KR20090067310A - Method of purifying p-coumaric acid from sasa quelpaertensis nakai, and cosmeceuticals and pharmaceutical composition using the purified p-coumaric acid - Google Patents

Abstract

A method for purifying p-coumaric acid from Sasa quelpaertensis Nakai is provided to suppress the activation of alpha-MSH and tyrosinase and have skin whitening effect. A method for purifying p-coumaric acid from Sasa quelpaertensis Nakai comprises: a step of extracting the Sasa quelpaertensis Nakai with ethanol at 10°C-70°C; a step of compression-concentrating the extract; a step of fractioning the concentrate with solvent to obtain fraction; a step of eluting the fraction and resolving with ethanol; and a step of recrystallizing. A cosmetic composition for skin whitening comprises 0.01-10 weight% of purified p-coumarilic acid as an active ingredient.

Description

Method of purifying p-coumarinic acid from Jeju jeokdae and functional cosmetics and pharmaceutical composition using the para-coumarinic acid {Method of purifying p-coumaric acid from Sasa quelpaertensis Nakai, and cosmeceuticals and pharmaceutical composition using the purified p-coumaric acid}

The present invention jeju jeori ( Sasa quelpaertensis Nakai) from the active ingredients, the p-coumarin acid (p -coumaric acid) purification and the purified p of the - related to a composition for functional cosmetics containing coumarin acid (p -coumaric acid), more specifically the Purification method is the jeju jeoldae quelpaertensis Extraction step of extracting Nakai) with ethanol at a temperature of 10 ° C. to 70 ° C .; Concentrate manufacturing step of producing a concentrated solution by reducing the extract solution obtained in the extraction step; Fractionating the concentrate with a solvent and concentrating under reduced pressure to obtain a fraction; An eluting solution preparation step of eluting the fractions by filtration to elute with time, dissolving the eluate in ethanol to obtain an eluting solution; Purified by coumarin acid (p -coumaric acid) the purification method and purification method, such as the p - -, and the eluted solution of ethanol (ethanol) comprises a recrystallization step that the re-crystallization to obtain crystals consisting of by p coumarin acid (p - Coumaric acid) as an active ingredient, the active ingredient inhibits the activity of tyrosinase (tyrosinase) and alpha-MSH (α-MSH) to inhibit the production of melanin (melanin) to a whitening effect functional cosmetic composition .

In general, hyperpigmentation of the skin includes cases of internal factors occurring irrespective of ultraviolet rays such as hormonal abnormalities, genetic diseases, and external factors that are abnormal in the synthesis and distribution of melanin pigment due to excessive ultraviolet irradiation. Doing. Phenomenologically, an enzyme called tyrosinase acts, followed by a series of oxidation processes, resulting in an excess of polymer called melanin, which accumulates in the skin. The tyrosinase (tyrosinase) is a blackening of the skin made of tyrosine (a kind of amino acid) of dopa (DOPA, 3,4-dihydro xyphenylalanine), dopaquinone (DOPA-quinone, pheomelanin) intermediate of the production of melanin polymer It is the most important enzyme in the process. As described above, the action of tyrosinase in the production of melanin is absolute. Therefore, melanin production can be regulated by inhibiting the expression of this enzyme protein or inhibiting the activity of the expressed enzyme stimulated by ultraviolet light. The proper amount of melanin pigment in the skin keeps the skin healthy and absorbs ultraviolet rays (UV). However, the excess melanin pigment makes the skin black and the skin color uneven. do. In addition, because the excessive production of melanin (melanin) causes pigmented skin diseases such as blackening skin, blemishes, freckles, etc., studies on the whitening ingredients have been started to improve and treat the above diseases.

In the prior art, the invention relates to a whitening component that exhibits a skin whitening effect, and the invention of Japanese Patent Application Laid-Open No. 4-9320 and Korean Patent No. 0417874 using ascorbic acid. Although ascorbic acid has excellent safety and effectiveness, there have been many problems such as deterioration of stability when hydrated in water, reaction with oxygen in the air, metal ions in the formulation, and water. In addition, there is a Japanese Patent Application Laid-Open No. 6-192062 using the hydroquinone as the whitening ingredient, but the hydroquinone has a problem in that it cannot be used due to severe skin side effects.

In addition, skin whitening compositions containing plant extracts have been developed, but these extracts have a color problem, which is problematic in combination with other cosmetic ingredients. In addition, since the active ingredient showing the whitening effect of the extract is not exactly identified, there is a problem that it is difficult to expect the same effect when manufacturing the product. Moreover, there was a problem that the possibility of containing unwanted components in the plant extract could not be excluded.

The present invention is conceived to solve the above problems, Jeju Sasa (Sasa quelpaertensis Nakai) of the active ingredient from the p-method for purifying in order to refine the coumarin acid (p -coumaric acid) is the purification method is the Cheju Extraction step of extracting Sasa quelpaertensis Nakai with ethanol at room temperature, the extract obtained in the extraction step to produce a concentrate by depressurizing the extract solution, the concentrate is fractionated with a solvent and concentrated under reduced pressure fractions A fractionation step of obtaining, an eluate obtained by eluting the fractions, an elution solution preparation step of dissolving the eluate in ethanol, and a recrystallization step of obtaining crystals by recrystallizing the elution solution with ethanol; the formed p - coumarin acid (p -coumaric acid) purification and the purified p - coumarin acid (p -coumaric acid) Utilized to provide a functional cosmetic composition and a pharmaceutical composition.

Jeju Sasa (Sasa quelpaertensis Nakai) of p from the active ingredient according to the present invention for achieving the above object, - a process for purifying a coumarin acid (p -coumaric acid) is the Cheju Sasa (Sasa quelpaertensis Extraction step of extracting Nakai) with ethanol at a temperature of 10 ° C. to 70 ° C .; Concentrate manufacturing step of producing a concentrated solution by reducing the extract solution obtained in the extraction step; Fractionating the concentrate with a solvent and concentrating under reduced pressure to obtain a fraction; An eluting solution preparation step of separating and filtering the fractions to elute with time to obtain an eluate, and dissolving the eluate in ethanol to obtain an eluate; And a recrystallization step of obtaining crystals by recrystallizing the elution solution with ethanol.

Preferably, the extraction step is dried Jeju jeori ( Sasa) quelpaertensis Jeju dried sasa ( Sasa ) as the stem of Nakai quelpaertensis Nakai) 4 to 20 times the weight of 30% to 80% ethanol (ethanol) is characterized in that the extraction by adding.

Preferably, the concentrate is characterized in that the extract is concentrated to a concentration of 4 to 20 times the concentration of the extract.

Preferably, in the fractionation step, the concentrate is partitioned into methylene chloride, chloroform and normal hexane, and then the fractionated concentrate is ethyl acetate, methylacetate. Tate (methyl acetate), normal butanol (N-butanol) and isopropanol (isopropanol) is characterized by fractionation.

Preferably, the eluate is obtained by eluting a fraction through a column of column chromatography together with a mixed solution of methylene chloride and methanol.

Preferably, the mixed solution is characterized in that methylene chloride (methylene chloride) and methanol (methanol) is mixed in a weight ratio of 3: 1 to 1: 3.

Preferably, the recrystallization step is a crystal forming step of forming a crystal by drying the elution solution under reduced pressure; A crystalline dissolving step of preparing the crystalline solution by dissolving the formed crystals in ethanol; And a stationary step of filtering the crystalline solution and inducing crystallization by standing the filtered crystalline solution.

Preferably, the crystal melting step is characterized in that the ethanol (ethanol) concentration of 1 to 10%, the crystals are dissolved by heating at 50 ℃ to 80 ℃ in the ethanol (ethanol).

Preferably, the stationary step is characterized in that the filtrate filtered the crystalline solution induces crystallization at -80 ℃ to 40 ℃.

Then, the functional cosmetic composition according to the present invention for achieving the above object, is a p purified by any of the methods described in the above-coumarin and the acid (p -coumaric acid) as an active ingredient, wherein the effective component tyrosinase It is characterized by inhibiting the production of melanin by inhibiting the activity of the tyrosinase and alpha-MSH (α-MSH).

More preferably, the cosmetic composition is characterized in that it comprises an excipient used in the cosmetic and 0.01 to 10wt.% Of the purified p -coumaric acid ( p- coumaric acid).

In addition, the pharmaceutical compositions according to the present invention for achieving the above object, is a p purified by any of the methods described in the above-coumarin and the acid (p -coumaric acid) as a validity minutes, the effective component tyrosinase It is characterized by inhibiting the production of melanin by inhibiting the activity of the tyrosinase and alpha-MSH (α-MSH).

And a coumarin acid (p -coumaric acid); and a range of 0.01 to 10wt% and pharmaceutical excipients more preferably, the pharmaceutical composition is the purified p..

Jeju Sasa (Sasa quelpaertensis Nakai) from the active ingredients, the p in accordance with the present invention as described above, as described above in the above-coumarin acid (p -coumaric acid) functional cosmetic composition and a pharmaceutical composition using the above-mentioned effective ingredient and purification of According to the above, the purification method is jeju jeju ( Sasa) quelpaertensis Nakai) active ingredient, the p in - if done correctly identified word produced by the products of the coumarin acid (p -coumaric acid) has an effect that can be expected to a certain bleaching action. In addition, the active material purified by the purification method is excellent in the prevention of skin blackening of the skin cells as well as a very excellent safety material with little skin irritation has an excellent whitening effect.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the accompanying drawings, Figure 1 is a jeju jeoldae quelpaertensis Graph (A) and details showing the effects of stem extract and leaf extract of Nakai) on the inhibition of melanin production in cells under alpha-MSH (α-MSH) treatment (black bars) (B), Figure 2 is a jeju jeoldae quelpaertensis Stem extract of Nakai) and fractions of the extract of stem extract were produced with melanin (A) and tyrosinase in cells under alpha-MSH (α-MSH) treatment (black bars) (tyrosinase) is a graph showing the effect on the activity (B), Figure 3 is a graph (A) and the melanin (melanin) showing the effect of the respective eluate on melanin production induced by alpha-MSH (α-MSH) ) This is a graph (B) showing the molecular structure through ¹³ C-NMR spectrum of the active ingredient separated from the eluate that most inhibits the formation.

And, Figure 4 is a jeju jeoldae quelpaertensis Nakai extract, the jeju jeolla ( sasa) quelpaertensis Ethyl acetate fraction of the stem of Nakai), Sasa jeju quelpaertensis HPLC analysis of the active ingredient, authentic p- coumaric acid, isolated from the ethyl acetate fraction of the stem of Nakai), Figure 5 is a Sasa jejudae quelpaertensis The active ingredient separated from Nakai) p - coumarin acid (p -coumaric acid) is a graph showing the effect on cell viability at the alpha -MSH (α-MSH), processing oil (black spots) no (white point) condition, Fig. 6 Silver Jeju Sacks quelpaertensis Nakai) separated from the valid components p - coumarin of an acid (p -coumaric acid) and arbutin (arbutin), alpha -MSH (α-MSH), oil (black bars) no (white bars), tyrosinase (tyrosinase) in terms a degree of suppressing the expression analyzed by comparing the activity (a) and the protein content (B) of the enzyme and a graph Jeju Sasa (Sasa quelpaertensis Nakai) separated from the valid components p - coumarin acid (p -coumaric acid) and arbutin (C) is a graph showing the degree to which (arbutin) inhibits melanin production under the condition of (alpha) -MSH (α-MSH) treatment (round point) no (square point).

Further, Figure 7 is p - Melanin from coumarin acid (p -coumaric acid), and their structure and their structural and material of similar material to that alpha -MSH (α-MSH), processing oil (black bars) no (white bars) conditions (melanin) is a graph showing the effect on the production.

Jeju jejudae according to the present invention ( Sasa quelpaertensis Of the active ingredient from Nakai) p - coumarin acid (p -coumaric acid) the purification method the Cheju Sasa (Sasa quelpaertensis Extraction step of extracting Nakai) with ethanol at a temperature of 10 ℃ to 70 ℃, a concentrated liquid preparation step of producing a concentrated solution by decompressing the extract obtained in the extraction step, fractions the concentrated solution with a solvent and concentrated under reduced pressure A fractionation step of obtaining a fraction, the fractions are filtered and eluted with time to obtain an eluate, the elution solution preparation step of dissolving the eluate in ethanol (ethanol) and the elution solution to the ethanol (ethanol) recrystallization crystals It includes a recrystallization step to obtain a.

Jeju Sasa ( Sasa) quelpaertensis Nakai) belongs to the Gramineae as Jeju Sasa (Sasa quelpaertensis Nakai). Jeju Sasa ( Sasa) quelpaertensis Nakai is evenly distributed throughout Mount Halla, and the Sasa Jeju quelpaertensis The appearance of Nakai) is 20 ~ 80cm in height, 3 ~ 4mm in diameter, hairless and green, and the nodes are raised and the area around the nodes is purple. Jeju Sasa ( Sasa) quelpaertensis Nakai) 's upper lobe is 50 ~ 90mm long, with lobes at the end, and the base is round and 6mm in diameter. And, the jeju jeori ( sasa quelpaertensis Nakai) leaves are oval or oblong, 7 ~ 20cm long, 15 ~ 20mm wide, with acute tip, white rounded at the edge. The surface is light green, hairless and gray green with slight hairs.

In the extraction step, the jeju choridae (Sasa quelpaertensis Nakai) extracts the constituents of the dried Jeju Jojidae (Sasa quelpaertensisThe dried Jeju jeokdae as a stem of Nakai (Sasa quelpaertensis 30% to 80% of ethanol, 4-20 times the weight of Nakai), is added and extracted at a temperature of 10 ° C to 70 ° C. Jeju jejudae (Sasa quelpaertensis Take the stem of Nakai)Sasa quelpaertensis This is because among the leaves, stems and roots of Nakai), the most active substance having a whitening effect is present at the stem. Extracting with the ethanol is to increase the extraction efficiency without destroying the active ingredient. When the concentration of the ethanol is less than 30% or more than 80%, the active ingredient is not extracted or the active ingredient is destroyed, so it is preferable to extract in the concentration range of the ethanol.

After the extraction step is subjected to the concentrate production step. In the concentrate preparation step, the extract extracted with ethanol is concentrated under reduced pressure to obtain a concentrate. The concentrate concentrates the extract at a concentration of 4 to 20 times the concentration of the extract. The preparation of the concentrate is to make the fraction well after the dispersion of the extract evenly.

In the fractionation step, the fractionation step is to separate the concentrate into methylene chloride (methylene chloride), chloroform (chloroform) and normal hexane (N-hexane), and then the fractionated concentrate is ethyl acetate (ethyl acetate), methyl acetate Fractions are made with metyl acetate, normal butanol (N-Buthanol) and isopropanol. The methylene chloride (methylene chloride), chloroform (chloroform) and normal hexane (N-hexane) has a low polarity serves to selectively remove impurities such as chlorophyll, lipids, essential oils. The ethyl acetate (ethyl acetate), methyl acetate DE (metyl acetate), n-butanol (N-Buthanol) and isopropanol (isopropanol) the active ingredient, the p - of the polarity of the coumarin acid (p -coumaric acid) and similar scope it is suitable for obtaining coumarin acid (p -coumaric acid) effectively - so the p.

In the elution solution preparation step, the fractions are filtered and eluted with time to obtain an eluate. The eluate is dissolved in ethanol to obtain an eluate. The eluate is obtained by elution of the fractions through a column of column chromatography together with a mixed solution of methylene chloride and methanol. Sepadex LH20 may be used as the column chromatography. The Sephadex LH20 (Sephadex LH20) is a name given to the stationary phase Sephadex LH20 (Sephadex LH20) is used. Sephadex LH20 (Sephadex LH20) has an excellent property of separating according to the polarity of the material is suitable for the separation of the active ingredient. In addition, the Sephadex LH20 (Sephadex LH20) can be separated from the stationary phase without loss of the active ingredient since the combination with the active ingredient is reversible. The stationary phase is very economical because it can be used repeatedly after removing the contaminants by washing with methanol (methanol). However, if the active ingredient p -coumaric acid ( p- coumaric acid) can be separated and filtered is not limited to the column chromatography (column chromatography).

As a mobile phase of the column chromatography, the mixed solution is made by mixing methylene chloride and methanol in a weight ratio of 3: 1 to 1: 3. When the amount of methylene chloride increases, the time for which the active ingredient remains in the fixed phase is excessively long, which is inefficient. In addition, when the amount of methanol (methanol) is greater than the amount, the time that the active ingredient remains in the fixed phase is not enough, the separation between the materials is inefficient. Therefore, the weight ratio of methylene chloride and methanol is preferably within the above range.

The fraction has a different moving speed through the stationary phase due to the chemical and physical properties of the molecules and different kinds of the eluted eluate depending on the moving speed difference. The mobile phase and the stationary phase are only preferred examples in the present invention, and the mobile phase and the stationary phase are not limited thereto as long as the eluate can be easily obtained from the fraction. The eluate is prepared by mixing ethanol in the eluate.

The recrystallization step is a crystal forming step of making the crystals by drying the eluent under reduced pressure, the crystallization step of preparing a crystalline solution by dissolving the formed crystals in ethanol (ethanol) and the crystalline solution is filtered and the filtered crystals And a stationary step of inducing crystallization by allowing the solution to settle. The elution solution is dried under reduced pressure to undergo a crystal forming step of making a crystal, followed by a crystal melting step. In the crystal melting step, ethanol is in a concentration of 1 to 10%, and the crystals are dissolved by heating at 50 ° C. to 80 ° C. in the ethanol. In order to recrystallize the crystals dissolved in the ethanol goes through a stationary step. The stationary step is to induce crystallization of the filtrate from which the crystalline solution is filtered at -80 ° C to 40 ° C. The re-crystallization step is via re-crystallization removal of impurities in the eluted solution and the p pure active ingredient to be volatilized ethanol (ethanol) - it is necessary to make a coumarin acid (p -coumaric acid).

The present invention jeju jeori ( Sasa quelpaertensis The active ingredient is separated from the extract extracted from Nakai) and used in the functional cosmetic composition and pharmaceutical composition having the skin whitening effect. Sasa Jeju in the functional cosmetic composition and pharmaceutical composition ( Sasa quelpaertensis Nakai) blend of 0.01 to 10% by weight of crude extract of the stem (stem) is very excellent in the whitening effect. Most preferably, when the crude extract is blended with 0.1 to 2.0% by weight, the whitening effect is very excellent. And, if the crude extract instead of the ethyl acetate (ethyl acetate) fraction or the active ingredient p -coumaric acid ( p- coumaric acid) purified from the crude extract or when combined with the effect Is improved.

As an excipient used in the functional cosmetics, it may further contain arbutin, arbutin, kojic acid, rucinol, vitamin C, vitamin C derivatives, and the like, and retinol for the purpose of improving wrinkles. (retinol) and its derivatives, indol acetic acid and its derivatives, adenosine (adenosin), tocopherol and its derivatives, kyinitin and the like. In addition, the excipient may include components conventionally used in cosmetic compositions, and include, for example, conventional auxiliaries and carriers such as stabilizers, solubilizers, vitamins, pigments and flavorings.

Pharmaceutical excipients used in the composition of the medicament include diluents such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. that are commonly used. In addition, sterilized aqueous solution, water-insoluble solvents, suspension solvents, emulsions, etc. may be included, and non-aqueous solvents, suspension solvents, such as propylene glycol, polyethylene glycol, olive oil (olive oil) Vegetable oils such as, injectable esters such as ethyl oleate, and the like can be used. Witsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used as the base material of the suppository.

In addition, the pharmaceutical composition of the present invention Jeju jeju ( Sasa quelpaertensis The extract extracted from Nakai) can be used in combination with various carriers that are accepted as pharmaceuticals, such as physiological saline or organic solvents, including purified active ingredients, and to increase stability and absorption, glucose and sucrose. Or carbohydrates such as dexrine; Antioxidants such as ascorbic acid or glutathione; Chelating agents; It can be used with small molecule proteins or other stabilizers.

The functional cosmetic composition and the pharmaceutical composition of the present invention is purified p -coumaric acid ( p- coumaric acid) as an active ingredient, the active ingredient is the active ingredient tyrosinase and alpha-MSH (α-MSH (α-MSH) Inhibits the production of melanin. The alpha-MSH (α-MSH) plays an important role in the expression of tyrosinase in the synthesis of melanin. The alpha-MSH (α-MSH) activates adenylly cyclase, which binds to the MSH receptor on the cell membrane to increase intracellular cyclic AMP. The increased cAMP activates PKA and the activated PKA phosphorylates CREB again, eventually activating the MITF transcription factor. A promoter region of the melanin production-related enzyme gene and an activated MITF bind to express genes of the enzymes.

It has recently been known that tyrosinase proteins have been synthesized and then matured and activated by binding to, glycosylated or phosphorylated with copper. Tyrosine is oxidized to dopa (DOPA, 3,4-dihydro xyphenylalanine) through hydroxylation and then dopa (DOPA, 3,4-dihydro xyphenylalanine) to dopaquinone (DOPA-quinone, pheomelanin) to melamine Synthesis begins. These two reactions proceed by the enzymatic action of tyrosinase and are the major regulatory points in the melanin synthesis pathway. Dopaquinone (DOPA-quinone, pheomelanin) combined with cysteine or glutathione is 5-S-cysteinyl dopa (DOPA, 3,4-dihydro xyphenylalanine) and glutathionyl dopa (DOPA, 3,4-) dihydro xyphenylalanine) and subsequent staged reactions eventually form a reddish pheomelanin. At the same time as the formation of pheomelanin (pheomelanin), dopaquinone (DOPA-quinone, pheomelanin) becomes dopachrome (DOPAchrome) when there is no thiol compound (thio compound) becomes black black melanin (eumelanin). Melanosomes containing the melanin are moved to keratinocytes through dendritic processes of melanocytes. The result is the deposition of melanin pigments on the skin surface.

In the present invention, the tyrosine (tyrosine) hydroxylation reaction and dopa (DOPA, 3,4-dihydro xyphenylalanine), which occur in time series by inhibiting the tyrosinase activity is oxidized to dopaquinone (DOPA-quinone, pheomelanin) In conclusion, by inhibiting the reaction is to prevent melanin (melanin) synthesis.

The p - coumarin acid (p -coumaric acid) is a phenolic material that is commonly distributed in the plant. Melanin generation was used for this the cells promote melanin (melanin) generated by treatment with an alpha -MSH (α-MSH) (melanin ) To demonstrate the whitening efficacy of coumarin acid (p -coumaric acid) - p of the invention Inhibitory effect was evaluated. Referring to Experimental Example 3 and Experimental Example 4 of the second embodiment, which is one of the preferred embodiments of the present invention, melanin production was induced in cells after alpha-MSH (α-MSH) treatment. melanin) to produce p - coumarin acid (p -coumaric acid) it can be seen that this was inhibited significantly. This p - suggests that coumarin acid (p -coumaric acid) is to inhibit the signaling pathway in melanin (melanin) production-accelerating the process of alpha -MSH (α-MSH) inhibiting melanin (melanin) produced. In addition, it increased by alpha -MSH (α-MSH) tyrosinase (tyrosinase) protein expression is also p - suggests that coumarin acid (p -coumaric acid) is suppressed.

Hereinafter, referring to the drawings, as the first embodiment, which is one of the preferred embodiments of the present invention, Jeju Josa ( Sasa) quelpaertensis Look at the preparation of the extract of Nakai) and the active ingredient in the extract. In addition, look at the cell whitening effect of the active ingredient obtained in the extract as a second embodiment of one of the preferred embodiments of the present invention. In addition, as a third embodiment of one of the preferred embodiments of the present invention, a functional cosmetic and a pharmaceutical composition having a skin whitening effect using the active ingredient obtained from the extract will be described.

First Embodiment

Jeju Sasadae ( Sasa quelpaertensis Nakai) was collected from Jeju Island in July 2007. 70% ethanol was added to the dried plants, extracted at room temperature, and concentrated under reduced pressure. Thus, extracts of bamboo (stem) and leaf extracts were obtained in yields of 1.2% and 6%, respectively. 60 g of the bamboo (stem) extract was suspended in distilled water, and then solvent fractionated using methylene chloride, ethyl acetate, and normal butanol (N-Buthanol), and each fraction was concentrated under reduced pressure to obtain chloride. 1.7 g of methylene chloride, 1.5 g of ethyl acetate, 2.3 g of normal butanol, and 52 g of water were obtained, respectively.

Among these fractions, ethyl acetate fractions were separated from the ethyl acetate fractions, and the ethyl acetate fractions were separated from the methylene chloride column using a Sephadex LH20 column. chloride): 50 fractions were eluted at 10 ml each using methanol (1: 1) solvent. Each concentrated fraction was used again by dissolving ethanol, and especially the eleventh fraction which showed the highest activity was 40 mg of crystals through concentration and ethanol recrystallization. The separated active material was identified by the UV-vis, FAB-MS, NMR analysis of the structural characteristics.

Each extract and the separated active substance in the HPLC analysis are compared to the material p - coumarin acid with (p -coumaric acid) was diluted with ethanol (ethanol) and 5 ㎛ Waters XTerra RP-18 ( 4.6 mm × 150 mm) column (column ). The mobile phase was 0.5% formic acid (A) and acetonitrile (B) with a flow rate of 1.0 ml / min. Analytical gradient conditions are 0-6 minutes: 100% A, 6-10 minutes: 0-12% B, 10-35 minutes: 12-21% B, 35-40 minutes: 21-25% B, 40-50 minutes : 25 to 100%, 50 to 60 minutes: 100% B and the detector was measured at 280 nm.

< Experimental Example 1  -Active ingredient in the extract>

Referring to Figure 2, Jeju Josadae ( Sasa quelpaertensis Nakai) extract of bamboo (stem) area was divided into methylene chloride, ethyl acetate, normal butanol (N-Buthanol) and water fractions, and bamboo (stem) on cells that promoted melanin synthesis. Site extracts and four fractions were treated with 100 ㎍ / ml to evaluate the melanin production inhibitory effect. FIG. 2A shows the melanin production of cells in the presence or absence of alpha-MSH (α-MSH) treatment of leaf extract and stem extract of Sasa quelpaertensis Nakai. When the alpha-MSH (α-MSH) is treated, it is shown by a white bar graph when not treated with a black bar. As shown in FIG. 2A, higher activity was confirmed in the ethyl acetate fraction and the normal butanol fraction of the stem extract, and the effect was not confirmed in the methylene chloride fraction and the water fraction. Next, the activity of tyrosinase was measured using dopa (DOPA, 3,4-dihydro xyphenylalanine) as a substrate in the cell lysate. As shown in FIG. 2B as a result of the measurement, the alpha-MSH (α-MSH) was shown as a white bar graph when not treated with a black bar. Referring to FIG. 2B, when the activity of tyrosinase is increased by the alpha-MSH (α-MSH), the activity of the ethyl acetate fraction and the normal butanol fraction is increased. It shows a significant decrease.

Referring to FIGS. 3 and 4, the ethyl acetate fraction having the highest activity was obtained again by Sepadex LH20 column chromatography. The first fraction most effectively inhibited melanin production (FIG. 3A). This fraction was recrystallized with ethanol to obtain white crystals, which were subjected to spectroscopic analysis. The result is as follows. : UV, _max (Ethanol), 311.4 nm; FAB-MS, m / z, 165.2 (M + H ⁺ ); ¹ H-NMR (CD ₃ OD), ppm, 6.27 (1H, doublet, J = 16 Hz, H-8), 6.80 (2H, doublet, J = 9 Hz, H-3 / 5), 7.43 (2H, doublet, J = 9 Hz, H-2 / 6), 7.59 (H, doublet, J = 16 Hz, H-7); ¹³ C-NMR (CD ₃ OD),, ppm, 115.75 (C-8), 116.95 (C-3 / 5), 127.39 (C-1), 131.22 (C-2 / 6), 146.79 (C-7 ), 161.30 (C-4), 171.15 (C-9). Based on these results, it can be estimated that the active substance is trans -p- coumaric acid compared with [Book on active substance]. The structure of the trans -p- coumaric acid and the ¹³ C-NMR spectrum are shown in FIG. 3B.

HPLC confirmed the same by analyzing the retention time of the active material and the authentic p- coumaric acid separated from the extract (Fig. 4). Fingerprint HPLC of the ethyl acetate fraction of the leaf extract, bamboo (stem) extract, and bamboo (stem) extract of Sasa quelpaertensis Nakai is shown in FIG. 4. At this time, these p - coumarin acid (p -coumaric acid) content was determined to be 0.1%, 0.7% and 12%, respectively.

Second Embodiment

In a preferred embodiment of the second exemplary embodiment of the present invention one of the Cheju Sasa (Sasa quelpaertensis In order to examine the whitening effect of the active ingredient in the extract extracted from the stem of Nakai), the melanin production inhibitory activity, cell viability and tyrosinase (tyrosinase) inhibitory effect through the experimental example.

< Experimental Example 1  -Measurement of melanogenesis inhibitory activity>

Jeju jejudae of the present invention ( Sasa) quelpaertensis The effect of inhibiting melanin production of bamboo extracts and leaf extracts of Nakai) was investigated in mouse melanoma B16F10 cells that were promoted by melanin production by alpha-MSH (α-MSH). B16F10 cells are widely used for this purpose, and are relatively easy to experiment in vitro compared to normal human melanocytes (melanocytes).

The melanoma cells were B16F10, a mouse melanoma cell purchased from the American Type Culture Collection (ATCC). The cells were cultured at 37 ° C., 5% CO ₂ in DMEM medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 0.1 mg / ml streptomycin. Cultured in the incubator. In addition, the test materials were pretreated for 60 minutes by concentration, and then 100 nM of alpha-MSH (α-MSH) was added thereto and cultured for 48 hours. In order to measure the melanin content released after the culture, the culture solution was taken 200 μl and transferred to 96 well-microplate, and the absorbance was measured at a wavelength of 490 nm in the microplate reader. The morphology and degree of pigmentation were observed under a phase contrast microscope.

Referring to Figure 1, the results of the microscopic observation that the treatment of alpha-MSH (α-MSH) to promote the production of melanin (α-MSH) at 100 nM and the degree of melanin production and the degree of pigmentation deposited in the cell It was confirmed (Figure 1). The Jeju Sasa (Sasa quelpaertensis Nakai) Bamboo (stem) region extract and leaf extract 1.0 mg / ml in cells treated cells within the melanin (melanin), it was confirmed that the synthesis is inhibited significantly in Jeju Sasa (Sasa quelpaertensis Nakai) was more effective in the stem region extract (FIG. 1).

< Experimental Example 2  Cell viability measurement

The viability of the cells was measured using 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT), and the violet color absorbance of MTT was degraded by mitochondrial activity in the cells. The viability of the cells was measured by measuring. Various concentrations of the test substance were treated with the cells and incubated for 48 hours, followed by reaction for 3 hours by adding MTT at a concentration of 2 mg / ml. After the reaction was completed, the medium was removed, and 1 ml of isopropanol was added and allowed to stand for 30 minutes until the formazen crystals were dissolved. The reaction solution was transferred to a 96 well-microplate and absorbance was measured at a wavelength of 570 nm in a microplate reader. The measurement results are shown in FIG. 5, and FIG. 5 is a sasa jeju bar. quelpaertensis The active ingredient isolated from p Nakai) - coumarin acid (p -coumaric acid) exhibited the effects on cell viability in an alpha -MSH (α-MSH), processing oil (black spots) no (white point) conditions.

Referring to FIG. 5, the absorbance measurement showed no cytotoxicity by this material, and only negligible cytotoxicity by alpha-MSH (α-MSH) itself (FIG. 5). Therefore, Jeju Sasa ( Sasa) quelpaertensis P is an active ingredient of the extract extracted from Nakai) - coumarin acid (p -coumaric acid) is so harmful to the human body is usable as a whitening agent.

< Experimental Example 3  Cell extract preparation and tyrosinase  Active measurement>

Collect the cultured cells and add the cell lysate (10 mM Tris-Cl, pH 7.4, 120 mM NaCl, 25 mM KCl, 2 mM EGTA, 1 mM EDTA, and 0.5% Triton X-100, protease inhibitor cocktail) The cells were disrupted by standing on ice for 1 minute, and then centrifuged (14,000 x g, 15 minutes at 4 ° C), and the supernatant was taken and used for measuring enzyme activity. Tyrosinase activity was measured by the degree of tyrosinase oxidizing dopa (DOPA, 3,4-dihydro xyphenylalanine) to dopachrome. The cell extract (20 μg protein) and 1 mM DOPA were added to 0.1 M sodium phosphate buffer (pH 6.8) for 60 minutes at 37 ° C. The degree of production of dopachrome was measured at a wavelength of 490 nm in a microplate reader.

The p - coumarin acid (p -coumaric acid) of melanin (melanin) is then treated in cytolytic cells to compare the mechanism of action of suppressing the generation and arbutin (arbutin), to thereby obtain the solution. First, tyrosinase activity was measured using this solution using dopa (DOPA, 3,4-dihydroxyphenylalanine) as a substrate.

Referring to Figure 6A, Figure 6A is a jeju jeoldae quelpaertensis W coumarin acid (p -coumaric acid) and arbutin (arbutin) is in order determine whether inhibiting tyrosinase (tyrosinase) expressed in alpha -MSH (α-MSH) or without conditions the active ingredient, the p separated from Nakai) It shows the activity level of the tyrosinase (tyrosinase). The case where alpha-MSH (α-MSH) was treated is indicated by a white bar when the alpha-MSH was not treated. Referring to FIG 6A to the p - show that coumarin acid (p -coumaric acid) that reduce the activity of tyrosinase (tyrosinase) regardless of the presence or absence of alpha -MSH (α-MSH) in a concentration-dependent manner and . Arbutin also has a weak but significant inhibitory effect.

In addition, referring to Figure 6C, the 6C is the Jeju cooking rod ( Sasa quelpaertensis Nakai) the active ingredient, the p separation in-coumarin is an acid (graph p -coumaric acid) and arbutin (arbutin) is showing the degree of inhibition of melanin (melanin) generated in accordance with alpha -MSH (α-MSH) or without treatment. In the graph, when alpha-MSH (α-MSH) is treated, a square point is indicated when the alpha-MSH is not treated as a smooth point. Referring to the Figure 6C the absorbance measurement result p a widely separated, compared with arbutin (arbutin) used as a raw material of the whitening agent to the existing - melanin (melanin) generation inhibition of coumarin acid (p -coumaric acid) effect is 10 times greater than it High (Figure 6C).

< Experimental Example 4  - Western blot Tyrosinase through of  Protein Content Analysis>

In order to denature the protein, the cell extract (20 ㎍ protein) prepared in advance was mixed with Laemmli buffer and left at 95 ° C for 5 minutes, followed by electrophoresis on a 10% SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. After transfer, the membrane was reacted at 4 ° C. overnight using a primary antibody and then reacted at room temperature for 1 hour with a secondary antibody conjugated with blush and horseradish peroxidase. Analysis was performed using Amersham's chemiluminescence kit.

The results of analyzing the protein content of tyrosinase in the cell lysate through the western blot are shown in FIG. 6B. 6B is a sasa jeju ( sasa) quelpaertensis Of the active ingredients isolated from Nakai) p - coumarin acid (p -coumaric acid) and arbutin (arbutin) a tee to the extent of inhibiting tyrosinase (tyrosinase) expressed in alpha -MSH (α-MSH) or without conditions or when This is a graph analyzed by comparing the amount of protein of the enzyme (tyrosinase). The case where alpha-MSH (α-MSH) was treated is indicated by a white bar when the alpha-MSH was not treated. Referring to Figure 6B, the arbutin does not reduce the protein content of tyrosinase as previously reported. On the other hand, the p- coumarin acid (p -coumaric acid) is that the inhibition of the tyrosinase (tyrosinase) protein produced by inhibiting the expression of intracellular melanin (melanin) increased by alpha -MSH (α-MSH) Is showing. These results showed that p- coumarin acid (p -coumaric acid) and arbutin (albutin) has a different mechanism of action in inhibiting the production of melanin (melanin).

< Experimental Example 5  - p - coumaric acid Effect on Tyrosinase Activity of Other Compounds Structurally Similar to

3- (4-Hydroxyphenyl) propionic acid , cinnamic acid, caffeic acid is present to name p - is a compound similar to the structural coumarin acid (p -coumaric acid). The p - coumarin acid (p -coumaric acid) and other compounds structurally similar to the effect of the melanin (melanin) generated impact and tyrosinase (tyrosinase) activity in a process for alpha -MSH (α-MSH) cells p - were compared with coumarin acid (p -coumaric acid). The comparison is shown in FIG. FIG 7 is a p - shows the effect on generated coumarin acid (p -coumaric acid) and melanin in the structure, and these substances are alpha -MSH (α-MSH) or without processing conditions of the structurally similar materials (melanin) .

Referring to FIG. 7, p - coumarin acid (p -coumaric acid) 3- lacking a double bond in the side chain of (4-Hydroxyphenyl) propionic acid or benzene hydroxyl missing one cinnamic acid or hydroxy group on the benzene ring in ring one further addition of caffeic acid than p - coumarin acid (p -coumaric acid) this illustrates that there is a stronger inhibitory effect. Through these results, p - it can be seen that the coumarin acid (p -coumaric acid) structure is the structure suitable for inhibiting the intracellular melanin production of.

Third embodiment

The effective substance of this invention p purified by the purification method - using a coumarin acid (p -coumaric acid) and to present the preferred embodiment for the purpose of the cosmetic. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

< Example (1)-flexible lotion ( skin A) manufacturing 1>

Jeju Sasadae ( Sasa quelpaertensis Nakai) vs. crude extract 0.1% by weight, p- coumaric acid 0.1% by weight, glycerin 7.0% by weight, 1,3-butylene glycol 4.0% by weight, hyaluronic acid 0.3% by weight, allantoin 0.1% by weight, DL-panthenol 0.1 A weight percent, 0.02 weight percent EDTA-2Na, 8.0 weight percent ethanol, 0.4 weight percent polysorbate 20, octyldodeceth-16 0 flexible lotion was prepared.

< Example (2)-supple lotion ( skin A) manufacturing 2>

Jeju Sasadae ( Sasa quelpaertensis Nakai) purified ethyl acetate fraction 0.5% by weight, glycerin 7.0% by weight, 1,3-butylene glycol 4.0% by weight, hyaluronic acid 0.3% by weight, allantoin 0.1% by weight, DL-panthenol 0.1% by weight, 0.02 wt% EDTA-2Na, 8.0 wt% ethanol, 0.4 wt% polysorbate 20, octyldodeceth-16 0 flexible lotion was prepared.

< Example (3)-manufacture of nutritional creams>

Jeju Sasadae ( Sasa quelpaertensis Nakai) crude extract 2.0% by weight, p- coumaric acid 2.0% by weight, beeswax 0.7%, cetostearyl alcohol 0.5% by weight, glyceryl monostearate 1.1% by weight, paraoxybenzoic acid ester 0.3% by weight, polyoxyethylene Sorbitan triisostearate (20, EO) 1.1 wt%, cyclomethicone 1.0 wt%, dimethicone 0.3 wt%, jojoba oil 1.0 wt%, squalane 2.0 wt%, octyldodecanol 2.0 wt%, liquid paraffin 3.0 A nutritious cream was prepared by mixing a weight percent, 3.0 weight percent glycerin, 4.0 weight percent glycerin, 3.0 weight percent 1,3-butylene glycol, 0.08 weight percent Carvermo 940 and a trace amount of pigment, flavor and residual purified water.

< Example (4)-manufacture of nutritional creams>

Jeju Sasadae ( Sasa quelpaertensis Nakai) crude extract 2.0% by weight, Jeju Sasa ( Sasa) quelpaertensis Nakai) Purified ethyl acetate fraction 0.5 wt%, beeswax 0.7 wt%, cetostearyl alcohol 0.5 wt%, glyceryl monostearate 1.1 wt%, paraoxybenzoic acid ester 0.3 wt%, polyoxyethylene sorbitan tree 1.1% by weight of isostearate (20, EO), 1.0% by weight of cyclomethicone, 0.3% by weight of dimethicone, 1.0% by weight of jojoba oil, 2.0% by weight of squalane, 2.0% by weight of octyldodecanol, 3.0% by weight of paraffin, A nutrition cream was prepared by mixing 3.0% by weight of glycerin, 4.0% by weight of glycerin, 3.0% by weight of 1,3-butylene glycol, 0.08% by weight of Carvermo 940, and a small amount of pigment, flavor and residual purified water.

On the other hand, it is an object of the present invention to supplement the disadvantages of the prior art, and to provide a whitening agent of high purity purified from plant extracts safe for human body. More specifically, the present invention Jeju jejudae ( Sasa quelpaertensis Nakai) to a method for purifying the whitening component and its use. Melanin pigmentation in skin, such as blemishes and freckles, is most likely due to hereditary effects, but is also caused by non-genetic factors such as hormonal changes, chronic inflammation, aging, and ultraviolet light. Due to the pathological physiological or environmental factors, unwanted skin pigmentation contributed not only to the development of related cosmetics but also to the search for natural products or compounds that can inhibit them at each stage of pigmentation.

The present invention jeju jeori ( Sasa quelpaertensis Nakai) is to purify the active ingredient from the extract of the stem. The active ingredient is Jeju Sasadae ( Sasa quelpaertensis Nakai) is more present in the extract of the stem than the leaf extract of the active ingredient purified from the extract of the stem portion is melanin (melanin) production inhibitory action is more powerful. The active ingredient is p -coumaric acid ( p- coumaric acid), p -coumaric acid ( p- coumaric acid) is not cytotoxic and melanin (melanin) production inhibitory effect is superior to the known arbutin (arbutin).

In addition, the p - coumarin acid (p -coumaric acid) appears to the whitening effect by inhibiting the expression of the tyrosinase (tyrosinase) induced by alpha -MSH (α-MSH). How this time the p - coumarin acid (p -coumaric acid) alpha -MSH (α-MSH), but clear that inhibit the expression of the tyrosinase (tyrosinase) induced by tyrosine (tyrosine) and structurally similar to the p - It can be assumed that p- coumaric acid will compete with L-tyrosine when the expression of tyrosinase is regulated by alpha-MSH (α-MSH). An interesting result has been reported that tyrosine increases tyrosinase expression by increasing the binding capacity of MSH receptors that bind alpha-MSH (α-MSH). Therefore, p - is assumed that the - (coumaric acid p) tyrosine (tyrosine) and competitively any action with respect to the regulatory region of the MSH receptor coumarin acid.

In conclusion, the present invention is Jeju Sasadae ( Sasa quelpaertensis Nakai) tyrosinase (tyrosinase) active ingredient, the p of suppressing in-coumarin acid (p -coumaric acid) of p accurately identified by the purification and the purification method that - using a coumarin acid (p -coumaric acid) Drug Composition and functional cosmetic composition. Due to the purification method will be able to overcome the heterogeneity of the effects that appeared when using as a cosmetic using conventional plant extracts. In addition, it can be said that the invention can be applied to other plant extracts in various ways to be used by selecting only the active ingredient except the unwanted components in the plant extract.

As described above, the present invention has been described with reference to one embodiment shown in the drawings, but this is merely exemplary, and various modifications and equivalent other embodiments may be made by those skilled in the art. I will understand the point. Therefore, the true technical protection scope of the present invention will be defined by the technical spirit of the appended claims.

1 is jeju jeoldae ( Sasa quelpaertensis Graph (A) and details showing the effects of stem extract and leaf extract of Nakai) on the inhibition of melanin production in cells under alpha-MSH (α-MSH) treatment (black bars) (B);

2 is a jeju jeoldae quelpaertensis Stem extract of Nakai) and fractions of the extract of the stem extracts melanin production (A) and tyrosinase (A) of cells under alpha-MSH (α-MSH) treatment (black bars) without (white bars) conditions. tyrosinase) graph showing the effect on activity (B);

Figure 3 is a graph showing the effect of each of the eluate on the production of melanin (melanin) induced by alpha-MSH (α-MSH) and the effective separation from the eluate that most inhibits the production of melanin (melanin) Graph (B) showing the molecular structure through the ¹³ C-NMR spectrum of the component;

4 is a jeju jeoldae quelpaertensis Nakai extract, the jeju jeolla ( sasa) quelpaertensis Ethyl acetate fraction of the stem of Nakai), Sasa jeju quelpaertensis A graph showing the HPLC analysis of the active ingredient, authentic p- coumaric acid, isolated from the ethyl acetate fraction of the stem of Nakai);

5 is a jeju jeoldae quelpaertensis Nakai) the active ingredients isolated from p-coumarine acid (p -coumaric acid), a graph showing the effect on survival cells in alpha -MSH (α-MSH), processing oil (black spots) no (white point) condition;

6 is a jeju jeoldae quelpaertensis Nakai) separated from the valid components p - coumarin of an acid (p -coumaric acid) and arbutin (arbutin), alpha -MSH (α-MSH), oil (black bars) no (white bars), tyrosinase (tyrosinase) in terms A graph comparing the degree of inhibition of expression with activity (A) and protein amount (B) of the enzyme was compared with Sasa quelpaertensis Graph showing the degree of inhibition of melanin production from the active ingredient ( p -coumaric acid) and arbutin isolated from Nakai) under the condition of alpha-MSH (α-MSH) C); And

7 is a p - Melanin from coumarin acid (p -coumaric acid), and their structure and material of these structurally similar substances are alpha -MSH (α-MSH), processing oil (black bars) no (white bars) conditions (melanin ) It is a graph showing the effect on generation.

Claims (13)

Jeju Sasadae ( Sasa quelpaertensis A method for purifying a coumarin acid (p -coumaric acid), - p is an active ingredient from Nakai) The purification method is the jeju jeori ( Sasa quelpaertensis Extraction step of extracting Nakai) with ethanol at a temperature of 10 ° C. to 70 ° C .; Concentrate manufacturing step of producing a concentrated solution by reducing the extract solution obtained in the extraction step; Fractionating the concentrate with a solvent and concentrating under reduced pressure to obtain a fraction; An eluting solution preparation step of eluting the fractions by filtration to elute with time, dissolving the eluate in ethanol to obtain an eluting solution; And Coumarin acid (p -coumaric acid) purification method - p comprises a; recrystallization step that the eluting solution of ethanol (ethanol) obtained was recrystallized crystals. The method of claim 1, The extraction step is dried Jeju jeori ( Sasa quelpaertensis Jeju dried sasa ( Sasa ) as the stem of Nakai quelpaertensis Coumarin acid (p -coumaric acid) purification method - p characterized in that the extract was added to 4 to 20 times the 30% to 80% of ethanol (ethanol) of the weight of the Nakai). The method of claim 1, The concentrate is p, it characterized in that the concentration of the extract solution to a concentration of 4 to 20 times the concentration of the extract liquid-coumarin acid (p -coumaric acid) tablet method. The method of claim 1, In the fractionation step, the concentrate is partitioned into methylene chloride, chloroform, and normal hexane, and then the fractionated concentrate is ethyl acetate, methyl acetate, and methyl acetate. acetate), normal butanol (N-butanol) and isopropanol (isopropanol), characterized in that the p -coumaric acid ( p- coumaric acid) purification method. The method of claim 1, The eluent is methylene chloride (methylene cloride) and methanol (methanol) column, the fractions with a mixed mixed liquid chromatography (column chromatography) column (column) to be eluted by p, characterized in that to obtain the passage of - coumarin acid ( p -coumaric acid) purification method. The method of claim 5, wherein The mixture is dichloromethane (methylene cloride) and methanol (methanol) is 3: 1 to 1: p of the said mixture in a weight ratio of 3-coumarin acid (p -coumaric acid) tablet method. The method of claim 1, The recrystallization step is a crystal forming step of producing a crystal by drying the elution solution under reduced pressure; A crystalline dissolving step of preparing the crystalline solution by dissolving the formed crystals in ethanol; And P -coumaric acid ( P -coumaric acid) purification method characterized in that it comprises a; the step of filtering the crystalline solution and the crystallization solution to leave the crystals to induce crystallization. The method of claim 7, wherein The crystal melting step is ethanol (ethanol) is 1 to 10% concentration of the ethanol to the crystals (ethanol) and heated at 50 ℃ to 80 ℃ dissolved p to the characterized in that in-coumarin acid (p -coumaric acid ) Purification method. The method of claim 7, wherein The value p is a phase, characterized in that for inducing the crystallization of the filtrate was filtered the crystals dissolved solution at -80 ℃ to 40 ℃ - coumarin acid (p -coumaric acid) tablet method. Any one of claims 1 to 9 as described in any one of the p purified by a purification method, which of the anti-coumarin acid (p -coumaric acid) as an active ingredient, wherein the effective component tyrosinase (tyrosinase) with alpha -MSH ( Functional cosmetic composition that inhibits the production of melanin by preventing the activity of α-MSH). The method of claim 10, The cosmetic composition is a functional cosmetic composition, characterized in that it comprises 0.01 to 10wt.% Of the purified p -coumaric acid ( p -coumaric acid) and excipients used in cosmetics. Any one of claims 1 to 9 as described in any one of the p purified by a purification method, which of the anti-coumarin acid (p -coumaric acid) as an active ingredient, wherein the effective component tyrosinase (tyrosinase) with alpha -MSH ( Pharmaceutical composition which inhibits the production of melanin by preventing the activity of (alpha) -MSH). The method of claim 12, The pharmaceutical composition is the purified p - coumarin acid (p -coumaric acid) a pharmaceutical composition comprising a pharmaceutical excipient and 0.01 to 10wt%..

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