KR20070105416A - Composition for whitening comprising extracts from betula platyphylla var. japonica hara - Google Patents

Abstract

A composition for whitening the skin comprising the extract of Betula platyphylla var. japonica Hara is provided to inhibit melanin production, moisturize the skin, improve skin touch feeling and remove fat ingredient from the skin without skin irritation or toxicity. A composition for whitening the skin comprises the extract of Betula platyphylla var. japonica Hara which is prepared by pulverizing Betula platyphylla var. japonica Hara, heating the pulverized Betula platyphylla var. japonica Hara in water, extracting the hot water extract with methanol to obtain a crude extract of Betula platyphylla var. japonica Hara, and extracting the crude extract with dichloromethane, ethylacetate and butanol, sequentially.

Description

Composition for whitening comprising extracts from Betula platyphylla var. japonica Hara}

1 is a process chart for separating the active ingredient from the endothelium of the birch according to the present invention.

2 is a schematic view showing a step of separating a substance from an ethyl acetate dissolving unit.

3 is a further schematic diagram illustrating a process of separating a substance from an ethyl acetate melted portion.

4 shows the results of TLC confirmation of an isolated substance.

5 is a micrograph of the scales and dorsal fin melanin pigment of goldfish (black color).

The present invention relates to a whitening composition comprising the components extracted from the birch endothelium as an active ingredient, more specifically the birch endothelium comprises components separated by sequential extraction of hot water, methanol, dichloromethane and ethyl acetate It relates to a whitening agent composition.

The skin of the human body is a very important tissue that protects the human body while having direct contact with the external environment and has biochemical and physical functions. As there is a high risk of damage to the skin due to exposure to ultraviolet rays due to the destruction of the ozone layer along with the serious air pollution problem, the selection of functional products related to cosmetics or cosmetics is concerned with the side effects of clean and white skin. It is a very important issue from the point of view. Whitening cosmetics should be free of side effects, human-friendly, and safe to use on specific constitutions, atopic or sensitive skin. Plant natural products are not only excellent in safety but also contain various ingredients beneficial to the human body, and thus are preferred as ingredients that provide whitening and moisturizing effects. Thus, there have been efforts to find an extract that is excellent in safety and provides various functionalities from herbals, vegetables, fruits, flowers and the like which have a function of suppressing skin aging.

The whitening agent developed so far is mostly to reduce the amount of melanin by inhibiting the activity of tyrosinase, the enzyme that plays the most important role in melanin biosynthesis. Hydroquinone, hydroxyanisole, ascorbic acid derivatives, kojic acid, azelaic acid, corticosteroids, retinoids, also known as tyrosinase inhibitors (retinoids), arbutin, etc., but there are difficulties in use due to problems such as safety and economics. In particular, hydroxyanisole and hydroquinone have strong melanogenesis inhibitory activity, but at the same time cause side effects such as degeneration or lethality of pigment cells and impaired the original function of the cells. In addition, kojic acid has a problem of low inhibitory activity, discoloration in use, instability of the substance itself, and there is a need for a product having improved these problems.

The present inventors have diligently researched the functional substances beneficial to the human body from natural ingredients, and have separated and purified various components from the endothelium of the birch to investigate their functionalities. It has been found that it can provide a variety of beneficial functions in addition to the function to promote, moisturize the skin and the function of the fine art, and came to complete the present invention.

Accordingly, it is an object of the present invention to provide a whitening composition comprising substances purified from the endothelium of birch.

The present invention provides a whitening composition comprising the components of birch endosperm separated by sequential extraction of hot water, methanol, dichloromethane and ethyl acetate.

Birch ( Betula platyphylla var. Japonica Hara) is a deciduous broad-leaved arboreous tree belonging to the oak tree birch family. It is 20m high and the bark is white and peeled horizontally. Leaves are triangular oval-shaped, 5 ~ 7㎝ long, with single or double serrated teeth at the edge. A male and female flower in April-May, with female flowers facing up and male flowers hanging down like Isaac. The fruit ripens in September, and its wings are wider than the butterfly of the fruit. Fruit ear droops and is 4cm long, cylindrical. When the height of the tree is more than 5m, the flowers bloom and bear fruit, and the seeds can fly away because they are winged and light. It is a positive pump that is strong in the cold but prefers enough sunlight. The quality of the wood is good, does not rot and is less affected, so it is used for building materials, handiwork, sculptures. White bark is used to cover the fuel or roof and is also used as landscape water. Bark is used to treat Jinhae, expectorant and asthma, and sap is used to treat neuralgia, rheumatism, etc. It is distributed in Gangwon-do, North Pyongan, Hamgyeong-do, Japan, and China in our country.

Until now, the birch extract is known to have a whitening effect, but it has not been confirmed which ingredient has an effect, and the present invention has revealed these specific ingredients.

The birch extract component which is the main component of the whitening composition is extracted with crushed birch bark under hot water under normal conditions or as specifically shown in the examples of the present invention, and then extracted with methanol to obtain a crude extract, and then dichloromethane is continued. , Serial extraction with ethyl acetate and butanol can yield the desired compound fractions. Compounds identified as separate are as follows.

Compound BetD-1 was isolated from the dichloromethane dissolution site as shown in FIG. 1 and had a molecular weight of 296. The molecular composition was C: 77.00%, H: 6.80%, O: 16.20%, and had a molecular skeleton of diarylheptanoids. And the molecular formula is 1,7-bis- (4-hydroxyphenyl) -hept-4-en-3-one having C ₁₉ H ₂₀ O ₃ , having the structure of Formula 1.

Compound BetE-1 has a molecular weight of 328, a molecular composition of C: 58.53%, H: 7.37% O: 34.11%, and a molecular formula of C ₁₆ H ₂₄ O ₇ , 2-hydroxymethyl-6- [3- ( 4-hydroxyphenyl) -1-methyl-propoxy] -tetrahydro-pyran-3,3,5-triol having the structure of formula (2) This is a substance known as rhododendrin (= buturoside).

Compound BetE-531 has a molecular weight of 462, a molecular composition of C: 64.92%, H: 7.41%, O: 27.67%, and a molecular formula of C ₂₅ H ₃₄ O ₈ , 2-hydroxymethyl-6- {5- ( 4-hydroxy-phenyl) -1- [2- (4-hydroxyphenyl) -ethyl] -pentyloxy] -tetrahydropyran-3,4,5-triol having the structure of formula (3)

Compound BetE-34a has a molecular weight of 494, a molecular composition of C: 60.72% H: 6.93% O: 32.35%, a molecular formula of C ₂₅ H ₃₄ O ₁₀ , 2- {5- (3,4-dihydroxy Phenyl) -3-hydroxy-1- [2- (4-hydroxyphenyl) ethyl] pentyloxy} -6-hydroxymethyl-tetrahydro-pyran-3,4,5-triol, wherein Has the structure of.

Compound BetE-411 has a molecular weight of 594, a molecular composition of C: 60.59%, H: 7.12%, O: 32,29%, and a molecular formula of C ₃₀ H ₄₂ O ₁₂ , 2- (3,4-dihydrate Hydroxy-4-hydroxymethyl-tetrahydro-furan-2-yloxymethyl) -6- {5- (4-hydroxyphenyl) -1- [2- (4-hydroxyphenyl) -ethyl] pentyloxy } -Tetrahydropyran-3,4,5-triol, having the structure of Formula (5)

Compound BetE-36 has a molecular weight of 726, a molecular composition of C: 57.84%, H: 6.93%, O: 35.22%, and a molecular formula of C ₃₅ H ₅₀ O ₁₆ , 2- [4- (3,4-dihydrate Hydroxy-4-hydroxymethyl-tetrahydro-furan-2-yloxy) -3-hydroxy-4-hydroxymethyl-tetrahydro-furan-2-yloxymethyl] -6- {5- (4- Hydroxy-phenyl) -1- [2- (4-hydroxy-phenyl) -ethyl] pentyloxy} -tetrahydro-pyran-3,4,5-triol, having the structure of the following formula (6).

Compound BetE-551 has a molecular weight of 422, a molecular composition of C: 56.87%, H: 5.25%, O: 37.88%, and a molecular formula of C ₂₀ H ₂₂ O ₁₀ , 7- (3,4-dihydroxy- 5-hydroxymethyl-tetrahydro-furan-2-yloxy) -2- (3,4-dihydroxy-phenyl) -chroman-3,5-diol, having the structure of the following formula (7).

Compound BetE-33 has a molecular weight of 492, a molecular composition of C: 60.97%, H: 6.55%, O: 32.48%, a molecular formula of C ₂₅ H ₃₂ O ₁₀ , 2- [7-hydroxy-2- ( 4-hydroxy-3-methoxy-phenyl) -5- (3-hydroxy-propyl) -2,3-dihydro-benzofuran-3-ylmethoxy] -6-methyl-tetrahydrofuran-3, As a 4,5-triol, it has a structure of following formula (8).

As a result of testing the efficacy of the extracted ingredients, it is derived from natural plants and is not toxic and safe, and also activates the skin, gives moisturizing effect to the skin, and provides excellent whitening effect. Can be used.

The amount of the compounds used is appropriately 0.01 to 5.0% by weight based on the total weight of the whitening composition. At concentrations below 0.01% by weight it is difficult to expect effects, and at concentrations above 5.0% by weight it can cause skin irritation.

The whitening agent composition of the present invention is an auxiliary ingredient to enhance the whitening effect, mung bean, Schisandra chinensis, cucumber, Astronomical dong, Peony, thorny ogapi, Sukjihwang, deer antler, yellow flower, Baekbongryeong, Angelica, Yulmu, Licorice, Sangbaekpi, Ginkgo leaf, Ganoderma lucidum, Yulpi, It may optionally contain one or more plant extracts selected from the group consisting of red ginseng or mixtures thereof.

Mung bean extract maintains detoxification and skin elasticity in addition to whitening function. Mung bean ( Phase of Phaseolus aureus, Phaseolus radiatus , Mung bean) is a perennial herb of the dicotyledonous plant Rosaceae, Provitamin A (0.22mg%), B1 (0.53mg%), B2 (0.12mg%,) PP (1.8mg%) Abundant carbohydrates and proteins such as starch (66%), pentosan, galantan, glucose, raffinose, candy, and dextrin. Therefore, green beans are good for cleansing cleansing, pore clean, sebum secretion, whitening, oily acne and skin acne. Grind the mung beans finely and massage them with water to remove lipids from the skin, which is effective for acne and freckles, and also works well for pediatric dermatitis. When the active ingredient of mung bean is extracted separately, it may be powdered and then extracted by a conventional organic solvent extraction method using alcohol or the like.

Schizandra may be added as an ingredient that provides flexibility to the skin. Schisandra chinensis has a blood pressure lowering and antitussive expectorant action, which is not only widely used as a herbal medicine but also has a function of controlling gastric secretion and promoting bile secretion. In particular, Schisandra chinensis has a unique taste that combines sweetness, bitterness, sourness, spicy taste, salty taste, etc., and it is also used as a raw material for teas and spirit drinks. Schisandra chinensis can be milled and then extracted with water or alcohol. In addition, extraction by steam distillation may result in more desirable results.

Cucumber extract may be added as an additional ingredient that has a moisturizing and soothing effect. Cucumbers are about 95% water, low in calories, low in protein, lipids and sugars, and potassium, vitamin A and vitamin C. In addition, the potassium contained in cucumber acts to discharge excess salt into the body and promotes diuretic action, which is known to be good for patients with kidney disease or hypertension. Cucumbers are also known to be good when you're thirsty and sick, especially when you're in the heat. Cucumber does not need to be extracted by a special extraction method, it can be used juiced. Finely chop the cucumber, filter using filter paper (Wattman NO.2), and use the final filtration using a 0.45 μm filter (for microbial removal).

Asparagus cochinchinensis (Lour.) Merrill is a perennial herb belonging to the Liliaceae family . It is wild at the foot of mountains in the south coast of Korea or China. The outer skin is removed as a medicine. The medicinal herbs have a little peculiar smell and taste slightly bitter. The astronomical extract can be prepared by cutting it and then extracting it with 3-20 times the volume of water or 1-4 lower alcohols.

Peony ( Paeonia albiflora Pallas ) is a dry powdered root of peony belonging to the Ranunculaceae family, columnar to conical, 7-20 cm long, 10-25 cm in diameter, with large ones. The outer surface is brown to light grayish brown with clear vertical wrinkles, abutments and transverse coverings, and sometimes a dent on the left end. Folded face is powdery, dense, grayish brown in magnifier, light brown neck. The drug has an unusual scent and tastes sweet at first and slightly steamed and bitter later. Paeoniflorin, Paeonine, Benzoic acid, Tannin, Albiflorin are known as constituents, anti-inflammatory, antibacterial, bactericidal, anti-inflammatory, whitening, moisturizing, melanin pigment production It is effective in inhibiting, preventing and treating acne, has anti-inflammatory and analgesic effects, and is known to cure spots, freckles, acne or rough skin. Peony should be extracted with 3 ~ 10 times of water.

Gasiohgapi is carotene (carotene), Liguria Stryn (ligustrin) as the dried root and rhizome and bark of the deciduous shrub of Araliaceae of Zhao (Araliaceae) (. Acanthopanax senticosus ( RUPR et MAXIM) HAR MS), 7- methyl- It contains various glycosides such as 6,8-dimethylcoumaringlycoside, galactoside, and caryophyllenol, flavones, essential oils and polysaccharides. It contains. The pharmacological effects of psoriasis that have been discovered to date have been reported to be sedative, antistress, immune enhancing, smooth muscle relaxation, anti-inflammatory, rheumatoid, etc. In addition, psoriasis induces mental and physical vitality, tonic effect and nonspecific It is known to have no side effects such as insomnia, unlike ginseng because it shows increased resistance, prevents disease, has an effect on hypoglycemia, cancer, hypotension and does not excite people. Prickly pear extract can be obtained in a conventional manner by extraction with water or a conventional organic solvent when extracted alone.

Rehmannia glutinosa is native to China and the flowers are reddish purple with strong red light from June to July. This is an edible plant that usually uses roots. Raw herbs are usually raw, and dried and ripened sulfur is mature. It is called. It is one of the most widely used herbal medicines such as antipyretic, geothermal, cardiac, blood and nourishing tonic, including mandite, sugar, karten, cytosterol and vitamin A. Sukji sulfur is good to extract 3 to 10 times the amount of water.

Deer Antlers ( Cervi Parvum Corun ) are dried and harvested unhorned young horns of the Cervus Nippon Temminck Var.Mantcharicus Swinhoe, Cervus elaphus L. or other species belonging to the family Cervidae . It has been known as an excellent blood tonic. In the literature such as Dongbogam, deer antler has tonic action, view blood action, gangjeong action, analgesic action, growth growth promotion action, heart failure treatment action and hyperactivity action, fatigue recovery, physical vitality and kidney diuretic effect, etc. It is known to have a lot of efficacy. When the antler extract is prepared alone, it is finely pulverized and then extracted by heating with water as a solvent or by dipping about 5 times of dilute alcohol in fine antler to leave the rust solution at room temperature for 1 to 2 weeks. Prepare an antler extract.

Hwangjeong ( Polygonatum falcatum A. Gray) is a dried perennial herbaceous perennial herbaceous ginseng root, also known as Suninyeo (량 人 餘糧), has a non-toxic, sweet taste and comforts the five intestines. It removes wind and moisture, lightens the body, replenishes regular bones and bone marrow, prevents aging, protects Orochill, protects the spleen and stomach, makes the heart and lungs healthy, strengthens the musculature. It is a healthy medicinal herb that is said to be the foremost drug in the remedy, such as to endure cold, heat, and weakness. When yellow gold is extracted alone, it can be extracted in a conventional manner using an organic solvent of water and ethanol.

Baekbong-ryong (白 茯 笭) is a cell parasitic in the soil roots 5 to 9 years after harvesting the water, and has been widely used as a folk remedy to remove pigmentation such as spots. Polysaccharides, organic acids, proteins, vitamin D, lecithin, adenine, choline, fructose, minerals, etc. are contained in the skin to make it clear and shiny. Baekbongryeong should be extracted with 3 ~ 10 times of water.

Angelica is a perennial herb of the dicotyledonous cultivars of the dicotyledonous plant, and is used for pain, bleeding, hemostasis, and tonic.

Yulmu is also known as Uiju, Inmi and Sei (薏米). It is a yearly herb with a monocotyledonous rice plant. Seeds are called Euiin (薏苡仁), or eat as tea or diuretic, analgesic, jingyeong, tonic, so edema, neuralgia, rheumatism, bladder stones, etc. are used as medicine. Raw leaves are used as tea substitutes and roots are used for jaundice and neuralgia. The leaves on the stems are also used as feed. If you use it, you can remove the dead skin on the surface of dead skin and pores, and it enhances the metabolism of the skin and regenerates the skin in a short time. It has a great effect on skin troubles.

Licorice acts as a whitening ingredient for inhibiting melanin production. Licorice is a perennial plant of the dicotyledonous rosemary legume. Glycyrrhicin in licorice is effective for soothing the skin. Abundant flavonoids strengthen blood vessels. It is excellent for anti-inflammatory skin and acne because it has an excellent anti-inflammatory effect, and also has a sedative and healing effect. Morus bark refers to the mulberry root, which is a scale-like bark that removes the cork layer of the mulberry root bark. Epidermis is excellent in whitening, antibacterial, anti-inflammatory, moisturizing, soothing, anti-aging, anti-allergic and antiviral activity.

Ginkgo biloba is a seed (fruit) is edible, in one shot, the seeds are called encyclopaedia (白果) is prescribed by sea water, asthma, oil well (遺精), urine turbidity (小便 白濁), nourishment (滋養). The bark (樹皮) is called encyclopedia and the root is called encyclopaedia, coronary arteriosclerosis, chest pain, heart pain, heart system, high blood pressure, asthma, sea water and so on. Recently, ginkgo biloba has been used to extract components of blood circulation improvement and heart disease drugs.

Ganoderma lucidum mushroom, also called Bulchocho, is a mushroom of the fungus fungus Democratic grove. It is a year-round mushroom that has a gloss like lacquer on its lampshade and bag surface. In oriental medicine, tonic, Jinhae, small swelling (효능) and the efficacy, such as nervous breakdown, heart disease, high blood pressure, various cancers are used.

Yulpi is used to dry the skin of chestnut, and it has been used for folk remedies due to its effects on aging skin and blemishes from the old days. Above all, it is rich in tannin, which is excellent when used on large and oily skin with pores.

Red ginseng extract mainly plays a role in promoting cell growth. Preferably 6 year old red ginseng is suitable in terms of price and efficacy.

It is a matter of course that the use of the auxiliary components in combination with the appropriate adjustment of the components rather than when used alone may exhibit unexpected more synergy.

The above additional plant extracts can be obtained by extraction with water or by conventional organic solvent extraction methods or by conventional extraction methods well known in the art. The method of extracting the active ingredient of each is carried out by a well-known method, it is more preferable to prepare the desired composition by combining each extract in an appropriate mixing ratio, but in some cases it may save time to process them all at once. Therefore, in the case of treatment at once, each material is washed cleanly, and then chopped, and then combined, first extracted with an organic solvent such as ethanol, and then first extracted with a component dissolved in an organic solvent, the extracted extraction foil (抽出 粕) Second extraction with an organic solvent such as ethanol at a slightly low concentration (for example, 20 to 70%) to extract the remaining active ingredients dissolved in the organic solvent and water, and finally the first and second extraction By extracting only the components dissolved in water only by tertiary extraction with water, all components dissolved in water and organic solvent are extracted. The extraction temperature can be maintained at 30 ~ 100 ℃, extraction time is 1 hour to 1 week, in some cases at room temperature for about 6 months. The solvent content is preferably used 3 to 20 times by volume.

The composition ratio of the components in the composition according to the invention according to various experiments according to the weight of the raw material of the composition of the birch-derived compound: natural plant extracts in the range of 0.1 to 99.9: 99.9 to 0.1% by weight Adjust to use. Preferably, it is good to contain in 2 to 98:98 to 2% by weight. In particular, 2% to 98% of the birch extract components: blending with 0.1 to 5% of each herbal extract can produce the most desirable results.

The above composition ratio is set in consideration of the effective amount and side effects of each vegetable component, and if outside the range of the ratio there is a fear that the effect is reduced. Of course, even if the composition is out of this range, there is no problem inherent functionality except that the functionality is somewhat lowered, the composition of the plant extract of the present invention is not limited to the above-mentioned range, even if somewhat out of the range of the composition ratio of a certain level of quality If there is no problem in preparing a plant extract having a will be understood to be included in the scope of the present invention.

Since the auxiliary components contain extracts of natural substances, not chemical synthetic agents, as shown by the results of Examples and Test Examples described later, there is no irritation or toxicity on the skin, and thus, stability is excellent, and skin whitening effects are excellent. In addition to having a moisturizing effect on the skin and has a function such as improving skin feel and removing fat components, it can be used as a component of cosmetics or foods requiring such functionality.

The formulation of the food composition according to the present invention, when used as a whitening functional food, can be formulated in the form of a beverage such as oral, pouch, or drink according to a conventional method in the art.

The dosage of the food composition of the present invention is appropriately set according to the form of the preparation, the method of administration, the purpose of use, and the age, weight, and symptoms of the individual to be applied thereto, but the amount of the active ingredient contained in the preparation is generally not constant. For example, 1 mg-2000 mg / kg per adult. Of course, since the dose varies depending on various conditions, an amount smaller than the above range may be sufficient, or it may be necessary beyond the range.

The food composition according to the present invention may be prepared according to the form of the composition desired by conventional supplements or additives or sweeteners such as licorice, vitamin C, citric acid, nicotinic acid, sodium benzoate, aspartame, saccharin, pectin, malitol, sorbitol, xylitol, One or more ingredients selected from the group consisting of guar gum, skim milk powder, and oligosaccharides can be added to increase palatability or aesthetics.

They are suitably used at about 0.01 to 20% by weight based on the total weight of the composition of the present invention.

Drinking water is, for example, the medicinal herb combination pear extract in 0.01-20% by weight, licorice 0.01-2% by weight, citric acid 0.01-2% by weight, malic acid 0.01-2% by weight, taurine 0.1-2% by weight, 0.01-2% by weight of vitamin C, 0.01-2% by weight of vitamin B1, 0.01-2% by weight of biotin, 0.01-2% by weight of liquid fructose, or the like can be added alone or mixed to produce a functional beverage.

The whitening cosmetic composition of the present invention is not particularly limited in the formulation, for example, supple cosmetics, astringent cosmetics, nourishing cosmetics, eye cream, nutrition cream, massage cream, cleansing cream, cleansing foam, cleansing water, powder, You may manufacture in formulations, such as an essence and a pack.

Formulations of the cosmetics may be prepared by conventional methods well known in the cosmetic manufacturing field.

Functional cosmetics as described above are not limited to these as an auxiliary component to further improve the desired functionality, by combining an appropriate amount of rice snow, rice bran, seaweed powder, kelp powder, bamboo salt, rice bran, seaweed powder, bamboo charcoal, etc. Functionality can be further increased.

When these auxiliary components are added alone or in combination, their addition amount is suitably used in an amount of 0.1 to 10% by weight based on the total weight of the composition.

Excipients of these cosmetics may vary depending on the purpose of use, but are generally preservatives such as glycerin, butylene glycol, propylene glycol, alcohols or vegetable preservatives, purified water, moisturizers, bulking agents, creams, surfactants, pH adjusters, flavorings It is preferable to prepare according to the form of the desired formulation by mixing a suitable amount. It may also include conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments and flavors, and carriers. In addition, the whitening agent of the present invention can be used in combination with other physiologically active ingredients for the purpose of improving the function, in particular, synergistically improving the whitening effect, assisting in the whitening effect, and improving the absorbency. There is no particular limitation, but for example, other oral whitening agents that can expect synergistic effects, antioxidants that indirectly contribute to the whitening effect, oily ingredients to enhance the efficiency of the body by improving the absorption efficiency, nutritional enhancement And various vitamins, minerals, amino acids and the like.

They are suitably used at about 0.01 to 90% by weight based on the total weight of the composition of the present invention.

<Example>

Hereinafter, the present invention will be described in more detail with reference to the whitening cosmetic composition of the present invention, but these are presented for the purpose of illustrating the present invention and are not intended to limit the present invention in any way.

Example 1 Isolation of Active Ingredients from Birch Endothelial

5 kg of crushed birch bark was placed in 5 L of water and extracted at 100 ° C. for 2 hours. Then, 200 g of the hydrothermal extract obtained was extracted twice with 1 L of 95% methanol at room temperature to obtain 60 g of crude extract. These crude extracts were sequentially extracted with dichloromethane, ethyl acetate and butanol to obtain a fraction. The isolation process of these fractions is shown in FIG. Each aliquot was purified by thin layer chromatography (TLC; Silica gel 60 F254, developing solvent; toluene: ethyl formate: formic acid = 5: 4: 1, V / V; TEF) with UV and colorant 50% H ₂ SO ₄ . Confirmed. Subsequent separation was performed for the dichloromethane dissolving unit and the ethyl acetate dissolving unit, and these separation processes are shown in FIGS. 2 and 3. First, in order to isolate useful components from the ethyl acetate dissolving unit, Separdex LH-20 was first used as a filler and developed using elution solvent H ₂ O: MeOH = 1: 1 to obtain eight fractions. This was subsequently separated as shown in FIGS. 2 and 3 to isolate two substances from one in fraction 2, three in fraction 3, one in fraction 4 and fraction 5, respectively. Thin layer chromatography (TLC) on isolated materials showed Rf 0.95 for BetD-1 at ethyl acetate: acetone: water = 10: 10: 1, and the materials isolated at the ethyl acetate solubles It was confirmed that it appears in Rf 0.4 ~ 0.7. TLC results of the isolation material are shown in FIG. 4.

Example 2 Structural Identification of Birch Endothelial Extract Fraction

The fractions obtained in Example 1 and the same amount of methanol were mixed, then treated with a C18-type Sep-pak (Waters, USA) and filtered through a 0.2 μm injection filter (Satorius, Germany). The filtrate was injected into reverse phase HPLC (SHIMADZU) equipped with Phenomenex® synerg I 4μ POLAR-RP 80A (250 x 4.60 mm 4 micron) and eluted using a mixed solvent under the conditions shown in Table 1 below. Chromatography was then performed using 50 μl sample size and the eluate was observed at 290 nm with SPD-M10A VP searcher. The pump used LC-10AD, the Dectector used SPD-M10A VP, and the oven used SIL-10AD.

Minutes Flow rate (ml / min.) Elution solvent Solvent A (%) ` Solvent B (%) 0.0 1.0 50 50 40.0 1.0 20 80 60.0 1.0 20 80 A solvent: 5% Acetonitrile + 0.035% trifluroacetic acid B solvent: 50% Acetonitrile + 0.025% trifluroacetic acid

The obtained components were commissioned by the Seoul Institute of Basic Science for structural identification, and the mass spectrometry was performed using Jeol JMS-700 Mstation Mass Spectrometer, and the nuclear magnetic resonance (NMR) spectra were Varian UI500 and 500 MHz FT-NMR. Measurement was made using a spectrometer.

A total of eight substances were isolated from the endothelial methanol extract of Betula platyphylla Var. Japonica Hara, each of which contains one diarylheptanoid (BetD-1) and four diarylheptanoid glycosides (BetE-531, BetE-34a, BetE-411 and BetE-36) were the most isolated compounds with diarylheptanoid skeleton. Other substances were isolated from 1 butanoid glycoside (BetE-1), 1 flavonoid glycoside with xylose (BetE-551) and 1 neonignan (BetE-33). ¹ H-NMR spectral results of the compounds are omitted for convenience because of the enormous data available. Specific compounds are as described above.

Example 3: Preparation of Plant Extract

500 g of mung bean, Schisandra chinensis, astronomical dong, thorny ogapi, antler, yellow, yulpi were prepared, and then chopped and powdered and 5 times of alcohol was added and extracted at a temperature of 80 ° C. for 5 hours. Similarly, 500 g each of Peony, Sukji-hwang, Baekbong-ryeong, Ganoderma lucidum, Licorice, Ginkgo biloba, Angelica, Yulmu, and Sangbaekpi were prepared, and then sliced and powdered and 5 times of water was added and extracted at 80 ° C. for 5 hours. 500 g of cucumbers were juiced and then the extracts were combined. After the extraction, the remaining residue was added again 5 times with ethanol (95%) and extracted again in the same manner, and extracted again with 50% concentration of ethanol in the remaining extraction foil (抽出 粕). By extracting only the components dissolved only in water by tertiary extraction with only 5 times the amount of the residual extract to extract all of the components dissolved in water and organic solvent was combined each extract. The extraction temperature was maintained at 30 to 100 ° C., the extraction time was 1 hour to 1 week, and in some cases at room temperature for about 6 months. The solvent content was generally added in 5 times volume ratio. The liquid obtained by the above extraction was passed through 0.45 micrometer filter paper, and the organic component was distilled under reduced pressure and 23 Kg of final mixed extracts were obtained.

Example 4 Preparation of Drinks

10 mg to 2 kg of the birch extract obtained in Example 2, 10 mg to 2 kg of the plant extract obtained in Example 3 was added, 20 g of citric acid, 17 g of aspartame and 30 g of vitamin C were added thereto, and the rest was added to purified water. 10 L was prepared and pasteurized. As a result, the beverage of the present invention obtained a favorable response from the conventional beverage product in terms of mouth feel, taste and comprehensive evaluation.

Test Example 1: In Vitro Whitening Activity Test

Simple and time-saving assays for the primary selection of substances with the potential for whitening activity in each fraction separated above for the selection of substances with tyrosinase inhibitory activity using mushroom tyrosinase The method of picking was performed. Mushroom tyrosinase and L-Dopa reacted to form dopaquinone, and the fraction that inhibited mushroom tyrosinase activity was selected first.0.5 ml (1 mg / m) of mushroom tyrosinase and sample, respectively. ml), 0.1 ml (110 units / ml) and 0.2 ml (5 mM) of L-Dopa were added and reacted at 37 ° C. for 5 minutes and measured at 475 nm, the maximum absorption wavelength of dopaquinone. Dopa is synthesized as dopaquinone, and when tyrosinase activity is inhibited, it is a simple first-order search method using the principle that the synthesis amount of dopaquinone is relatively small.

Tyrosinase inhibition rate (%) =

A: Sample + Tyrosinase + L-DOPA

B: sample + phosphate buffer + L-DOPA

C: D.W + tyrosinase + L-DOPA

D: D.W + Phosphate Buffer + L-DOPA

In the case of the birch extract, the red color was inherent, and it was not suitable for confirming the result. During the experiment, the color was darkened in proportion to the concentration of the sample.

Alternatively, antioxidant activity was measured by the DPPH method. DPPH was used to measure the ability to erase radicals present in the sample. After mixing the appropriate concentration of DPPH and the sample diluted to each concentration it was reacted for 30 minutes at 37 ℃. Thereafter, the absorbed radicals can be measured by measuring the absorbance at 516 nm. As shown in Table 2, the activity of the birch endothelium hot water extract (BetHW-IB) and the endothelial hot water extract ethyl acetate fraction (BetHW-IB-2-E) showed the highest antioxidant activity, 70.6 and 78%, respectively.

Test Example 2 Measurement of Intracellular Tyrosinase Inhibitory Activity of B16 / F10

Whether the whitening active material absorbed into the cell inhibited the activity of the intracellular tyrosinase was measured as follows. Cells were added to 1 x 10 ⁵ cells / well in a 24 well plate and incubated for 24 hours at 37 ° C. and 5% CO ₂ . After replacing the medium, 20 μl of birch extract dissolved in DMSO was added to each well so that the final concentration was 0, 1, 10, 100, 200 ug / ml. After incubation for 48 hours, the medium was removed, washed twice with PBS, cells were removed with 0.25% trypsin, centrifuged, and washed twice. It was dissolved in 1.0% Triton X-100, 0.5% deoxycholic acid, 1.0 mM phenylmethylsulfonyl fluoride, and then disrupted at 4 ° C. for 4 hours. do. The cell suspension was allowed to settle by centrifugation at 5000 rpm, and then the supernatant was used as a crude tyrosinase extract. Add 50 μl supernatant + 50 μl L-DOPA (or 100 mM catechol) + 100 μl 0.1 M sodium phosphate buffer to react in a 37 ° C. incubator for 5 minutes and then at 475 nm with a microplate ELISA reader (El 312e, Bio-Tek). Absorbance was measured.

Tyrosinase activity of kojic acid, birch endothelial ethanol insoluble component (BetHW-IB-1), birch endothelial ethyl acetate fraction (BetHW-IB-2-E) was used as a control group. 61.9 and 63.3 were identified as having high activity with inhibition rates of 69, 32.1 and 36.7%, respectively.

Test Example 3 Measurement of Intracellular Melanin Synthesis of B16 / F10

The synthesis process of melanin in vivo is 3,4-dihydroxyindolephenylamine (L-DOPA) through tyrosine as a substrate and hydroxylation by tyrosinase, which is phenylalanine-3,4 Oxidized to quinone (L-dopaquinone). Dopaquinone is known to form melanin polymer through 5,6-dihydroxyindole after conversion to dopachrome, a relatively unstable intermediate.

The measurement of melanin levels was carried out using a modified method of Hosoi et al. (Hosoi J., Abe E., Suda T. and Kuroki T.: Regulation of melanin synthesis of B16 mouse melanoma cells by 1 a-25-dyhydroxyvitamin D3 and retinoic acid.Cancer Res /. 45, 1474 (1985).). The cells were added to 1 x 10 ⁵ cells / well in a 24 well plate and incubated at 37 ° C. and 5% CO ₂ for 24 hours. After replacing the medium, 20 μl of sample birch extract dissolved in DMSO was added to each well so that the final concentration was 0, 1, 10, 100, 200 ug / ml. After 48 hours of incubation, the medium was removed, washed twice with PBS, cells were removed with 0.25% trypsin, centrifuged, and washed twice. After the cells were allowed to settle by centrifugation, the supernatant was removed, 150 μl of 1N NaOH was added, and boiled for 1 hour in an 80 ° C. water bath to release intracellular melanin. After cooling at room temperature, the absorbance of 405 nm, which is the maximum absorption wavelength of melanin, was measured in a microplate ELISA reader (El 312e, Bio-Tek), and obtained from a standard straight line prepared using synthetic melanin (Sigma Co.). It was calculated as a percentage of the amount of melanin.

Intracellular melanin synthesis of birch extracts and solvent fractions was measured. The intracellular melanin of the birch endothelial hot water extract (BetHW-IB) and the methanol fraction of the birch endothelial hot water extract (BetHW-IB-2) as shown in Table 2 Melanin synthesis was inhibited at 67.8 and 68%, and kojic acid used as a control was 94.4%, showing an inhibition rate of about 5.6%, indicating that the inhibition of intracellular melanin synthesis was low.

Test Example 4: Toxicity test on skin cells in vitro

To investigate the cytotoxicity of tyrosinase inhibitors, SK-MEL-31 cell line, a human normal melanocyte cell line, was purchased from Korea Cell Line Bank, and the MTT test was performed based on Mosmann's method. carried out by the IC ₅₀ value was determined. The cytotoxic effects of the drugs were investigated by 3,4,5-dimethyl-thiazole-3 and 5-diphenyl tetrazolium bromide (MTT) assays, in which live cells can degrade tetrazolium salts. It is based on preventing intracellular accumulation.

After diluting the melanocyte cell solution, add 180 µl to a 96-well microtiter plate, and incubate for 72 hours at 37 ° C. and 5% CO _2. After titration, the OC ₅₄₀ value in the MTT treatment ranges from 0.4 to 0.7. The cell number was obtained.

Melanosite cell solution was diluted to the proper concentration determined in the preliminary experiment, and 180 µl was added to the 96-well micro titer plate, and the cells were incubated for 24 hours under 37 ° C. and 5% CO ₂ air conditions. After 24 hours of incubation, each birch extract was dissolved in DMSO on a plate inoculated with cells, and 20 μl was added to each well to add a final concentration of 0, 1, 10, 100, 200 μg / ml. Cisplatin (sigma co.) Was used as a positive control. 48 hours of continuous incubation at 37 ° C., 5% CO ₂ conditions. MTT stock solution (5 mg / ml) was dissolved in PBS and filtered through a 0.22 μm filter to remove formazan crystals and stored in the dark at room temperature. Remove the medium from the plate cultured for 48 hours, wash with PBS, add 180 μl of medium, add 20 μl of MTT solution to each well, and incubate for 4 hours at 37 ° C. and 5% CO _2. Was removed and shaken in a microplate shaker until formazan was completely dissolved by adding 150 μl of DMSO, and then absorbance was measured at a wavelength of 540 nm using a microplate ELISA reader (El 312e, Bio-Tek). .

The percentage of control Absorbance was calculated from the eight wells of the 96-well microtiter plate for the mean OC ₅₄₀ value of each column. This percentage corresponds to the cell survival rate compared to the control. In addition, the 50% inhibitory concentration IC ₅₀ value is defined as the concentration of a substance that reduces the absorbance at 540 nm by 50%. The IC ₅₀ value of each substance is used as an indicator of cytotoxicity. As a result of confirming the cytotoxicity of the birch extract and each solvent fraction as shown in Table 2, it was confirmed that all of the IC50 values were not toxic except for the outer skin ethanol extract, and the outer skin ethanol extract (BetEt-OB) It was found to be toxic at 29 ug / ml.

Table 2 summarizes the results of the above test example.

designation Extraction Antioxidant activity Cytotoxicity (IC 50 = µg / ml) Whitening effect (200㎍ / ml) Tyrosinase Synthesis (%) Z Melanin Synthesis (%) Kojic acid > 200 31 94.4 BetHW-IB Hydrothermal 70.6 > 200 80 67.2 BetHW-IB-1 MeOH insol 67.5 > 200 61.9 94.7 BetHW-IB-2 MeOH sol 62.1 > 200 76 68 BetHW-IB-2-E EtoAc 78 > 200 63.3 79.3 BetHW-IB-2-B Buthanol 71 > 200 72.4 88.7 BetHW-IB-2-R Residue 51 > 200 88.4 76.6 BetHW-OB Hydrothermal jacket 52.9 > 200 67 78 BetHW-OB-1 MeOH insol 38.6 > 200 102 180 BetHW-OB-2 MeOH sol 50.3 > 200 75 90 BetHW-W Neck heat 17.6 > 200 102 106 BetEt-IB Naeethanol 56 > 200 88 92 BetEt-OB Outer Ethanol 44 29 98 96.2

As a result of confirming the activity of the solvent fraction of the birch endothelium extract, the activity of the EtoAC fractions was good overall, and based on this, it was subsequently separated and purified to identify the single substances. The table above is the test data for the EtoAC fractions.

Experimental Example 5: In vivo whitening effect test using black eye goldfish

In order to confirm the food potential of the sample having a whitening activity, the activity was measured using a black black fish (jet black gold fish) with a black body all over. Black-tailed goldfish was purchased and allowed to acclimate for 2 days, and then NaCl was added to 0.9%. One week later, after removing the salt from the carp cuticle, each 11-12 animals were added to the control, benzoic acid, birch endothelial hot water extract methanol solubles (BetHW-IB-2) added to 50mg / ml and treated. After two weeks of cultured crucian carp, each was taken out and paralyzed with an electric shock of 100 V. Pectoral region, lateral region scales, dorsal fins were removed and lyophilized, and used for melanin content analysis. Lyophilized scaly scales were used as they were, and the dorsal fin was ground with a mortar and used as a sample. 1N NaOH corresponding to 250 times (v / w) of each sample was added thereto, mixed for 15 minutes, heated in a bath (100 ° C), cooled, and absorbed at 420 nm. The melanin standard curve was a synthetic melanin reagent as a sample. The absorbance was measured for each concentration under the same conditions. In addition, each of the scales and fins were cut at a certain site to observe melanin under a microscope (Microphot-FX, Nikon, Japan).

Microscopic results of the administration group and the control group ingested birch extract was confirmed that the number and size of the melanin pigment in both the scale (scale) and dorsal fin (dosal fin) was reduced. Melanin quantification resulted in a 47% reduction in scales and 23% decrease in dorsal fin (Table 3). Table 3 shows the effect of benzoic acid on the melanin content of the scales and dorsal fins of goldfish (black).

Melanin content (㎍ / g)             scales Dorsal fin Control 233.8 + 29.8 (100 + 12.7) 764.1 + 85 (100 + 11.1) Inventive Compound (BetHW-IB-2) 125.6 + 48.4 (53 + 34.1) 594.7 + 82.9 (77.1 + 13.6)

Since the natural cosmetic composition according to the present invention contains an extract of a natural substance other than a chemical synthetic agent, there is no irritation or toxicity to the skin, so it has excellent stability, inhibits the production of melanin, has a skin whitening effect, and a moisturizing effect, thus feeling the skin. It can be used in cosmetic products requiring such functionality because it provides several beneficial functions such as improving and removing fat components.

Claims (3)

A whitening composition, which is separated and purified from the endothelium of birch, and contains at least one of the following compounds as a main component. 1,7-bis- (4-hydroxyphenyl) -hept-4-en-3-one, 2-hydroxymethyl-6- [3- (4-hydroxyphenyl) -1-methyl-propoxy] -tetrahydro-pyran-3,3,5-triol, 2-hydroxymethyl-6- {5- (4-hydroxy-phenyl) -1- [2- (4-hydroxyphenyl) -ethyl] -pentyloxy] -tetrahydropyran-3,4,5- Triol, 2- {5- (3,4-Dihydroxyphenyl) -3-hydroxy-1- [2- (4-hydroxyphenyl) ethyl] pentyloxy} -6-hydroxymethyl-tetrahydro-pyran- 3,4,5-triol, 2- (3,4-Dihydroxy-4-hydroxymethyl-tetrahydro-furan-2-yloxymethyl) -6- {5- (4-hydroxyphenyl) -1- [2- (4- Hydroxyphenyl) -ethyl] pentyloxy} -tetrahydropyran-3,4,5-triol, 2- [4- (3,4-Dihydroxy-4-hydroxymethyl-tetrahydro-furan-2-yloxy) -3-hydroxy-4-hydroxymethyl-tetrahydro-furan-2-yl Oxymethyl] -6- {5- (4-hydroxy-phenyl) -1- [2- (4-hydroxy-phenyl) -ethyl] pentyloxy} -tetrahydro-pyran-3,4,5-tri All, 7- (3,4-Dihydroxy-5-hydroxymethyl-tetrahydro-furan-2-yloxy) -2- (3,4-dihydroxy-phenyl) -chroman-3,5-diol , And 2- [7-hydroxy-2- (4-hydroxy-3-methoxy-phenyl) -5- (3-hydroxy-propyl) -2,3-dihydro-benzofuran-3-ylmethoxy] -6-methyl-tetrahydrofuran-3,4,5-triol. According to claim 1, It is further selected from the group consisting of mung bean, Schisandra chinensis, cucumber, astronomical dong, peony, thorny ogapi, sukjihwang, antler, yellowfin, baekbongryeong, tangwi, yulmu, licorice, sangbaekpi, ginkgo biloba, ganoderma lucidum, yulpi, red ginseng Whitening composition, characterized in that it comprises one or more extracts of vegetable origin. The composition of claim 1, which is provided in the form of a food.

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