JP5627585B2 - Skin whitening composition containing an extract, fraction or compound derived from anthracnose - Google Patents

Description

  The present invention relates to an extract, fraction, or composition for skin whitening containing a compound, more specifically, an ethanol extract obtained by extracting kanakugi with ethanol, a solvent fraction obtained therefrom, or The present invention also relates to a composition for skin whitening comprising a compound separated and purified therefrom as an active ingredient.

  Research on bioactive substances from natural products is actively progressing, and plant resources are widely used as therapeutic and preventive agents for diseases or health aids. As natural antioxidants, α-tocopherol, vitamin C, carotenoids, flavonoids, and the like are known. Substances having such an antioxidant effect are widely distributed in animals and plants, and many studies have been conducted. The field carried out is plant-derived substances. As a result of biological experiments, plant-derived secondary metabolites and the like were found to suppress and eliminate the generation of free radicals and active oxygen to prevent cell damage due to oxidation.

  Most natural antioxidants reported to date have been derived from plants and are known to be primarily polyphenolic compounds, especially flavonoids, which are lipid oxidation, active oxygen scavenging and oxidative stress Since it has the effect of preventing aging and preventing and delaying cancer and heart disease, it is used in many fields such as current foods, pharmaceuticals, and cosmetics. Moreover, melanin is a high-molecular natural pigment of phenols widely present in animals, plants, and microorganisms as an important defense means for protecting a living body from ultraviolet rays.

  Melanin enhances the viability of ultraviolet rays, dryness, extreme temperatures, etc., and improves the quality of coffee, tea, tobacco, etc., but excessive melanin synthesis forms stains, freckles, skin spots on the human body and causes skin aging. It is known to promote and participate in the induction of skin cancer. The biosynthesis of melanin pigment is regulated by various enzymes including tyrosinase enzyme. Among them, tyrosinase acts on the oxidation of dihydroxyindole after the initial biosynthesis process in which tyrosine is transferred to L-dopaquinone. To do. Therefore, research to find tyrosinase activity inhibitors occupies an important part in the development of whitening agents, and hydroquinone and 4-hydroxyanisole are known as tyrosinase inhibitors that have been known subsequently. , Ascorbic acid derivatives, kojic acid, azelaic acid, corticosteroid, retinoids, arbutin, catechin, 3,4,5-trimethoxycinnamate thymol ester (3,4,5 -trimethoxy cinnamate thymol ester), but there are many problems in their safety and economics, and they are difficult to use.

  Lindera erythrocarpa (Lindera erythrocarpa) is a plant belonging to the family Lauraceae, and more than 1,500 species of 45 genera are distributed around the world, and it is known that 12 species of 6 genera grow in Korea. . The anthracnose is a hermaphrodite, with pale yellow flowers blooming from April to May and a red fruit of about 8 mm in September. It is a deciduous tree with a height of 5 meters that grows naturally in the warmer regions of Japan and China, including the southern region of the Republic of Korea. The dried fruit has a unique aroma and bitterness and is used in Japan as a gastrointestinal and neuralgia analgesic. In addition, ethanol extract extracted from leaf and fruit of citrus leaves and lucidone, which is one of the cyclopentadione compounds, have been reported to have a high anti-inflammatory effect (Wang et al., 2008), Cyclopentadione compounds isolated from anthracnose have been reported to have an anticancer effect (Oh et al., 2005; Korean Patent 10-0658519).

  An object of the present invention is to provide a composition for skin whitening containing an extract of kanakugi, a solvent fraction thereof, a cyclopentadione compound separated therefrom and a derivative thereof as an active ingredient.

  In view of the above, the present inventors obtained an ethanol extract, sequential fractions using a solvent, a cyclopentadione compound, or a derivative thereof using a Linda erythrocarpa in Jeju, South Korea. By searching for antioxidant activity and treating B16F10 mouse melanoma cells and RAW264.7 cells to investigate melanin biosynthesis inhibitory activity, they are useful for skin whitening functional cosmetics, foods or pharmaceutical compositions The present invention was completed by confirming that it could be used as a resource.

  The composition for skin whitening containing an extract, fraction or compound derived from an anthracnose of the present invention is an arbutin already known as a whitening agent by containing a cyclopentadione compound or a derivative thereof as an active ingredient. It has a very excellent effect of showing higher melanin inhibitory activity than (arbutin).

FIG. 1 is a flow chart schematically showing a process of obtaining an ethanol extract and a solvent fraction thereof from kanakugi according to the present invention. FIG. 2 shows the results of HPLC analysis for the an extract of cornflower ethanol of the present invention. FIG. 3 shows the result of HPLC analysis for the n-hexane fraction of the extract of the Anemone ethanol of the present invention. FIG. 4 shows the results of HPLC analysis for the methylene chloride fraction of the extract of the anthracnose ethanol of the present invention. FIG. 5 shows the results of HPLC analysis for the ethyl acetate fraction of the an extract of anthracnose ethanol of the present invention. FIG. 6 shows the results of HPLC analysis of the n-butanol fraction of the extract of the present invention. FIG. 7 shows the results of HPLC analysis for the water fraction of the extract of the anthracnose ethanol of the present invention. FIG. 8 shows the results of high performance liquid chromatography for quantitative analysis of Compound 1. FIG. 9 shows the ¹ H, ¹³ C-NMR spectrum of Compound 1. FIG. 10 shows the results of high performance liquid chromatography for quantitative analysis of Compound 2. FIG. 11 shows the ¹ H, ¹³ C-NMR spectrum of Compound 2. FIG. 12 shows the results of high performance liquid chromatography for quantitative analysis of Compound 3. FIG. 13 shows the ¹ H, ¹³ C-NMR spectrum of Compound 3. FIG. 14 shows the results of high performance liquid chromatography for quantitative analysis of Compound 4. FIG. 15 shows the ¹ H, ¹³ C-NMR spectrum of Compound 4. FIG. 16 shows the results of high performance liquid chromatography for quantitative analysis of Compound 5. FIG. 17 shows the ¹ H, ¹³ C-NMR spectrum of Compound 5. FIG. 18 shows the results of high performance liquid chromatography for the quantitative analysis of Compound 6. FIG. 19 shows the ¹ H, ¹³ C-NMR spectrum of Compound 6. FIG. 20 shows the results of high performance liquid chromatography for the quantitative analysis of Compound 7. FIG. 21 shows the ¹ H, ¹³ C-NMR spectrum of compound 7. FIG. 22 shows the results of high performance liquid chromatography for the quantitative analysis of Compound 8. FIG. 23 shows the ¹ H, ¹³ C-NMR spectrum of Compound 8. FIG. 24 shows the results of high performance liquid chromatography for the quantitative analysis of Compound 9. FIG. 25 shows the ¹ H, ¹³ C-NMR spectrum of compound 9. FIG. 26 is a graph showing the cell viability of B16F10 cells expressed by an anthrax ethanol extract and its solvent fraction. The data at this time is the mean ± SD value obtained after three measurements. FIG. 27 is a graph showing the melanin production inhibitory activity expressed by an extract of anemone ethanol and its solvent fraction in B16F10 cells. The data at this time is the mean ± SD value obtained after three measurements. FIG. 28 is a graph showing the cell tyrosinase inhibitory activity expressed by an anemone ethanol extract and its solvent fraction in B16F10 cells. The data at this time is the mean ± SD value obtained after three measurements. FIG. 29 is a graph showing the cell viability represented by lucidone in B16F10 cells. The data at this time is the mean ± SD value obtained after three measurements. FIG. 30 is a graph showing the cell viability represented by methyllinderone in B16F10 cells. The data at this time is the mean ± SD value obtained after three measurements. FIG. 31 is a graph showing the cell viability represented by canakugiol in B16F10 cells. The data at this time is the mean ± SD value obtained after three measurements. FIG. 32 is a graph showing melanin production inhibitory activity expressed by lucidone in B16F10 cells. The data at this time is the mean ± SD value obtained after three measurements. FIG. 33 is a graph showing the melanin production inhibitory activity represented by methyllinderone in B16F10 cells. The data at this time is the mean ± SD value obtained after three measurements. FIG. 34 is a graph showing the melanin production inhibitory activity represented by canakugiol in B16F10 cells. The data at this time is the mean ± SD value obtained after three measurements. FIG. 35 is a graph showing tyrosinase inhibitory activity expressed by lucidone in B16F10 cells. The data at this time is the mean ± SD value obtained after three measurements. FIG. 36 is a graph showing the influence of the compound of the formula 10 derived from anthracnose of the present invention on the cell viability of B16F10 cells. FIG. 37 is a graph showing the effect of the compound of the chemical formula 10 derived from anthracnose of the present invention on melanin production suppression in B16F10 cells. FIG. 38 shows an aspect in which the compound of the chemical formula 10 derived from anthracnose of the present invention suppresses the expression of tyrosinase, TRP-1 and TRP-2 genes in B16F10 cells. At this time, C means a control group, and Arb means arbutin.

  In one embodiment, the present invention provides a composition for whitening skin, which contains an extract of kanakugi or a solvent fraction thereof as an active ingredient.

  In the present invention, the extract of the anemone tree includes a solvent selected from water, a lower alcohol such as ethanol and methanol, or a mixed solvent thereof, preferably one extracted with ethanol. In the present invention, the extract includes an extract obtained by an extraction process, a diluted solution of the extract, a concentrated solution, a dried product obtained by drying the extract, or a roughly purified product thereof, or Any one of the purified products will be included.

  As an embodiment of the present invention, the extract of the anemone is dried with hot air and pulverized by depositing in 70% ethanol, leaching by stirring at room temperature for 3 days, filtered, It can be obtained by concentration under reduced pressure.

In the present invention, the solvent fraction of the kanakuginoki extract is obtained by suspending the kanakuhinoki extract in distilled water, and then, from a non-polar solvent, n-hexane (n-hexane; n-Hex), methylene chloride (CH). ₂ Cl ₂ ), ethyl acetate (EtOAc), and n-butanol (n-BuOH).

As an example of the present invention, a solvent fraction of an extract of kanakuginoki is obtained by suspending an extract of kanakuginoki ethanol in distilled water and using a separatory funnel to remove n-hexane (n-Hex), methylene chloride (CH ₂ Cl ₂ ), ethyl acetate (EtOAc), and n-butanol (n-BuOH) were used for fractionation, followed by filtration and concentration under reduced pressure to obtain a fraction for each solvent.

  As another embodiment, the present invention provides a skin whitening composition containing, as an active ingredient, one or more compounds selected from the group consisting of compounds represented by the following chemical formulas 1 to 10.

  In the present invention, the compounds and the like can be used after being separated and purified from citrus trees. Moreover, in this invention, except the compound of Chemical formula 10, among the said compounds, a commercial product can be obtained and used, or it can also manufacture and use by a well-known synthesis method.

  As a preferred embodiment of the present invention, the composition of the present invention is a cosmetic composition.

  Ingredients contained in the cosmetic composition of the present invention include kanagawa extract as an active ingredient, its solvent fraction, or other components usually used in cosmetic compositions in addition to compounds separated therefrom, for example, Conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and perfumes, and carriers can be included. In addition, the cosmetic composition may further include a skin absorption promoting substance in order to enhance the effect.

  The cosmetic composition of the present invention can be manufactured as any dosage form conventionally manufactured in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, It can be formulated in powders, soaps, surfactant-containing cleansings, oils, powder foundations, emulsion foundations, wax foundations, sprays, and the like, but is not limited thereto. Specifically, it can be produced in the form of soft lotion, nutritional lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

  When the dosage form of the present invention is a paste, cream or gel, the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc or Zinc oxide or the like can be used.

  When the dosage form of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder is used as a carrier component. In particular, in the case of a spray, chlorofluorohydrocarbon is further used. ), Propylene / butane or propellants such as dimethyl ether.

  When the dosage form of the present invention is a solution or an emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, There are benzyl alcohol, benzyl benzoic acid, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid ester.

  When the dosage form of the present invention is a suspension, water, ethanol or a liquid diluent such as propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester are used as a carrier component. Such suspending agents, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth can be utilized.

  When the dosage form of the present invention is a surfactant-containing cleansing, the carrier component is aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, Sarcosine, fatty acid amide ether sulfates, alkylamide betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters can be utilized.

  In a preferred embodiment of the present invention, the composition of the present invention is a pharmaceutical composition.

  When the composition of the present invention is manufactured as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention is a lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate Gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like are not limited thereto. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting system, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative and the like in addition to the above-described components.

  The pharmaceutical composition of the present invention can be administered by various routes such as oral or parenteral, and can be administered, for example, orally or transdermally. However, it is preferably applied to the transdermal administration of parenteral administration, more preferably to the topical application route by application.

  Suitable dosages of the pharmaceutical composition of the present invention include the formulation method, mode of administration, patient age, weight, sex, morbidity, food and drink, administration time, route of administration, excretion rate and response sensitivity. Various prescriptions can be made depending on various factors. The oral extract of the present invention, the solvent fraction thereof, or the compound isolated therefrom, in the case of an oral dosage form, 5 to 30 mg / kg as an adult standard, preferably 10 mg / kg once to several times a day. In the case of an external preparation, it may be applied 1 to 5 times per day in an amount of 1.0 to 3.0 ml per day as an adult standard, and it should be continued for 1 month or more.

  The pharmaceutical composition of the present invention can be obtained by a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the invention belongs, and a pharmaceutically acceptable carrier and / or excipient. Can be manufactured in a unit volume form by being formulated with a sucrose, or can be manufactured in a multi-volume container. At this time, the dosage form is compatible with pharmaceutical preparations including powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and other oral dosage forms, ointments, creams and other external preparations. Any form can be used and can further comprise a dispersant or stabilizer.

  As a preferred embodiment of the present invention, the composition of the present invention is a food composition.

  When the composition of the present invention is produced as a food composition, it includes not only kanaku extract, fractions thereof, or compounds separated therefrom as active ingredients, but also ingredients that are usually added during food production, for example, Contains protein, carbohydrates, fats, nutrients, seasonings and flavors. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, etc .; disaccharides such as maltose, sucrose, oligosaccharides, etc .; and polysaccharides such as normal sugars and xylitol such as dextrin, cyclodextrin, etc. Sugar alcohols such as sorbitol and erythritol. Natural flavoring agents [thaumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) can be used as flavoring agents.

  In another embodiment, the present invention provides a compound of Formula 10 below.

  In the present invention, the compound represented by the chemical formula 10 can be separated from the anemone tree, preferably from the thin skin portion of the anemone tree.

  In another embodiment, the present invention provides a method for separating a compound of Formula 10 from asterisks.

As a preferred embodiment, the method for separating the compound of Formula 10 may include the following steps:
Water Kanakuginoki, lower alcohols C ₁ -C _4, or the steps of extracting at a selected from a mixed solvent thereof The solvent obtain Kanakuginoki extract;
Fractionating the anthracnose extract with hexane, methylene chloride, ethyl acetate and butanol to obtain each solvent fraction;
Performing fractionation by subjecting the anthracnose solvent fraction to primary silica gel column chromatography using hexane and ethyl acetate as a solvent gradient as elution solvents; and eluting the primary silica gel column chromatography fraction. Performing a secondary silica gel column chromatography using a mixed solvent of hexane and ethyl acetate as a solvent to separate a novel polymethoxyphenol compound of Formula 10;

  In the present invention, the anemone tree is a thin skin portion of the anemone tree.

In the present invention, the Kanakuginoki extract water, ethanol, lower alcohols C ₁ -C _4, such as methanol or a solvent selected from a mixed solvent thereof, preferably obtained by extraction with ethanol. In the present invention, the extract includes an extract obtained by an extraction process, a diluted solution of the extract, a concentrated solution, a dried product obtained by drying the extract, or a roughly purified product thereof, or Any one of the purified products can be included.

  As an embodiment of the present invention, the extract of the anemone is dried with hot air and pulverized by depositing in 70% ethanol, leaching by stirring at room temperature for 3 days, filtered, It can be obtained by concentration under reduced pressure.

In the present invention, the solvent fraction of the kanakuginoki extract is obtained by suspending the kanakuhinoki extract in distilled water, and then, from a non-polar solvent, n-hexane (n-hexane; n-Hex), methylene chloride (CH). ₂ Cl ₂ ), ethyl acetate (EtOAc), and n-butanol (n-BuOH).

As an example of the present invention, a solvent fraction of an extract of kanakuginoki is obtained by suspending an extract of kanakuginoki ethanol in distilled water and using a separatory funnel to remove n-hexane (n-Hex), methylene chloride (CH ₂ Cl ₂ ), ethyl acetate (EtOAc), and n-butanol (n-BuOH) were used for fractionation, followed by filtration and concentration under reduced pressure to obtain a fraction for each solvent.

  Hereinafter, the configuration and effects of the present invention will be described more specifically with reference to examples. However, these examples are only exemplary descriptions of the present invention, and the scope of the present invention is limited to these examples. It is not limited to.

Example 1: Manufacture of kanakugi extract The plant sample linden (Lindera erythrocarpa) used in the present example was used after being sold by the extract bank of Jeju High-Tech Industry Promotion Agency, Republic of Korea.
First, linden erythrocarpa Makino. Was washed with running water, dried with hot air at 40 ° C. for 3 days, and pulverized with a pulverizer. 200 g of the dried sample was deposited in 70% ethanol and stirred for 3 days at room temperature to leach out. The leachate was filtered with a filter, and the leach filtration process was repeated three times. The filtrate was concentrated under reduced pressure. As a result, 60 g of ethanol extract was obtained.

Example 2: Separation of Solvent Fraction Derived from Kanakhinoki Extract In this example, the solvent used for sample extraction was Merk Co. Junsei Co. Hyman Co. A company product was used.
Solvent fractionation with respect to the ethanol extract of citrus was performed as follows.
First, 42 g of 60 g of 70% ethanol extract prepared in Example 1 was suspended in distilled water, and using a separatory funnel, n-hexane (n-Hex), methylene chloride ( After fractionation using CH ₂ Cl ₂ ), ethyl acetate (EtOAc), and n-butanol (n-BuOH), filtration and concentration under reduced pressure were performed, and fractions n-Hex 8.56 g, CH _{2 Cl 2 1.47g, EtOAc 4.14g,} n-BuOH 10.2g, to give a H 2 O 14.58 g (see Figure 1).
HPLC analysis was performed on each extract and fraction obtained as described above. The results are shown in FIGS.

  In subsequent experiments, powdered fractions of hexane, methylene chloride and ethyl acetate were dissolved in 100% ethanol, butanol and water fractions were dissolved in 100% ethanol and 1 × Phosphate Buffered Saline. : PBS, pH 7.4) was mixed at a ratio of 1: 1 and dissolved completely, and then used after filtration.

Example 3 Separation of Compound from Solvent Fraction of Kanacinoki Ethanol Extract and Identification of Structure Ethyl acetate fraction layer (4.0 g) was separated using celite with methylene chloride, ethyl ether, ethyl acetate and acetone. Fractions were separated, and fractions were separated using ethyl ether fractions on normal phase silica gel using chloroform and methanol as developing solvents. Using methanol, compound 1 was obtained in fraction-1, and compound 2 was obtained in fraction-3. From fraction-2, compound 3 and compound 4 were obtained using 30% aqueous methanol using Prep-HPLC.

  Ten fractions were obtained using normal phase silica gel with hexane and ethyl acetate as solvent gradient conditions with a methylene chloride fraction layer (2.53 g). Fraction-2 was purified using normal phase silica gel with hexane and ethyl acetate as developing solvents to give compound 5, and from fraction-5 using normal phase silica gel with chloroform and methanol as developing solvents 6 was obtained. Compound 7 was obtained from fraction-6 using the recrystallization method, and compound 8 was obtained from fraction-7 using the recrystallization method. From fraction-8, normal phase silica gel was used to obtain two fractions using chloroform and methanol as developing solvents. Among them, compound-9 was obtained as fraction-8-2. Nine obtained compounds were confirmed using HPLC analysis and NMR, and as a result, it was confirmed that they were purely separated.

  Finally, 200 g of oleander leaves were extracted with 70% ethanol to obtain 60 g of extract, from which 9 compounds were purely separated and purified.

  The yield based on each separated fraction and compound was as shown in Table 1 below.

Using a nuclear magnetic resonator (Bruker Co. 500 MHz, NMR), ¹ H, ¹³ C, COZY, HMQC, and HMBC spectra were obtained, and the structures were determined by comprehensively analyzing these spectra, and high performance liquid chromatography ( HPLC) was used for quantitative analysis.
The results of high performance liquid chromatography and ¹ H, ¹³ C-NMR spectrum of each compound are shown in FIGS.
As a result, Compound 1 was identified as quercetin of the following chemical formula 1.

  Compound 2 was identified as quercitrin of formula 2 below.

Compound 3 was identified as quercetin-3-O-arabinofuranoside of Formula 3 below.

Compound 4 was identified as kaempferol-3-O-rhamnopyranoside of formula 4 below.

  Compound 5 was identified as caffeic acid ethyl ester of formula 5 below.

  Compound 6 was identified as cinamic acid methyl ester of formula 6 below.

  Compound 7 was identified as lucidone of formula 7 below.

  Compound 8 was identified as methyl linderone of formula 8 below.

  Compound 9 was identified as kanakugiol of formula 9 below.

Experimental Example 1: Measurement of antioxidant activity of an extract and fractions of the present invention 1) 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical elimination experiment Measurement of electron donating ability Was measured according to the DPPH free radical elimination method by Blosis method. Specifically, 100 μl of various concentrations of samples dissolved in ethanol were dispensed into 96-well plates (well plates), the same amount of 0.4 mM DPPH solution was added and allowed to stand at room temperature for 10 minutes, and the absorbance was measured at 517 nm. It was measured. At this time, butylated hydroxy anisole (BHA) was used as a positive control group. The DPPH free radical scavenging activity was calculated from the following mathematical formula 1 and expressed as the sample concentration (IC ₅₀ ) expressed when the DPPH absorbance decreased by 50%.

In the above formula,
A _sample = absorbance of the reaction solution to which the sample is added,
A _control = absorbance of the reaction solution to which ethanol was added instead of the sample.

Table 2 below shows the results for the antioxidant activity of the ethanol extract of kanakugi and sequential fractions. The free free radical scavenging activity of DPPH increased in a concentration-dependent manner in both the ethanol extract and the sequential fractions depending on the treatment concentration. Of the sequential fractions, ethyl acetate fractions showed higher activity than BHA, which is a control group for DPPH free radical scavenging activity, and the remaining fractions excluding methylene chloride and hexane fractions were also remarkable. IC ₅₀ values at this time were 7.43 μg / ml (ethyl acetate), 16.88 μg / ml (ethanol), 18.54 μg / ml (butanol), 66.77 μg / ml, respectively. It was indicated as ml (water) (Table 2).

2) Xanthine oxidase inhibition and peroxide scavenging activity measurement Uric acid production by xanthine oxidase was measured by absorbance increased at 290 nm, and allopurinol (Sigma) was used as a control group did. The amount of superoxide was measured at 560 nm by the nitroblue tetrazolium (NBT) reduction method. For the reaction solution, prepare various concentrations of each sample, 0.5 mM xanthine and 1 mM EDTA in 100 μl of 200 mM phosphate buffer (pH 7.5), and add 50 mU / ml xanthine oxidase. Induced the production of uric acid. The peroxide scavenging activity was caused to react by adding 0.5 mM NBT to the reaction solution. Xanthine oxidase inhibition and peroxide scavenging activity is indicated by the sample concentration (IC ₅₀ ) expressed when the absorbance of the produced uric acid and peroxide decreases by 50%. After that, the average value was obtained.

The results for the xanthine oxidase inhibitory activity and the peroxide radical scavenging activity of the anthracnose ethanol extract and sequential fractions are also shown in Table 2. Both an extract of ethanol and sequential fractions showed xanthine oxidase inhibitory activity in a concentration-dependent manner, and hexane fraction (> 1000 ug / ml) and methylene chloride (IC ₅₀ 86.21 μg / ml) had slightly lower activities. However, the IC ₅₀ values of other fractions and the like were 5.55 to 9.79 μg / ml, indicating high inhibitory activity (Table 2). Also in the peroxide radical scavenging activity, the IC ₅₀ value is 16.86 μg / ml (ethyl acetate), which is slightly lower than the control group allopurinol (IC ₅₀ = 3.82 μg / ml). (Table 2).

  As already reported, these effects are the effects of preventing and delaying the prevention of aging, cancer and heart disease by preventing lipid oxidation, scavenging of active oxygen and preventing oxidative stress. These effects are utilized in many fields such as foods, pharmaceuticals, and cosmetics. Therefore, Kanakugi can be usefully used for the prevention of many oxidative stresses and injuries that occur in vivo, based on its antioxidant effect.

Experimental Example 2: Cytotoxicity measurement using MTT assay (assay) of an extract and fraction of the present invention The B16F10 mouse melanoma cell used in this experimental example was purchased from the American Cell Line Bank (ATCC). It was. B16F10 cells in DMEM medium containing 10% fetal bovine serum (FBS, Gibco), 1% Antibiotic-Antimycotic (Gibco), L-glutamine and sodium bicarbonate ( Gibco) was cultured in a 37 ° C., 5% CO ₂ cell incubator. The RAW 264.7 cell is a murine macrophage cell line, which is sold by KCLB (Korean Cell Line Bank), 10% FBS, 100 unit / ml penicillin, 100 μg / ml. The cells were cultured in a 5% CO ₂ cell incubator at 37 ° C. using DMEM medium (Gibco) containing streptomycin.

B16F10 cells were seeded at 2 × 10 ⁴ cells per well in a 24-well plate and cultured in a 5% CO ₂ cell incubator at 37 ° C. for 24 hours, after which the sample was 12.5 μg / ml in each well. , Treated at concentrations of 25 μg / ml, 50 μg / ml, and 100 μg / ml and cultured for 72 hours. 200 μl of MTT solution produced at a concentration of 2 mg / ml was added thereto, and the cells were cultured under the same culture conditions for 4 hours. The medium was removed, and the cells were washed twice with PBS. DMSO (200 μl) was added to each well, and the absorbance was measured at 570 nm using an ELISA reader (μQuant, USA).

  To examine the effects of Lindera erythrocarpa ethanol extract and sequential fractions on cell viability of B16F10 cell line, 12.5 μg / ml, 25 μg / ml, 50 μg / ml and 100 μg / ml After treatment with various concentrations up to 3 days and culturing, cell viability was observed by MTT method. As can be seen from FIG. 2, when the cell viability of the control group was set to 100%, among the concentrations of kanakugi ethanol extract and respective sequential fractions, the concentrations were almost 12.5, 25, 50, and 100 μg / ml. Between 81 and 103%, there was a slight decrease or increase compared to the control group. Thus, compared with the control group, the concentrations of 12.5 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, etc. were slightly different, but did not show any noticeable change. It was confirmed that the citrus ethanol extract and sequential fractions of the present invention had low cytotoxicity and could be used as a whitening agent.

Experimental Example 3 Measurement of Melanogenesis Inhibitory Activity of Anthracnose Extracts and Fractions of the Present Invention A 24-well plate was inoculated with 2 × 10 ⁴ B16F10 cells per well and maintained at 37 ° C. and 5% CO _{2 for} 24 hours. After culturing in the cell incubator, the sample was treated in each well at a concentration of 12.5 μg / ml, 25 μg / ml, 50 μg / ml, and 100 μg / ml, and after the sample treatment for 3 days, the medium was removed. Cells were washed twice with PBS. After adding 500 μl of 1N NaOH per well and dissolving at 56 ° C. for 30 minutes, the absorbance was measured at 405 nm using an ELISA reader (μQuant, USA).

  3 days at various concentrations up to 12.5 μg / ml, 25 μg / ml, 50 μg / ml, and 100 μg / ml of the anthracnose ethanol extract and sequential fractions observed as non-cytotoxic in Example 2 above After the treatment, melanin production inhibitory activity was measured. As can be seen from FIG. 3, when treated with 100 μg / ml of a synthetic substance, arbutin, which is a control group and is known as a whitening agent, 31% melanin production inhibitory activity was exhibited, and 12.5 μg / ml and 25 μg / ml, respectively. When treated to concentrations of 50 μg / ml and 100 μg / ml, the kanaku ethanol extract showed an inhibitory activity of 0.6%, 7.9%, 31.6%, 45.8%. Moreover, 33%, 49%, 24%, 31%, and 28% at the concentration of 100 μg / ml of each sequential fraction and the like showed a higher melanin inhibitory activity than the control group, and were extremely outstanding as a whitening agent in natural plants. Results are shown. Therefore, it was confirmed that the anthracnose ethanol extract and sequential fractions of the present invention have low cytotoxicity while reducing melanin production and can be used as a whitening agent.

Experimental Example 4 Measurement of Tyrosinase Inhibitory Activity of Extracts and Fractions of the Anthracnose Extract of the Present Invention A 24-well plate was inoculated with 2 × 10 ⁴ B16F10 cells per well and cultured at 37 ° C. and 5% CO ₂ for 24 hours. After culturing in a vessel, the samples were treated in the respective wells at concentrations of 12.5 μg / ml, 25 μg / ml, 50 μg / ml, and 100 μg / ml, and after 3 days of sample treatment, the medium was removed to remove the cells. After washing twice with PBS, the cells in the well were centrifuged to form a cell precipitate, and lysis buffer (0.1 M sodinm phosphate buffer, 0.2 mM PMSF, 1% Triton X-100) was added. The cells were left on ice to destroy the cells and centrifuged, and then the upper layer solution was taken and used for enzyme activity measurement. Each sample was placed in a reaction solution (12.5 mM L-Dopa, 1.5 mM L-Tyrosine, 67 mM sodium phosphate buffer (pH 6.8)) and allowed to react at 37 ° C. for 1 hour, followed by ELISA reader (μQuant, USA). The absorbance was measured at 405 nm.

  Inhibition of the final amount of melanin after treatment with kanakugi ethanol extract and sequential fractions indicates that it is related to the activity of enzymes involved in melanin synthesis. Therefore, tyrosinase activity in B16F10 cells was reduced to 12 respectively. As a result of treatment with various concentrations of 5 μg / ml, 25 μg / ml, 50 μg / ml, and 100 μg / ml for 3 days, 19% tyrosinase inhibitory activity was obtained when treated with arbutin as shown in FIG. And sequential fractions at 12.5 μg / ml, 25 μg / ml, 50 μg / ml, and 100 μg / ml concentrations respectively, the ethanol extract was 13.9%, 15.9%, 36.8%, It showed a high inhibitory activity of 42.5%. In addition, each fraction showed 40%, 21%, 19%, 41%, and 26% at a concentration of 100 μg / ml, indicating a higher tyrosinase inhibitory activity than the control group. From these results, it was determined that one of the main causes was that the decrease in melanin production by the anthracnose ethanol extract and sequential fractions in the B16F10 cell line was due to tyrosinase inhibitory activity.

Experimental Example 5: Cytotoxicity measurement of the cyclopentadione compound of the present invention derived from anthracnose using MTT assay 1) Lucidone (Compound 1)
To examine the effect on cell viability of the B16F10 cell line using a single substance isolated as lucidone, up to 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, and 10 μg / ml After culturing after treatment at various concentrations for 3 days, cell viability was observed by MTT method. As can be seen from FIG. 29, when the cell survival rate of the control group was 100%, the caffeic acid concentration was 965, 106, almost 10% at a concentration of 1.25, 2.5, 5, 10 μg / ml. Slightly decreased or increased compared to the group. In this way, the concentrations of 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, and 10 μg / ml, etc., were slightly different from the control group, but did not show any noticeable changes. It was confirmed that the single substance isolated as lucidone of the present invention has low cytotoxicity and can be used as a whitening agent.

2) Methyllinderone (compound 2)
To examine the effect on cell viability of the B16F10 cell line using a single substance isolated as methyllinderone, up to 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, and 10 μg / ml After treatment with various concentrations of for 3 days and culturing, cell viability was observed by MTT method. As can be seen from FIG. 30, when the cell viability of the control group was 100%, the caffeic acid concentration was almost 93 to 99% at 1.25, 2.5, 5, and 10 μg / ml. There was a slight decrease compared to the group. In this way, the concentrations of 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, and 10 μg / ml, etc., were slightly different from the control group, but did not show any noticeable changes. It was confirmed that the single substance isolated as methyllinderone of the present invention has low cytotoxicity and can be used as a whitening agent.

3) Kanakugiol (compound 3)
To examine the effect on cell viability of the B16F10 cell line using a single substance isolated as canakgiol, up to 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, and 10 μg / ml After culturing after treatment at various concentrations for 3 days, cell viability was observed by MTT method. As can be seen from FIG. 31, when the cell viability of the control group was 100%, the caffeic acid concentrations of 1.25, 2.5, 5, and 10 μg / ml were almost 91 to 96%, compared with the control group. Slightly decreased. In this way, the concentrations of 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, and 10 μg / ml, etc., were slightly different from the control group, but did not show any noticeable changes. Thus, it was confirmed that the single substance isolated as kanakugiol of the present invention has low cytotoxicity and can be used as a whitening agent.

Experimental Example 6: Measurement of Melanin Inhibition Effect of Cyclopentadione Compound Derived from Anthracnose of the Present Invention 1) Lucidone (Compound 1)
To measure the melanogenesis inhibitory effect on the B16F10 cell line using a single substance isolated as lucidone, a variety of up to 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml and 10 μg / ml respectively. After treatment at the concentration for 3 days, the melanogenesis inhibitory activity was measured.
As a result, as can be seen from FIG. 32, 23.5% of melanin production inhibitory activity was exhibited when treated with 50 μg / ml of arbutin, which is a control group and is known as a whitening agent, and 1.25 μg / ml and 2.5 μg, respectively. Lucidone showed high inhibitory activity of 3%, 19%, 37%, 53% when treated to concentrations of 5 / g, 5 μg / ml and 10 μg / ml. Therefore, it showed higher melanin inhibitory activity than the control group arbutin, and showed good results as a whitening agent in natural plants.

2) Methyllinderone (compound 2)
To measure the melanogenesis inhibitory effect on the B16F10 cell line using a single substance isolated as methyl Linderon, various up to 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml and 10 μg / ml respectively After treatment at various concentrations for 3 days, melanin production inhibitory activity was measured.
As a result, as can be seen from FIG. 33, 23.5% of melanin production inhibitory activity was exhibited when treated with 50 μg / ml of arbutin which is a control group and is known as a whitening agent, and 1.25 μg / ml and 2.5 μg, respectively. Methyllinderone showed high inhibitory activity of 1%, 14%, 38% and 62% when treated to concentrations of 5 / g, 5 μg / ml and 10 μg / ml. Therefore, it showed higher melanin inhibitory activity than the control group arbutin, and showed good results as a whitening agent in natural plants.

3) Kanakugiol (compound 3)
To measure the melanogenesis inhibitory effect on the B16F10 cell line using a single substance isolated as canakgiol, a variety of up to 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml and 10 μg / ml respectively. After treatment at the concentration for 3 days, the melanogenesis inhibitory activity was measured.
As a result, as can be seen from FIG. 34, 23.5% of melanin production inhibitory activity was exhibited when treated with 50 μg / ml of arbutin, which is a control group and is known as a whitening agent, and 1.25 μg / ml and 2.5 μg, respectively. / Canalgiol showed high inhibitory activity of 2%, 16%, 29% and 61% when treated to concentrations of 5 μg / ml and 10 μg / ml. Therefore, it showed higher melanin inhibitory activity than the control group arbutin, and showed good results as a whitening agent in natural plants.

Experimental Example 7: Measurement of Tyrosinase Inhibitory Activity of Cyclopentadione Compound from Anthracnose of the Present Invention Tyrosinase activity in B16F10 cells was 3 at various concentrations of 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, and 10 μg / ml, respectively. As a result of the daily treatment, as shown in FIG. 35, the control group showed 19% tyrosinase inhibitory activity when treated with arbutin, and lucidone concentrations of 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, and 10 μg / ml, respectively. When treated with, tyrosinase inhibitory activity was higher than that of the control group, which was 10%, 17%, 26.4%, and 48.9%. From these results, it was determined that one of the main causes was that the decrease in melanin production by the lucidone single compound isolated from cynomolgus in the B16F10 cell line was due to tyrosinase inhibitory activity.

Example 4: Manufacture of an extract of leaves and thin skin of cypress leaves and separation of solvent fraction therefrom A terrestrial plant sample, Lindera erythrocarpa, was used for the Jeju High-Tech Industry in South Korea. The solvent used for the sample extraction, which was sold after being sold by Shinkoin Extract Bank, was manufactured by Merck Co., Ltd. A company product was used.
The fresh leaves of kanakugiki were washed with running water, dried with hot air at 40 ° C. for 3 days, and pulverized with a pulverizer. A 200 g dry powder sample was deposited in 70% ethanol and extracted by stirring for 24 hours at room temperature for 3 days, and this leachate was filtered through a filter. After the leaching filtration process was repeated three times, the obtained filtrate was concentrated under reduced pressure. Of 60 g of the obtained extract (70% ethanol extract), 42 g was suspended in distilled water, and n-hexane (n-Hex), methylene chloride (CH ₂ Cl ₂ ), ethyl using a separatory funnel. Fractionation using acetate (EtOAc) and n-butanol (n-BuOH), followed by filtration and concentration under reduced pressure, 8.56 g of fractions by solvent, 1.47 g of methylene chloride, 1.47 g of methylene chloride, 4.14 g of acetate, 10.2 g of n-butanol, and 14.58 g of water were obtained.
On the other hand, the thin skin of kanakugiki was dried by the same method as that for the leaves, pulverized, and extracted with 68.2 g of 70% ethanol extract obtained under the same conditions using 560 g of the dry powder sample. Fractionation was performed to obtain each fraction.

Example 5: Separation of a compound from an anemone solvent fraction and identification of structure As a filler used in the separation process of this example, normal-phase silica gel 60 (0.063-0.200 mm, Merck Co.) was used. As an instrument used in the analysis process, HPLC (Alliance 2695, Waters Co.) was used, and XTerra (registered trademark) C18 3.5 μm 4.6 × 100 mm was used. As a detector used for analysis, A Waters 2998 model PDA was used. As the solvent and reagent used in the experiment, HPLC grade was used, and as the equipment used for structural analysis, NMR (Nuclear Magnetic Resonance) was a JNM-LA500 of Burker (German Co.). the methanol -d _4, using chloroform -d _1.

Fractionation is performed under normal phase silica gel (hexane / ethyl acetate) (v / v) and solvent gradient conditions on methylene chloride fraction layer (2.5 g) of the leaves of the ankle tree obtained in Example 4 above. 10 fractions were obtained. Of these, compound 10 (21.8 mg) was obtained from fraction-6 using the recrystallization method.
On the other hand, fractionation was carried out under normal-phase silica gel (hexane / ethyl acetate) (v / v) and solvent gradient conditions on the thin-layered methylene chloride fraction layer (7.0 g) obtained in Example 4 above. , 8 fractions were obtained. Among the fractions, fraction-2 was used for chromatography under normal phase silica gel (hexane / ethyl acetate) (3/2) conditions. Among them, fraction 2-2 to compound 12 (59.6 mg) Compound 11 was obtained from fraction-2-3.

Compounds 10 to 12 obtained as described above were subjected to compound analysis and structure confirmation via HPLC, NMR, (1D, 2D NMR), and LR / HR FAB-MS. The content in the ethanol extract was confirmed.
As a result, compound 10 was identified as the cyclopentadione series lucidone, compound 11 was the same series of methyllinderone, and compound 12 was modified from the cyclopentadione series of formula 10 below. Was identified as a novel compound (N2) that has not been reported to date. The IUPAC name of the novel compound is 2,3,4,5-tetramethoxy-6- (1-methoxy-3-phenylpropyl) phenol [2,3,4,5-tetramethoxy-6- (1-methoxy- 3-phenylpropyl) phenol], and the general name was named jeju-polymethoxy-phenol. 1D and 2D NMR data of Compound 12 are shown in Table 3 below.

Characteristics of compounds 1) IUPAC name: 2,3,4,5-tetramethyl-6- (1-methoxy-3-phenylpropyl) phenol
2) Color: Dark green 3) Scent: Slightly decaying odor 4) Solubility: Organic solvent such as methanol, acetone, chloroform 5) Distribution only in thin skin of cypress 6) Generic name: jerju-polymethy-phenol

  Table 4 below shows the yields of the ethanol extract of kanakugi and leaves and fractions of each solvent and the compound of Formula 10.

  As can be seen from Table 4 above, the compound of Formula 10 was not found in the leaves, but was observed as high as 0.71% in the thin skin.

  The single compound separated from the anemone was completely dissolved by adding DMSO solvent, and then filtered and used in the subsequent experiments.

Experimental Example 8: Cytotoxicity measurement of compound of formula 10 of the present invention using MTT assay In order to investigate the effect on the cell viability of B16F10 cell line using a novel compound not currently known, Such an experiment was conducted.
B16F10 mouse melanoma cells used in this experiment were obtained from the American Cell Line Bank (ATCC). B16F10 cells were cultured at 37 ° C. using DMEM medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco), 1% antibiotic (Antibiotic-Antimycotic) (Gibco), L-glutamine and sodium bicarbonate. The cells were cultured in a 5% CO ₂ cell incubator.
B16F10 cells were seeded in a 24 well plate at 2 × 10 ⁴ cells per well and cultured for 24 hours in a 37 ° C., 5% CO ₂ cell incubator, and then the sample was contained in each well at 5 μg / ml, The cells were treated for 3 days at various concentrations up to 10 μg / ml, 20 μg / ml, and 30 μg / ml and cultured for 72 hours. 200 μl of MTT solution produced at a concentration of 2 mg / ml was added thereto, and cultured for 4 hours under the same culture conditions to remove the medium, and the cells were washed twice with PBS. 200 μl of DMSO was added to each well, and the absorbance was measured at 570 nm using an ELISA reader (μQuant, USA).

  As a result, as can be seen from FIG. 36, when the cell survival rate of the control group was 100%, the compound of the chemical formula 10 was almost 97 to 103% at the concentrations of 5, 10, 20, and 30 μg / ml. Slightly decreased or increased compared to the group. Thus, compared with the control group, the concentrations of 5 μg / ml, 10 μg / ml, 20 μg / ml, and 30 μg / ml were slightly different, but did not show any noticeable change. It was confirmed that the compound of Chemical Formula 10 has low cytotoxicity and can be used as a whitening agent.

Experimental Example 9 Measurement of Melanogenesis Inhibitory Activity of Compound of Formula 10 of the Present Invention A 24-well plate was inoculated with 2 × 10 ⁴ B16F10 cells per well and incubated at 37 ° C. in a 5% CO ₂ cell incubator for 24 hours. After culturing, the sample was treated in each well and treated for 3 days, after which the medium was removed and the cells were washed twice with PBS. After adding 500 μl of 1N NaOH per well and dissolving at 56 ° C. for 30 minutes, the absorbance was measured at 405 nm using an ELISA reader (μQuant, USA).

  As a result, as can be seen from FIG. 37, when treated with 50 μg / ml of arbutin which is a control group and is known as a whitening agent, 33.6% of melanin production inhibitory activity was exhibited. When treated with ml, 10 μg / ml, 20 μg / ml, and 30 μg / ml, high inhibitory activity of 19%, 32%, 51% and 59% was exhibited. Therefore, it showed a melanin inhibitory activity approximately twice as high as that of the control group arbutin, showing good results as a whitening agent in natural plants.

Experimental Example 10: Measurement of mRNA Expression Inhibitory Activity Involved in Melanogenesis of Compound of Formula 10 of the Present Invention B16F10 mouse melanoma cells used in this experimental example were sold from the American Cell Line Bank (ATCC). B16F10 cells were treated with DMEM medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco), 1% antibiotic (Antibiotic-Antimycotic) (Gibco), L-glutamine and sodium bicarbonate at 37 ° C., 5 ° C. Cultured in a% CO ₂ cell incubator.
The cultured cells were washed 2-3 times with PBS, and then total RNA extraction was separated using TRIzol-reagent (Invitrogen, USA). The cells were homogenized by adding TRIzol-reagent, followed by adding chloroform and centrifuging (12,000 rpm, 15 min). Add the same amount of isopropanol to the supernatant, centrifuge (12,000 rpm, 10 min) to precipitate RNA, wash with 75% diethylpyrocarbonate (DEPC) -treated ethanol, and then dry. And dissolved in distilled water treated with DEPC. The RNA was quantified by measuring the absorbance at 260 nm, and RNA having an A260 / A280 nm ratio in the range of 1.6 to 1.9 was used in the experiment. For cDNA synthesis, Improm-II ^™ cDNA kit (Promega, USA) was used, 1 μg of total RNA (total RNA) was used as oligo (dT) primer (oligo (dT) primer), dNTP (0.5 μM), 1 unit RNase inhibitor. The reaction was stopped by heating with Improm-II ^™ reverse transcriptase (2 U) at 25 ° C. for 5 minutes, 37 ° C. for 60 minutes, and 70 ° C. for 10 minutes. Polymerase chain reaction (PCR) is used to amplify tyrosinase, TRP-1, TRP-2, β-actin from synthesized cDNA, 1 μl of cDNA, 4 μM of 5 ′ and 3 ′ primer, 10 × buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.1% Triton X-100), 250 μM dNTP, 25 mM MgCl ₂ , 1 unit Taq polymerase (Promega, USA) were mixed and mixed with 3rd distilled water. After the final 25 μl, it was performed using a Perkin-Elmer Thermal Cycler. At this time, PCR conditions were 94 ° C./30 seconds, 50-55 ° C./45 seconds, and 72 ° C./45 seconds 20-25 times, and the product generated by PCR was 1.2% agarose gel (agarose gel) and stained with ethidium bromide to identify specific bands.
Melanocytes are known to be involved in a process in which NO generated from keratinocytes and the like by ultraviolet rays increases melanin production through the cGMP pathway, increases tyrosinase and TRP-1, and induces tyrosinase activity mRNA expression. ing. RT-PCR was performed to confirm whether the melanin-suppressing effect of a single compound isolated from an anemone at a concentration that does not show toxicity in B16F10 mouse melanoma cells is due to the suppression of mRNA expression. The aspect of expression suppression of TRP-1 and TRP-2 genes other than tyrosinase known as a main enzyme for melanin production was confirmed.

As a result, as can be seen from FIG. 38, it was shown that the expression levels of tyrosinase and TRP-1 mRNA decreased in a concentration-dependent manner. In the normal melanin synthesis process, TRP-2 is a carboxylated derivative of dopachrome 5,6-dihydroxyindole-2-carboxylic acid (DHICA). However, as a result of confirming the expression of the TRP-2 mRNA gene, the TRP-2 mRNA expression was constant regardless of the concentration.
Therefore, it was confirmed that the novel compound isolated from the anemone tree inhibits melanin synthesis by decreasing mRNA expression of tyrosinase and TRP-1 regardless of TRP-2 (FIG. 38). Based on the above results, it was confirmed that the novel compound isolated from kanakugi had excellent melanin pigment formation inhibition and tyrosinase inhibition effects on B16F10 black seed cells. I found out.

  As described above with reference to the above Examples and Experimental Examples, the composition for skin whitening containing the extract, fraction or compound of anthracnose of the present invention is effective for the cyclopentadione compound or its derivative. By containing it as an ingredient, it exhibits excellent melanin production inhibitory activity and tyrosinase inhibitory activity, so that it can be usefully used in the cosmetics, pharmaceutical and food industries.

Claims (10)

Skin whitening composition containing only the compound represented by the following chemical formula 10 as an active ingredient:
.   The composition for skin whitening according to claim 1, wherein the compound is separated and purified from kanakugi.   The skin whitening composition according to claim 1 or 2, wherein the skin whitening composition is a cosmetic composition.   The skin whitening composition according to claim 1 or 2, wherein the skin whitening composition is a pharmaceutical composition.   The skin whitening composition according to claim 1 or 2, wherein the skin whitening composition is a food composition. Compound represented by the following chemical formula 10:
.   The compound according to claim 6, wherein the compound of Chemical Formula 10 is isolated from anthracnose.   The compound according to claim 6, wherein the compound represented by Chemical Formula 10 is isolated from a thin skin of anthracnose. Water Kanakuginoki, lower alcohols C ₁ -C _4, or the steps of extracting at a selected from a mixed solvent thereof The solvent obtain Kanakuginoki extract;
Fractionating the above citrus extract with hexane, methylene chloride, ethyl acetate and butanol to obtain each solvent fraction;
Performing the first silica gel column chromatography fractionation of the anthracnose solvent fraction using hexane and ethyl acetate as a solvent gradient in a solvent gradient; and the primary silica gel column chromatography fractionation. The method of isolate | separating the compound of Claim 6 from a cypress tree including the step which performs a secondary silica gel column chromatography using the mixed solvent of hexane and ethyl acetate as an elution solvent.   The method according to claim 9, wherein the anemone is a thin skin portion of the anemone.