KR0145940B1 - Novel antimycotic compound process for the purification thereof and antimycotic composition comprising same - Google Patents

Abstract

The present invention relates to an antifungal substance KRIBB-BP004 of the following structural formula (I) isolated from garlic (Alium sativum L.), a method for separating and purifying it from garlic, and an antifungal composition comprising KRIBB-BP004 as an active substance.

Description

Novel antifungal substances, methods for purification thereof and antifungal compositions comprising the same

1 shows the absorbance analysis spectrum of KRIBB-BP004 by ultraviolet-visible spectroscopy,

Figure 2 shows the absorbance analysis spectrum of KRIBB-BP004 by ratio recording infrared spectroscopy,

3 shows the EI-MS molecular weight analysis spectrum of KRIBB-BP004 by VG70-VSEQ mass spectrometer,

4 shows the 1 H-NMR spectrum at 500 MHz of KRIBB-BP004 by Brooker AMX-500,

5 shows the ¹³ C-NMR spectrum at 125 MHz of KRIBB-BP004 by Brooker AMX-500,

6 shows HOMOCOSY NMR analytical spectrum at 500 MHz of KRIBB-BP004 by Brooker AMX-500,

7 shows the HETEROCOSY NMR analytical spectrum at 500 MHz of KRIBB-BP004 by Brooker AMX-500.

The present invention relates to a novel antifungal substance, a method for purifying the same, and a pharmaceutical composition comprising the same. More specifically, the antifungal substance KRIBB-BP004 isolated from garlic (Alium sativum L.), its purification method and activity from garlic An antifungal composition comprising an effective amount of KRIBB-BP004 as a substance.

Recently, as the residual toxicity of organic synthetic fungicides and their environmental pollution have become a serious social problem, there is a need for the development of low-toxic or pollution-free agricultural fungicides and food and pharmaceutical antifungal agents containing pollution-free antifungal substances isolated from natural resources. It is increasing.

Garlic has been reported in many countries since the late 19th century, and since 1960s, biological research on the treatment of cardiovascular disease and antibacterial and antifungal activity has been actively conducted. In particular, garlic has been used mainly as a dietary supplement in Eastern Europe and Asia, but in Germany, garlic preparations have recently been sold as one of the top prescription drugs. In addition, commercially-made garlic extracts are widely used in the treatment of fungal infections in China, and substances such as allicin, diallyl disulfide, and diallyl trisulfide exhibit antifungal activity. (LD Lawson, ACS Symposium Series 534, Human Medicinal Agents from Plants, Chapt. 21, Bioactive Organosulfur Compounds of Garlic Products, pp 306-330 (1993); and Hunan Medical College, Clin. Med. J., 93, 123-126 (1980)).

Therefore, the present inventors extracted a new antifungal substance KRIBB-BP004 different from the previously reported substance from garlic in the process of searching for the antifungal substance from the plant.

It is an object of the present invention to provide novel antifungal substances.

Another object of the present invention is to provide a method for separating and purifying the antifungal substance from garlic.

Another object of the present invention is to provide a pharmaceutical composition comprising the antifungal substance as an active ingredient.

In accordance with the above object, the present invention provides a novel antifungal substance KRIBB-BP004 extracted from garlic.

According to another object of the present invention, in the present invention KRIBB-BP004 from garlic, comprising the step of extracting the crushed garlic with a mixture of water and acetone and ethyl acetate to obtain a crude extract and purifying the crude extract by chromatography Methods of separation and purification are provided.

According to this still another object, the present invention provides an antifungal composition comprising an effective amount of KRIBB-BP004 as a sulfur component.

The present invention is described in detail as follows.

The novel antifungal substance of the present invention is a compound of formula (I) designated KRIBB-BP004:

A summary of the physicochemical properties of the compound is shown in Table 1 below.

KRIBB-BP004 can be produced by separating and purifying from extracts of crushed garlic, and on the other hand, also by chemical synthesis. To produce KRIBB-BP004 from garlic, the crude extract obtained by sequentially crushing garlic with a mixture of water and acetone and ethyl acetate was subjected to silica gel column chromatography, prep-TLC, and high pressure liquid chromatography (HPLC). ), KRIBB-BP004 is isolated and purified.

Meanwhile, KRIBB-BP004 exhibits antifungal activity, and its antifungal activity can be determined by performing a bioassay on an agar plate using Trichophyton mentagraphytes as a test bacterium. As reference material, KRIBB-BP004, which is purely isolated, is used and its antifungal activity is expressed by the diameter of the low-ring ring that inhibits the growth of the test bacteria. In addition, KRIBB-BP004 with a purity of 95% or higher exhibits a minimum inhibitory concentration (MIC) of about 10-50 μg / ml for human pathogens.

Accordingly, the present invention provides an antifungal composition comprising an effective amount of KRIBB-BP004 or a derivative thereof as an active ingredient.

Such pharmaceutical compositions may include pharmaceutically acceptable excipients, carriers, diluents, etc. together with KRIBB-BP004 or derivatives thereof, and the formulations of the compositions may be prepared according to known methods using readily available ingredients. can do. 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 (solid or in liquid media), soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders And the like.

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 compositions of the present invention may be administered orally or parenterally, and the active compounds are effective for a wide range of dosages. For example, daily dosages of active compounds typically range from about 1 to 500 mg per kg of body weight. However, the amount of compound actually administered will be determined by the physician in light of the relevant circumstances, including the condition of the disease to be treated, the route of administration chosen, the age, weight and response of the individual patient, and the severity of the disease, and thus the range of administration may be It is not intended to limit the scope of the invention in any way.

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

Example 1

Isolation and Purification of KRIBB-BP004 from Garlic

Garlic purchased from Uiseong-gun, Gyeongsangbuk-do, Korea was removed from the roots and skins and then crushed with a green juice. 8 kg of water: acetone mixture (1: 1 (v / v)) was added to 2 kg of crushed garlic, and the mixture was stirred in a refrigerating chamber at 4 ° C. for about 12 hours and then filtered. Extraction was repeated twice with a mixture of water and acetone, and the extracts were collected and concentrated under reduced pressure.

The concentrated extract was made into 500 ml of hydrated state, and extracted by adding 500 ml of ethyl acetate three times. The extracted ethyl acetate layer was dried under reduced pressure to obtain 8 g of a tan oily substance.

160 g of silica gel (Merck, USA, product no. 9385) was charged to a column (4.5 x 20 cm) using a solution of hexane-ethyl acetate (7: 1 (v / v)), followed by 16 g of the oily substance obtained above. Was adsorbed onto silica gel to fill the column top. The adsorbed oily substance was fractionated by eluting stepwise from the hexane-ethyl acetate (7: 1 (v / v)) solution gradually increasing the ratio of ethyl acetate.

The fraction containing the active substance was dried under reduced pressure and then hexane-ethyl acetate (10: 1 and 2: 1 (v / v) was prepared using preparative thin layer chromatography (prep-TLC, thickness 1mm, Merck, USA, product number 13895). ) And the active fractions were separated. Fractions containing the active material were scraped, eluted with methanol, dried under reduced pressure, and finally purified using HPLC (Perkin-Elmer, S410). As an HPLC column, Phenomenex Ultracarb 10 ODS (250 x 21.2 mm, 10 μm) was used, and the active material was eluted with 85% methanol at a flow rate of 4 ml per minute to 230 mm. The peak was confirmed in the ultraviolet (UV) absorption wavelength. The antifungal activity of the absorbed peaks showed that the peak eluted at 22 minutes of retention time (Rt) showed antifungal activity. Finally, the pure antifungal substance KRIBB-BP004 obtained by HPLC was 7 mg.

In the above, the presence of KRIBB-BP004 in the extract and the partially purified sample was TLC (TLC plate: silica gel 60 F of Merck, thickness 0.25mm, developing solvent: hexane-ethyl acetate (2: 1 (v / v)) The Rf value of KRIBB-BP004 in the TLC was 0.42, and the part showing antifungal activity was tricopytone mentagro as in Example 3 below. Pits were identified by bioautogram with the test bacteria.

Example 2

Structure Determination of KRIBB-BP004

Various instrumental analyzes were performed to determine the structure of KRIBB-BP004.

1) UV (Visible) absorbance analysis

1.0 mg of the final purified KRIBB-BP004 was dissolved in 100 ml of 100% methanol, and the absorption wavelength was analyzed using an ultraviolet-vis spectrophotometer (Shimazu, UV-265), and the absorption peaks appeared at 205 mm and 239 mm. It confirmed (FIG. 1).

2) Infrared (IR) absorbance analysis

2 mg of KRIBB-BP004 was dissolved in chloroform, applied to an AgBr window, dried, and analyzed by a ratio recording infrared spectrometer (Bio-Rad Digilab Division, FTS-80). The material is 1311 cm And 1122cm Indicates the presence of a sulfone group at 987 cm With 933 cm Shows an absorption peak indicating the presence of an allyl group at (figure 2).

3) Molecular weight analysis

Molecular weights were measured by the High-Resolution Electron Impaction (HREI) -MS method using the VG70-VSEQ Mass Spectrometer (Vacuum Generator, UK). Ion molecule peaks appeared (Figure 3). CHOS was obtained from the molecular formula of KRIBB-BP004 by the molecular weight 250.0334 by the said measurement and the mathematical calculation method.

4) nuclear magnetic resonance (NMR) analysis

KRIBB-BP004 was completely dried, dissolved in CDCl, and placed in a 5 mm diameter tube, and subjected to NMR analysis with a Brooker AM-500. H-NMR is 500 MHz, C-NMR was measured at 125 MHz, H spectrum, Analysis of the C spectrum, 2D-HOMOCOSY spectrum and 2D-HETEROCOSY spectrum (4, 5, 6 and 7 degrees) revealed that the active substance had two sulfone groups and three allyl groups.

Therefore, when the analysis results are summarized and combined, it can be seen that the structure of the antifungal active substance found by the present inventors is the same as the above formula (I).

Example 3 Antifungal Activity Assay of KRIBB-BP004

Antifungal activity of KRIBB-BP004 was determined by performing a bioassay on agar plates using Trichophyton mentagraphytes as the test bacteria. As a reference material, purely isolated KRIBB-BP004 was used, and its antifungal activity was expressed by the diameter of the low-ring ring that inhibited the growth of the test bacteria.

That is, after inoculating the test spores on a plate containing potato-dextrose agar medium (200 g of potato per liter, 20 g of vinegar dextrose, 15 g of agar, final pH 5.6 at 25 ° C.), and then sterile stainless steel cup was plated. The garlic extract filtered through a 0.22 μm sterile Millipore filter was placed in the cup and incubated for 48 hours at 25 ° C. to measure the diameter of the growth resistant ring. When 50 µl of purely isolated 1 mg / ml KRIBB-BP004 solution was injected, the diameter of the growth-lowering ring was 40 mm.

Example 4 Determination of Minimum Developmental Concentration Concentration for Human Pathogens

The eleven fungi listed in Table 2 were treated with potato-dextrose agar (PDA) medium, Sabouraud's agar medium (10g of neopeptone (Neopeptone, Difco), 40g of bactodextrose, bacto agar) 15 g, final pH 5.6 at 25 ° C. and YM agar medium (3 g Bacterial Yeast Extract, 3 g malt extract (Difco), 5 g Bacterium peptone, 10 g Bactose dextrose 10 g, 20 g Bacterium agar, final pH 6.2 at 25 ° C.) Inoculate and incubated at 24 to 30 ° C. for 1 to 14 days, and then 5 ml of distilled water is added and spores 10 Suspension at a concentration of CFU / ㎖ was used as a bacterial solution.

After diluting the KRIBB-BP004 solution two times in succession, it is mixed with various fungi medium and dispensed on a flat plate of 5 cm diameter so as to have a final concentration of 100 to 0.195 µg / ml, respectively. When the medium is solidified, the inoculated bacteria solution prepared above is inoculated with 1 platinum for each plate, incubated for 1 to 4 days at 24 to 30 ° C, and then visually observed to determine the concentration at which growth of bacteria is suppressed. Determined.

The minimum growth inhibition concentration (MIC) measurement results for the fungi of KRIBB-BP004 are shown in Table 2 below.

Formulation Example: Preparation of pharmaceutical formulation containing KRIBB-BP004

(1) Preparation of capsule

Hard gelatin capsules were prepared using a pharmaceutical composition comprising the following ingredients:

The ingredients were mixed thoroughly and filled into hard gelatin capsules in a total amount of 500 mg.

(2) Preparation of tablets

Tablets each containing 500 mg of active ingredient were prepared as follows:

Active ingredient, starch and cellulose are passed through No. 45 mesh sieve and mixed thoroughly. An aqueous solution containing polyvinylpyrrolidone was mixed with the powder obtained, and then the mixture was passed through No. 14 mesh sieve. The granules obtained were dried at 50 ° C. and passed through a No. 18 mesh sieve. Sodium carboxymethyl starch, magnesium stearate and talc, which were previously passed through a No. 60 mesh sieve, were added to the granules, mixed, and compressed into tablets to obtain tablets of 500 mg each.

(3) Preparation of aerosol

An aerosol solution was prepared comprising the following ingredients:

The active compound is mixed with ethanol and fragrance and the mixture is added to some propelant 22, cooled to -30 [deg.] C. and transferred to a fill container. The required amount is then transferred to a stainless steel vessel and diluted with the remaining amount of propane. Subsequently, the valve part is attached to the container.

Claims (4)

Compound KRIBB-BP004 of Structural Formula (I) exhibiting antifungal activity: A process for purifying KRIBB-BP004 comprising extracting mashed garlic with a mixture of water and acetone and ethyl acetate in order to obtain a crude extract and purifying the crude extract by chromatography. The method of claim 2, wherein the chromatography step is performed by silica gel column chromatography, preparative thin layer chromatography (prep-TLC) and high pressure liquid chromatography (HPLC) in series. An antifungal composition comprising an effective amount of KRIBB-BP004 or a pharmaceutically acceptable salt thereof as an active ingredient, together with a suitable pharmaceutical excipient.