KR19990024810A - Antifungal composition containing catechin and method for preparing catechin compound - Google Patents

Abstract

The present invention relates to novel uses of catechin compounds having the general formula (1) as antifungal agents. These catechin compounds show antifungal activity by inhibiting the activity of chitin synthase II. The present invention also relates to a process for producing new catechins, wherein the compound is obtained from bark of yew, a new material.

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Description

Antifungal composition containing catechin and method for preparing catechin compound

The present invention relates to novel uses of catechins as antifungal agents. The present invention also relates to a novel preparation for preparing the compound from the bark of yeast which is a new source.

Recently, visceral mycosis is increasing due to opportunistic infection caused by AIDS, long-term use of immunosuppressants or anticancer drugs after organ transplantation. Research on cell wall and cell membrane synthase inhibitors of fungi as antifungal agents has been continued for a long time, but there are only a limited number of compounds that are effective as antifungal agents. Moreover, the need for new antifungal agents has long been emerging due to the high toxicity and side effects. .

Conventionally, antifungal agents such as polyenes, azoles, and arylmethyl compounds have been developed and used as antifungal agents acting on cell membranes of fungi, but antifungal agents acting on cell membranes have high toxicity due to low fungal selectivity and serious side effects.

Therefore, fungi have been studied for a long time as a selective target for the development of antifungal agents having higher fungal selectivity, taking into consideration that cell walls exist unlike mammalian cells. The main components of fungal cell walls consist of the polysaccharides chitin and glucan (D. J. Frost et al., J. Antibiot., 48, 306-310 (1995)). Chitin is a homopolymer in which N-acetyl-D-glucosamine is composed of β-1,4 bonds and is synthesized by chitin synthase I, II and III (WJ Choil et al., Proc. Natl. Acad. Sci. , 90, 80-94 (1994)). As the properties and functions of these chitin synthase are known, chitin synthase II is involved in the formation of the chitin synthase and chitin synthetase III is involved in the formation of septa by developing strains that selectively produce each enzyme using molecular biology techniques. The search for compounds that inhibit the progress is ongoing. So far, polyoxins and nicomycin have been reported as chitin synthase inhibitors (J. Nagatsu et al., Agric. Biol. Chem., 29, 848-854 (1965) and HP Friedler et al., J. Chem, respectively). Tech.Biotechnol., 32, 271-280 (1982).

The inventors of the present invention, while searching for a new chitin synthase inhibitor, found that a known substance, catechin, has an activity of inhibiting chitin synthase II, thus completing the invention of a new use of these compounds as an antifungal agent. In addition, it has been found that the compound can be obtained from a yeast that is not known to contain the compound in the past, thereby completing a new manufacturing method for producing the compound from the yew.

Catechin is a known compound (Merch Index 10th Edition, page 266, pp. 1884), conventionally antihypertensive effect (JS Choi, J. Nat. Prod., 54, 218-224 (1991)), antioxidant activity ( H. Mangiapane et al., Biochem. Pharm., 43, 445-450 (1992) and A. Pannala et al., BBRC, 232, 164-168 (1997)), antimutagenic activity (DK Monteith, Mutation Research, 240, 151-158 (1990)) and anti-cancer promoting effects (N. Matsumoto et al., Jpn. J. Cancer Res., 87, 1034-1038 (1996)), etc. There has been no report of antifungal activity as an inhibitor.

It is an object of the present invention to provide new antifungal compositions which inhibit the activity of chitin synthase II involved in the chitin synthesis of fungi.

Another object of the present invention is to provide a method for preparing a catechin compound from a new source.

1 shows the ¹ H-nuclear magnetic resonance (NMR) spectrum of catechin.

Figure 2 shows the ¹ H-NMR spectrum of epicatechin (epicatechin).

3 shows the ¹³ C-NMR spectrum of catechin.

The present invention achieves the above object by providing an antifungal composition comprising a catechin compound having the formula (1) and a pharmaceutically acceptable carrier.

Formula 1

Where Or to be.

The present invention also achieves the above object by providing a process for preparing the catechin compound of formula (1) from bark of attention, a novel source that is not known as a source of catechin in the prior art.

Hereinafter, the present invention will be described in more detail.

A known compound, catechin, is based on UDP- [ ¹⁴ C] GlcNAc (N-acetyl-D-glucosamine) as a substrate (WJ Choi et al., Anal. Biochem., 219, 368-372 (1994)). As a result of testing the inhibitory activity of chitin synthase II, the compounds showed stronger inhibitory activity than conventional polyoxins.

Catechin compounds show the inhibitory activity of chitin synthase II, an enzyme essential for the formation of fungal cell membranes. For example, the catechin of the trans form having the formula (2) has an IC ₅₀ value of 39 μg / ml, and the catechin, also called epicatechin with the formula (3), whose cis type has a IC ₅₀ value of 44 μg / ml. They inhibit the activity of chitin synthase II and can therefore be used in pharmaceutical compositions as antifungal agents.

Such pharmaceutical compositions may include pharmaceutically acceptable excipients, carriers, diluents, etc. together with catechin as the active ingredient, and the formulations of the compositions may be prepared according to known methods using readily available ingredients. have. In preparation of the formulation, the active ingredient can be mixed with the carrier, diluted with the carrier, or contained in a carrier in the form of a capsule, sachet, paper or other container. If the carrier serves as a diluent, it may be a solid, semisolid or liquid substance which acts as a vehicle, excipient or medium for the active ingredient. Thus, the formulations may be tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (in solid or in liquid media), soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders, etc. It may be in the form of.

Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, Cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may further comprise lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components. Compositions of the present invention can be formulated using techniques known in the art to release the active ingredient rapidly, continuously or delayed after administration to a patient.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and may be administered at least once a day to be an active ingredient of 10 to 15 mg / kg body weight, preferably 12 to 13 mg / kg body weight. Dosage may be appropriately adjusted depending on the conditions to be treated, including the condition to be treated, the route of administration, the age, sex, weight, health condition, diet, time of administration, rate of excretion, drug mixture and severity of the disease, The dosage range does not limit the scope of the invention in any aspect. The catechin compound may be used as an antifungal agent by formulating into a unit dosage form or a multiple dosage form such as tablets, capsules, powders, granules, suspensions, emulsions or parenteral preparations by conventional methods.

The catechin compounds can be obtained according to known methods as known materials, but the inventors have completed a new method for obtaining the compounds from the bark of yew, a new source.

Hereinafter, the method of manufacturing a catechin compound from the bark of attention is demonstrated.

The dried bark of interest is added with an organic solvent such as methanol, chloroform or ethyl acetate to extract an organic solvent layer containing the active substance, and then partially purified by reverse phase chromatography to obtain an active substance. At this time, LiChroprep RP-18 (Merck) may be used as the reverse phase filler, and a mixed solution of methanol and water (1: 1) may be used as the developing solvent. The obtained product is then purified by silica gel column chromatography. At this time, a solvent in which methylene chloride and methanol are mixed in a ratio of 80:20 to 90:10, preferably 85:15 can be used as the elution solvent.

The active fraction obtained above is then subjected to high performance liquid chromatography to purify pure catechin compound. In this case, C18 may be used as a filler, and a solvent in which methanol and water are mixed at a ratio of 2: 8 to 4: 6, preferably 3: 7 may be used as the developing solvent.

According to the present invention, catechin of formula 2 and epicatechin of formula 3 can be obtained in yields of 30 mg and 50 mg, respectively, from 1 kg of bark of yeast.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 Isolation and Purification of a Kitechin Compound

5 kg of completely dried bark of bark was extracted twice with 20 L of 95% methanol twice for 5 days, and the filtrates were collected and concentrated under reduced pressure to obtain an extract. The fraction dissolved in methanol was added to a column packed with C18, and the active fraction was partially purified by reversed phase chromatography using a mixed solution of methanol and water (1: 1) at a flow rate of 3.5 ml / min as a developing solvent. Purified. Subsequently, the obtained active fraction was adsorbed on a column filled with silica gel (Merch, Art No. 9385?), And silica gel column chromatography was performed with an elution solvent mixed with methylene chloride and methanol in a ratio of 85:15 as a developing solvent. The active fraction was separated.

Finally, HPLC was performed using an eluent mixed with methanol and water at a ratio of 3: 7 using Phenomenex Ultracarb 10 ODS (250 x 21.2 mm), which was finally filled with C18. Eggplants were purified. Finally, pure compounds catechin and epicatechin were obtained in yields of 30 mg and 50 mg, per kg of bark of yeast, respectively.

Example 2 Structure Analysis of Active Inhibitors

The activity inhibitor of chitin synthase II isolated and purified in Example 1 was analyzed to determine the structure as follows.

Ultraviolet (UV) absorbance was measured using an ultraviolet-visible spectrometer (Shimadzu, UV-265), and infrared (IR) absorbance was measured using a ratio-recording infrared spectrometer (Bio-Rad Digilab Division, FTS-80). The molecular weight was determined using a VG70-SEQ mass spectrometer to determine the molecular weight and molecular formula by measuring HRCI (High resolution MS. Also, ¹ H, ¹³ C using a nuclear magnetic resonance (Varian 300 MHz, Brucker 400 MHz NMR) , Cozy, HMQC, HMBC, NOESY spectra were obtained, and these spectra were synthesized and analyzed to determine their structure.

Catechin Epicatechin Appearance Brown powder Brown powder Molecular formula C ₁₅ H ₁₄ O ₆ C ₁₅ H ₁₄ O ₆ Molecular Weight 290 290 Melting Point (℃) 210 240 Availability MeOH, Acetone, DMSO MeOH, Acetone, DMSO Insoluble Nucleic acid, chloroform Nucleic acid, chloroform

Example 3 Measurement of Inhibitory Activity of Chitin Synthetase II

Determination of the inhibitory activity of chitin synthase II was carried out using UDP- [ ¹⁴ C] GlcNAc (N-acetyl-D-glucosamine) (400,000 cpm / μmol) as a substrate (Won-Ja Choi et al. , Anal.Biochem., 219, 368-372 (1994)). 32 mM Tris-HCl (pH 8.0), 1.6 mM cobalt acetate, 1.1 mM UDP- [ ¹⁴ C] GlcNAc, 20 μl of chitin synthase II coenzyme solution, 14 μl of sample to be tested, 2 μl of trypsin ) (2 mg / mL) was added and pre-reacted at 30 ° C. for 15 minutes.

2 μl of trypsin inhibitor (4 mg / mL) was added to the reaction solution, which was allowed to stand on ice for 10 minutes, followed by 3 μl of 0.8 M N-acetylglucosamine to add a total volume of 50 μl. After reacting at 30 ° C. for 90 minutes. 1 mL of 10% trichloroacetic acid was added to the reaction solution to stop the reaction, followed by filtration using GF / C filter paper. Subsequently, the filter paper was dried, and then 3 mL of cocktail was added to measure radioactivity using a liquid scintillation couter. The inhibitory activity of chitin synthase II was calculated as follows.

At this time, the blank sample was reacted without adding the sample and chitin synthase II, and the control sample was reacted without adding the sample to the reaction solution.

The results of measuring the inhibitory activity (IC ₅₀ ) of inhibiting the activity of chitin synthase II by 50% by catechin and epicatechin in the same manner are shown in Table 2 below. Here, as a comparative group, polyoxin D (E. Cabib, Antimicrob. Agents Chemother., 35, 170-173 (1991)), which is known as an inhibitor of the conventional chitin synthase II, was used. It can be seen that catechin and epicatechin are 1.6-1.8 fold stronger in inhibiting chitin synthase II.

compound IC ₅₀ (μg / ml) Catechin 39 Epicatechin 44 Polyoxins 70

As such, catechin compounds can be used as antifungal agents, respectively, by inhibiting the activity of chitin synthase II, which can be prepared from a new source of bark of yew.

Claims (2)

An antifungal composition comprising the catechin compound of Formula 1 and a pharmaceutically acceptable carrier. Formula 1 Where Or to be. A method of preparing a catechin compound represented by the following Chemical Formula 1, comprising extracting a dried bark of interest with an organic solvent and performing reverse phase chromatography, silica gel chromatography, and high performance liquid chromatography (HPLC). Formula 1 Where Or to be.