KR100197865B1 - Inhibitors against tyrosinase activity and melanin formation - Google Patents

Abstract

The present invention provides a method for preparing MR-93C, MR-93D and MR-93E compounds represented by the following structural formulas (I), (II) and (III) and MR-93C, MIR-93D or MR-93E, or derivatives thereof It relates to a tyrosinase activity and delanin production inhibitor comprising as an active ingredient.

Description

Tyrosinase Activity and Melanin Inhibitors

The present invention relates to inhibitors of tyrosinase activity and melanogenesis and inhibitors of melanogenesis and methods for preparing the same. More specifically, tyrosinase activity and melanin containing a compound MR-93C, MR-93D or MR-93E that can inhibit the activity of melanin by inhibiting the activity of tyrosinase, an enzyme involved in melanogenesis. It relates to a method of producing MR-93C, MR-93D and MR-93E by culturing production inhibitors and Trichoderma microorganisms.

Melanin is converted from tyrosine to dopa, dopaquinone by the action of tyrosinase present in pigment cells, and is produced via dopachrome. This melanin is present in the skin and has an important function of protecting the body from ultraviolet rays. However, since the excessive production of melanin is known to play a significant role in the formation of blemishes, freckles, and the like, promote skin aging, and induce skin cancer, the development of drugs for the purpose of preventing melanin overproduction has been actively progressed in recent years.

As a whitening agent, p-metoxyphenol, hydroquinone, kojic acid or arbutin are already used, but they are weak in activity or cause denaturation or lethality of pigment cells. It can also cause side effects, such as impairing the cell's original function. On the other hand, vitamin C and derivatives thereof are used for the purpose of inhibiting melanin production, but they also have the disadvantage of low tyrosinase inhibitory activity. Therefore, there is a need for the development of inhibitors that exhibit tyrosinase inhibitory activity in a small amount and inhibit melanin biosynthesis.

The present inventors solved the problems of existing melanogenesis inhibitors, and continued research to develop new highly active tyrosinase activity and melanogenesis inhibitors, MR-93 belonging to trichoderma genus fungus Three kinds of inhibitors, that is, MR-93C, MR-93D, and MR-93E, were isolated from the culture medium of the strains and developed tyrosinase activity and melanogenesis inhibitors including them.

MR-93C (1- (1,4,5-trihydroxy-2-isocyanopent-2-enyl) ethanol) and MR-93D (4-hydroxy-8-isocyano-1-oxaspiro [4.4] Cyclonon-8-en-2-one) has been separated from the complex state with a rhodium-containing reagent by Boyd et al. (J. Chem. Soc). Perkin Trans. 1, 1461-1465 (1991). MR-93E is also a known compound produced by Trichoderma hamatum (Can, J. Microbiol. 28, 1252-1260 (1982)), and its synthesis is also known (J. Chem. Soc. Chem. Commun., 816-817 (1985). However, since MR-93C and MR-93D were separated into complexes rather than in pure form, and there were no reports on the inhibition of tyrosinase activity and the melanin production activity of the above substances, the tyrosinase activity of the above substances. The present invention has been completed by revealing the inhibitory and melanogenesis inhibitory activity.

It is an object of the present invention to provide novel tyrosinase activity and melanogenesis inhibitors containing compounds MR-93C, MR-93D or MR-93E which exhibit tyrosinase activity inhibition and melanin production inhibition activity.

Another object of the present invention is to cultivate a microorganism capable of producing the inhibitory compound, and to separate the compounds showing inhibitory activity of tyrosinase activity and melanogenesis from the culture medium, tyrosinase activity and melanin inhibitory compound It is to provide a way to produce.

Hereinafter, the present invention will be described in detail.

The present invention relates to tyrosinase activity and melanogenesis inhibitors containing a compound of formula (I), (II) or (III).

MR-93C: 1- (1,4,5-trihydroxy-2-isocyanopent-2-enyl) ethanol

MR-93D: 4-hydroxy-8-isocyano-1-oxaspiro [4.4] cyclonon-8-en-2-one

MR-93E: 3- (3-isocyanocyclopentene-1-ylidene) propanoic acid

The compounds MR-93C, MR-93D, and MR-93E may be isolated from the culture medium after culturing the Trichoderma sp. MR-93 strain, and the microbial properties of the Trichoderma sp. MR-93 strain are shown in Table 1 below. .

Trichoderma spp. MR-93 strain used to prepare the MR-93C, MR-93D and MR-93E compounds of the present invention was deposited on June 9, 1994 to the Genetic Bank of Korea Institute of Science and Technology (KCTC). Deposited as No. KCTC 0114BP.

When trying to produce MR-93C, MR-93D and MR-93E using Trichoderma spp. MR-93 strain, the strain is usually cultured in a medium containing nutrients used by microorganisms. Nutrient sources include sucrose and lactose as carbon sources, and peptone, cornsteep liquor and potassium nitrate as nitrogen sources. Depending on other needs, magnesium sulfate and other inorganic salts may be added.

As the culture method, a culture method under aerobic conditions, in particular immersion culture method is suitable, the culture is carried out at 26-30 ℃, preferably 26 ℃.

After the incubation is completed, the culture medium is filtered to remove the cells, and the filtrate may be purified by a process such as adsorption chromatography, ethyl acetate extraction, gel filtration chromatography, high pressure liquid chromatography.

Identification of MR-93C, MR-93D, and MR-93E during and during the culturing process was based on melanogenesis inhibition and tyrosinase inhibitory activity of Streptomyces bikiniensis NRRL B-1049. Do it together. First, streptomyces Bikinisis NRRL B-1049 was incubated for 2 weeks in VDYA-agar medium (V-8 juice 200ml, glucose 2g, yeast extract 2g, calcium carbonate 1g, agar 20g, distilled water 800ml, pH 7.2) for 2 weeks. The spore suspension is then prepared with sterile water. Suspension suspension in ISP No.7 medium (15g of glycerol, 0.5g of tyrosine, 1g of asparagine, 0.5g of potassium phosphate, 0.5g of sodium chloride, 0.01g of iron sulfate, 1L of distilled water, 1ml of microsaline solution, 20g of agar) to which yeast extract was added After application, the paper plate soaked with the sample on the surface of the medium, and after 48 hours incubation at 28 ℃ by comparing the size of the resulting ring can be determined melanin production inhibition.

In addition, in order to confirm the tyrosinase inhibitory activity, the sample was placed in a microplate, 01M phosphate buffer (pH 6.5) and 1.5 mM L-tyrosine solution were added, and then the tyrosinase enzyme solution was added to the microplate. The absorbance was measured at 490 nm using a microplate reader, the plate was reacted at 37 ° C. for 10 minutes, and the absorbance was measured at 490 nm again to calculate the inhibition rate (%) against tyrosinase. It is determined by the concentration of inhibitor with an inhibition rate of 50%.

On the other hand, derivatives having the same or similar tyrosinase inhibitory activity and melanogenesis inhibitory activity as MR-93C and MR-93D can be prepared by known chemical synthesis methods, and the production method or structure thereof can be changed. It is obvious to those skilled in the art and when the effects as inhibitors are similar, they are also included in the scope of the present invention, and the production of such derivatives is Trichoderma sp. MR-93 strain or its mutants, fusions or It may also be obtained by culturing gene recombinants.

Tyrosinase activity inhibitors and melanin production inhibitors used in the present invention can be used in medicines, quasi-drugs, cosmetics, etc., the application amount may vary depending on the control used. At low concentrations of MR-93C, MR-93D and MR-93E, the inhibition of tyrosinase activity and the inhibition of melanogenesis were better than that of conventional inhibitors.

In addition, acute toxicity test results of isonitrile antibiotics (Fujiwara et al., Agric. Biol. Chem. 46 (7). 1803-1809 (1982)) having the same or similar structure as the inhibitors of the present invention, It is expected that the tyrosinase activity and melanogenesis inhibitor of the present invention will show similar acute toxicity results from the fact that the LD is about 20-300 mg / kg upon intravenous injection.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Example 1

Fermentation production

As the medium and production medium, sucrose 2%, lactose 1%, peptone 0.3%, sodium chloride 0.5%, corn steep liquor 0.3%, potassium nitrate 0.23%, magnesium sulfate 0.05%, inorganic salt solution 0.1% Containing medium was used. As a species culture, seed cultures which were divided into 200 ml of 5 1 l triangular flasks each were sterilized at 121 ° C for 20 minutes, and the tetrahedral 1-2 platinum of Trichoderma sp. MR-93 strain (KCTC 0114BP) was added thereto. After inoculation was incubated for 2 days at 25 ℃. 1 L of the seed culture solution was inoculated into a 50 L incubator containing 35 L of production medium sterilized at 121 ° C. for 1 hour, and incubated at 25 ° C. for 6 days at a stirring speed of 200 rpm.

Example 2

Isolation and Purification of MR-93C and MR-93D

The culture medium of Example 1 was filtered through a filter paper to remove the cells and culture medium was obtained. 35 liters of the filtrate was passed through a Diaion HP-20 column (Nippon Rensui Co., Japan), and the column was washed with water, eluted with 30% methanol to desorb the bound material, and the eluate was reduced to 500 ml volume. Concentrated. The aqueous solution was extracted three times with the same amount of ethyl acetate, and the ethyl acetate layer was reconcentrated.

This concentrate was dissolved in 4 ml of 50% methanol and subjected to gel filtration chromatography (eluate: 50% methanol) on a Sephadex LH-20 column (Pharmacia, Sweden) to collect the active fractions. The collected fractions were concentrated under reduced pressure and eluted with a 1-100% methanol concentration gradient through an MCI-gel column (Mitsubishi Kasei Corporation, Japan). The eluate was subjected to high pressure liquid chromatography (column: PLRP-S (Phenomenex, 100 μs, 8μ), flow rate: 1.5 ml / min, 220 nm detection, eluent: 10% acetonitrile) to obtain MR-93C at a residence time of about 18 minutes. MR-93D was purely separated around 70 minutes of residence time. The solvent was removed under a reduced pressure dryer, and the residue was freeze-dried to obtain 1 mg of MR-93C and 0.8 mG of MR-93C as brown powders.

Example 3

Isolation and Purification of MR-93E

The culture medium of Example 1 was filtered through a filter paper to remove the cells and culture medium was obtained. 3L of the culture filtrate was passed through a Diaion HP-0 column (Nippon Rensui Co., Japan), the column was washed with water, eluted with 30% methanol to desorb the bound material, and the eluate was reduced to 500 ml volume. Concentrated. This aqueous solution was extracted three times with the same amount of ethyl acetate, and the ethyl acetate layer was concentrated again.

The concentrate was dissolved in 2 ml of 50% methanol and subjected to gel filtration chromatography (eluate: 50% methanol) on a Sephadex LH-20 column (Pharmacia, Sweden) to collect fractions. The collected fractions were subjected to high pressure liquid chromatography (column: PLRP-S (Phenomenex, 100 μs, 8μ), flow rate: 1.5 ml / min, 277 nm detection, eluent: 10% acetonitrile) to obtain MR-93E at a retention time of about 10 minutes. Pure separation. The solvent was removed with a vacuum dryer, and the residue was freeze-dried to obtain 0.8 mg of MR-93E as a brown powder.

Example 4

Structure Determination of MR-93C, MR-93D and MR-93E

NMR spectrum (Varian UNITY 300) was used for structural analysis of the compounds of the present invention, and NMR spectra were measured using DO as a solvent. Molecular weight was determined by ESIMS (electrospray ionization mass spectroscopy, VG Quattro 400 mass spectrometer), UV spectrum (Shimazu UV-260) was measured using distilled water or methanol as a solvent, IR characteristics are FR-IR (Laser Precision Analytical) , IFX-65s).

As a result, the physicochemical characteristics of the active substances MR-93C, MR-93D and MR-93E produced by the Trichoderma species MR-93 strain were as shown in Tables 2, 3 and 4.

For example, MR-93D is a brown powder with a maximum absorption peak at UVτ (HO) max 229nm, and ESIMS analysis shows that (M + Na) at 202m / z due to the charge of isocyanide. The peak was observed and the molecular weight was found to be 179. The IR spectrum showed a specific peak of isocyanide group near 2100 cm and a peak of lactone ring near 1600 cm. In addition, as a result of structural analysis by NMR, the molecular formula was determined as CHNO. Also, H-NMR spectrum, H- H COSY spectrum, 3-CH was identified at 3.10 ppm (dd, J = 6, 18), 2.60 ppm (dd. J = 3, 18), 6-CH at 6.19 ppm (s), and 8-CH. And 9-CH were observed around 2.1-2.8 ppm. Based on the above physicochemical characteristics and structural analysis results, MR-93D is 4-hydroxy isolated from Boyd et al. (J. Chem. Soc. Perkin, Trans. 1, 1461-1465 (1991)). It was identified as -8-isocyano-1-oxaspiro [4.4] cyclonon-8-en-2-one.

Example 5

Tyrosinase Inhibitory Activity of MR-93C, MR-93D and MR-93E

15 μl of the sample was placed in each hole of 96-well microplate, 150 μl of 0.1 M phosphate buffer (pH 6.5) and 25 μl of 1.5 mM L-tyrosine solution were added, followed by enzyme solution (tyrosinease). , 7 μl of sigma) was added, and the absorbance was measured at 490 nm using a microplate reader (manufactured by Biorad). After reacting the plate for 10 minutes at 37 ° C., the absorbance was again measured at 490 nm, and the inhibition rate for tyrosinase (%) was calculated by the following equation, and the IC value was the concentration of the inhibitor whose enzyme activity inhibition rate reached 50%. Determined.

IC ₅₀ values using MR-93C, MR-93D, MR-93E, kojic acid, tropolone, hydroquinone and arbutin as inhibitors were shown in Table 1 below.

Example 6

Determination of Melanin Inhibitory Activity of MR-93C, MR-93D and MR-93E

First, streptomyces Bikinisis NRRL B-1049 was incubated for 2 weeks in VDYA-agar medium (V-8 juice 00ml, glucose 2g, yeast extract 2g, calcium carbonate 1g, agar 20g, distilled water 800ml, pH 7.2) for 2 weeks. After the spore suspension was prepared in sterile water. ISP No. 7 medium containing 0.2% yeast extract (1.5 g of glycerol, 0.5 g of tyrosine, 1 g of asparagine, 0.5 g of potassium phosphate, 0.5 g of sodium chloride, 0.01 g of iron sulfate, 1 l of distilled water, 1 ml of trace salt solution, 20 g of agar) 0.2 ml each of the spore suspension was applied, and a paper disc (0.8 cm) moistened with the sample was placed on the surface of the medium and incubated at 28 ° C. for 48 hours to compare the size of the resulting ring. The results of investigating melanogenesis inhibitory activity using MR-93C, MR-93D, MR-93E, paramethoxyphenol, kojic acid and hydroquinone as samples were shown in Table 2 below.

As can be seen in the above examples, MR-93C, MR-93D, MR-93E show excellent tyrosinase inhibitory activity and melanin production inhibitory activity even at low concentrations.

Although the invention has been described in detail by the specific embodiments above, it will be apparent to those skilled in the art that many modifications and substitutions are possible without departing from the spirit and scope of the invention as defined in the claims. Substitutions are also included within the scope of the present invention.

Claims (1)

Tyrosinase activity and melanogenesis inhibitors comprising a compound represented by the following structural formulas (I), (II) or (III), or derivatives thereof as an active ingredient;