KR20140020488A - Novel flaniostatin compound and a composition comprising thereof - Google Patents

Abstract

The present invention provides: flaniostatin, which is a novel compound; a solvent solution thereof; a hydrate thereof; salt thereof; and a composition containing thereof as an effective component. The composition which contains flaniostatin, the solvent solution thereof, the hydrate thereof or the salt thereof as an effective component can be used for: the control of tyrosinase activity; the control of melanin generation; the preventing, treating, or curing of diseases caused by the excessive generation of melanin; and whitening.

Description

TECHNICAL FIELD The present invention relates to a novel planiostatin compound and a composition comprising the same as an active ingredient.

The present invention relates to a novel flaniostatin compound having tyrosinase inhibitory activity and a composition comprising the compound as an active ingredient.

In melanocytes among the cells constituting the skin epidermis, melanin synthesis is increased by the exposure of the sunlight, and increased melanin has a beneficial effect of protecting the skin from external stimuli. However, it is also effective in preventing erythema or pigmentation, , Skin cancer induction, toxicity by melanin precursors, and promotes apoptosis, causing harmful or cosmetic problems to the human body.

Melanin is produced in the melanosome through tyrosinase as a major enzyme and the amino acid tyrosine is converted to 3,4-dihydroxyphenylalanine (DOPA) and DOPAquinone. By inhibiting the activity of the major enzyme, tyrosinase, to inhibit melanin synthesis, inhibition of melanin synthesis and whitening effect can be induced [Kim et al., Korean J. Medicinal Crop . Sci . 16 , 255-260, 2008]. Therefore, the inhibitory activity of tyrosinase is recognized as a useful evaluation method for development of melanin synthesis inhibitor and skin whitening agent.

Kojic acid and arbutin are used as a whitening agent having tyrosinase inhibitory activity, but cytotoxicity and mutagenic side effects have been reported. Therefore, it is necessary to develop new drugs having melanin biosynthesis inhibitory activity and few side effects.

Disclosure of the Invention The present invention provides a novel substance having a tyrosinase inhibiting activity and a melanin formation inhibiting activity.

In order to solve the above problems, the present invention provides a novel glycoside compound, flanriostatin, a hydrate thereof, a solvate thereof and a salt thereof.

Planiostatin provided through this patent application has a tyrosinase inhibitory activity, a melanin formation inhibitory activity, and a whitening activity. The composition comprising planiostatin can be used as a cosmetic composition, a pharmaceutical composition, a food or a food additive composition for inhibition of tyrosinase, inhibition of melanin formation, whitening effect.

Figure 1 is an HPLC profile of purified water extract.
Figure 2 is the ESI-Mass spectrum of planiostatin.
3 shows IR spectrum of planiostatin.
4 is a ¹ H-NMR spectrum of planiostatin (CDCl ₃ ).
5 is a ¹³ C-NMR spectrum of planiostatin (CDCl ₃ ).
Figure 6 is the HMQC spectrum of planiostatin.
Figure 7 is a ¹ H- ¹ H COZY spectrum of planiostatin.
8 is the HMBC spectrum of planiostatin.
9 is a graph showing tyrosinase inhibitory activity of planiostatin. Assay solutions include plnio statin or arbutin at various concentrations and 1500 units / mL tyrosinase, 1.5 mM tyrosine. Each experimental value is the mean ± SD of three experimental results.
Fig. 10 shows the cell viability of B16-F1 melanoma cell line by treatment with planiostatin. (A) MTT reductioin assay (B) LDH release assay.
Fig. 11 is a photograph showing the morphological changes observed in the B16-F1 melanoma cell line after treatment with planiostatin for 24 hours. (A: control group, B: 10 μM treated group, and C: 50 μM treated group).
FIG. 12 shows the cell death effect of planiostatin on the B16-F10 melanoma cell line after staining with hoechst 33342. A: Control group, B: 10 μM treated group, C: 50 μM treated group.

The present invention relates to a novel compound having tyrosinase inhibitory activity, flannostatin. The inventors of the present invention separated and identified a novel substance that inhibits the activity of tyrosinase from the leaf extract of Cudrania tricuspidata Bureau and named it as flaniostatin (Examples 1 and 2). Platyostatin had a more potent inhibitory effect on tyrosinase activity than artbutin used as a skin lightening agent (Example 3). In addition, it was confirmed by the MTT reduction assay, the LDH release assay, the morphological change of the cells, and the hoechst 33342 staining test that the platyostatin does not have cytotoxicity to the B16-F1 melanoma cell line (Examples 4 to 7) .

 These results indicate that platyostatin is superior to tyrosinase inhibitory activity and melanin formation inhibitory activity and has excellent safety against B16-F1 melanoma cell line. Therefore, existing tyrosinase inhibitors having side effects such as gene mutation, instability and skin cancer induction due to toxicity And that it is possible to do so.

Accordingly, the present invention provides, in one embodiment, flannostatin, a hydrate thereof, a solvate thereof, and a pharmaceutically acceptable salt thereof represented by the following formula (1), which is a novel compound.

[Formula 1]

Planiostatin can be produced by organic synthesis or by separation from natural products. For example, planiostatin can be isolated and purified by alcohol extraction, extraction with an organic solvent, and chromatography. Specifically, for example, the following steps may be included, but are not limited thereto.

1) Extracting the leaves with alcohol. The alcohols used for extracting the leaves of Cucumis japonica are aliphatic alcohols having 1 to 6 carbon atoms, and specifically, methanol, ethanol, isopropanol, butanol and the like can be used. In the concrete examples of the present invention, methanol is used as the aliphatic alcohol, but even if other aliphatic alcohols exemplified above are used, the desired extract of the present invention can be sufficiently obtained.

2) The step of extracting the leaf alcoholic extract of cedarwood with organic solvent and water. The organic solvent used herein may be selected from aliphatic alcohols having 1 to 6 carbon atoms, aliphatic hydrocarbons having 1 to 10 carbon atoms, aliphatic halogenated hydrocarbons having 1 to 10 carbon atoms, and esters having 2 to 10 carbon atoms.

3) Separation of the components of the distribution extract by a chromatographic method. The chromatography used here may be silica gel column chromatography. For the eluent used in the silica gel column chromatography, for example, a mixture of chloroform and methanol may be used. The silica gel column chromatography eluate may be further subjected to additional steps such as HPLC.

There are no particular limitations on the extraction of alfalfa leaf, dispensing by organic solvent, the amount of solvent used in the chromatography process, application method, and the like, as described above, It is also possible to change this. In addition, the crude extract may be further purified by repeating several times as described above according to need. In order to determine the chemical structure of the tyrosinase activity inhibitory substance isolated from the leaf extract of Curcuma longa, an optical analyzer such as high-resolution ESI-mass, UV, and NMR can be used.

In one embodiment, the present invention provides a method for producing a planiastatin compound from a leaf of Cudrania tricuspidata Bureau comprising the above-described process.

The planiastatin compound of the present invention includes planiostatin, a salt compound thereof, a solvate, and a hydrate thereof.

Examples of salts of planiostatin include pharmaceutically acceptable salts such as acid addition salts and quaternary ammonium salts. Examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate and phosphate, and organic acid salts such as oxalate, maleate, fumarate, lactate, malateate, tartrate, And organic acid salts such as acid salts. Examples of quaternary ammonium salts include lower alkyl halides such as methyl iodide, methyl bromide, ethyl iodide and ethyl bromide; Lower alkyl sulfonates such as methyl methanesulfonate and ethyl methanesulfonate; And quaternary ammonium salts such as lower alkyl aryl sulfonates such as methyl-p-toluene sulfonate. The present invention also provides a solvate or hydrate of planiostatin. Solvates include, but are not limited to, dioxane, dimethylacetamide, dimethylformamide, cyclopentanone, picoline, and the like.

As an embodiment of the present invention, there is provided a composition having tyrosinase inhibitory activity comprising planiastatin, a hydrate thereof or a solvate thereof, or a salt thereof as an active ingredient, a kit comprising the same, and a method for inhibiting tyrosinase activity using the composition do.

In another embodiment of the present invention, there is provided a composition having melanogenesis inhibitory activity comprising planiastatin, a hydrate thereof or a solvate thereof, or a salt thereof as an active ingredient, a kit containing the melanin inhibitory activity, . ≪ / RTI >

Furthermore, as one embodiment of the present invention, there is provided a composition having a whitening effect comprising planiastatin, a hydrate thereof or a solvate thereof, or a salt thereof as an active ingredient, a kit containing the same, and a whitening method using the same.

As another embodiment of the present invention, there is provided a pharmaceutical composition for preventing, ameliorating, or treating diseases caused by tyrosinase hyperactivity or melanin excess production, comprising planiastatin, a hydrate thereof or a solvate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient A kit comprising the same, and a method of preventing, ameliorating, or treating a disease caused by excessive tyrosinase activity or melanin overgrowth by administration or administration thereof. Diseases caused by the excessive production of melanin include erythema, pigmentation, skin blackening, skin aging, skin cancer, and cell death due to melanin precursors, but are not limited thereto. Plagiostatin may be used for this purpose, either by systemic administration (e.g., oral administration) and topical administration (e.g., by skin application), respectively, or concurrently.

The compounds, compositions and kits of the present invention may be administered to a person in need of prevention or treatment of diseases due to overexpression or hyperactivity of tyrosinase and may be administered to in vitro tests requiring inhibition of tyrosinase expression or hyperactivity .

In the present invention, planiostatin may be contained in an amount of 0.0005 to 30% by weight, preferably 0.01 to 10% by weight, based on the whole amount of the composition, but it can be controlled as desired according to the purpose. The optimum dose of the pharmaceutical composition of the present invention is appropriately determined according to age, individual differences, symptoms, etc. However, when administered to humans, the dose is usually 0.01 to 100 mg / kg, preferably 0.1 to 10 mg / kg, The amount can be administered once a day or divided into several times.

The compositions of the present invention may be used in the form of solids, solutions, emulsions, dispersants, micelles, liposomes, ointments, and the like, and may include organic or inorganic carriers or excipients suitable for oral or parenteral application. The compositions of the present invention are generally for tablets, powders, pellets, capsules, pills, suppositories, solutions, emulsions, suspensions, solutions, jelly, injections and any other form suitable for use. It can be mixed with a nontoxic pharmaceutically acceptable carrier. Usable carriers include solid, semisolid or liquid glucose, lactose, gum arabic, gelatin, mannitol, starch paste, magnesium trisilicate salt, talc, corn starch, keratin, colloidal silica, potato starch, urea, chain Medium length triglycerides, dextran and other carriers suitable for use in the preparation of the formulations are included. In addition, auxiliaries, stabilizers, thickeners, colorants and flavoring agents can be used.

Planiostatin has a melanin formation-inhibiting activity and is low in cytotoxicity, and thus can be used in a cosmetic composition. And can be used particularly as a cosmetic composition for skin whitening. The cosmetic composition of the present invention may contain 0.001 to 90% by weight, preferably 0.01 to 70% by weight, of planiastatin relative to the total weight. In addition, the cosmetic composition of the present invention may further contain vitamins, peptides, polysaccharides, lipids, etc. in addition to planiastatin. In addition, the cosmetic composition of the present invention may further contain a preservative component, a moisturizer, a surfactant, a pigment, an ultraviolet absorber, But are not limited to, an inhibitor, a plant extract, a pH adjuster, an alcohol, a flavoring, purified water, and the like.

The cosmetic composition of the present invention may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, oil, wax, powder, spray, soap, cleansing, , But is not limited to.

The present invention also provides a food composition or a food additive composition for inhibiting tyrosinase activity, inhibiting melanin formation, containing a planiastatin compound as an active ingredient. The present invention also provides a food composition or food additive composition for the prevention and improvement of diseases caused by melanin overproduction. The food composition or food additive composition comprising the planiastatin of the present invention can be suitably used alone or in combination with other food or food ingredients according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use thereof. In general, planiostatin may be added in an amount of 0.0001 to 30% by weight, preferably 0.1 to 10% by weight based on the raw material in the production of food or beverage, It is possible. There is no particular limitation on the kind of the food. Examples of the food include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Drinks, tea, drinks, alcoholic beverages, and vitamin complexes.

Hereinafter, the present invention will be described in detail with reference to examples and production examples. However, it should be understood that the present invention is not limited thereto.

Experimental material

Cudrania used in the experiment The leaves of tricuspidata Bureau were grown in Sancheong - gun, Gyeongnam province. DMEM media and penicillin, streptomycin, fetal bovine serum (FBS) and horse serum (HS) were purchased from Gibco-BRL (Grand Island, NY, USA) and used to assess tyrosinase inhibitory activity (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide used for the evaluation of cytotoxicity against tyrosinase, tyrosine, arbutin and skin cell lines ) Was purchased from Sigma Chemical Co. (St Louis, Mo, USA) and LDH (lactate dehydrogenase) release assay kit were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All of the various solvents and reagents used in the other studies were of grade 1 or higher.

Cell culture and treatment

The cell line used in this experiment was B16-F1 murine melanoma, which was purchased from the Korean Cell Line Bank (KCLB). For cell culture, 10% FBS, 5% HS and 100 units / ml penicillin, 100 mg / ml streptomycin and 2 mg / ml NaHCO ₃ were added to the DMEM medium and 95% And cultured at 37 ° C in a 5% CO ₂ incubator (MCO-18AIC, Sanyo, Osaka, Japan).

Statistical processing

All data were processed using the SPSS-PC + statistical package. One-way analysis of variance (ANOVA) was performed to determine the percentage and mean ± standard deviation (SD) of each item. To compare the cytotoxicity of planiostatin, the F value was calculated. Using Scheffe's test, . Statistical significance was assessed at a level of less than 5%.

< Example  1> Of planiostatin  Separation and purification

Leaves of Cudrania tricuspidata Bureau), 6 L of methanol was added, and the mixture was extracted at room temperature for 3 days. 1 filter paper (Advantec, Tokyo, Japan). The extraction filtrate was extracted at 40 ° C with a rotary evaporator (EYELA N-1000, Tokyo, Japan). In order to separate the leaf extracts from the leaves according to the polarity and nonpolar solvent conditions, 2 L of water was added, and the mixture was divided into hexane layer and water layer using hexane. Diethyl ether and ethyl acetate ethyl acetate) was added and extracted.

After confirming tyrosinase inhibitory activity in the ethyl acetate layer, the silica gel column chromatography was carried out under a solvent condition of CHCl ₃ : MeOH = 5: 1. Each fraction was monitored by silica gel TLC and the tyrosinase inhibitory activity was assessed by bioassay.

The active substance isolated by silica gel column chromatography was identified as a single substance by HPLC (high performance liquid chromatography). When the mobile phase was eluted with 40% methanol using an ODS column, the active substance could be separated in about 26 minutes (Fig. 1) and named as flaniostatin.

< Example  2> Of planiostatin  Structure determination

In order to determine the structure of the planiostatin, the ESI-MS spectrum was measured in a positive mode. As a result, [M + Na] ⁺ was observed at m / z 499.1 and the molecular weight was found to be 476 (FIG. 2). The IR spectra were able to predict hydroxyl at 3400 cm ^-1 and carbonyl at 1400 cm ^-1 (Figure 3).

As a result of ¹ H-NMR spectroscopy, three methylene proton, 4.25 ppm anomeric proton, 3.1 to 4.3 ppm oxygenated methine proton, and oxygenated methine proton at 2.9 ~ 4.0 ppm and 3.6 ~ 3.9 ppm, respectively, methylene proton. However, since methyl proton was not present, ¹ H-NMR spectrum showed that plnio statin was a compound of the flavonoid glycoside system (FIG. 4).

^As a result of ¹³ C-NMR spectrum, one carbonyl carbon was observed at 180.4 ppm, anomeric carbon at 103.2 ppm, and aromatic carbon at about 120 ppm, suggesting the presence of isoflavonoid derivatives. A number of oxygenated carbons were observed between 61.3 and 76.7 ppm (Fig. 5).

As a result, the NMR chart of Table 1 was completed. From these results, it can be deduced that six hydroxyl groups, three methylene groups, and 11 methine groups exist and are linked by ester bonds.

The HMQC spectrum was analyzed to determine the presence or absence of quaternary carbon of planiostatin and the linkage between H and C, and all proton-bearing carbon ( ¹ J _{C -H} ) could be identified (FIG. 6) , And ¹ H- ¹ H COZY spectra. Three partial structures including isoflavones and sugars were identified (Fig. 7).

In addition, the HMBC spectrum was analyzed to confirm the linkage of the three sub-structures. From the analysis of the HMBC spectrum, it was found that 123.6, 157.3 ppm carbon from methine proton at 7.26 ppm, 122.4 ppm carbon from 6.75 ppm methine proton, 7.88 ppm 103.9, 109.0 and 167.1 ppm carbon from 6.21 ppm methine proton, 159.7, 109.0 and 167.1 ppm carbon from the methylene proton at 2.91 and 1.85 ppm, respectively, at carbon of 180.4, 123.0 and 157.0 ppm from methine proton, 3.90 , A long-range correlation was observed from the methylene proton at 3.62 ppm to the carbon at 159.7, 103.2 ppm, and from the methine proton at 4.25 ppm to the carbon at 68.2 ppm, thereby determining the chemical structure of the AC ring of the isoflavone glycoside ).

From the above results, it was possible to connect three partial structures identified by the ¹ H- ¹ H COZY spectrum, and as a result, the chemical structure of the planiostatin of formula 1 having the molecular formula of C ₂₃ H ₂₄ O ₁₁ can be determined there was.

[Formula 1]

Flaniostatin is a novel substance in the isoflavone glycoside series, which has not been reported so far, and is isoflavone and sugar linked by methylene to form an ester bond. It is the first time that isoflavone is bound to a sugar by methylene It is a known combination method.

< Example  3> Of planiostatin Tyrosinase  Measurement of inhibitory activity

To each well of a 96-well plate, add 100 μl of plasniostatin, 140 μl of 0.05 M sodium phosphate buffer (pH 6.8), 20 μl of tyrosinase enzyme (1500 units / ml mushroom tyrosinase) and 40 μl of substrate (1.5 mM tyrosine) After incubation at 37 ° C for 15 minutes, the absorbance was measured at 490 nm using an ELISA reader. Methanol was used as a control, and arbutin was used as a positive control.

Arrhythin and planiostatin were treated at concentrations of 10, 50, 100 and 500 μM, respectively, and the activity of tyrosinase was measured. As a result, platyostatin showed tyrosinase inhibitory effects of 22.4, 25.0, 31.4 and 51.0% Showed inhibitory effects of 9.8, 17.4, 24.6 and 39.5%, respectively (Fig. 9). From these results, it was confirmed that platyostatin has a higher tyrosinase inhibitory activity than arbutin.

< Example  4> MTT reduction  Measurement of cell viability by analysis

MTT reduction assay was performed to determine the effect of ploniostatin on the survival of melanoma cell line B16-F1. B16-F1 cells were plated at a concentration of 1 × 10 ⁵ cells / ml in each well of a 96-well plate and cultured for 24 hours at 37 ° C in a 5% CO ₂ incubator. Planiostatin was added to the final concentration Were treated with B16-F1 cell lines at 1, 5, 10, and 50 μM, respectively, for 24 hours. Then, 10 μl of MTT (5 mg / ml) solution dissolved in PBS buffer was added to each well, and formazan formation was confirmed by reacting for 30 minutes, and the medium was completely removed. To dissolve the formazan formed on the well bottom, 100 μl of DMSO was added, and the absorbance was measured at 540 nm using an ELISA reader (Model 680, BioRad, USA). Control cells cultured without treatment with planiostatin were incubated with 100 %, The relative cell viability was calculated (Fig. 10A).

The cell viability was 96.4, 99.1, 101.2, and 100.9%, respectively, when treated with 1, 5, 10, and 50 μM of plnio-statin, respectively. And did not show cytotoxicity against the cells.

< Example  5> LDH  Cytotoxicity by emission measurement

The cytotoxicity of planiostatin to the B16-F1 cell line was confirmed by the LDH release assay. B16-F1 cell line is 1 × 10 ⁵ cells / 100 μl each in a concentration of 96 ml and dispensed into each well of the well plate ₂ CO After incubation for 24 hours in an incubator, the cells were treated with B16-F1 cell line so that final concentrations of planiostatin were 1, 5, 10, and 50 μM, respectively, and cultured for 24 hours. 50 μl of the supernatant of the culture was dispensed into a new 96-well plate, and 50 μl of LDH reagent was added to the culture, allowed to stand at room temperature, and reacted for 10 minutes. When the reaction was completed, the reaction was stopped by adding 100 μl of stop solution, 1 N HCl, and the absorbance was measured at 540 nm using an ELISA reader. For the measurement of LDH release, 50 μl of 0.5% Triton X-100 solution was added, and the cells were shaken at 40 rpm for 10 min. Then, 50 μl of LDH reagent was added to the cell wall. At the end of the reaction, the reaction was stopped by adding 100 μl of stop solution, 1 N HCl, and the absorbance was measured at 540 nm. The percentage of cytotoxicity by LDH was calculated by calculating the value of LDH liberated from the cell to the total LDH of the culture medium and living cells (FIG. 10B).

As a result, LDH release was 32.2% in control and 31.9, 31.8, 32.1, and 33.1%, respectively, when treated with plnio statin.

< Example  6> Planiostatin  Observation of morphological changes of cells by

When the cells are sensitized to the external environment, they are injured and at the same time, morphological changes are induced by induction of cell death. Therefore, morphological changes of B16-F1 cell line were observed using an inverted microscope in order to investigate whether or not the effect of flonyostatin on the morphological changes of cells.

B16-F1 cell line was added to each well at 2 × 10 ⁵ cells / ml in a 6-well plate, incubated at 37 ° C in a 5% CO ₂ incubator for 24 hours, and plinyothatin was added to a final concentration of 10, 50 μM After incubation for 24 hours, the morphological characteristics of the B16-F1 cell line were photographed (100-fold) using a phase-contrast microscope (Nikon, Japan).

When cells are killed, not only the number of whole cells is reduced due to cytotoxicity but also the protrusions almost disappear, leading to a large morphological change of the cells. In the group treated with 10 and 50 μM of each of the control and planiostatin groups, (Fig. 11).

< Example  7> Planiostatin  Cell by Whether death induction  ( Hoechst  33342 staining)

When the cells are damaged, the apoptotic body appears around the nucleus and apoptosis is induced. To determine whether planiostatin induces apoptosis, nuclei were stained with fluorescence microscope using hoechst 33342, a fluorescent dye specifically binding to nuclear DNA in B16-F1 cell line.

The cells were cultured for 24 h at 2 × 10 ⁵ cells / ml in a 6-well plate and treated for 10, 50 μM final concentration of plnio-statin. After washing twice with PBS buffer, fixed with 10% formalin for 2 hours, washed again with PBS buffer and stained with hoechst 33342 (10 μM) for 30 min. After staining, the cells were washed with PBS buffer and observed with fluorescence microscope.

As a result of treatment with planiostatin, it showed an oval nucleus with no effect. Thus, it was confirmed that planiastatin does not cause damage to the nucleus of the cell (FIG. 12), and it was confirmed that B16-F1 cell line does not show toxicity.

Hereinafter, one embodiment of the formulation of the present invention is illustrated, but the present invention is not limited thereto.

< Formulation example  1> Manufacture of nutrition cream

The mixture was mixed with 0.5% by weight of planiostatin, 6% by weight of prolylene glycol, 4% by weight of glycerin, 5% by weight of liquid paraffin, 3% by weight of squalane, 1.5% by weight of polysorbate 60, Cream.

< Formulation example  2> Ointment system

1 g of plnio statin, 1.5 g of dexpanthenol, 1.0 g of stearic acid, 5.0 g of liquid paraffin, 4.0 g of solder, 3.0 g of cetanol, 13.0 g of propylene glycol, 1.5 g of triethanolamine, 0.025 g of dibutylhydroxytoluene, g, polysorbate 800.1 g, and purified water 63.0 g were blended according to a conventional ointment preparation method to prepare ointments.

< Formulation example  3> Drink

The purified water was mixed in a total amount of 100% by weight with 0.1% by weight of planiostatin, 10% by weight of fructose, 1% by weight of citron, and 0.2% by weight of sodium citrate.

Claims (7)

Flaniostatin represented by the following formula (1), a hydrate thereof, a solvate thereof or a salt thereof.
[Chemical Formula 1]

A composition having tyrosinase inhibitory activity or melanin production inhibitory activity comprising a flaniiostatin represented by the following formula (1), a hydrate, a solvate thereof, or a salt thereof as an active ingredient.
[Chemical Formula 1]

A pharmaceutical composition for preventing or treating a disease caused by tyrosinase overactivity or melanin overproduction, which comprises flaniiostatin represented by the following formula (1), a hydrate, a solvate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient .
[Chemical Formula 1]

Food composition for preventing or ameliorating diseases caused by tyrosinase overactivity or melanin overproduction, which comprises a flaniiostatin represented by the following formula (1), a hydrate, a solvate thereof, or a pharmaceutically acceptable salt thereof as an active ingredient .
[Chemical Formula 1]

1. A cosmetic composition comprising flanriostatin represented by the following formula (1), a hydrate thereof, a solvate thereof, or a salt thereof as an active ingredient.
[Chemical Formula 1]

6. The whitening cosmetic composition according to claim 5, A step of extracting alfalfa leaf, a step of extracting by organic solvent, and a step of chromatography.
[Chemical Formula 1]

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