JP4686720B2 - Tyrosinase activity inhibitor and whitening cosmetic containing the same - Google Patents

Description

  The present invention relates to a tyrosinase activity inhibitor, and more particularly to a tyrosinase inhibitor that effectively inhibits tyrosinase related to melanin production and a whitening cosmetic using the same.

  Currently, many whitening cosmetics are on the market for the purpose of preventing sunburn, spots, freckles and the like on the skin. These whitening cosmetics contain components that prevent the production of melanin, which is a pigment substance, and many of them are tyrosinase activity inhibitors that inhibit tyrosinase, which is the rate-limiting enzyme for melanin production.

  As typical tyrosinase inhibitors, kojic acid and arbutin are known. Among these, kojic acid is produced in cultured fermentation products of Aspergillus oryzae, and its tyrosinase inhibitory activity is high. However, there are reports of carcinogenicity by oral ingestion and side effects such as leukocyte depletion, and it is safe. It is hard to say that sex is sufficiently secured. Arbutin is a compound that can be obtained from an extract of walnut and bilberry, and its safety has been almost confirmed. However, there is a problem that its tyrosinase inhibitory activity is weaker than that of kojic acid.

  Accordingly, development of a new tyrosinase activity inhibitor having excellent activity and high safety has been demanded.

  As a result of intensive search to solve the above-mentioned problems, the present inventors have found that a rum that is a waste product after the production of rum has tyrosinase inhibitory activity. As a result of further research, a substance responsible for this tyrosinase inhibitory activity was found and the present invention was completed.

で表される化合物を有効成分とするチロシナーゼ活性阻害剤である。 That is, the present invention provides the following formula (I)

The tyrosinase activity inhibitor which uses the compound represented by these as an active ingredient.

  The present invention is also characterized in that the rum is subjected to a purification treatment combining two or more treatments selected from styrene synthetic adsorbent treatment, silica gel column chromatography treatment, gel filtration treatment and liquid chromatography treatment. It is a manufacturing method of the compound represented by Formula (I).

  Compound (I), which is an active ingredient of the tyrosinase activity inhibitor of the present invention, is a substance having a stronger tyrosinase activity inhibitory action than, for example, widely used arbutin. Moreover, since it is obtained from sugarcane that has been used as a food for a long time, it is also highly safe.

  Moreover, since it can be obtained from the waste of rum, the cost is very low.

  Therefore, the tyrosinase activity inhibitor of the present invention can be used as a whitening component in a skin external preparation, a whitening cosmetic, or the like.

  The compound obtained by the above formula (I) of the present invention is a known compound named Imperanene (4-hydroxy- [2- (4-hydroxy-3-methoxyphenyl) ethenyl] -3-methoxy-benzenpropanol). It is.

  Since the glucose glycoside of imperanene has been separated from brown sugar in the past, the above compound was produced by hydrolysis of the imperanene glycoside derived from sugarcane during the production of rum, and contained in rum Conceivable.

  The above compound (I) (Imperane) is combined with various appropriate column chromatography in consideration of its properties, for example, styrene-based synthetic adsorbent treatment, silica gel column chromatography treatment, gel filtration treatment and liquid chromatography. It can be separated and obtained from the rum by subjecting it to a purification treatment combining two or more treatments selected from the treatments. An example of specific means for obtaining compound (I) from such a rum is as follows.

  First, the rum is centrifuged and the supernatant is obtained as a filtrate. The filtrate is adsorbed on a styrene synthetic adsorbent and then eluted with a suitable solvent. Examples of the styrene-based synthetic adsorbent used include Amberlite XAD type (made by Rohm and Haas) and HP type (made by Mitsubishi Kasei), and elution solvents include ethanol, Mention may be made of lower alcohols such as methanol, acetone or a mixture of this with water.

  Next, from the fractions obtained as described above, a fraction having strong tyrosinase inhibitory activity is selected, adsorbed onto a silica gel column, and then eluted with an appropriate solvent. In this chromatographic treatment, examples of the solvent to be used include mixed solvents such as ethyl acetate-methanol and chloroform-methanol, and these may be used stepwise or gradients with different mixing ratios. preferable.

  Furthermore, among the fractions obtained by the silica gel column treatment, a fraction having strong tyrosinase inhibitory activity is selected, and this is subjected to gel filtration. In this gel filtration, TOYOPEARL HW type (manufactured by Tosoh Corporation), Sephadex LH type (manufactured by Pharmacia), etc. are used, and the solvent is a mixture of lower alcohol such as methanol and water. Is used

  Finally, a fraction having strong tyrosinase inhibitory activity is selected from the fractions obtained by the above gel filtration treatment, and this is treated by liquid chromatography. In this liquid chromatograph, a reverse-phase column such as LiChroprep type (manufactured by Merck) is used, and as a solvent, a mixture of lower alcohol such as methanol and water is used.

In addition, since compound (I) of this invention has absorption in _OD450 vicinity, it can monitor by measuring the light absorbency of an ultraviolet part in each process.

  The tyrosinase activity inhibitor of the present invention obtained as described above can be used by blending with a skin external preparation or a whitening cosmetic composition according to a conventional method.

  For example, in the case of whitening cosmetics, for example, about 0.0001 to 10% by mass is blended with a known cosmetic base, and cosmetics, emulsions, creams, and pastes may be prepared according to conventional methods.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not restrict | limited at all by these Examples.

Reference example 1
Inhibitory Activity Measurement Method Using Human-derived Crude Tyrosinase (1) Preparation of Crude Tyrosinase Solution Human-derived tyrosinase was prepared from a permanent pigment producing strain HMV-II derived from malignant melanoma. That is, 10% fetal bovine serum was added to an equal volume mixed medium (DMEM / Hum F-12) of Dulbecco's modified Eagle's medium and Ham's F12 medium, subcultured and cultured at 37% at 5% carbon dioxide concentration. It was. Cells were seeded in a 90 mm dish at a density of 3 × 10 ⁵ cells, added with 100 μM final concentration of dopa, and cultured for 10 days. On days 5 and 8, the medium was replaced with a medium containing a final concentration of 100 μM dopa.

Cultured cells are collected by trypsin treatment, and 1 ml of PBS (-) containing 1% Triton X-100 is added to 1 × 10 ⁷ cells, and sonication is performed at 4 ° C. for 60 minutes to destroy the cells. did. This cell lysate is centrifuged at 12000 rpm for 30 minutes, 0.05 M phosphate buffer (pH 6.8) is added in an equal amount, and centrifuged at 12000 rpm for 10 minutes, and the supernatant is used as a crude tyrosinase solution. Using.

(2) Tyrosinase inhibitory activity Tyrosinase inhibitory activity was performed using a 96-well microplate. Add 50 μl of the sample solution prepared to each concentration, 40 μl of 1.5 mg / ml l-dopa solution and 100 μl of 0.05 M phosphate buffer (pH 6.8), and after pre-incubation at 37 ° C. for 10 minutes. Then, 50 μl of the crude tyrosinase solution was added and stirred. Thereafter, the reaction was carried out at 37 ° C. for 90 minutes, and the absorbance at 470 nm was measured with a microplate reader.

Example 1
Method for Acquiring Tyrosinase Inhibiting Substance from Rum Sake The rum sake remaining after rum distillation at Helios Sake Brewery Co., Ltd. on November 25, 2004 was used as a raw material. This rum was stored frozen until separation.

  800 ml of the above rum was centrifuged (12,000 rpm, 4 ° C., 20 minutes), and the supernatant was subjected to suction filtration (ADVANTEC No. 5B). The filtrate was passed through a glass column (150 ml) packed with Amberlite XAD-1180, and then 600 ml of purified water, 25% methanol, 50% methanol and 100% methanol were successively flowed.

  The 100% methanol eluate was concentrated by evaporation (yield 1.04 g), passed through a silica gel column (Wakogel C-100, column 300 ml) and adsorbed, and then ethyl acetate-methanol was mixed. Elution was carried out with 600 ml each of eluents with ratios of 10: 0, 7: 3, 5: 5, 3: 7 and 0:10, and each eluate was divided into 5 fractions (Fr1 to Fr5). I drew it.

  Among these, Fr1 (168 mg) having a high tyrosinase inhibitory activity obtained a fraction eluted with 70% methanol by an HLB cartridge (manufactured by Waters), and this was further added to a glass column packed with TOYOPEARL HW-40C ( The solution was passed through and adsorbed through a column size of 1.2 cm × 80 cm, and then eluted with a gradient using 60% methanol (120 ml) and 80% methanol (120 ml). The eluate was fractionated by 3 g, and the eluate was monitored by the absorbance in the ultraviolet region.

  Of these, the peak Fr1-5 (11 mg) eluting from 25 to 27 showed tyrosinase inhibitory activity, and this fraction was further eluted with 40% methanol on a LiChroprep RP-18 column (Waters). Fractionation was performed at 3 g each, and the separation status was monitored by measuring the absorbance in the ultraviolet region. As a result, strong tyrosinase inhibitory activity was observed at the largest peak (Fr1-5-2, 5.2 mg) near 80.

  Fr1-5-2 in which activity was recognized was analyzed by HPLC. The HPLC conditions were Develosil ODS MG-5 (4.6 × 250 mm, Nomura Chemical) for the column, a 30 minute linear gradient from 70% methanol to 100% methanol for the eluent, a flow rate of 0.7 ml / min, and a detection of 300 nm. As a result, it was confirmed that the tyrosinase-inhibiting active substance was obtained as a single peak at an elution time of 6.59 min, and the same fraction was purified as a single component. The yield of the same compound based on rum was calculated as 6.5 ppm.

Example 2
Structural analysis of tyrosinase inhibitory active substance About the tyrosinase inhibitory active substance obtained in Example 1, various two-dimensional NMR spectra such as UV-VIS spectrum, ¹ H-NMR, ¹³ C-NMR, HMQC, HMBC, and electrospray ion ( ESI) The structure was determined by analyzing the mass spectral data. As a result, the separated compound was identified as the known compound imperanene (molecular weight 330, C ₁₉ H ₂₂ O ₅ ).

Example 3
Human Tyrosinase Inhibitory Activity of Isolated Compounds The identified impellerene was measured for human tyrosinase inhibitory activity with arbutin and kojic acid as a comparison. As a result, the ID ₅₀ of impellerene was calculated to be 1.11 mM, which was a weak value compared to 0.35 mM of kojic acid, but an activity stronger than 1.43 mM of arbutin was observed.

  In addition, when the absorbance value when the substrate concentration and the inhibitor concentration were variously changed was graphed and a line Weber-Burk plot was prepared, the obtained three regression lines were collected on the Y axis. This indicates that the mechanism of inhibition of tyrosinase activity of impellerene is inhibition of competition with the substrate dopa.

  The inhibitory activity of human tyrosinase of Compound (I) (Imperane) used in the present invention was stronger than that of arbutin.

Therefore, the tyrosinase-inhibiting active agent of the present invention can be advantageously used as an active ingredient in external preparations for skin and whitening cosmetics.

Claims (5)

The following formula (I)

The tyrosinase activity inhibitor which uses the compound represented by this as an active ingredient.   The tyrosinase inhibitory activator according to claim 1, which is obtained from rum mash.   The rum is obtained by a purification treatment combining two or more treatments selected from a styrene-based synthetic adsorbent treatment, a silica gel column chromatography treatment, a gel filtration treatment and a liquid chromatography treatment. 3. The tyrosinase inhibitory activator according to item 2.   A skin external preparation containing the tyrosinase-inhibiting activator according to any one of claims 1 to 3.   A whitening cosmetic comprising the tyrosinase-inhibiting activator according to any one of claims 1 to 3.