KR20220002078A - Skin whitening composition comprising crataegus pinnatifida extract or fractions thereof - Google Patents

Abstract

The present invention relates to a composition for skin whitening, comprising an extract of Crataegus pinnatifida as an active ingredient. Since the extract of Crataegus pinnatifida of the present invention has excellent stability and can effectively inhibit melanin production through a mechanism of inhibiting the expression and activity of tyrosinase, the composition of the present invention comprising the same as an active ingredient exhibits an excellent whitening effect through inhibition of melanin production without irradiation to skin, thereby being usefully used as a functional cosmetic composition and a health functional food composition.

Description

Composition for skin whitening comprising hawthorn extract or a fraction thereof {SKIN WHITENING COMPOSITION COMPRISING CRATAEGUS PINNATIFIDA EXTRACT OR FRACTIONS THEREOF}

The present invention relates to a skin whitening composition comprising a hawthorn (Crataegus pinnatifida) extract or a fraction thereof, and more specifically, to a skin whitening cosmetic composition for inhibiting melanin production by including a hawthorn fruit extract as an active ingredient, a pharmaceutical It relates to a medical composition.

As the exposure of the skin to UV rays is increasing due to environmental pollution, the deposition of skin color due to skin aging is getting worse. In addition, as interest in skin pigmentation is increasing for cosmetic reasons, research to discover more stable and effective whitening materials is being actively conducted. Tyrosine, a kind of amino acid, is oxidized by tyrosinase to form dopa and dopaquinone, which in turn is 5,6-dihydroxy indole (5,6-dihydroxy indole, indole). It is automatically oxidized to 5,6-quinone) and finally to produce a melanin polymer by polymerization. Looking at related studies, research on materials that inhibit tyrosinase activity to inhibit melanin synthesis (arbutin, kojic acid, licorice extract, mulberry extract, etc.) , Research on UV-blocking materials (titanium dioxide, gamma oryzanol, oxybenzone, etc.), research on materials that are toxic to melanocytes (vitamin C, etc.) Research on removal materials (vitamin E, vitamin C) is being conducted. However, most of these are insufficient in effectiveness or incomplete in formulation, their use is limited in terms of skin stability, and high dependence on imports for raw materials. Recently, safer and more effective melanogenesis inhibitors have been developed. Many studies to be done are required. Therefore, research on functional whitening materials using various natural product extracts as replacement raw materials for whitening has also been actively conducted, and sagebrush, chrysanthemum extract, yulpi, mung bean, etc. are being used as whitening raw materials, and the development of an excellent alternative whitening agent is urgently needed.

On the other hand, hawthorn is round and red in appearance, and has white spots. The outer surface is yellowish brown or grayish reddish brown with many fine wrinkles. On one side, there is a recessed place with a diameter of 5 mm, and sometimes calyxes remain around it, and on the other side, a peduncle or mark remains. On the side cut sideways, there are 5 ovary chambers, and each chamber contains 1 seed. The seeds are divided into several balls, 6-8 mm long, light brown on the outside, hard, and hard in quality. Other names are Sansa (酸査), Sanlihong (山里紅), Red Fruit (紅果), Buksansa (北山査), Red Sweets (赤瓜子), Sanjohong (山棗紅), Red Sweets (紅果子), Yanggu Ruler (羊仇子), Billiard Ruler (棠子), Copycat (茅), Sanli Gua (山里果), Narrative (鼠査), Narrative (鼠), Yanggu (羊), Red Thread (赤爪實), Heir, It is also called Guja (子) and Gyemae (梅). The flowers of hawthorn are white in May, and the fruits are pomegranate, round and have white spots. In oriental medicine, it has been known to have effects such as stomach, digestion, convergence, and pain relief. Pharmacological ingredients include caffeic acid, citric acid, corosolic acid, crataegolic acid, euscaphic acid, hyperin, hypero Side (hyperoside), procatechuic acid (procatechuic acid), pyrogallol (pyrogallol), oleanolic acid (oleanolic acid), ursolic acid (ursolic acid), vitexin (vitexin), etc. have been reported. (Korean Patent No. 10-0570120) effect, antibacterial (Kim Jung-hyun et al., 2010) effect, and circulatory system disease (Ryu, HY et al., 2007) improvement effect, etc. have been reported.

As a prior document related to hawthorn, Korean Patent Application Laid-Open No. 10-2005-0079256 discloses a composition for the prevention and treatment of allergic diseases to a mixture containing eoseongcho, bokbunja mixed extract, and hawthorn additionally, and a prior thesis [Li, C. et al., 2011] disclosed that the water fraction of hawthorn hawthorn has an anti-inflammatory effect, but the fact that the fraction obtained by refractionation of hawthorn extract with an organic solvent can be used as a skin whitening agent or pigmentation improving agent has not yet been has not been disclosed until now.

The main object of the present invention is to provide a cosmetic composition for skin whitening comprising, as an active ingredient, a hawthorn extract having melanin production inhibition and extracellular secretion inhibitory activity.

Another object of the present invention is to provide a hawthorn-specific extraction method that can most effectively exhibit the skin whitening effect.

In order to achieve the object of the present invention as described above, the present invention provides a composition for skin whitening comprising an extract of Crataegus pinnatifida or a fraction thereof as an active ingredient.

The composition for skin whitening of the present invention can inhibit tyrosinase expression or inhibit melanin production, and the composition is formulated according to a conventional method known in the art, and is used as a cosmetic composition, a food composition, a pharmaceutical composition or a quasi-drug composition. can be used

The hawthorn (Crataegus pinnatifida) fruit extract of the present invention can be prepared by any one or more methods selected from the group consisting of reduced pressure high temperature extraction, hot water extraction, reflux extraction, hot water extraction, room temperature extraction, ultrasonic extraction and steam extraction method, but limited thereto it is not going to be

According to a preferred embodiment of the present invention, the fraction can be prepared by concentrating the hawthorn extract under reduced pressure and fractionating it with ethyl acetate, and the concentration of the ethyl acetate in the fractionation is 12.5 μg/mL to 100 μg/ mL, 12.5 μg/mL to 50 μg/mL, 12.5 μg/mL to 25 μg/mL, 25 μg/mL to 100 μg/mL, 50 μg/mL to 100 μg/mL, 150 μg/mL to 200 μg/mL, 200 μg/mL to 250 μg/mL mL.

The present invention also provides a pharmaceutical composition for preventing or treating skin hyperpigmentation disease, comprising an extract of Crataegus pinnatifida fruit or a fraction thereof as an active ingredient.

The fraction of the present invention can be prepared by concentrating the hawthorn extract under reduced pressure and fractionating it with ethyl acetate, and the concentration of the ethyl acetate in the fractionation is 12.5 μg/mL to 100 μg/mL, 12.5 μg/mL to 50 μg/mL, 12.5 μg/mL to 25 μg/mL, 25 μg/mL to 100 μg/mL, 50 μg/mL to 100 μg/mL, 150 μg/mL to 200 μg/mL, 200 μg/mL to 250 μg/mL.

According to an embodiment of the present invention, the hyperpigmented skin diseases that can be treated according to the composition of the present invention include freckles, hereditary contralateral dyspigmentation, reticular hyperpigmentation, nevus squamous, nevus pigmentosa, melasma, senile pigmentation. , vitiligo, Badenburg's syndrome, post-inflammatory pityriasis pityriasis, water droplets hypomelanosis, melasma and dandruff.

The present invention also provides a method for preparing a composition for skin whitening.

According to the present invention, a) hot water extraction of hawthorn fruit, b) concentrating the extract prepared according to step a) under reduced pressure, c) using ethyl acetate for the product of step b) After fractionation, there is provided a method for preparing a composition for skin whitening comprising a fraction of Crataegus pinnatifida fruit, comprising the steps of concentrating under reduced pressure and d) lyophilizing the product of step c).

According to an embodiment of the present invention, the concentration of ethyl acetate during fractionation in step c) is 12.5 μg/mL to 100 μg/mL, 12.5 μg/mL to 50 μg/mL, 12.5 μg/mL to 25 μg/mL , 25 μg/mL to 100 μg/mL, 50 μg/mL to 100 μg/mL, 150 μg/mL to 200 μg/mL, 200 μg/mL to 250 μg/mL.

The composition according to the present invention can be usefully used as a cosmetic composition, a food composition, a quasi-drug composition and a pharmaceutical composition for inhibiting melanin synthesis by including a hawthorn extract.

According to the present invention, it is possible to prepare the most effective hawthorn extract and its fractions for inhibiting melanin synthesis and for skin whitening.

FIG. 1A is a graph showing cell viability after treating B16F1 cells with hawthorn extract by concentration, and FIG. 1B is a graph showing cell viability after treatment with hawthorn extract and α-MSH (0.2 μM) at each concentration at the same time.
2 is a graph showing the content of melanin in B16F1 mouse melanoma cells by using a spectrophotometer and simultaneously processing hawthorn extract and α-MSH (0.2 μM) for each concentration.
3 is an image showing the color change of a cell pellet and a cell culture medium after treatment with a compound according to an embodiment of the present invention.
4 is a graph showing the content of B16F1 mouse melanoma cells treated with hawthorn extract and α-MSH (0.2 μM) at different concentrations at the same time, and the content of extracellular melanin using a spectrophotometer.
5A is a graph measuring tyrosinase activity after treating hawthorn extract by concentration to α-MSH and B16F1 cells, and FIG. 5B is a graph showing hawthorn extract by concentration treated with α-MSH (0.2 μM) and B16F1 This is an image confirming the expression of the protein (anti-MITF antibody) in the cell.
6 shows the efficacy of inhibiting mRNA expression of melanin synthesis-related products of hawthorn extract in B16F1 mouse melanoma cells induced with α-MSH according to an embodiment of the present invention. FIG. 6A is an image showing the mRNA expression level of tyrosinase when processing hawthorn extract, and FIG. 6B is an image showing the mRNA expression level of MITF.
7 is a schematic diagram showing a method for preparing a fraction using a hawthorn fruit extract according to an embodiment of the present invention.
8 is a graph showing the melanin content of B16F1 melanoma cells treated with a water extract or α-MSH (0.2 μM) fraction of hawthorn fruit according to an embodiment of the present invention. Data are expressed as mean ± SD (n = 3), with a significant difference of ^### p < 0.001 compared to the control group and ^* p <0.05, ^*** p < 0.001 compared to the α-MSH treated group. . In FIG. 8 , HF denotes a hexane fraction, CF denotes a chloroform fraction, EF denotes an ethyl acetate fraction, BF denotes a butanol fraction, WF denotes a water fraction, and CE denotes a hawthorn fruit extract.
9 is a graph showing the melanin content in B16F1 melanoma cells treated with a water extract of hawthorn fruit or an α-MSH (0.2 μM) ethyl acetate fraction according to an embodiment of the present invention. Data are presented as mean ± SD (n = 3), significant difference compared to control group is ^### p < 0.001, and significant difference compared to α-MSH treated group is ^** p < 0.01, ^*** p < 0.001 is displayed
10 is a graph confirming the effect of hawthorn fruit extract and its fractions on tyrosinase activity of B16F1 cells according to an embodiment of the present invention. Tyrosinase activity was determined in B16F1 cells in the absence or presence of α-MSH (0.2 μM). Data are expressed as mean ± SD (n = 3), significant difference compared to the control group is expressed as ^### p <0.001, and significant difference compared to the α-MSH treated group ^{is expressed as ***} p <0.001. In FIG. 10 , HF denotes a hexane fraction, CF denotes a chloroform fraction, EF denotes an ethyl acetate fraction, BF denotes a butanol fraction, WF denotes a water fraction, and CE denotes a hawthorn fruit extract.
11 is a graph confirming the effect of hawthorn fruit ethyl acetate fraction on tyrosinase activity in B16F1 cells according to an embodiment of the present invention. Tyrosinase activity was determined in B16F1 cells in the absence or presence of α-MSH (0.2 μM). In Figure 11, B16F1 cells were exposed to various concentrations of ethyl acetate fractions (12.5, 25 or 50 μg/mL) and hawthorn fruit extract for 72 h. In FIG. 11 , the data are expressed as mean ± SD (n = 3), a significant difference compared to the control group is ^### p <0.001, and a significant difference compared to the α-MSH treatment group ^{is expressed as ***} p <0.001.
12 is an image confirming the effect of hawthorn fruit ethyl acetate fraction on tyrosinase protein expression in B16F1 cells according to an embodiment of the present invention. In Figure 12, the cellular protein level of tyrosinase was investigated by Western blot analysis, where the data are expressed as mean ± SD (n = 3), and significant difference compared to control is ^### p < 0.001, a significant difference compared to the α-MSH treatment group ^{is expressed as *} p <0.05, ^*** p <0.001.

Hereinafter, the present invention will be described in more detail.

As described above, the existing inhibitors of melanin production have limitations in that the whitening effect is insignificant or lacks safety on the skin, so an alternative that can provide an excellent effect is required.

The 'hawthorn' of the present invention is a hawthorn fruit, which is a small tree belonging to the genus Rosaceae, is called hawthorn or hawthorn. The taste is sour, but the color is bright and it has a moderate sweetness, so it was made into jelly, canned food, alcohol, and tea.

The present inventors found that hawthorn fruit extract or a fraction thereof can provide a whitening effect by inhibiting tyrosinase activity and significantly inhibiting its melanin production under conditions that are not toxic to melanin-producing cells (Example) 1 to 4) were confirmed and the present invention was completed.

The hawthorn (Crataegus pinnatifida) extract of the present invention is characterized in that it exhibits a skin whitening effect by inhibiting the production of melanin in melanin-producing cells or inhibiting extracellular secretion. Specifically, it was confirmed that the hawthorn extract inhibited melanin synthesis and extracellular secretion in melanoma cells of mice (see FIGS. 2 to 4 ).

Accordingly, the present invention can provide a composition for whitening comprising a hawthorn 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.

The hawthorn fruit extract of the present invention may be prepared by using water, an organic solvent, or a mixture thereof as a solvent. The solvent is preferably water or a C1 to C4 lower alcohol, more preferably water, methanol, ethanol or propanol, and most preferably water may be used as the solvent.

The hawthorn fruit extract may be included in the composition at a concentration of 10 to 500 μg/ml, but preferably at a concentration of 300 to 500 μg/ml.

The hawthorn fruit extract may be prepared by any one or more methods selected from the group consisting of reduced pressure high temperature extraction, hot water extraction, reflux extraction, hot water extraction, room temperature extraction, ultrasonic extraction and steam extraction method, preferably reflux extraction method It is preferable to manufacture with After the extraction process, a purification process for increasing the purity of the extract, such as filtration, centrifugation or concentration process, may be additionally included. The concentration may be made by any one or more methods selected from the group consisting of precipitation concentration, evaporation concentration, reduced pressure concentration, ultrafiltration, reverse osmosis, and centrifugal separation, preferably concentration under reduced pressure.

The fraction of the hawthorn extract may be prepared after fractionation of the hawthorn extract using at least one selected from the group consisting of hexane, chloroform, ethyl acetate, butanol and water as a solvent, and the solvent during fractionation is ethyl acetate Most preferred.

The composition of the present invention may be prepared as a cosmetic composition, a food composition or a quasi-drug composition according to a conventional method widely used in the art.

The cosmetic composition of the present invention contains a hawthorn extract or a fraction thereof as an active ingredient, and a basic cosmetic composition (lotion, cream, essence, face wash such as lotion, cream, essence, cleansing foam and cleansing water, pack, body oil) together with a dermatologically acceptable excipient; It may be prepared in the form of a color cosmetic composition (foundation, lipstick, mascara, makeup base) 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 for the purpose of improving physical properties, thickeners, inorganic salts, synthetic polymer materials, and the like may be included. For example, when preparing a face wash and soap with the cosmetic composition of the present invention, it can be easily prepared by adding the hawthorn 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 a hawthorn 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 and vitamins for the purpose of improving physical properties such as fragrances, chelating agents, pigments, antioxidants, and preservatives may be additionally added.

When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal fiber, vegetable fiber, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. This can be used.

When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydro It may contain a propellant such as carbon, propane/butane or dimethyl ether.

When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene fatty acid esters of glycol, 1,3-butylglycoloil, glycerol aliphatic esters, polyethylene glycol or sorbitan.

When the formulation of the cosmetic composition of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.

When the formulation of the cosmetic composition of the present invention is surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate as carrier components , fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolin derivative or ethoxylated glycerol fatty acid ester, and the like may be used.

The content of the hawthorn extract or a fraction thereof contained in the cosmetic composition of the present invention is not limited thereto, but 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 whitening effect cannot be expected, and if it exceeds 10% by weight, there may be difficulties in safety or formulation.

The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food, food additives and feed, and includes animals including humans or livestock. target for eating. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a health food, the hawthorn fruit extract or a fraction thereof of the present invention may be prepared and consumed in the form of tea, juice, and drink, or granulated, encapsulated and powdered to be consumed. In addition, it can be prepared in the form of a composition by mixing the hawthorn fruit extract or a fraction thereof of the present invention with a known substance or active ingredient known to have a whitening effect.

Food compositions of this type 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) Corned 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 hawthorn fruit extract or a fraction thereof.

In addition, the nutritional supplement is not limited thereto, but may be prepared by adding hawthorn fruit extract or a fraction thereof to capsules, tablets, pills, and the like. In addition, although not limited to this as a health functional food, for example, hawthorn fruit extract or a fraction thereof is prepared in the form of tea, juice, and drink and liquefied, granulated, encapsulated and powdered so that it can be consumed (health drink). can be ingested. In addition, in order to use the hawthorn fruit extract or a fraction thereof 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 may be prepared in the form of a composition by mixing hawthorn fruit extract or a fraction thereof with an active ingredient known to have a whitening effect.

When the composition for skin whitening of the present invention is used as a health drink composition, 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.

The hawthorn fruit extract or a fraction thereof may be contained as an active ingredient in a food composition for whitening, and the amount is an effective amount to achieve a whitening effect, but is not particularly limited, but 0.01 to 100% by weight based on the total weight of the composition and 0.01 to 50% by weight is preferred. The food composition of the present invention can be prepared by mixing hawthorn fruit extract or a fraction thereof with other active ingredients known to have a whitening effect.

In addition to the above, the health food of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, It may contain glycerin, alcohol or a carbonation agent and the like. In addition, the health 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.

When the hawthorn extract of the present invention or a fraction thereof is used as a quasi-drug additive, the composition may be added as it is or used together with other quasi-drug ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prophylactic, health or therapeutic treatment).

Preferably, the quasi-drug composition may be a shower foam, detergent soap, hand wash or ointment.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating skin hyperpigmentation disease comprising a hawthorn extract.

The skin hyperpigmentation disease is melanoma, hyperpigmentation, such as freckles, hereditary contralateral dyspigmentation, reticular hyperpigmentation, nevus, such as congenital pigmentation such as nevus squamous, nevus pigmentosa, etc., and melasma, senile pigmentation, etc. acquired pigmentation disorders such as acquired pigmentation, and hereditary skin diseases such as vitiligo, Badenburg syndrome, acquired skin diseases such as post-inflammatory pityriasis pityriasis, unexplained water droplet hypomelanosis, melasma, skin diseases caused by medication such as minocycline, bleomycin, busulfan, zidovudine, and skin diseases that have spread through infection, such as rhinitis and the like.

The “prevention” refers to any action that reduces the frequency or degree of occurrence of pathological phenomena in animals. Prevention may be complete or partial. In this case, it may mean a phenomenon in which the disease caused by the skin deposition in the individual is reduced compared to the case where the composition is not used.

The "treatment" refers to any action that clinically intervenes to change the natural process of a subject or cell to be treated, and may be performed while a clinical pathology is in progress or to prevent it. The desired therapeutic effect is to prevent the occurrence or recurrence of a disease, alleviate symptoms, reduce any direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, alleviating or temporarily ameliorating the condition, or improving the prognosis.

The pharmaceutical composition may form a pharmaceutically acceptable acid addition salt according to a method conventional in the art. The free acid may be an organic acid or an inorganic acid, for example, the inorganic acid may be hydrochloric acid, hydrobromic acid, sulfuric acid, or phosphoric acid, and the organic acid is citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, Fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, benzenesulfonic acid, salicylic acid , nicotinic acid, isonicotinic acid, picolinic acid, glutamic acid or aspartic acid, but is not limited thereto. In addition, the pharmaceutical composition may further include commonly used carriers, excipients and diluents.

The composition may be administered in the form of oral delivery or parenteral delivery. The kaurenoic acid derivative compound may be administered systemically or locally, and the administration may include oral administration and parenteral administration. When the compound is administered as a composition, it may be formulated with a suitable amount of a pharmaceutically acceptable vehicle or carrier to provide an appropriate dosage form.

On the other hand, the pharmaceutical composition comprising the hawthorn extract may further include a carrier, excipient and diluent used in the preparation of the pharmaceutical composition. The carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

In addition, the composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, and sterile injection solutions.

The solid preparation for oral administration includes tablets, pills, powders, granules, capsules, and the like, and the solid preparation includes at least one excipient in the hawthorn extract and its fractions, for example, starch, calcium carbonate, sucrose, lactose. , or it can be prepared by mixing gelatin and the like. In addition to the above excipients, lubricants such as magnesium stearate and talc may be used.

As the liquid formulation for oral administration, suspensions, internal solutions, emulsions, syrups, etc. may be used, and in addition to simple diluents such as water and liquid paraffin, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. have.

A sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a lyophilized formulation, and a suppository may be used in the formulation for parenteral administration. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin oil, and glycerogelatin may be used.

The pharmaceutical composition comprising the hawthorn extract may be administered to a subject in a pharmaceutically effective amount. The “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 is dependent on the patient's type, severity, drug activity, and drug Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field.

The pharmaceutical composition 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 singly or multiple times. Taking all of the above factors into consideration, it is preferable to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent 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.

[Example 1]

Preparation of hawthorn extracts and fractions

<1-1> Preparation of hawthorn extract

The hawthorn extract (001-112) used in this study was obtained from the Korea Plant Extracts Bank (Daejeon, Korea) of the Korea Research Institute of Bioscience and Biotechnology. The above hawthorn was collected in Yongin-si, Gyeonggi-do in 2002. Add 1L of methyl alcohol 99.9% (HPLC grade) to the plant (60g) dried in the shade and use an ultrasonic extractor (ultrasonic extractor, SDN-900H, SD-ULTRASONIC CO., LTD) at room temperature for 30 cycles (40KHz, 1500 W, 15 min. ultrasonication-120 min. standing per cycle). After filtration and drying under reduced pressure, a hawthorn extract (20.40 g) was obtained. The received hawthorn extract (001-112) was dissolved in dimethyl sulfoxide (DMSO) at a high concentration (200 mg/ml), and then dissolved in sterile tertiary distilled water (100, 250, 500 μg/ml) and used in the experiment.

<1-2> Preparation of fractions of hawthorn extract

The hawthorn extract prepared in Example <1-1> was suspended in 100 ml of distilled water, the same amount of hexane (100 ml) was added, shaken, and then equilibrated to collect the hexane-soluble parts and concentrate the process 3 times in total to obtain a hexane fraction. secured. A chloroform fraction was obtained by sequentially performing the same method, and finally a water fraction was obtained.

[Example 2]

<2-1> Cell culture

B16F1, a mouse melanoma cell line, was used for the cells, and the cells were purchased from the Korean cell line bank (Korea Cell Line Bank). Using DMEM medium containing 10% fetal bovin serum (FBS) and 1% penicillin streptomycin, it was maintained in an incubator at 37 °C containing 5% CO₂. For maintenance, cells were subcultured once every two days.

<2-2> Confirmation of cytotoxicity

To confirm the cell viability of the hawthorn extract against the mouse melanoma cell line B16F1, MTT assay was performed. B16F1 cells were spread in a 24-well cell culture plate at a density of 2.5×10⁴. These cells were treated with hawthorn extract (100, 250, 500 μg/ml) and standard kojic acid (1 mM) for each concentration, and stimulated in the presence or absence of α-MSH (0.2 μM) at 37 °C for 48 hours. made it After 48 hours, 20 μL of MTT reagent (80 μg/ml) was put into each well and reacted for 1 hour at 37° C. under dark conditions. The medium containing the MTT reagent was removed, and 400 μL of DMSO was added to dissolve intracellular formazan. For absorbance measurement, 200 μL was transferred to a 96 well plate, and absorbance was measured at 552 nm. Cell viability was expressed as a percentage of the control group (see FIG. 1).

Referring to Figure 1, it can be confirmed whether hawthorn extract shows cytotoxicity in B16F1 melanoma cells. FIG. 1A is a graph showing cell viability after treating B16F1 cells with hawthorn extract by concentration, and FIG. 1B is a graph showing cell viability after treatment with hawthorn extract and α-MSH (0.2 μM) at each concentration at the same time.

Specifically, in FIG. 1A, B16F1 cells were treated with hawthorn extract (100, 250, 500 μg/ml) and standard kojic acid (1 mM) for 48 hours. In FIG. 1B , B16F1 cells were simultaneously treated with hawthorn extract (100, 250, 500 μg/ml), kojic acid (1 mM), and α-MSH (0.2 μM) as standards. The control group was treated with the MTT reagent (80 μg/ml, dark condition) after 48 hours using a medium that was not treated with the sample, and then the absorbance was measured to indicate the cell viability.

Referring to A and B of Figure 1, it can be seen that when the hawthorn extract was treated by concentration and when α-MSH was treated at the same time, it did not show any particular toxicity compared to the control group.

[Example 3]

<3-1> Melanin contents measurement (melanin content measurement in cells)

To measure the intracellular melanin content, B16F1 cells were spread on a 60 mm cell culture dish at a density of 5×10⁴. These cells were treated with hawthorn extract (100, 250, 500 μg/ml) and standard kojic acid (1 mM) for each concentration, and stimulated with α-MSH (0.2 μM) at 37° C. for 72 hours. After 72 hours, the cell pellet was collected and dissolved by reacting at 80° C. for 1 hour with 1M NaOH containing 10% DMSO. The content of melanin was shown by measuring the absorbance at 405 nm.

<3-2> Melanin contents measurement (extracellular melanin content measurement)

To measure the extracellular melanin content, a cell culture medium was used. After treatment with hawthorn extract (100, 250, 500 μg/ml) and standard kojic acid (1 mM) at 37 °C for 72 hours, B16F1 cells were stimulated with α-MSH (0.2 μM) and then cell cultured After transferring 150 μL of the medium to a 96-well plate, absorbance at 405 nm was measured and indicated.

)를 동시에 처리하고, 세포 내 멜라닌을 분광광도계를 이용하여 그 함량을 나타낸 그래프이며, 도 4는 동일한 방식으로 세포 외 멜라닌을 분광 광도계를 이용하여 함량을 나타낸 그래프이다. 도 3은, 세포 펠렛(pellet)과 세포배양 배지에 실시예 3에 따라 산사 추출물을 처리한 후 색의 변화를 나타낸 이미지이다. 구체적으로, 도 2 및 4에서는 B16F1 마우스 흑색종 세포에 농도별 산사 추출물 (100, 250, 500 μg/ml)과 표준물질인 kojic acid (1 mM) 그리고 α-MSH (0.2 μM)를 동시에 처리하고, 72시간 후에 세포외/세포내 멜라닌을 분광광도계를 이용하여 그 함량을 나타내었다. 405nm에서 흡광도를 측정하여 대조군의 %로 멜라닌의 함량을 나타내었다. 도 3에서는 세포 펠렛(pellet)과 세포배양 배지 색이 산사 추출물을 처리한 후 변화하는 것을 변화 강도와 함께 나타낸다.The content of melanin in FIGS. 2 and 4 was expressed as a percentage of the control group. Figure 2 shows the concentration of hawthorn extract and α-MSH (0.2 μm) in B16F1 mouse melanoma cells.

) is treated simultaneously, and is a graph showing the content of intracellular melanin using a spectrophotometer, and FIG. 4 is a graph showing the content of extracellular melanin using a spectrophotometer in the same manner. Figure 3 is an image showing the change in color after treating the hawthorn extract according to Example 3 to a cell pellet and a cell culture medium. Specifically, in FIGS. 2 and 4, B16F1 mouse melanoma cells were simultaneously treated with hawthorn extract (100, 250, 500 μg/ml) and standard materials kojic acid (1 mM) and α-MSH (0.2 μM). , the content of extracellular/intracellular melanin was measured using a spectrophotometer after 72 hours. Absorbance was measured at 405 nm to indicate the content of melanin as a percentage of the control. In FIG. 3, changes in the color of the cell pellet and the cell culture medium after treatment with the hawthorn extract are shown together with the change intensity.

Melanin content measurement results according to Example 3 are shown in FIGS. 2 to 4 . 2 to 4, it can be confirmed by comparing the hawthorn extract and the standard material (kojic acid) in α-MSH-induced B16F1 mouse melanoma cells to determine whether the hawthorn extract inhibits the production of melanin, the final product of the melanin biosynthesis process. have. The production of melanin was induced in the α-MSH alone treatment group, and it can be confirmed that the content of extracellular/intracellular melanin was reduced to a level similar to that of the control group in a concentration-dependent manner when the hawthorn extract was treated. The decrease in melanin can be confirmed by the color of the cell pellet and the cell culture medium after treatment with the hawthorn extract. Therefore, it is shown that hawthorn extract is effective in reducing the production of melanin, the final product of the melanin biosynthesis process.

[Example 4]

Western blot analysis (protein expression analysis)

B16F1 cells were spread on a 60 mm cell culture dish at a density of 5×10⁴, and hawthorn extract (100, 250, 500 μg/ml) and standard kojic acid (1 mM) were mixed with α-MSH (0.2 μM) and They were reacted together at 37 °C for 72 hours. After 72 hours, the remaining medium and other residues were washed twice with phosphate buffered saline (PBS). Cell pellets were collected, and proteins were extracted with a lysis buffer (RIPA buffer) containing a protein inhibitor. The extracted protein was quantified by DC protein assay, and the quantified protein was separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) and nitrocellulose (NC) membranes. The membrane was blocked with 5% non-fat dry milk dissolved in Tris-buffered saline-Tween 20 solutions (TBS-T), and reacted with a primary antibody at 4 °C for 24 hours. After 24 hours, the reaction was carried out at room temperature for 1 hour, rinsed with TBS-T, and then reacted with horseradish peroxidase secondary antibody (anti-mouse and anti-rabbit IgG) at room temperature for 2 hours. Protein bands were visualized using western HRP substrate and ImageQuant LAS 500 (GE Healthcare Life Science, USA).

The protein expression analysis according to Example 4 is shown in FIG. 5 . 5A is a graph measuring tyrosinase activity after treating hawthorn extract by concentration to α-MSH and B16F1 cells, and FIG. 5B is a graph showing hawthorn extract by concentration treated with α-MSH (0.2 μM) and B16F1 This is an image confirming the expression of the protein (anti-MITF antibody) in the cell. Specifically, in FIG. 5A, after treatment for 72 hours in the presence or absence of hawthorn extract (100, 250, 500 μg/ml), standard kojic acid (1 mM) and α-MSH (0.2 μM) by concentration, The tyrosinase activity was measured, and in FIG. 5B, hawthorn extract (100, 250, 500 μg/ml) by concentration, kojic acid (1 mM), and α-MSH (0.2 μM) as a standard substance were present for 3 hours in the presence or absence. After treatment, protein expression was confirmed (anti-MITF antibody).

MITF is a microphthalmia-associated transcription factor, a transcription factor known to regulate the expression of various genes essential for melanin synthesis in melanocytes. In addition, it is involved in the regulation of melanocyte differentiation, pigmentation, proliferation and survival. The signaling pathway related to MITF regulates the production of melanin, and when melanocyte-stimulating hormone (α-MSH) produced from ultraviolet light binds to the Melanocortin 1 receptor (MC1R), it is essential for melanin synthesis through a series of signal transduction processes. It induces the synthesis of melanin by increasing the expression of genes tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein 2. Among them, tyrosinase is the main enzyme regulating the production of melanin. ① Oxidation of L-tyrosine to Dopaquinone ② Oxidation of L-tyrosine to L-Dopa ③ 5,6-dihydroxyindole (DHI) to indole-2-carboxtlic acid-5, It is involved in oxidation to 6-quinone (IQ).

Referring to FIG. 5 , the activity of tyrosinase was induced by α-MSH, and it was confirmed that the activity was decreased in a concentration-dependent manner by treating the hawthorn extract for each concentration. In addition, protein expression of MITF was also induced by α-MSH, which confirmed that the expression was reduced by treating the hawthorn extract. Therefore, hawthorn extract inhibits the enzymatic activity of tyrosinase, which plays an important role in melanin synthesis, and suppresses the protein expression of MITF, indicating that it is effective in reducing melanin production.

[Example 5]

Reverse-Transcription Polymerase Chain Reaction analysis (mRNA expression analysis)

B16F1 cells were spread at a ^{density of 3Х10 5} in a 60 mm cell culture dish. After 24 hours, hawthorn extract (100, 250, 500 μg/ml) and standard kojic acid (1 mM) were pre-treated and 1 hour later, α-MSH (0.2 μM) was treated. After reacting at 37°C for 36 hours, 1 mL of Trizol reagent (ambion) was added to the dish from which the culture medium was removed, and after reacting for 5 minutes, it was recovered in a 1.5 mL tube. Cholroform (sigma aldrich, USA) 200 μL was added, and the solution was vortexed until it became light pink. After the solution was mixed, it was reacted for 5 minutes, and the solution was separated by centrifuge (12000rpm, 10 minutes, 4℃). After carefully collecting 500 μL of the supernatant (aqueous layer, aqueouse phase) from among the separated layers, it was transferred to a 1.5 mL tube. 500 μL of iso-prophyl alcohol (sigma aldrich, USA) was added, mixed by inverting, and then reacted for 5 minutes. The RNA was precipitated by centrifuge (12000rpm, 10 minutes, 4℃), and to remove the remaining organic solvent, 1 mL of 75% Ethanol DEPC-treated water was added and centrifuge (12000rpm, 5 minutes, 4℃) was performed twice. did After waiting for the pellet to dry, DEPC-treated water was added to dissolve the pellet.

To quantify the extracted RNA, the concentration was measured using a QuickDrop Micro-Volume Spectrophotometer (molecular devices, US). After measuring the RNA concentration to be 2500 ng/μl, add a random primer and Oligo dT primer (Promega, USA) to attach the primer first, then goScript™ 5X reaction buffer, MgCl2, dNTP, GoScript™ Reversr transcriptase ( Promega, USA) was additionally added to finally synthesize double-stranded cDNA. 80 μl of DEPC was added to the synthesized cDNA and diluted to 500 ng/μl.

To proceed with PCR, put 5X Green Gotaq reaction buffer, Forward primer, Reverse primer, dNTP, MgCl2, Gotaq DNA polymerase, and template (cDNA) into a PCR tube and use ①tyrosinase (Tm:55 ℃) with a thermal cycler (BIO-RAD, USA). , 107bp, forward primer:5'-CTC TGG GCT TAG CAG TAG GC-3', reverse primer: 5'-GCA AGC TGT GGT AGT CGT CT-3') ②MITF(Tm:55 ℃, 273bp, forward primer:5 '-CAG CC ATAA ACG TCA GTG TG-3', reverse primer: 5'-GAG TGA GCA TAG CCA TAG GG-3') ③GAPDH (PCR was performed according to the conditions. After PCR, 1.5% agarose gel (Biosesang, seungnam, KOREA) to confirm expression.

6 shows the effect of inhibiting mRNA expression of melanin synthesis-related products of hawthorn extract in B16F1 mouse melanoma cells induced with α-MSH according to Example 5. FIG. 6A shows the mRNA expression level of tyrosinase when treating hawthorn extract, and FIG. 6B shows the mRNA expression level of MITF. In FIG. 6A , α-MSH (0.2 μM) was treated 1 hour after pretreatment with hawthorn extract (100, 250, 500 μg/ml) and standard kojic acid (1 mM) for each concentration. After reacting for 36 hours, RT-PCR was performed. Referring to FIG. 6 , the mRNA of α-MSH tyrosinase and MITF was expressed, and it can be confirmed that the expression is decreased in a concentration-dependent manner of the hawthorn extract. This indicates that the hawthorn extract has the effect of suppressing the mRNA level of products involved in melanin synthesis and finally inhibiting the synthesis of melanin.

Summarizing the above examples of the present invention, the hawthorn extract showed the inhibition of mRNA levels of Tyrosinase and MITF, which play an important role in the biosynthesis of melanin in B16F1, a mouse melanoma cell induced by α-MSH. Inhibition of this level inhibited the tyrosinase enzyme activity, protein expression, and MITF protein expression, and it can be confirmed that the reduction of melanin, the final product, was achieved. This showed higher inhibition of tyrosinase activity and mRNA level than the standard kojic acid. Therefore, it indicates that the hawthorn extract can be used as a raw material for cosmetics for whitening, a composition for skin whitening for suppression of melanin production, and a pharmaceutical composition for preventing or treating skin hyperpigmentation disease.

Hereinafter, the whitening activity according to the method of extracting hawthorn fruit is confirmed.

[Experimental Example 1]

Statistical processing

All experimental values were expressed as mean ± standard deviation. Statistical analysis was processed by analysis of variance (ANOVA) using GraghPad 5.0, and the significance limit was <0.05, followed by post-hoc analysis by Duncan's multiple range test. carried out.

Experimental Example 1-1. Preparation of hydrothermal extract of hawthorn fruit

A hot water extract was prepared using hawthorn fruit, distilled water 10 times the weight of 100 g was added, extracted at 95 ° C. for 2 hours, filtered to filter out the residue, concentrated under reduced pressure, and freeze-dried to use as a hot water extract. Extracted in the same way The hot water extract was concentrated under reduced pressure to prepare 200 ml and used for the organic solvent fraction.

Experimental Example 1-2. Preparation of organic solvent fraction of hawthorn fruit hot water extract

Fractions were obtained by adding 200 ml of an organic solvent three times to 200 ml of hawthorn fruit hot water extract. As organic solvents, n-hexane, chloroform, ethyl acetate, and butanol were sequentially used for fractionation. When the organic solvent layer and the water layer were separated by standing at room temperature for each solvent step, only the organic solvent layer was recovered, concentrated under reduced pressure, and then freeze-dried and used as a sample of the organic solvent fraction (see FIG. 7 ).

As a result, 100 g of hawthorn fruit was extracted with water to obtain 5.8 g of a water extract, and the water extract obtained by water extraction of 100 g of hawthorn fruit was concentrated under reduced pressure to prepare 200 mL of hot water extract, which was then mixed with hexane, chloroform, ethyl acetate, butanol and water. The fractions were concentrated under reduced pressure and then lyophilized to obtain 0.041 g of a hexane fraction, 0.652 g of chloroform, 0.464 g of an ethyl acetate fraction, 3.724 g of a butanol fraction, and 2.014 g of a water fraction.

Experimental Example 1-3. Effect of hawthorn hot water extract and fractions on the inhibition of melanin synthesis

Melanocytes (B16F1) ^{were inoculated into 5Х10 4} cells in a 60mm dish. The next day after inoculation, the sample and the positive control group (kojic acid, 1 mM) were treated, and the remaining groups except the Control were treated with α-MSH (α-melanin stimulating hormone, 0.2 μM) that induces melanin production. After 72 hours, cells were recovered, dissolved in 1N NaOH containing DMSO at 80° C. for 20 minutes, and then intracellular melanin was measured at absorbance at 405 nm. The melanin production amount of each group was compared based on 100% of the control not treated with α-MSH.

8 and 9, the melanin content was measured using melanoma cells in order to confirm the whitening effect of the hot water extract of hawthorn fruit and the organic solvent fraction according to the above method. As a result of measuring the melanin content in the hexane, chloroform, ethyl acetate, butanol and water fractions of the hawthorn hot water extract and the hot water extract, at a treatment concentration of 50 μg/mL, ethyl acetate fraction > hawthorn water extract > hexane fraction > butanol fraction > In the order of water fraction> chloroform fraction, the melanin synthesis inhibition rate was excellent (see FIG. 8 ). Melanin synthesis was inhibited in the organic solvent fractions except for the chloroform fraction, but the melanin content of the chloroform fraction was increased more than that of the α-MSH-treated group. Thereafter, the ethyl acetate fraction, which had the best inhibition of melanin synthesis among the fractions, was treated at concentrations of 50 μg/mL, 25 μg/mL, and 12.5 μg/mL, and hawthorn hot water extract was used as a comparison group at 50 μg/mL and 100 μg/mL. mL and kojic acid, a positive control, was treated with 1 mM, and then the melanin content was compared. When α-MSH, which induces melanin production, was treated alone, the melanin content increased to 175% compared to the control, and when water extracts of hawthorn berries were treated at concentrations of 50 μg/mL and 100 μg/mL, 161%, 150 % melanin content was suppressed (see FIG. 9). In particular, when the ethyl acetate fraction was treated at concentrations of 12.5 μg/mL, 25 μg/mL, and 50 μg/mL, the melanin content was inhibited to 156%, 147%, and 117%, respectively. When the water extract and ethyl acetate fraction of hawthorn fruit were treated at the same concentration of 50 μg/mL, the ethyl acetate fraction inhibited melanin synthesis by 20% compared to the water extract, and the effect of inhibiting melanin production compared to kojic acid, a positive control. This was better.

Experimental Example 1-4. Measurement of tyrosinase activity of hawthorn hot water extracts and fractions

The melanocytes (B16F1) was treated after a sample and a positive control kojic acid inoculated with ⁴ 5Х10 density on 60mm dish, it processes the α-MSH to induce melanin synthesis in the other groups except the Control. After 72 hours, cells were recovered and protein was extracted using a lysis buffer. After quantifying the protein, the same amount of protein for each group was put in a 96-well plate, and L-dopa (2mM) was added thereto, followed by reaction at 37° C. for 1 hour, and absorbance was measured at 450 nm. The tyrosinase activity of each group was measured based on 100% tyrosinase activity of the control not treated with α-MSH.

As a result of measuring the tyrosinase activity of hawthorn hot water extracts and fractions, the tyrosinase activity was 386 when only a-MSH was treated with 100% of the untreated control group at a non-toxic concentration of 50 μg/mL. %, and 353%, 330%, 242%, 288%, and 350% of tyrosinase activity were exhibited in the hexane fraction, chloroform fraction, ethyl acetate fraction, butanol fraction, and water fraction, respectively. In particular, the ethyl acetate fraction was superior to the water extract of hawthorn fruit or the kojic acid-treated group, which is a positive control, in inhibiting tyrosinase (see FIG. 10 ). Therefore, thereafter, the ethyl acetate fraction was treated at concentrations of 50 µg/mL, 25 µg/mL, and 12.5 µg/mL, and the hawthorn hot water extract was treated at 50 µg/mL and 100 µg/mL as a control group and kojic as a positive control. Tyrosinase activity was measured using 1 mM acid. As a result, the group treated with α-MSH increased tyrosinase activity by 474% compared to the control group, and when the ethyl acetate fraction was treated at concentrations of 12.5 μg/mL, 25 μg/mL, and 50 μg/mL, the tyrosinase activity was each 397%, 373%, and 344% were reduced to 83.6%, 78.6%, and 72.5% compared to the α-MSH treatment group (see FIG. 11 ). In addition, when the hawthorn water extract was treated at concentrations of 50 μg/mL and 100 μg/mL, it was reduced to 429% and 391%, respectively, to 90.3% and 82.4% compared to the α-MSH-treated group. This means that when the water extract of hawthorn fruit and the ethyl acetate fraction were treated at the same concentration of 50 μg/mL, the ethyl acetate fraction was superior to the water extract in the inhibition of tyrosinase activity by about 17.8%, and the water extract was treated at a concentration of 100 μg/mL. It was superior by about 10%, and the tyrosinase activity inhibitory effect was superior to that of kojic acid used as a positive control.

Experimental Example 1-5. Tyrosinase Protein Expression Analysis Using Western Blotting Method

B16F1 cells ^{were inoculated into a 60mm dish at a density of 5Х10 4} , and the next day samples were treated with kojic acid and melanin-stimulating hormone α-MSH, respectively, and cultured for 3 days. After the cultured cells were washed with phosphate buffered saline, the cells were collected, a lysis buffer was added, and the cells were lysed by placing them on ice for 15 minutes, and the supernatant obtained by centrifugation at 13,000Хg for 15 minutes was used as a protein. After quantifying the protein content by the Bradford assay, the same amount of protein was separated by 12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and then the protein was transferred to a polyvinylidene difluoride (PVDF) membrane. Blocking was performed with TBS-T (tris buffered saline-tween20) containing 5% nonfat skim milk for 1 hour. After that, it was washed 3 times with TBS-T, added anti-tyrosinase (1:1,000) and reacted at 4°C for one day, followed by sufficient washing, and secondary antibody (Horseradish Peroxidase) attached ( 1:1,000) and reacted at room temperature for 1 hour. After washing 3 times for 10 minutes with TBS-T, color development was performed by ECL (enhanced chemiluminescence) method.

Claims (12)

A composition for skin whitening comprising an extract of hawthorn (Crataegus pinnatifida) fruit or a fraction thereof as an active ingredient. According to claim 1,
The composition inhibits the expression of tyrosinase or inhibits the production of melanin, a composition for skin whitening. According to claim 1, wherein the hawthorn (Crataegus pinnatifida) fruit extract is prepared by any one or more methods selected from the group consisting of reduced pressure high temperature extraction, hot water extraction, reflux extraction, hot water extraction, room temperature extraction, ultrasonic extraction, and steam extraction method. The composition for skin whitening. According to claim 1,
The fraction was prepared by concentrating the hawthorn fruit extract under reduced pressure and fractionating it with ethyl acetate, a composition for skin whitening. 5. The method of claim 4,
The concentration of the ethyl acetate in the fractionation is 12.5 μg / mL to 100 μg / mL, skin whitening composition. According to claim 1, wherein the composition is any one or more selected from the group consisting of a cosmetic composition, a food composition and a quasi-drug composition, skin whitening composition. A pharmaceutical composition for preventing or treating skin hyperpigmentation disease, comprising an extract of Crataegus pinnatifida fruit or a fraction thereof as an active ingredient. 8. The method of claim 7,
The fraction is a pharmaceutical composition for preventing or treating skin hyperpigmentation disease, which is prepared by fractionation with ethyl acetate after concentrating the hawthorn extract under reduced pressure. 8. The method of claim 7,
The concentration of the ethyl acetate in the fraction is 12.5 μg/mL to 100 μg/mL, a pharmaceutical composition for preventing or treating skin hyperpigmentation disease. 8. The method of claim 7,
The skin hyperpigmentation disease is, freckles, hereditary contralateral dyspigmentation, reticular hyperpigmentation, squamous nevus, nevus pigmentosa, melasma, senile pigmentation plaque, vitiligo, Badenburg syndrome, post-inflammatory pneumoconiosis, water drop melanosis, melasma and A pharmaceutical composition for preventing or treating skin hyperpigmentation disease comprising at least one of the diseases selected from the group consisting of rhizomes. In the method for preparing a composition for skin whitening,
a) hydrothermal extraction of hawthorn fruit;
b) concentrating the extract prepared according to step a) under reduced pressure;
c) fractionating the product of step b) using ethyl acetate, followed by concentration under reduced pressure; and
d) A method for preparing a composition for skin whitening comprising a fraction of Crataegus pinnatifida fruit, comprising the step of freeze-drying the product of step c). 12. The method of claim 11,
The concentration of the ethyl acetate during fractionation of step c) is 12.5 μg/mL to 100 μg/mL, the method for preparing a composition for skin whitening.

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