JP2003516358A - Melanin synthesis inhibitory compound and composition containing the melanin synthesis inhibitory compound - Google Patents

Abstract

(57) [Summary] The present invention relates to a melanin synthesis inhibitory compound containing gomisin N or γ-schizandrin, and a composition containing the compound. The composition of the present invention has an excellent melanin synthesis inhibitory effect without showing cytotoxicity. The present invention also relates to a Gomiko extract having excellent melanin synthesis inhibitory effect without showing cytotoxicity.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a compound having a melanin synthesis inhibitory effect and a composition containing the compound.

[Prior Art] When the skin is exposed to sunlight, a melanin pigment is synthesized in order to prevent ultraviolet rays.
The skin becomes dark due to the appearance of the melanin pigment on the surface of the skin.

The melanin is a complex of a black pigment and a protein as a biopolymer substance of phenols widely distributed in nature. The melanin is physically and chemically yellow-pheomelanin showing a reddish brown color (pheomelanin), brown-black eumelanin (eumelanin), and trichochrome having similar characteristics (trichochrome), is roughly divided into three types, All of their biosynthetic pathways are tyrosinase (EC1.14.
It is reported to be carried out by the action of 18.1). Human skin color is determined by the type and amount of the pigment, and overproduction of the pigment is known to form freckles on the human skin, promote skin aging, and induce skin cancer. . Also, in food, vegetables,
May reduce fruit and fish quality.

Melanin synthesis inhibitors have been applied in the fields of pharmaceuticals, cosmetics, foods, etc., as therapeutic agents for skin diseases, skin whitening agents, brown spot inhibitors for foods, etc., and the demand for them has increased rapidly in recent years. It's starting.

Studies on melanin synthesis inhibitors have mainly been focused on the development of tyrosinase inhibitors, and as a result, various whitening agents have been developed, but various problems have been raised.

4-Hydroxyanizole and hydroquinone have been applied topically for the treatment of spots and hypertension during pregnancy. Although these compounds show a strong inhibitory activity on melanin production, they have side effects such as degeneration or death of pigment cells and damage to the original functions of cells, so they are used only in some countries. Is allowed. Kojic acid [5-hydroxy-2- (hydroxymethyl-γ-pyrone), arbutin (hydroquinone-β-D-glucopyranoside), etc. are also used as an anticorrosive agent for food, as a whitening agent for cosmetics and pharmaceuticals, together with ascorbic acid. Although it is the main active ingredient used as an ingredient, it has the disadvantages of low inhibitory activity, discoloration during use and instability of the substance itself.

Korean Patent No. 228741 discloses that schizandra extract is added to a solid food or drink to inhibit the activity of tyrocyanase and inhibits the activity of glucosyltransferase to have a whitening effect on teeth and a dental caries preventive effect. It describes a solid food or drink in which an extract of Gomizai is included.

On the other hand, gomisin N and γ-schizandrin are mutual stereoisomers, and their structural formulas are as follows.

[0009]

[Chemical 1]

[0010]

[Chemical 2]

Gomisin N inhibits the activity of cholesterol acyltransferase (Pl
anta med.1999, 65: 74-76), chitinase (chiti), a cell wall synthase of fungi.
nase) II activity has been reported to be inhibited (Journal of Pharmaceutical Sciences, 1995, 43 (4): 509-515).
.

[0012]   Today, the physiological activity of γ-schizandrin is unknown.

[Problems to be Solved by the Invention] An object of the present invention is to provide an extract of Schisandra moriensis having melanin inhibitory activity.

An object of the present invention is to provide a compound that inhibits the synthesis of melanin, and to provide a composition that inhibits the synthesis of the melanin.

[Means for Solving the Problems] The present invention relates to a melanin synthesis inhibitor containing a garlic extract. The schizandra extract of the present invention preferably contains gomisin N or γ-schizandrin. The present invention also relates to a melanin synthesis inhibitor containing gomisin N or γ-schizandrin.

The present inventors have discovered that gomisin N and γ-schizandrin have almost no cytotoxicity and an excellent whitening effect.

That is, since gomisin N and γ-schizandrin have much lower tyrosine inhibitory activity than conventional substances having a whitening effect, for example, kojic acid and arbutin, the melanin activity of gomisin N and γ-schizandrin is It is understood that it is not a mechanism by the inhibition of tyrosinase.

Further, it can be seen that gomisin N and γ-schizandrin do not inhibit the oxidative process of melanin synthesis stage initiated from tyrosine because of its low antioxidant activity. Gomisin N and γ-schizandrin do not show cytotoxicity to cells and selectively inhibit only pigment synthesis.

Gomisin N and γ-schizandrin can be extracted from organisms or produced by synthetic and other known methods, for example, gomisin N and γ-schizandrin can be extracted from Schisandra chinensis. You can

The composition containing gomisin N and γ-schizandrin according to the present invention can be used as a melanin synthesis inhibitor, and can also be used as a cosmetic, a pharmaceutical whitening agent and a food browning agent. The specific composition of the composition according to the present invention can be easily determined by those skilled in the art.

The activity measuring method used for confirming the activity of the whitening agent in the present invention is as follows.

Measurement of Inhibitory Ability of Tyrosinase Activity To measure the inhibitory ability of tyrosinase activity, tyrosine (Sigma, USA) was added to 0.1 ml of tyrosine (Sigma Co., USA) in 1.5 ml of phosphate buffer (pH 6.8) whose temperature was previously adjusted in a 35 ° C. water bath. added at a concentration of mg / ml,
Tyrosinase (EC 1.14.18.1; 4,400 units / mg powder, Sigma) 110 units / ml was added. Here, after adding samples diluted according to concentration and reacting at 37 ° C for 10 minutes, 475 nm
The value obtained by measuring the absorbance with SAbs, the value obtained by adding the distilled water instead of the enzyme solution to measure the absorbance is BAbs, and the value obtained by adding the distilled water instead of the extracted sample to measure the absorbance with CA
The inhibition rate was calculated by the following formula with bs.

[0023]

[Equation 1]

Measurement of Sulfation Activity The sulfation activity of each compound was measured by dissolving 0.1 mg of each compound in 0.1 ml of methanol and adding to 5 ml of ethanol containing 58 mg of linoleic acid. (pH 7.0) 5 ml and distilled water are added to adjust to 12 ml, and the mixture is left to stand in a culture medium at 40 ° C. twenty four
After a lapse of time, 7 ml of 75% ethanol was reacted with 0.1 ml of the reaction solution, 150 μl of 0.02M FeCl ₂ solution and 150 μl of 30% ammonium thiocyanate for 3 minutes, and the absorbance was measured at 500 nm.

Measurement of inhibitory activity against Streptomyes bikiniensis melanin synthesis S. bikiniensis KCTC 9172 200 ml V-8 juice, glucose 2 g, yeast extract 2
Inoculate Papavizas VDYA slant medium at pH 7.2 containing g, CaCO ₃ 1 g, 2 g and bacto agar distilled water 800 ml. After adding 2 ml of distilled water to the surface of the slant culture cultivated at 28 ° C for 2 days, spores formed on aerial mycelia were scraped off with a platinum loop to prepare a spore suspension.

[0026] 1.5% glucose, 0.5% L-tyrosine, L- asparagine _{0.1%, K 2 HPO 4 0.05} %, MgSO 4 · 7H 2 O 0.05%, NaC1 0.05%, FeSO 4 × 7H 2 O 0.001%, Bakutoaga ( bacto agar)
Bacterial yeast extract (bacto-yeast extract) in tyrosine agar medium composition containing 2%
0.2% is added to make a solid medium and sterilized, and then a plate medium is prepared in a petri dish. Add 0.4 ml of S. bikiniensis spore suspension per agar plate and apply evenly,
Place an 8 mm diameter paper disk soaked with 1 l of sample and incubate at 28 ° C for 48 hours.

The size of the zone produced by the inhibition of melanin production is measured on the back side of the plate. As a control group, p-hydroxyanisole was used.

Measurement of melanin synthesis inhibitory activity of Aspergillus niger spores A. niger KCTC 2118 was added to potato dextrose agar medium (potato 20%, dextrose 2%, agar 1.5%; PDA medium) at a temperature of 37 ° C. After culturing for 6 days at 10 ° C., 10 ml of 0.01% Tween 80 ™ solution is added to the surface of the plate to scrape the spores formed on the hyphae to make a spore suspension. After adding the spore suspension to PDA medium at 5%, add 1 ml of medium to 24 wells, solidify and dissolve the sample in 50 μl of ethanol and add it directly to the surface of the plate. After culturing for 2 days, the spore pigment synthesis was visually observed. Adjust the number of spores per ml of spore suspension to 1.5 x 10 ⁵ CFU. P-Hydroxyanisole was used as a control group.

Measurement of melanin synthesis inhibitory activity of melanoma cells B16 mouse melanoma cells were treated with 10% FCS (fetal chalf serr) at a mobility of 5 × 10 ³ cells / ml.
um) suspended in DMEM (Dulbescco's Modified Eagle's Medium, Gibco BRL) medium containing (um), 5 ml of suspended cells (Falcon, Becton dickin)
son) and adding black samples by concentration, and then at 37 ° C under 5% CO ₂ -95% air conditions.
Culture at. After culturing for 4 days, the cells adsorbed on the bottom of the flask were washed with phosphate buffered saline (PBS), and then the cells were detached with 0.05% trypsin and 0.53 mM EDTA solution, and 1500 rpm 10 After centrifuging and collecting in a tube, the color of the cells is visually compared with the control group supplemented with arbutin.

The cells obtained by centrifugation are treated with 5% trichloroacetic acid (TCA), stirred and centrifuged, and then melanin is precipitated. The melanin is washed with phosphate buffered saline. Then, add 1 ml of sodium hydroxide 1N and boil for 10 minutes to dissolve melanin, and measure the absorbance at 400 nm with a spectrophotometer to express the amount of melanin produced per unit cell number (10 ⁶ cells) as a method of expressing the absorbance Then, the melanin production amount relative to the control group was calculated as the inhibition rate (%) to determine the IC ₅₀ .

Hereinafter, examples according to the present invention will be described, but the scope of the present invention is not limited to these examples.

Example 1. Separation of Melanin-Inhibiting Substance 10 mL of methanol was added to a sample obtained by crushing 500 g of Schisandra chinensis and extracted twice with shaking at room temperature for 48 hours. After extraction, it was filtered with filter paper and concentrated under reduced pressure at 50 ° C. to obtain 298 g of concentrated extract. To this, 2 L of a mixture of hexane: water: methanol = 10: 9: 1 (V / V / V) was added to fractionate into a hexane layer and an aqueous layer. A hexane layer extract (21.4 g) showing melanin synthesis inhibitory activity of A. niger spores was subjected to silica gel column chromatography (solvent: benzene: acetone, 98: 2 → 3: 7 (V / V)) to give 200 ml each. All 1
Two fractions were obtained. Fractions 2 to 5 showing melanin synthesis inhibitory activity of A. niger spores were subjected to measurement of melanin synthesis inhibitory activity of melanoma cells.
It showed melanin synthesis inhibitory activity without decreasing the cell number up to the concentration of μg / ml. Fraction 3 (3.
1 g) was subjected to reverse phase (hereinafter abbreviated as RP) column chromatography (70% methanol → 100% methanol). A total of 25 fractions of 50 ml each were obtained. Among them, 14 highly active fractions were developed by preparative TLC (hexane: ethylene acetate = 6: 1), and only the band showing the melanin synthesis inhibitory activity of A. niger spores was collected. A portion of the collected active band was analyzed by preparative HPLC (column: Vydac ODS, 5 μm, 250 × 10 mm I.D.
D, flow rate: 2 ml / min, UV detector: 220 nm, solvent: 70% acetonitrile (aceto
nitrile)) to separate the two compounds and named them SHC1 and SHC2, respectively. Table 1 shows the results of analysis of ¹ H-NMR and ¹³ C-NMR spectra of the two finally separated substances.

[0033]

[Table 1]

SHC1 and SHC2 were all colorless oils and all had similar molecular weights on EI-MS of m / z 400. Based on such results of experiments, SHCl and SHC2 all, a three-dimensional an isomer having a molecular formula of C ₂₃ H ₂₈ O _6, SHC1 the Gomisin N (gomisin N), SHC
2 was estimated to be γ-schizandrin. By comparing the results of HPLC analysis of standard forms of gomisin N and γ-schizandrin with the physical and chemical properties and NMR data of gomisin N and γ-schizandrin recorded in the literature, SHC1 is gomisin N and SHC2 Was confirmed to be γ-schizandrin.

Example 2. Measurement of tyrosinase inhibitory activity Gomisin N and γ-schizandrin obtained from Example 1 were diluted by 1/2 from the concentration of 200 ppm to measure tyrosinase inhibitory activity, and a positive control group, kojic acid, arbutin, p-hydroxyl. The activity was compared with anisole. The results are shown in Fig. 1. In Fig. 1, the black triangles indicate kojic acid, the black squares indicate arbutin, the black circles (●) indicate p-hydroxyanisole, the white circles (○) indicate γ-schizandrin, and the white squares (□) indicate gomisin N. As shown in the figure, kojic acid shows an inhibition rate of 50% or more at a concentration of about 10 ppm,
Arbutin showed a 50% or more inhibition rate at a concentration of 25 ppm and p-hydroxyanisole at a concentration of 50 ppm, but gomisin N and γ-schizandrin showed a weak activity at a concentration of 200 ppm or more.

Example 3. Measurement of Antioxidant Activity Gomicin N and γ-schizandrin separated from Example 1 were added to reaction solutions containing 100 μg each of linolenic acid, and antioxidant activity was measured 24 hours later. As a control group, an experiment was performed using ascorbic acid, α-tocopherol, and p-hydroxyanisole. The results are shown in Fig. 2. In FIG. 2, A is ascorbic acid, B is α-tocopherol, C is p-hydroxyanisole, D is gomisin N, and E is γ-schizandrin. Experimental results show 72% ascorbic acid after 24 hours
The antioxidative activity of α-tocopherol and p-hydroxyanisole was 41% respectively.
, 38%, but gomisin N and γ-schizandrin showed 24% and 22%, respectively.

Example 4. Streptomyces bikiniensis
Of Melanin Synthesis Inhibitory Activity Against Streptomyces bikinie of Gomisin N and γ-Schizandrin Isolated from Example 1
The melanin synthesis inhibitory activity against nsis was measured, and the results are shown in Table 2. As a result, as shown in Table 2, γ-schizandrin was used as a potent melanin production inhibitor in comparison with p-hydroxyanizole, which has been clinically tested in skin cancer patients.
Although the activity was slightly weaker at a high concentration of μg or more, it showed almost similar activity at a concentration of about 20 μg, and gomisin N had a slightly weaker activity than p-hydroxyanisole and γ-schizandrin, but 12.5 μg The activity was exhibited up to the concentration of.

[0038]

[Table 2]

Example 5. Aspergillin synthesis inhibitory activity PDA medium added to a spore suspension of A. niger was dispensed into a 24-well plate, solidified, and then gomisin N and γ-schizandrin isolated from Example 1 were respectively attached to the surface. 500μ
After stepwise dilution with ethanol from a concentration of g / ml and treatment, spore pigment synthesis was visually observed, and the results are shown in FIG. In FIG. 3, C is 100% ethanol, H is p-hydroxyanisole, G is gomisin N, and S is γ-schizandrin. After culturing at 37 ℃ for 2 days, both gomisin N and γ-schizandrin were 12
It exhibited activity at a concentration of 0 μg / ml, and 60 μg / m in the control group treated with p-hydroxyanisole.
It showed strong activity even at a concentration of l. However, Gomisin N and γ-schizandrin were 500μ
Compared to the inhibition of spore pigment synthesis without antibacterial activity against A. niger even at a concentration of g / ml or higher, p-hydroxyanizole showed that A.niger in the concentration-treated groups of 500 μg / ml and 250 μg / ml. Cytotoxicity was observed.

Example 6. Measurement of melanin synthesis inhibitory activity B16 mouse melanoma cells were treated with gomisin N and γ-schizandrin at 10 μg / ml and 20
The cells were treated at a concentration of μg / ml, cultured, and the cells were centrifuged and visually observed.
At the concentration of g / ml, arbutin showed a stronger activity than that of the 100 μg / ml concentration-treated group used as a positive control group, and no cytotoxicity was observed at a concentration of 100 μg / ml or more. The results are shown in Fig. 4 (SHC1: Gomisin N, SHC
2: γ-schizandrin). The substance separated from Schisandra chinensis was judged to have a strong melanin synthesis inhibitory effect without damaging cells.

Further, when melanin was extracted from cells treated with gomisin N, the amount of melanin produced was measured to calculate the IC ₅₀ , and the value was 10 μg / ml, and the IC ₅₀ value of the arbutin was 300 μg. At / ml, Gomisin N is found to be a powerful whitening compound that inhibits melanin synthesis.

Example 7. Measurement of allergy induction After applying 15 mg each of Gomisin N, 8 mm fin chamber
40 healthy men and women were attached to the front part of the arm for 24 hours and then removed. 2
After a lapse of time, the degree of skin reaction is visually observed and CTFA (The Cosmetic, Toiletry
and Fragrance Association Inc.), the skin irritation degree was calculated by dividing all the judgment values of the subjects and then dividing by the number of subjects.

[0043]

[Table 3]

When the degree of skin irritation was about 3.0, no allergic reaction occurred, but the degree of skin irritation of gomisin N was 2.0.

As described above, the melanin synthesis inhibitory substance and the composition containing the melanin synthesis inhibitory substance according to the present invention have an excellent whitening effect and can reduce cell toxicity.

In addition, the melanin synthesis inhibitory substance according to the present invention and the composition containing the melanin synthesis inhibitory substance can be used for cosmetics, medicated whitening agents, quasi-drugs, food brown spot inhibitors and the like. effective.

[Brief description of drawings]

FIG. 1 is a graph showing the inhibitory effect of tyrosinase activity of gomisin and γ-schizandrin according to the present invention.

[Fig. 2]   5 is a graph showing the antioxidant activity of gomisin N and γ-schizandrin.

FIG. 3 is a photograph showing aspergillin synthesis inhibitory activity of gomisin N and γ-schizandrin.

FIG. 4 is a photograph showing the inhibitory activity of gomisin N and γ-schizandrin on the in vitro cell culture synthesis.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 17/00 A61P 17/00 C07D 317/70 C07D 317/70 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM , AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP , KE, KG, KP, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Jun, Hyun, Oh Republic of Korea , Taejong 305-301, Yousung-ku, Bonmung-dong 161-5 (72) Inventor Lee, Chang, Ho South Korea, Taejong 302-181, Souk, Naedon 220-2, Lotte apartment 106- 1201 (72) Inventor Kim, Yoon, Ho, Taejeong, Republic of Korea 30 5-390, Yousung-ku, Junmin-dong, Choengnare Apartment 108-202 (72) Inventor Hong, Seung, Sue South Korea, Daejeong 305-390, Yousung-ku, Junmin-dong 462-2, Choungnare Apartment 109- 404 (72) Inventor Lee, Hyun-soo Korea, Seoul 137-040, Seochok, Banpo Dong 550-18, Jewo House 101 F Term (Reference) 4C083 AA111 CC01 EE16 4C086 AA01 AA02 BA13 MA01 MA04 NA14 ZA89 ZC02 4C088 AB65 BA08 CA14 NA14 ZA89 ZC02

Claims (8)

[Claims] 1. A melanin synthesis inhibitory composition containing gomisin N or γ-schizandrin. 2. The composition according to claim 1, wherein the composition is a whitening agent. 3. The composition of claim 2, wherein the composition is cosmetic. 4. The composition of claim 2, wherein the composition is a pharmaceutical whitening agent. 5. The composition according to claim 2, wherein the composition is a food browning agent. 6. The composition according to claim 1, wherein the composition is derived from Schisandra chinensis. 7. Gomishin containing Gomiza extract and in an amount sufficient for a whitening effect
A composition containing N, which has a whitening effect. 8. A gomisin N-containing composition produced by a step of obtaining a fraction from a garlic extract by column chromatography, and collecting the gomisin N-containing fraction from the obtained fraction.