KR20090018459A - Whitening cosmetic compositions containing extract of isodon inflexus - Google Patents

Abstract

A cosmetic composition containing the extract of Isodon inflexus is provided to inhibit production of melanin and improve use safety by using natural product or compound extracted therefrom, thereby whitening the skin safely and effectively. The skin-whitening cosmetic composition contains 0.0001-10 wt.% of the extract of Isodon inflexus or the solvent fraction of Isodon inflexus extract including excisanin A represented by the chemical formula(1) which is prepared with the extraction solvent selected from methanol, ethanol, hexane, ethylacetate, dimethyl formamide, tetrahydrofuran, chloroform and diethylether.

Description

Whitening cosmetic compositions containing extract of Isodon inflexus

The present invention is an acid peppermint ( Isodon inflexus ) relates to a whitening cosmetic composition containing the extract as an active ingredient. The present invention also provides a method for separating and purifying an excisanin A compound represented by Chemical Formula 1 having melanogenesis inhibitory activity from an acidic peppermint extract. The excisanin A compound and the acidic peppermint extract of the present invention are excellent in inhibiting melanin production and are expected to be used as natural whitening agents for skin diseases of excessive pigmentation.

Mountain pepper ( Isodon inflexus ) is a perennial herb of the dicotyledon and moss family. Its origin is distributed in Korea, Japan and China and grows in mountainous areas. It grows to 40-100cm in height, stems stand upright, gathered, many branches are split, and the whole hairs grow. The leaves are opposite each other, triangular egg-shaped, 3-6cm long, 2-4cm butterfly, and the bottom is like a wing of petiole with dull teeth at the edge. Flowers bloom in June-August with a blue-purple color, and hang on the stems with a pickled inflorescence. Flower diameter is 8-10mm in length, and the whole becomes a large flower ear. Calyxes are bell-shaped, hairy, and divided into 5 branches, the fragments being narrow triangles. Corollas are shaped like lips, upper lip facing up, split into 5 branches, and lower lip is convex. Two of the four stamens are long, with one pistil and the pistil split into two branches. Fruits are small nuts in the calyx and ripen in September to October. Young shoots are eaten as herbs.

There have been no studies on the identification of active substances having activity related to whitening in acidic peppermint. Therefore, the present inventors have investigated the active substances related to natural whitening agent in natural products. The whitening effect was confirmed through the inhibition of melanin production in melanocytes. From this, a material having a whitening effect was separated and examined for its chemical properties, thereby confirming that it was excisanin A, thereby completing the present invention.

Structure of the active ingredient excisanin A isolated from the extract of organic acidic peppermint.

Man's desire for beauty is essential for modern people who want to have time for life. In Korea, since the Cosmetic Act was implemented separately, research and development has been actively conducted with much interest in functional cosmetics. In particular, Asians have long been considered as a symbol of beauty because of their desire for white and fine skin.

Human skin color is determined by factors such as the environment, race, and sex and various factors such as the amount of melanin, carotene and hemoglobin, but the main cause associated with hyperpigmentation of the skin is due to the ideal increase in melanin pigment in the epidermis. Melanin is biosynthesized from tyrosin in melanocytes in melanocytes in the basal layer of the epidermis and metastasizes to peripheral keratinocytes (VJ Hearing and TM Ekel, Mammalian tyrosinase, Biochem. J. , 157, 549, 1976). Melanin plays an important role in preventing skin damage from UV rays (Y. Mishima, S. Hatta, and Y. Ohyama, Induction of melanogenesis suppression: cellular pharmacology and mode of differential action, Pigment Cell Res ., 1, 367 , 1998, H. Matubara, Inhibitoryh effect of lichen metabolites and their synthetic analogues on melanin biosynthesis in cultered B-16 mouse melanoma cells, Nateral Product Sciences, 4, 3, 1998) It causes cosmetic problems and, on the contrary, too little production leads to skin lesions such as vitiligo. Melanocytes are activated by ultraviolet rays, inflammatory reactions of the skin, hormonal abnormalities, genetic diseases, chemical stimuli, etc., proliferate and differentiate, causing excessive melanin pigmentation, which in turn causes skin pigmentation. In particular, the stimulation for ultraviolet rays, which is a representative factor for increasing the melanin pigment in the skin, has been increased as the desire for destruction of the ozone layer and the desire for leisure activities have increased recently. The path to skin pigmentation caused by UV light is not clear, but is mainly due to factors secreted from keratinocytes. Factors secreted by stimulated keratinocytes include endothelin-1 (ET-1), α-melanocyte stimulating hormone (α-MSH), adrenocorticotropic hormome (ACTH), and nitric oxide (NO ⁻ ). ET-1 stimulates the protein kinase C (PKC) pathway and cAMP pathway in melanocytes to increase melanocyte formation and α-MSH and ACTH also promote skin pigmentation through cAMP pathway (Imokawa G, Kobayashi T, Miyagishi M, Higashi K, Yada Y: The role of endothelin-1 in epidermal hyperpigmentation and signaling mechanism of mitogenesis and melanogenesis.Pigment Cell Res 10: 218-28, 1997, Thody AJ, Graham A: Does alpha-MSH have a role in regulating skin pigmentation in humans? Pigment Cell Res 11: 265-74, 1998). Pro-opiomelano-cortin (POMC), a precursor of α-MSH, also binds to melanocortin-1 (MC-1) receptors in melanocytes, like α-MSH, to increase cAMP activity and promote melanin production (Sungbin Im, Moro O, Peng F, Medrano EE, Cnelius J, Babcokc G, Nordlund JJ, Abdel Malek ZA: Activation of the cyclic AMP pathway by α-melanotropin mediated the response of human melanocytes to ultraviolet B radiation.Cancer RES 58: 47-54 , 1998, Romero-Grailler C, Aberdam E, Clement M, Ortonne JP, Bailotti R: Cycle AMP a Key Messenger in the Regulation of Skin Pimentation.Pigment Cell Res. 13: 60-69. 2000).

In functional whitening cosmetics, the whitening effect is induced through inhibition of excessively generated melanin pigment, inhibition of pigmentation through UV protection, and reduction of the produced melanin pigment. Phenolic derivatives such as hydroquinone and resorcinol, L-ascrobic acid and its derivatives, and arbutin, lactic acid, glucosamin, and tunicamycin have been developed as inhibitors of melanin production, but due to skin irritation and safety problems, only a limited amount is used. Many (H. Matubara, Inhibitory effect of lichen metabolites and their synthetic analogues on melanin biosynthesis in cultured B-16 mouse melanoma cells, Natural Product Sciences, 4,3,1998).

As people's lives get better, the desire for well-being grows stronger, and the demand for self-realization and the interest in beauty soar. As a result, the desire for purchasing high-functional cosmetics that emphasizes effectiveness rather than a simple concept of cosmetics It is getting bigger. In particular, there is an increasing tendency to obtain such effects from human-friendly and harmless natural ones rather than artificial ones, and thus the use of natural raw materials is increasing not only in cosmetic raw materials but also in food and pharmaceutical systems, and the researches that follow are increasing. have.

The purpose of the present invention is to develop new whitening cosmetics through the development of safer and more effective natural materials through the use of harmless and environmentally friendly materials, and to provide a healing agent for skin pigment changes. do. It is another object of the present invention to provide a method for separating excisanin A from acidic peppermint, and for the first time reveals that this material has a whitening function.

In order to achieve the above object, the present invention provides an acidic peppermint extract having melanin production inhibitory activity by extracting the acidic peppermint with methyl alcohol and other organic solvents. In another aspect, the present invention provides a solvent fraction of the acidic peppermint extract having a whitening activity extracted by extracting the ground portion of acidic peppermint with 80% methanol, and then added again an organic solvent such as hexane, ethyl acetate, butanol, and the like.

As the extraction solvent of the present invention, lower alcohols such as methanol and ethanol, hexane, ethyl acetate, DMF (dimethyl foramide), THF (Tetrahydrofuran), chloroform, diethyl ether and the like can be used, and preferably methanol or ethanol is preferable. .

In addition, the present invention is immersed in acidic peppermint starch with 80% methanol (water, methanol mixed solution) and repeatedly extracted and concentrated to obtain a methanol extract and then suspended in 20% methanol and organic solvents such as hexane, ethyl acetate and butanol In turn, a corresponding solvent extraction fraction is prepared.

The present invention also provides a method for separating and purifying excisanin A from acidic peppermint extract using silica gel chromatography and prep-HPLC.

The inventors found that the substance separated and purified by the above procedure was an active substance showing activity such as a whitening effect, and confirmed that the chemical structure was excisanin A by investigating hydrogen and carbon nuclear magnetic resonance spectra.

The cosmetic composition for whitening containing the acidic peppermint extract of the present invention or the solvent fraction of the acidic peppermint extract as an active ingredient may be provided in the form of general cosmetic water, cream, essence, mask pack, or the like. The solvent fraction of the acidic peppermint extract preferably contains 1 × 10 ⁻⁴ to 10 wt% of the total weight of the cosmetic composition. When it is contained 0.0001% by weight or less, the whitening effect of the present invention is hardly expected, and when it is contained by 10% by weight or more, there is a problem in formulation.

As excisanin A, it is preferable to contain 1 * 10 <^-5> -1 weight% of the total weight of a cosmetic composition. It is difficult to expect the whitening effect of this invention when it contains 1 * 10 <^-5> weight% or less.

The acid and peppermint extract of the present invention and the excisanin A compound, which is an active ingredient of the peppermint extract, exhibit excellent melanin production inhibitory effect and can be widely used as a safe and effective whitening agent derived from natural plants in the future.

In addition, the peppermint extract obtained according to the present invention can be widely applied to the cosmetics as well as the pharmaceutical field.

The present invention is extracted repeatedly by immersing the ground portion of acidic peppermint in 80% methanol to obtain an acidic peppermint methanol extract having a whitening effect. In addition, the methanol extract of acid and mint is extracted again using an organic solvent hexane, ethyl acetate, butanol and the like to provide an extraction fraction having a whitening activity.

The present invention provides an acidic ethylacetate fraction with excellent whitening activity by fractionating the ground portion of acidic peppermint with 80% methanol and fractionating it with an organic solvent using a concentrated methanol extract.

The present invention provides a composition having a whitening activity using the acidic peppermint extract as an active ingredient. Specifically, the extract may be used as a composition for whitening cosmetics and pharmaceuticals by inhibiting melanin production in vivo.

The whitening active ingredient of the acidic peppermint extract was determined to be effective in extracting melanin from melanocytes of mice using an acidic peppermint methanol extract. The fraction showed the melanin production inhibitory effect outstanding.

The present invention was able to obtain a single material using silica gel column chromatography and prep-HPLC in the ethyl acetate fraction extraction showing the excellent whitening efficacy. In addition, hydrogen and carbon nuclear magnetic resonance spectroscopy was carried out to confirm that this compound is excisanin A.

The present invention by measuring the ability to control the expression of genes involved in the melanogenesis inhibitory effect and biosynthetic process using melanocytes melan-a cells and B16 cells to investigate the whitening efficacy of the extract of the acidic peppermint containing excisanin A The effect was confirmed.

Example 1 Preparation of Solvent Fractions from the Ground Part of Acid Peppermint

2240.7 g of acidic peppermint outpost native to Jeju Island were collected, trimmed, and finely crushed. After stirring and extracting three times with 10 L of 80% methanol, the mixture was concentrated under reduced pressure to obtain 320 g of methanol extract. 10 g of the dried crude product was suspended in 1 L of 20% methanol, and then, 1 L of hexane was added to separate the hexane layer. This was repeated three times to obtain a hexane fraction layer. Ethyl acetate 1L was added to the remaining water layer to separate the ethyl acetate layer, and this was repeated three times to obtain an ethyl acetate fraction layer. Finally, 1 L of butanol was added to the water layer three times, and the mixture was partitioned into a butanol layer and a water layer. The remaining methanol crude extracts were repeatedly used in a solvent fraction.

The obtained fractions were concentrated under reduced pressure to yield 7.752 g of ethyl acetate fraction, which was then fractionated using solvents sequentially through a column filled with celite. As a result, 0.168 g of hexane fraction, 4.298 g of dimethyl chloride fraction, 2.149 g of ethyl acetate fraction and 0.63 g of methanol fraction were obtained.

In order to investigate the whitening effect of the peppermint extract, the effect of inhibiting melanin production of each fraction obtained in Example 1 was investigated using two mouse melanocytes.

Experimental Example 1: Melanogenesis Inhibition Experiment

The melan-a cell is a non-tumorigrinc mouse melanocyte cell line that has most of the properties of normal mouse melanocytes, but is immortalized and can be easily used in experiments. Therefore, the degree of inhibition of melanin production of the sample extract was measured by using melan-a cells. Melan-a cells were plated at 1 × 10 ⁵ cells / mL in 24 well plates using RPMI 1640 medium (Gibco, USA) medium, and the samples were treated and incubated in a 37 ° C. 10% CO ₂ incubator for 3 days. After removal of the plate medium, the cells were harvested, 1N NaOH was added to completely dissolve the cells, and measured by enzyme linked immunosorbant assay (ELISA) reader at 450 nm.

Make standard soultion using synthetic melanin and measure the absorbance by putting sample and standard soultion in 96 well plate. Melanin concentrations were determined from standard concentration curves prepared with synthetic melanin.

In order to determine whether a significant effect on the melanocyte B16 mouse melanocyte of the sample extract was measured, the degree of inhibition of melanogenesis was measured using B16 cells. B16 melanocytes were plated at 1 × 10 ⁵ cells / well in 6well plates using Dulbecco's modified eagle medium (DMEM), and then the samples were treated and incubated in a 37 ° C. 5% CO ₂ incubator for 3 days. After removal of the plate medium, the cells were harvested, 1N NaOH was added to completely dissolve the cells, and measured by ELISA reader at 450 nm.

Viability measurement of cells (MTT assay)

Determination of 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, a tetrazole (MTT) was performed by modifying Mosmann's method. When measuring the amount of melanin, the sample was treated in the same manner with duplicate plates, and after incubation, formazan (formazan) formed by treating MTT (50 mg / mL) reagent (0.1 mg) was dissolved in DMSO, followed by ELISA reader at 540 nm. Measured and compared with control.

  Determination of Melanin and Cell Activity of Organic Solvent Fractions in melan-a Cells Inhibition of melanin contents (%) (50㎍ / mL) MTT (%) (50 μg / mL) crude extract 30.3 ± 3.1 8.7 ± 2.4 hexane 47.4 ± 2.2 82.0 ± 2.0 EtOAc 83.4 ± 1.7 95.4 ± 2.1 BuOH -1.19 ± 3.3 -5.89 ± 1.8 H ₂ O 2.6 ± 3.4 42.3 ± 3.1 arbutin 43.3 ± 1.8 -0.8 ± 3.2

As shown in Table 1, it showed a good effect at the level of almost inhibiting the proliferation of cells at the concentration of 50 ㎍ / mL in the state of acidic extract, melanin by the result of inhibiting cell proliferation at the same concentration in the fractionation layer Inhibition of melanin production was confirmed by diluting the sample to a concentration that does not affect cell proliferation and treating it with melan-a cells and B16 cells. A negative sign in the table indicates that it promotes melanogenesis.

  Effect of Ethyl Acetate Fraction on Melanin Production in Mouse Skin Cells cell type EtOAC Treatment Concentration (µg / mL) Inhibition of melanin contents (%) MTT (%)  melan-a cell EtOAc (10) 82.6 ± 3.2 10.2 ± 3.3 EtOAc (5) 66.3 ± 2.3 6.1 ± 3.0 arbutin 48.3 ± 2.8 -3.7 ± 2.5  B16F1 EtOAc (10) 76.1 ± 6.1 -1.1 ± 2.2 EtOAc (5) 64.8 ± 8.5 -5.0 ± 9.7 arbutin (500) 37.0 ± 2.2 6.5 ± 2.8 hydroquinone (1) 65.8 ± 4.7 7.4 ± 7.7  B16F10 EtOAc (10) 60.7 ± 3.7 4.7 ± 0.2 EtOAc (5) 42.9 ± 2.6 1.4 ± 0.9 arbutin (100) 12.5 ± 7.4 4.3 ± 3.8 hydroquinone (0.5) 39.2 ± 0.2 12.7 ± 0.7

As shown in Table 2, the ethyl acetate fraction extract showed excellent melanin production inhibitory effect in both melan-a and B16 cells even at concentrations that did not affect cell proliferation.

Example 2 Separation and Structure Determination of Whitening Materials from Acid Peppermint

In the solvent fraction obtained from the acidic starch outpost of Example 1, an ethyl acetate fraction having excellent whitening activity was isolated according to the following procedure. 1, 2, and 3 show the process of fractionation and separation.

Ethyl acetate-hexane fractions were fractionated over a 7.752g 4.4783g of the first celite dimethyl chloride 0.04g 3.29g fraction, ethyl acetate fraction 1.03g, 0.24g of a result of the methanol fraction obtained preferentially performed in the melanogenesis inhibition test, CH ₂ Cl ₂ Pure silica gel (Merck) was eluted with developing solvent CHCl ₃ and MeOH with a gradient of 0-100% by weight on a glass column (3 × 24 cm). 1.5307 g of the obtained 10% was again charged with a silica gel (3 × 30 cm) by using a developing solvent (CHCl ₃ : MeOH = 10: 1), followed by filling with silica gel to obtain 6 fractions. Of these, 0.2343 g of F4, which exhibits a good melanin inhibitory effect, was filled with pure silica gel in a glass column (3 × 35 cm) by suspension with a developing solvent (CHCl ₃ : MeOH = 9: 1), and five fractions were obtained. Among them, 0.1405 g of F4-4, which has a good melanin inhibitory effect, was developed on a glass column (1.5 × 35 cm) with pure silica gel (CH ₂ Cl ₂ : CHCl _3). : 10 fractions were obtained after the suspension was filled with Acetone = 1: 1: 1). Among them, f6 to f8 showing melanin inhibitory activity were collected and H (excisanin A) was isolated using prep-HPLC (FIG. 4).

Among the single materials separated by Prep-HPLC, H was found to be excisanin A in the hydrogen nuclear magnetic resonance spectrum and carbon nuclear magnetic resonance spectrum, which is consistent with Nihon Kagakkai, Chemistry Letters, 1981, 6, 753-756, 1972. It was.

Experimental Example 2 Whitening Effect of Fraction Layers Passed Through a Normal Phase Column

The fractions obtained while passing through each column in Example 2 were concentrated to measure the melanin production inhibitory effect using melan-a cells.

  Effect of Celite Fractions on Melanin Production Melan-a (µg / mL) Inhibition of Melanin contents (%) MTT (%) Hexane (25) 21.8 ± 2.9 3.9 ± 13.5 CH ₂ Cl ₂ (50) 95.1 ± 2.8 95.4 ± 0.5 EtOAc (50) 87.1 ± 1.9 13.4 ± 6.0 MeOH (50) 62.1 ± 12.6 4.4 ± 5.2 Arbutin (50) 48.9 ± 5.7 7.0 ± 6.2 Hexane (12.5) 32.2 ± 0.5 16.8 ± 5.2 CH ₂ Cl ₂ (10) 89.4 ± 3.5 49 ± 11.5 EtOAc (10) 54.6 ± 6.8 2.5 ± 3.1 MeOH (10) 16.7 ± 5.1 2.1 ± 1.1 Arbutin (10) 22.1 ± 4.0 2.4 ± 2.7 Hexane (6.25) 37.3 ± 0.5 3.7 ± 2.2 CH ₂ Cl ₂ (5) 77.5 ± 0.9 13.5 ± 0.4 EtOAc (5) 31.5 ± 3.4 10.5 ± 1.8 MeOH (5) 0.8 ± 2.4 5.9 ± 1.8 Arbutin (5) 7.8 ± 5.1 2.7 ± 0.4

As shown in Table 3, the dichloromethane fractionation layer exhibited excellent melanin production inhibitory effect even at low concentrations as a result of treatment with cells lowered to no concentration of cytotoxicity.

Figure 5 shows the melanin amount in the absence of inhibitor (C), CH ₂ Cl ₂ and EtOAc fraction and the degree of inhibition of melanin production of arbutin (Ar) as a positive control. Fractions are much better than arbutin.

  Effect of Fractions of Dichloromethane Extract on Melanin Production MeOH content (relative to CHCl ₃ ) (%) Treatment concentration 5㎍ / mL (melan-a cell) Treatment concentration 1 ㎍ / mL (melan-a cell) Inhibition of Melanin contents MTT Inhibition of Melanin contents MTT 0 16.4 ± 3.5 0.95 ± 0.95 -1.1 ± 1.3 -2.8 ± 5.7 10 59.5 ± 1.3 3.25 ± 6.55 10.7 ± 13.4 -4.95 ± 0.25 20 80.0 ± 3.8 17.4 ± 18.4 38.3 ± 9.9 -3.5 ± 1.2 30 63.1 ± 2.8 6.75 ± 1.25 22.5 ± 0.9 -7.65 ± 2.45 40 33.4 ± 9.3 9.25 ± 2.55 5.7 ± 9.3 -1.85 ± 1.85 50 29.4 ± 22.9 10.95 ± 8.25 5.4 ± 5.4 1.6 ± 0.6 60 -3.1 ± 23.2 11.75 ± 9.05 7.7 ± 3.9 0 ± 0 70 22.5 ± 4.7 10.2 ± 6.8 18.3 ± 4.2 -2.7 ± 6.2 80 15.7 ± 4.0 3.95 ± 1.25 17.8 ± 4.5 1 ± 2.7 90 17 ± 2.5 6.25 ± 3.55 8.7 ± 8.3 3.3 ± 1.3 100 2.3 ± 2.7 15.2 ± 1 9.3 ± 14.8 10.1 ± 1.2 arbutin 50 38.4 ± 9.9 -1.85 ± 1.3

As shown in Table 4, it was confirmed that excellent melanin production inhibitory effect was observed even at low concentrations in the 10% and 20% fractions.

  Effect of 10% Fractions on Melanin Production Melan-a (µg / mL) Inhibition of Melanin contents (%) MTT (%) F1 (5) -43.4 ± 1.5 9.1 ± 0.7 F2 (5) 87.2 ± 1.7 8.4 ± 3.2 F3 (5) 86.8 ± 1.4 9.8 ± 1.2 F4 (5) 85.1 ± 1.4 7.6 ± 1.8 F5 (5) 17.4 ± 2.2 0.2 ± 2.7 F6 (5) 3.9 ± 3.5 5.9 ± 4.7 F1 (1) -23.3 ± 2.0 9.4 ± 3.8 F2 (1) 25.4 ± 5.5 7.7 ± 7.5 F3 (1) 23.7 ± 2.7 8.7 ± 2.7 F4 (1) 57.5 ± 3.1 6.9 ± 5.8 F5 (1) 9.3 ± 4.0 9.4 ± 1.2 F6 (1) -0.1 ± 1.1 10.9 ± 4.0 Arbutin (50) 49.3 ± 3.3 3.1 ± 2.5 Arbutin (10) 24.9 ± 5.6 3 ± 7.5 Arbutin (5) 13.2 ± 6.5 2.3 ± 6.7

Table 5 shows the melanin production inhibitory effect of the 6 fractions obtained by re-fractionation of 10% fractions through normal silica gel, measured by concentration using melan-a cells. Good effects were also investigated in F2 and F3.

  Effect of F4 Fractions on Melanin Production Melan-a (µg / mL) Inhibition of Melanin contents (%) MTT (%) F4-4 (5) 85.5 ± 0.7 12.6 ± 1.3 F4-4 (1) 51.6 ± 2.0 6.7 ± 1.7 Arbutin (50) 39.7 ± 2.6 4.9 ± 1.5 Arbutin (5) 11.1 ± 4.8 4.8 ± 2.1 Arbutin (1) 10.7 ± 3.6 4.1 ± 3.1

In Table 6, the fourth fraction of the fraction obtained in 10% was again measured the melanin production inhibitory effect in melan-a cells at a concentration that does not affect the cell proliferation of the fourth fraction of the five fractions obtained through the column It was confirmed that there is an excellent effect compared to the arbutin used as a control.

  Effect of Excisanin A on Melanin Production compound melan-a B16F1 B16F10 concentration (㎍ / mL) Inhibition of Melanin contents (%)  MTT (%) Inhibition of Melanin contents (%)  MTT (%) Inhibition of Melanin contents (%)  MTT (%) H (1) 67.9 ± 0  2.5 ± 1.2 26.9 ± 9.6 1.8 ± 3.7 56.7 ± 0.1 6.1 ± 0.3 Arbutin (100) 29.4 ± 0.3 -6.0 ± 0.6 13.9 ± 0.5 -0.6 ± 0.2 0.9 ± 6.3 4.3 ± 3.8 Hydroquinone (0.5) 74.1 ± 3.4 -4.3 ± 3.3 30.4 ± 0.9 12.8 ± 0.8 39.2 ± 0.2 12.7 ± 0.7

As a result of measuring the melanin production inhibitory effect on the different melanocytes of the substance H isolated in Table 7, the melanin production inhibitory effect is lower than that of the ethyl acetate fraction or the next step fraction, but shows superior efficacy compared to the control group, The better melanogenesis effect of the fraction is believed to be due to the synergistic effect of excisanin A and other compounds.

Example 3: Melanin Production Inhibition Effect on UV B Irradiation

Measurement of melanin production inhibitory effect by UV B irradiation

Melan-a cells were plated at 1 × 10 ⁵ cells / mL in a 24 well plate using RPMI medium, and the samples were treated and incubated in a 37 ° C. 10% CO ₂ incubator for one day. After irradiating with UV B 10mJ / cm ² for ² days from the next day, exchange it with a new medium containing the sample, incubate for one more day, remove the plate medium, harvest the cells, add 1N NaOH, and completely dissolve the cells 450 nm Measured by ELISA at and compared with control.

Make standard soultion using synthetic melanin and measure the absorbance by putting sample and standard soultion in 96 well plate. Melanin concentrations were determined from standard concentration curves prepared with synthetic melanin.

Viability measurement of cells (MTT assay)

MTT quantification was performed by modifying Mosmann's method. When measuring the melanin amount, the plate was duplicated and the sample was treated in the same way. After incubation, formazan formed by treatment with MTT (50 mg / mL) reagent (0.1 mg) was dissolved in DMSO and measured by ELISA at 540 nm. It was.

RT-PCR

Melan-a cells were plated at 4.5 × 10 ⁵ cells / well on a 60 mm plate using RPMI medium, and the samples were treated and incubated in a 37 ° C. 10% CO ₂ incubator for one day. After irradiating with UV B 10mJ / cm ² for ² days from the next day, exchange with a new medium containing the sample, incubate for one more day, remove the plate medium, and extract RNA using Tri-zol solution (Invitrogen, USA). It was. 1 μL total RNA was heated at 70 ° C 5min, 4 ° C 5min, 37 ° C 60min, and 10min at 70 ° C with oligo (dT) ₁₈ primer, dNTP (0.5 μM), 1 unit RNase inhibitor, M-MuLV reverse transcriptase (2U) CDNA was synthesized. Polymerase chain reaction (PCR) was performed with 2μL cDNA, 10 × buffer (10mM Tris-HCl, pH 8.3, 50mM KCl, 0.1% Triton X-100), 250μM dNTP, 1unit Taq polymerase (Promega) to amplify primers from synthesized cDNA. , USA) was amplified by distilled water at 25 μL, followed by denaturation at 94 ° C for 45s, annealing at 45s for 45s, and extension for 90s at 72 ° C for 25-30 cycles. The primers used are as follows.

  Primers of Melanin Synthesis-Related Genes Used in RT-PCR Serial number Gene name DNA base sequence (5′-3 ′) One Actin  R: TGG AAT CCT GTG GCA TCC ATG AAA C F: TAA AAC GCA GCT CAG TAA CAG TCC G 2 Tyrosinase  R: GGC CAG CTT TCA GGC AGA GGT F: TGG TGC TTC ATG GGC AAA ATC 3 TRP-1  R: GCT GCA GGA GCC TTC TTT CTC F: AAG ACG CTG CAC TGC TGG TCT 4 TRP-2  R: GTT GCT CTG CGG TTA GGA AG F: TGT GCA AGA TTG CCT GTC TC 5 Mitf  R: TGA TGA TCC GAT TCA CCA GA F: AGC GTG TAT TTT CCC CAC AG 6 MC1-R  R: CCA GGA AGC AGA GAC TGG AC F: ACT CCA ATG CCA CCT CTC AC 7 c-Kit  R: ATC CCG ACT TTG TCA GAT GG F: CAC GTT TTT GAT GGT GAT GC R: reverse F: froward

In Figure 6 it was confirmed that the acidic ethyl acetate fraction layer significantly inhibited mRNA expression of mitf, tyrosinase in a concentration-dependent manner. Excisanin A significantly inhibited the expression of mitf, tyrosinase and c-kit mRNA at 1 ㎍ / mL, and suppressed MC1r and TRP-1, but had little effect on TRP-2. It was confirmed that it does not have. In general, it can be seen that it has superior inhibitory activity than the control group arbutin (Ar) and hydroquinone (HQ).

In FIG. 7, Western blotting confirmed the degree of inhibition of tyrosinase expression of excisanin A isolated from the acid and mint EtOAc layers, and showed a strong inhibitory effect at the protein level compared to the control groups arbutin and hydroquinone.

  Effect of Excisanin A on Melanin Biosynthesis of Mouse Skin Cells by UVB Irradiation melan-a B16F1 B16F10  Substance (μg / mL) Inhibition of Melanin contents (%)  MTT (%) Inhibition of Melanin contents (%)  MTT (%) Inhibition of Melanin contents (%)  MTT (%) H (1) 43.9 ± 10.2 -1.6 ± 4.9 43.1 ± 9.1 1.7 ± 1.7 60.0 ± 1.8 1.5 ± 0.4 Arbutin (100) 16.5 ± 9.1 -5.7 ± 2.6 24.9 ± 3.2 -4.3 ± 2.6 19.9 ± 1.9 6.6 ± 0.9 Hydroquinone (0.5) 76.5 ± 12.1 20.0 ± 7.6 53.4 ± 8.3 1.3 ± 4.4 40.4 ± 5.7 17.7 ± 1.1

Table 9 is a measure of the inhibition of melanin production after two days of irradiation with UV B 10mJ / cm ² , showing similar or better effects than hydroquinone used as a control as before the irradiation, and superior effect to arbutin. Confirmed. However, it was also confirmed that the synergistic effect of the active substances in the previous step was confirmed by confirming that the melanin production inhibitory effect was relatively lower than the previous step fraction.

1 is an extractive separation system diagram of an acid-mineral EtOAc fraction layer.

2 is an exploded view of F4

3 is a flow diagram of the separation of excisanin A from F4-4

4 shows the results of HPLC analysis of f6 of the fraction F4-4.

Figure 5 is a photograph showing the melanogenesis inhibition of dichloromethane and EtOAc fraction extract

FIG. 6 shows the results of regulation of gene expression related to melanin biosynthesis of acidic peppermint solvent fraction and H.

FIG. 7 shows western blotting results in melan-a cells of acid and mint EtOAc fraction layers.

Claims (6)

A whitening cosmetic composition containing a solvent extract of an acidic peppermint extract or an acidic peppermint extract as an active ingredient. The cosmetic composition for whitening according to claim 1, wherein the acidic peppermint extract or the solvent fraction of the acidic peppermint extract contains 1 × 10 ⁻⁴ to 10 wt% of the total weight of the composition. The cosmetic composition for whitening according to claim 1 or 2, wherein the active ingredient of the solvent extract of the peppermint extract or the peppermint extract is excisanin A represented by the following formula (1). [Formula 1]

The cosmetic composition for whitening according to claim 3, wherein the excisanin A contains 1 × 10 ⁻⁵ to 1 wt% of the total weight of the cosmetic composition. The cosmetic composition for whitening according to claim 1, wherein the solvent fraction of the acidic peppermint extract is an ethyl acetate fraction. The acidic peppermint starch was immersed in a mixed solution of water and methanol, and extracted repeatedly one or more times, concentrated to obtain a methanol extract, and then suspended in 20% methanol and extracted three times with hexane to obtain a hexane fraction layer. A method of preparing a solvent fraction of an acidic peppermint extract having a whitening effect, characterized in that acetate and butanol are sequentially added and repeated extraction three times to obtain a solvent fraction corresponding thereto.

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