 14,000 for 30 min. Centrifuged at xg. After protein quantification by Bradford dye binding assay (Bio-Rad Laboratories, USA) using bovine serum albumin as a standard, the supernatant (40 μg/ml) was mixed with 100 μl of L-DOPA (2 mg/ml). ), put in 0.1 M sodium phosphate buffer (pH 6.8), and incubated at 37°C for 1 hour.

Because rodent tyrosinase is similar to human tyrosinase, several studies have used B16F10 melanoma cells of choice to test tyrosinase inhibition.

As a result, as shown in [Fig. 3], in this study, B16F10 cells treated with α-MSH alone significantly increased the relative cellular tyrosinase activity (189.4%) compared to untreated control cells (100%). did it In cells treated with STR-E (10 μg/ml), STR-E-C (10, 20 and 30 μg/ml) and STR-E-EA (2 and 3 μg/ml) compared to α-MSH treated control Tyrosinase activity was significantly inhibited by 34.1%, 44.4%, 49.6%, 59.4%, 38.3%, and 47.6%, respectively. STR-E (5 μg/ml) and STR-E-EA (1 μg/ml) inhibited cellular tyrosinase activity by 20.1% and 18.9%, but statistically significant between each treatment and the α-MSH treated control group. There was no difference.

Analysis of the effect of inhibiting melanin synthesis

B16F10 melanoma cells (2×10 5 cells/well) were cultured in the presence of α-MSH (500 nM) at 37° C. for 24 hours, followed by various concentrations of STR-E (5 and 10 μg/ml), STR-E-C (10, 20 and 30 μg/ml) and STR-E-EA (1,2 and 3 μg/ml), or arbutin (30 μg/ml). After incubating the plates in a CO incubator at 37 °C for 72 h, the cells were washed twice with 1x PBS, treated with Trypsin-EDTA, collected by centrifugation, and then incubated at 80 °C in 1N NaOH with 10% DMSO for 1 h. did. Absorbance was measured at 405 nm using a microplate reader.

To determine the inhibitory effect of STR-E, STR-E-C and STR-E-EA on melanin synthesis, each sample at a given concentration was treated with α-MSH-stimulated B16F10 melanoma cells.

As a result, as shown in [Fig. 4], arbutin (30 μg/ml), STR-E-C (10, 20 and 30 μg/ml) and STR-E-EA compared to the α-MSH treatment control group (133.3%) (2 and 3 μg/ml) melanin content of churagu increased by 28.5%, 16.8%, 17.8%, 27.4%, 16.8%, and 25.7%, respectively. STR-E (5 and 10 μg/ml) and STR-E-EA (1 μg/ml) treatment group slightly inhibited melanin synthesis, but there was no statistically significant difference between each treatment group and the α-MSH treatment control group.

Effect of STR-E-C on melanogenic protein expression in α-MSH-stimulated B16F10 melanoma

B16F10 melanoma cells (2 x 10 ⁵ cells/well) were cultured in the presence of α-MSH (500 nM) for 24 hours, followed by various concentrations of STR-EC (10, 20 and 30 μg/ml) or arbutin ( 30 μg/ml) were further treated in a CO ₂ incubator at 37° C. for 72 hours. Cells were lysed with RIPA buffer (Thermo Fisher Scientific, USA), incubated on ice for 1 h, and centrifuged at 14,000 x g for 30 min. After protein quantification by Bradford dye binding assay (Bio-Rad Laboratories, USA), cell lysates were adjusted with lysis buffer (20 μg/ml). After protein separation by 10% SDS-PAGE, the gel was rinsed with TBS (20 mM Tris, pH 7.4, 137 mM NaCl). The isolated protein was electrophoretically transferred to a polyvinylidene difluoride membrane at 4°C for 16 hours at a constant voltage. After blocking in TBS-T (TBS + 0.1% Tween 20) containing 5% skim milk at 37 °C for 4 hours, the membrane was diluted 1:200 with mouse polyclonal anti-MITF antibody (Santa Cruz Biotechnology, USA) , 1:500 diluted mouse polyclonal anti-tyrosinase antibody (Santa Cruz Biotechnology, USA) and 1:1,000 diluted mouse monoclonal anti-β-actin antibody (Santa Cruz Biotechnology, USA). After washing three times with TBS-T, horse peroxidase-linked anti-mouse IgG diluted 1:5,000 was treated. Bound antibodies were detected using an enhanced chemiluminescence (ECL) kit (ThermoFisher Scientific, USA). Band intensities were determined using a Chemi-doc Alliance Q9 Micro (UVITEC Ltd., UK) according to the manufacturer's instructions. An anti-β actin antibody was used to assess the equivalent loading of the amount of protein.

To elucidate the mechanism of inhibition of the sample on melanogenesis, STR-E-C was treated with α-MSH-stimulated B16F10 melanoma cells for 72 h. Protein levels of MITF and tyrosinase were evaluated by SDS-PAGE and Western blot analysis.

As a result, as shown in [Fig. 5] and [Fig. 6], both proteins were significantly decreased in melanoma cells stimulated with α-MSH in a concentration-dependent manner. As expected, STR-E-C exhibited an inhibitory effect on MITF expression and its downstream enzyme, tyrosinase, at the translational level through MITF inactivation in α-MSH-stimulated B16F10 melanoma cells.

Claims (6)

A cosmetic composition for skin whitening comprising a Sophora tonkinensis root extract or a fraction thereof.
[Claim 2] The cosmetic composition according to claim 1, wherein the extract is extracted using water, an organic solvent, or a mixture thereof as a solvent.
[Claim 2] The cosmetic composition according to claim 1, wherein the fraction of the biceps root extract is fractionated using at least one selected from the group consisting of nucleic acid, chloroform, ethyl acetate, butanol, and water as a solvent.
A pharmaceutical composition for preventing or treating skin hyperpigmentation-related diseases, comprising Sophora tonkinensis root extract or a fraction thereof.
5. The method of claim 4, wherein the hyperpigmentation-related disease is freckles, senile spots, liver spots, melasma, brown or dark spots, solar pigmentation spots, cyanic melasma, hyperpigmentation after drug use, gestational brown spots (gravidic) chloasma) and a pharmaceutical composition, characterized in that at least one selected from the group consisting of hyperpigmentation after injury or inflammation.
A health functional food composition for preventing or improving skin hyperpigmentation-related diseases comprising Sophora tonkinensis root extract or a fraction thereof.

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