KR0154497B1 - Process for preparation of melanin formation inhibiting compound having tyrosinase activity - Google Patents

Abstract

The present invention is to cultivate trichoderma species MR-304 strain (KCTC 0123BP), remove the cells from the culture, and then separate the MR-304-1 compound represented by the following structural formula (I) from the filtrate It is related with the manufacturing method of MR-304-1 compound containing.

The compound has an action of inhibiting tyrosinase activity and melanin production.

Description

Method for preparing tyrosinase activity and melanin inhibitor

The present invention provides a compound MR-304-1 (methyl 3- (1,5-dihydroxy-3-isocyanocyclopent-3-enyl) prop-2 having a tyrosinase activity and an inhibitor of melanogenesis. -Enoate) and a tyrosinase activity and melanin production inhibitor comprising the same. More specifically, compound MR- which can inhibit melanin production by culturing Trichoderma MR-304 strain (KCTC O123BP) and inhibiting the activity of tyrosinase, an enzyme involved in melanogenesis. A method for producing 304-1 and a tyrosinase activity and melanogenesis inhibitor composition containing the same compound.

Melanin is converted from tyrosine to dopa, dopaquinone by the action of tyrosinase present in the 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 overproduction of melanin is known to form blemishes, freckles, etc., promote skin aging, and play an important role in inducing skin cancer, recently, development of a drug for the purpose of preventing melanin overproduction has been actively conducted.

As a whitening agent, p-methoxyphenol, hydroquinone, and kojic acid are already used, but they are weak in activity, cause denaturation or lethality of pigment cells, and inactivate cell functions. May cause side effects such as damage. In addition, vitamin C and derivatives thereof are used for the purpose of inhibiting melanin production, but they also have disadvantages of low tyrosinase inhibitory activity. Therefore, there is a need for the development of inhibitors that exhibit tyrosinase inhibitory activity in small amounts and inhibit melanin biosynthesis.

The present inventors searched for a tyrosinase inhibitor and a melanin inhibitor that meet the above objectives from a microbial culture, and as a result, MR-304-1 from a culture medium of the MR-304 strain belonging to the genus Trichoderma To isolate and develop tyrosinase activity and melanin inhibitors containing the same.

MR-304-1 (Methyl 3- (1,5-dihydroxy-3-isocyanocyclopent-3-enyl) prop-2-enoate) is a known substance and is known by Baldwin et al. It has been synthesized (J. Chem. Soc. Commun., 816-817 (1985)) and has been isolated in the form of a complex with a reagent containing rhodium (J. Chem. Soc. Perkin Trans. 1, 1461 -1465 (1991)). However, the inhibitory effect of tyrosinase activity and melanin production of MR-304-1 has not been reported. Accordingly, the present inventors have completed the present invention by discovering that the substance has an action of inhibiting tyrosinase activity and inhibiting melanin production.

Accordingly, an object of the present invention comprises culturing Mr. 304 strain of Trichoderma genus (KCTC 0123BP), removing the cells from the culture, and then separating the MR-304-1 compound of formula (I) from the filtrate. And it provides the manufacturing method of the compound of following structural formula (I).

MR-304-1: Methyl 3- (1,5-dihydroxy-3-isocyanocyclopent-3-enyl) prop-2-enoate

Another object of the present invention is to provide a tyrosinase activity and melanogenesis inhibitor composition comprising an effective amount of the compound Mr-304-1 of the formula (I) or a derivative thereof.

Hereinafter, the present invention will be described in detail.

According to the present invention, the compound MR-304-1 may be isolated from the culture medium after culturing the MR strains of Trichoderma genus, and the microbial characteristics of Mr. 304 strains of Trichoderma spp. same.

Mr. 304 strain of Trichoderma genus of the present invention was deposited with the Korea Institute of Science and Technology Genetic Bank (KCTC) as October 1, 1994 Accession No. KCTC 0123BP. The MR-304 strain, like other fungi, is easily changed in appearance, but the mutant strain (naturally occurring or induced) of MR-304 strain. All of the bacteria of the genus Trichoderma with the productivity of Mr-304-1, even if it is a fusion or genetic recombinant, are included in the scope of the invention.

In order to produce MR-304-1 using the Trichoderma genus MR-304 strain, the strain is cultured in a medium containing a nutrient source normally used by microorganisms. Nutrients include sucrose, lactose and the like as carbon sources, and peptone, corn steep liquor, potassium nitrate and the like as nitrogen sources. If necessary, it may be effective to add magnesium sulfate and other inorganic salts.

As a culture method, a culture method under aerobic conditions, in particular immersion culture method is suitable. The culture is carried out at a temperature range of 26 to 30 ℃.

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

Identification of Mr-304-1 during culturing and purification can be performed as follows based on melanin inhibition and tyrosinase inhibitory activity of Streptomyces bikiniensis NRRL B-1049. First, Streptomyces bikiniensis NRRL B-1049 was prepared in VDYA-agar medium (200 ml of V-8 juice, 2 g of glucose, 2 g of yeast extract, 1 g of calcium carbonate, 20 g of agar, 800 ml of distilled water, pH 7.2). After culturing for 2 weeks to produce spores, a spore suspension is prepared with sterile water.

ISP No. 7 medium with 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, 20 g of agar) ) By applying 0.2 ml of a spore suspension to each plate, and then laminating a paper plate moistened with a sample on the surface of the medium and incubating at 28 ° C. for 48 hours to determine the inhibition of melanin production.

In addition, in order to confirm the tyrosinase inhibitory activity, the sample was placed in a microplate, 0.1M phosphate buffer (pH 6.5) and 1.5mM L-tyrosine solution, and then the tyrosinase solution was added to the microplate lye. The absorbance was measured at 490 mm using a microplate reader, the plate was reacted at 37 ° C. for 10 minutes, and the absorbance was measured at 490 mm again to calculate the inhibition rate (%) against tyrosinase. It is determined by the concentration of inhibitor with an inhibition rate of 50%.

The inhibition of melanin biosynthesis of B16 melanoma cells (b-16 melanoma cells) is determined as follows. 5 x 10 B16 melanoma cells Cells / mL concentration Roll in suspension containing 10% fetal calf serum (Dulbescco's Modified Eagle's Medium). Suspension cells are placed in a tissue culture flask and incubated at 37 ° C. under 5% CO-95% air conditions. After 4 days of incubation, the cells are washed with phosphate buffered saline (PBS) and trypsinized to remove cells attached to the bottom. The cells are collected in a tube, washed with PBS, and a minimum inhibitory concentration is obtained.

On the other hand, derivatives having the same or similar tyrosinase inhibitory holiness and melanogenesis inhibitory activity as those of MR-304-1 can also be prepared by known chemical synthesis methods. 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. Such derivatives include Trichoderma sp. MR-304 strain or its mutants, transfusions, or genetic recombinants. It can also be obtained through the culture of.

The tyrosinase activity and melanin production inhibitor of the present invention can be used in medicines, quasi-drugs, cosmetics, etc., and can be prepared to contain an effective amount of Mr-304-1 or a derivative thereof and a pharmaceutically useful carrier, and its applied amount Silver may vary depending on the dosage form used and the like. MR-304-1 has better tyrosinase activity inhibition and melanin production inhibition activity than conventional inhibitors even at low concentrations.

In addition, acute toxicity test results of isonitrile antibiotics (Rujiwara et al., Agrc, Bool. Chem., 46 (7), 1803-1809 (1982)) having the same or similar structure as the inhibitor of the present invention, intraperitoneal injection into rats It is expected that the tyrosinase activity and melanogenesis inhibitor of the present invention will show similar toxicity results from the fact that the city LD is about 20 to 300 mg / kg.

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

Production of MR-304-1

Step 1) Culture

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 seed culture, seed media dispensed in two 500 ml Erlenmeyer flasks each 100 ml were sterilized at 121 ° C. for 20 minutes, and the tetrahedral 1-2 platinum strains of Trichoderma Mr-304 strain (KCTC 0123BP) were added thereto. Inoculated and shaken at 25 ° C. for 2 days. 150 ml of the seed culture solution was inoculated into a 5 petri incubator containing a 3 liter production medium sterilized at 121 ° C. for 1 hour, and the resultant was incubated at 25 ° C. for 6 days at 300 rpm stirring speed.

Step 2) Separation and Purification

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

The concentrate was dissolved in a small amount of 30% methanol and then subjected to gel chromatography (eluate 30% methanol) on a Sephadex LH-20 column (Pharmacia, Sweden) to collect the active fractions. The collected fractions were subjected to high pressure liquid chromatography (column: YMC-Pack ODS-AM (YMC Co. Japan, 250 X 10mmI.D.S-5 μm, 120A), flow rate: 1.5ml / min, 220nm detection, eluent 60% Acetonitrile) and MR-304-1 was purified purely near the residence time of 13 minutes.

The solvent was removed with a reduced pressure dryer and then lyophilized to obtain 0.4 mg of MR-304-1 as a brown powder.

Example 2

Structure Determination of MR-304-1

NMR spectroscopy (Varian UNITY 300) was used for structural analysis of the compounds of the present invention, and NMR spectra were measured using CDOD as a solvent. The molecular weight was determined by ESI-MS (electrospray ionization mass spectroscopy, VG Quattro 400 mass spectrometer), UV spectrum (Shimazu UV-260) was measured using methanol as a solvent.

As a result, the physicochemical characteristics of the active substance MR-304-1 produced by the Trichoderma species MR-304 strain were shown in Table 2 below.

MR-3-4-1 was brown powder and had a maximum absorption peak at 211 nm in the ultraviolet region when methanol was used as a solvent. The ESI-MS analysis resulted in (M + na) at 232 m / z due to the charge of isocyanide. ^{A +} peak was observed, confirming the molecular weight 209. In addition, as a result of structural analysis by NMR, the molecular formula was determined to be C10H11NO4, and the structure of MR-304-1 was the same as that of Formula (Ⅰ) as a result of confirming the structure by HMBC (heteronucler multiple bond coherence) and NOE (nuclear overhauser effect). Could know.

Example 3

Determination of Tyrosinase Inhibitory Activity of MR-304-1

15 μl of the sample was placed in each hole of a 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 (tyrosinase, Sigma). 7 μl was added and the absorbance was measured at 490 nm using a Microplate reader (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 ₅₀ value of the inhibitor was 50% of the enzyme activity inhibition rate. Determined by concentration.

A: Absorbance before reaction of the sample containing the inhibitor

B: absorbance after reaction of the sample with inhibitor

C: Absorbance before reaction of sample without inhibitor

D: Absorbance after reaction of sample without inhibitor

IC ₅₀ values of MR-304-1, kojic acid, and arbutin as samples were shown in Table 3 below.

Example 4

Measurement of Melanin Inhibitory Activity of MR-304-1

First, streptomyces Bikinisis NRRL B-1049 was incubated for 2 weeks in VDYA-agar medium (V-8 wntm 200ml, glucose 2g, yeast extract 2g, calcium carbonate 1g, agar 20g, distilled water 800ml, pH 7.2) for 2 weeks. After the production of the spore suspension was prepared in sterile water. ISP No. 7 medium with 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, 20 g of agar) 0.2 ml each of the spore suspension was applied to the plate), and a paper disc (0.8 cm) moistened with the sample was placed on the surface of the medium, and then cultured at 28 ° C. for 48 hours. The results of investigating melanogenesis inhibitory activity using MR-304-1, kojic acid and hydroquinone as samples were shown in Table 4 below.

Example 5 Effect of MR-304-1 on Melanin Biosynthesis Inhibition of Melanoma Cells

B16 melanoma cells were suspended in medium containing 10% fetal bovine serum (Dulbescco's Modified Eagle's Medium) at a concentration of 5 × 10 ³ cells / ml. Suspended cells were placed in a tissue culture flask and incubated at 37 ° C. under 5% CO ₂ -95% air conditions. After 4 days of incubation, the cells were washed with phosphate buffered saline (PBS) and trypsinized to remove cells adhered to the bottom. Cells were collected in tubes and washed with PBS to obtain the minimum inhibitory concentration (MIC). The results of investigating MIC values using MR-304-1, kojic acid, and arbutin as samples were shown in Table 5 below.

As can be seen in the above examples, MR-304-1 exhibits excellent tyrosinase inhibitory activity and melanin production inhibitory activity even at low concentrations.

Although the present invention has been described in detail by the above-described feature embodiments, it will be apparent to those skilled in the art that many modifications and substitutions are possible, especially 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 (2)

MR-, comprising culturing Mr-304 strain of Trichoderma genus (KCTC 0123BP), removing the cells from the culture, and then separating the MR-304-1 compound of formula (I) from the filtrate 304-1 Preparation of Compounds. A tyrosinase activity and melanogenesis inhibitory composition comprising an effective amount of the compound MR-304-1 of the formula (I) or a derivative thereof as defined in claim 1.