KR0145944B1 - Tyrosinase inhibitor produced by agrocybe cylindracea - Google Patents

Abstract

The present invention is a compound of the following formula Geri-ME006 produced from Agocybe cylindracea, a type of mushrooms, and exhibiting tyrosinase activity and melanin inhibitory activity. The present invention relates to a tyrosinase activity and a melanogenesis inhibitor composition comprising the compound or a derivative thereof as an active ingredient, wherein Geri-ME006 exhibits excellent tyrosinase and melanogenesis inhibitory activity:

Description

Tyrosinase Inhibitors Produced from Agrocybe cylindracea and Methods for Preparing the Same

1 is a hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum of the Geri-ME006 compound,

2 is the carbon nuclear magnetic resonance ( ¹³ C-NMR) spectrum of the Geri-ME006 compound.

The present invention relates to a novel tyrosinase activity inhibitor and a method for preparing the same, and more particularly, to inhibit melanin production by inhibiting tyrosinase activity and produced from a mushroom, Agocybe cylindracea. Indole-based novel compound Geri-ME006, a method for preparing the compound comprising separating and purifying from willow, and a tyrosinase activity and melanogenesis inhibitor composition comprising the compound or a derivative thereof as an active ingredient will be.

Melanin is converted from tyrosine to dopa and dopaquinone by the action of tyrosinase present in pigment cells, and is produced via dopachrome and the like. Melanin is present mainly 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, para-methoxyphenol (p-methoxyphenol), hydroquinone, kojic acid or arbutin is already used, but they are weak in activity or cause degeneration or death of pigment cells. It can also cause side effects, such as impairing the cell's original function. 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 have searched for a tyrosinase activity inhibitor that meets the above objectives, and isolated Geri-ME006, a substance that inhibits tyrosinase activity, from a willow mushroom, which is a kind of mushrooms, to reveal its chemical structure, It has been developed tyrosinase activity and melanin inhibitors containing the same.

It is an object of the present invention to provide novel tyrosinase activity inhibitors.

Another object of the present invention is to provide a method for preparing the inhibitor, including separating and purifying the inhibitor from a willow bunch.

Still another object of the present invention is to provide a tyrosinase activity and melanogenesis inhibitor composition comprising the inhibitor or a derivative thereof as an active ingredient.

In accordance with the above object, in the present invention, a novel compound Geri-ME006 (6-hydroxyindol-3-carboxaldehyde), which exhibits tyrosinase inhibitory activity, and a fruiting body of willow There is provided a method for preparing the compound, including separating and purifying the compound by extraction, chromatography, and the like. The present invention also provides a tyrosinase activity and melanogenesis inhibitor composition comprising the inhibitory compound or a derivative thereof as an active ingredient.

The present invention is described in more detail as follows.

The novel tyrosinase activity inhibitors of the invention are named Geri-ME006 as indole-based compounds having the structure (I):

Geri-ME006: 6-hydroxyindole-3-carboxaldehyde

The compound Geri-ME006 can be obtained by separating and purifying from the fruiting body of the willow bunch, for example, by treating the willow bunch with various solvents and extracting it, and then purifying by silica gel column chromatography and high pressure liquid chromatography. . In the extraction, it is preferable to sequentially use methanol, chloroform, ethyl acetate, butanol, and the like, and the ethyl acetate layer is concentrated and used for subsequent chromatography.

In the process of separating and purifying Geri-ME006, the degree of inhibition of tyrosinase activity was traced. The method of confirming the inhibitory activity was as follows. The sample was placed in a microplate, 0.1M phosphate buffer (pH 6.5) and 1.5mM L-tyrosine solution were added, and then the tyrosinase solution was added to measure absorbance at 490 nm using a microplate reader. The plate was allowed to react at 37 ° C. for 10 minutes, and then absorbance was measured at 490 nm to calculate the inhibition rate (%) against tyrosinase, and the IC ₅₀ value was determined as the concentration of the inhibitor whose enzyme activity inhibition rate reached 50%. Decide

Meanwhile, Geri-ME006 may be prepared by a known chemical synthesis method, and derivatives having the same or similar tyrosinase inhibitory activity and melanogenesis inhibitory activity as Geri-ME006 may be prepared by the same method. Changes in the method or structure will be apparent to those of ordinary skill in the art and, when the effects as inhibitors are similar, they are also within the scope of the present invention, and such derivatives may be obtained through cultivation of willows. There will be.

Tyrosinase activity and melanogenesis inhibitors of the present invention can be used in medicines, quasi-drugs, cosmetics, etc., can be prepared to contain an effective amount of Geri-ME006 or its derivatives and pharmaceutically useful carriers, the amount of the application It may vary depending on the formulation. Geri-ME006 has better inhibition of tyrosinase activity and melanogenesis at lower concentrations than conventional inhibitors.

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

Separation and Purification of Geri-ME006

10 kg of willow pines sold from Daejeon Metropolitan City were cut into 2cm size, extracted with 100% methanol for 24 hours, concentrated under reduced pressure, and extracted sequentially using chloroform, ethyl acetate and butanol. Among them, the ethyl acetate layer was concentrated and dissolved in a small amount of a mixed solvent (chloroform: methanol = 100: 1), followed by adsorption chromatography using a silica gel column (70-230 mesh, Merck) (chloroform: methanol = 100: 1, 50: Eluting with a mixed solvent of 1, 10: 1) to elute the active substance. The concentrated active fraction was dissolved in a small amount of chloroform: methanol = 30: 1 solvent, eluted with a mixed solvent of chloroform: methanol = 30: 1, 20: 1 on a silica gel column (230-400 mesh, Merck), and then the active fraction was separated. Collect high pressure liquid chromatography (column: Senshu pak ODS-5251-SH (Senshu Co. Japan, 20 X 250 mm), flow rate: 9.9 mL / min, 210 nm detection, eluent 27% methanol) to obtain a residence time of about 16 minutes. A new compound, Geri-ME006, was purified purely.

The solvent was removed by concentration under reduced pressure, and then freeze-dried to obtain 3.2 mg of Geri-ME006 as a brown powder.

Example 2

Physicochemical Properties of Geri-ME006

In order to determine the physicochemical properties of the compounds of the present invention, as the solvent dew Trojan methanol (CD ₃ OD) using the hydrogen nuclear magnetic resonance to tetramethylsilane as a reference material by NMR analyzer (Varian UNITY 300) ⁽¹ H- NMR) and carbon magnetic resonance ( ¹³ C-NMR) spectra were measured and the results are shown in FIGS. 1 and 2, respectively. The molecular weight was determined by EI-MS (Hewlett Packard MS Engine 5989A), and the UV spectrum (Kontron Uvicon 930) was measured using methanol as a solvent.

As a result, the physicochemical characteristics of the active substance Geri-ME006 isolated from the willow are shown in Table 1 below.

Geri-ME006 exhibited maximum absorption peaks at 208, 240, 276, and 308 nm in the ultraviolet region when methanol was used as a brown powder and EI-MS analysis showed (M + H) at 162 m / z. The peak was observed and confirmed as molecular weight 161. In addition, as a result of structural analysis by NMR, the molecular formula was determined as CHNO, and the structure of Geri-ME006 was as shown in the following Formula (I).

Example 3

Tyrosinase Inhibition Activity of Geri-ME006

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 Tyrosinase enzyme solution (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 with inhibitor

B: Absorbance after reaction of the sample with inhibitor

C: Absorbance before reaction of sample without inhibitor

D: Absorbance after reaction of the sample without inhibitor

Using the Geri-ME006, kojic acid and arbutin as samples, the effective concentration (IC ₅₀ ) value showing tyrosinase inhibitory activity was investigated. As shown in Table 2, the IC ₅₀ value of Geri-ME006 was 1.5 µl / Ml. This value is much lower than that of kojic acid (10 µl / ml) and arbutin (15.0 µl / ml), which are currently used as tyrosinase inhibitors. The tyrosinase inhibitory activity of Geri-ME006 is higher than that of conventional inhibitors Excellent.

As can be seen in the above examples, Geri-ME006 shows excellent tyrosinase inhibitory activity and melanin production inhibitory activity even at low concentrations.

Although the present 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 particularly 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 (3)

Tyrosinase activity and melanogenesis inhibitors Geri-ME006, derivatives thereof and pharmaceutically acceptable salts thereof represented by the following structural formula (I): A method for preparing a Geri-ME006 compound comprising treating a willow (Agocybe cylindracea) with a solvent to extract the Geri-ME006 compound and then purifying by silica gel column chromatography and high pressure liquid chromatography. A tyrosinase activity and melanogenesis inhibitor composition comprising an effective amount of Geri-ME006 or a derivative thereof as an active ingredient.