KR0185033B1 - Antibiotics extracted from coprinus atramentatius - Google Patents

Abstract

The present invention relates to novel sesquiterpenoid compounds of formula (I) and formula (II).

[Formula I]

[Formula II]

The present invention relates to a method for preparing new sesquiterpenoid compounds of Structural Formula (I) and Structural Formula (II) by isolating and purifying from a culture solution of manure mycelium mycelium.

The present invention also relates to the use of a pharmaceutical composition comprising the new sesquiterpenoid compound obtained from manure mycelium mycelium as an active ingredient as an antibiotic. The present invention also relates to an extract of manure myrtle mushroom and its culture solution, a preparation method thereof and an antibiotic using the extract as an active ingredient.

Description

New antibiotic produced by strain of Coprinus atramentatius

The present invention relates to novel sesquiterpenoid compounds of the following formulas (I) and (II).

[Formula I]

[Formula II]

The present invention relates to a method for preparing a new sesquiterpenoid compound of Structural Formula (I) and Structural Formula (II) by isolating and purifying from a culture solution of manure mycelia (Coprinus atramentatius).

The present invention relates to a homeostasis comprising the new sesquiterpenoid compound as an active ingredient.

More particularly, the present invention relates to the use of a novel sesquiterpenoid compound obtained from manure mycelium mycelium and its derivative as an inducer, as an antibiotic, wherein the pharmaceutical composition of the present invention It can be usefully used because it shows higher antimicrobial activity to strains resistant to eggplant antibiotics.

The present invention relates to an extract of manure myrtle mushroom and its culture solution, a method for producing the same, and an antibiotic comprising the manure myrtle mushroom extract as an active ingredient.

Basidiomycetes are higher grade fungi that contain more than a thousand species, and so far one hundred species of antibiotics have been isolated. However, current research achievements are weak compared to the large number of species and broad distribution of basidiomycetes, so it is possible to study antibiotics using basidiomycetes.

Looking at the metabolic process characteristics of basidiomycetes, it can be seen that the secondary metabolites produced by these strains have characteristics not seen in other fungi. Typically, terpene-based antibiotics are produced by the development of shikimic acid and mevalonic acid pathways similar to those of higher plants. In particular, these terpene-based materials have excellent antibacterial effects, and thus may be usefully used for agriculture or medicine.

In addition, antibiotics produced by basidiomycetes have various activities such as anti-tumor, immune regulation, blood pressure drop, blood sugar drop, antithrombosis, and nervous system action in addition to the above antibacterial action, and are currently used as a resource to search for new substances. It is attracting attention.

[Prior art]

To date, antibiotics isolated from basidiomycetes were first detected in marsmic acid in deciduous mushrooms in 1945.Since then, 20 kinds of polyethylene-based substances including lentinamycin were found in shiitake mushrooms. Confirmed. In addition, several species of terpenoids, including allurodiscal, in the culture of Allurodiscus mirabilis, also belong to the genus Calvatia craniformis Aromatic compounds, such as calvatic acid, have been isolated from the culture medium of) and have been reported to exhibit antimicrobial activity.

The present inventors collect, classify and identify basidiomycetes native to Korea in order to develop new antibiotics, purely isolated mycelia from their fruiting bodies, and incubated them. The present invention was completed by investigating the antimicrobial activity of human and plant pathogens of the components contained in the culture medium of mycelium and by opening new medicinal or agricultural antibiotics from mushrooms through the separation, purification and structural analysis of active substances.

Specifically, an object of the present invention is to provide a new sesquiterpenoid compound of structural formula (I) and structural formula (II) having a new substituent in the sesquiterpenoid compound.

[Formula I]

[Formula II]

The present invention provides a method for preparing new sesquiterpenoid compounds of formula (I) and formula (II) using manure myrtle mushroom.

Specifically, the compound of the present invention is extracted by concentrating the culture solution of manure myrtle strain with methanol, and then extracted with ethyl acetate and concentrated again, dissolved in a mixed solvent of chloroform and methanol, and prepared by performing adsorption chromatography.

It is an object of the present invention to provide a pharmaceutical composition as an active ingredient comprising a novel sesquiterpenoid compound of formula (I) or formula (II) as an active ingredient.

In addition, the pharmaceutical composition of the present invention can be used as an anti-tumor agent, an immunomodulator, a blood pressure lowering agent, a hypoglycemic agent, an antithrombotic agent and a neuromodulator.

It is an object of the present invention to provide extracts containing structural formula (I) and structural formula (II) compounds having antimicrobial activity from manure myrtle strains and cultures thereof, methods for their preparation, and the use of the extracts as antibiotics.

1 shows the hydrogen nuclear magnetic resonance spectrum ( ¹ H-NMR) of the compound of formula (I).

2 shows the carbon nuclear magnetic resonance spectrum ( ¹³ C-NMR) of the compound of formula (I).

3 shows the hydrogen nuclear magnetic resonance spectrum ( ¹ H-NMR) of the compound of formula II.

4 shows the carbon nuclear magnetic resonance spectrum ( ¹³ C-NMR) of the compound of formula II.

The present invention is described in detail as follows.

In order to open new antibiotics in basidiomycetes, YMG media were obtained by obtaining the corprinus atrimentarius ASI 20013, a manure mushroom, from the Genetic Resources Conservation Center of the National Academy of Agricultural Science and Technology. (Yeast extract 0.4%, malt extract 1.0%, glucose 0.4%, pH 6.0) and the like, and the culture solution is used for separation and purification of antibiotics and the like.

The culture supernatant of manure myrtle strain was adsorbed using a diaion HP-20 column, washed with 30% methanol, eluted with 70% methanol, and concentrated. The concentrate was extracted again with ethyl acetate, concentrated and dissolved in a mixed solvent of chloroform: methanol, and then subjected to adsorption chromatography with a solvent of various chloroform: methanol ratio using a silica gel column (70-230 mesh, manufactured by Merck). The active fractions are concentrated.

The active fraction obtained above is dissolved in a small amount of methanol and ODS column chromatography is carried out in a solvent of methanol to obtain two active fractions A, B, which are divided and concentrated respectively. Each of the following active fractions was prepared by preparative TLC chromatography with a mixed solvent of chloroform: methanol, where fraction A recovered the band at an RF value of 0.35 and fraction B at an RF value of 0.4. As a result, the compounds of formulas (I) and (II) can be obtained from each fraction.

In order to assay the active fraction in the separation and purification of the compounds of the present invention, the sample in the purification process is dissolved in a certain amount of methanol and then dried on a paper disk. The dried disc is placed on an antibiotic-resistant Staphylococcus aureus (KCTC 1928) plate and incubated, and the activity of the compound is assayed by the formation of the resulting growth ring.

The physicochemical properties of the structural formula (I) and the structural formula (II) compounds isolated and purified above are as shown in Example 2.

In the present invention, in order to assay a new antibiotic, Escherichia coli BE 1186, Salmonella typhimurium KCTC 1925, Staphylococcus aureus KCTC 1916 (Staphylococcus aureus KCTC 1916). Staphylococcus aureus KCTC 1928 6051), Pyricularia oryzae KCTC 1939, and Candida albicans KCTC 1940 yeast.

Strains used in the present invention are incubated at the optimum medium and temperature for each strain according to their characteristics. Each strain cultured in a suitable medium is mixed with fresh medium cooled to 40-50 ° C. again, and 5 ml of each medium is preliminarily coagulated in a coagulated plate and used for assaying antibiotics.

At this time, the antimicrobial activity was measured using the agar diffusion method, etc. First, the bacteria isolated from the fruiting body were inoculated with a potato-dextrose liquid medium, incubated at an appropriate temperature, and the cells were filtered. do. The culture filtrate and the cells obtained at this time are immersed in acetone, and the extract obtained by filtration on Whatman filter paper is taken up, absorbed by paper disk (Toyo Co.) and dried. The paper disk is placed on a plate with assay bacteria and incubated according to the culture conditions of each assay, and then the diameter of the growth resistant ring formed is measured to assay the antibacterial activity.

In addition, in order to determine the efficacy of the compounds of the formulas (I) and (II) as homeostasis, the minimum growth concentration of the microorganisms is measured using an agar dilution method or the like. (Hewitt, W., Theory and Applcation of Microbiological Assay, AP, 1989). As a result, the structural formula (I) and (II) compounds were Staphylococcus aureus KCTC 1916 strain, Staphylococcus aureus KCTC 1928 strain, Escherichia coli BE 1186 strain among the strains to be tested, pepper bacterium bacterium, and vegetable gray It is confirmed that the fungus shows a low growth inhibitory concentration of about 1-25 ppm.

In particular, considering that the Staphylococcus aureus KCTC 1928 strain used as the disclosed strain is a strain resistant to antibiotics such as penicillin, kanamycin, chromamphenicol, tetracycline and macrolide system, the structural formula of the present invention ( The compounds I) and (II) show higher activity against strains resistant to the above antibiotics. This is a result of measuring activity using the Staphylococcus aureus KCTC 1916 strain as a comparison group.

In the pharmaceutical use of the concentrates and tablets of the present invention, it can be prepared by methods known in the pharmaceutical art, and can be used in the formulation of tablets, capsules, powders, solutions, suspensions, injections and the like using appropriate excipients. Can be.

A pharmaceutical composition comprising a pharmaceutically acceptable salt or excipient is prepared and administered orally or intravenously in an amount of 1 mg to 2 mg per kg body weight.

Hereinafter, the present invention will be described in detail by way of examples.

The following examples illustrate the present invention in detail, and the content of the present invention is not limited by the examples.

Example 1

Cultivation of Manure Strains

Obtained Corinus atramentarius ASI 20013 strain, a manure mushroom that is preserved at the National Institute of Agricultural Science and Technology, Center for Genetic Resources Conservation, Rural Development Administration. %, Malt extract 1.0%, glucose 0.4%, pH6.0) was inoculated in 500ml and then incubated for 3 days by shaking at 120rpm at 28 ℃. 30L of the same culture solution was added to a 50L fermenter, 120 ml of the cultured species was added thereto, shaken at 200 rpm at 28 ° C. for 3 days, and the culture solution was used for separation and purification.

Example 2

Antimicrobial Activity Assay of Strain Culture

In order to assay the antimicrobial activity of the strain culture, mushroom cells were inoculated in YMG (liquid medium) and incubated for 10 days at 28 ° C. The cell culture medium was then filtered and the culture filtrate and cells obtained in this process were 70% acetone. It was then immersed for 12 hours, and then it was filtered through Whatman No. 1 filter paper to obtain an extract, and the extract was absorbed into a paper disk (manufactured by Toyo Co., Ltd.) having a diameter of 100 µl each, and dried.

The dried paper discs were placed on a plate with assay bacteria and incubated according to the culture conditions of each assay bacteria. Growth inhibitory ring is formed according to the activity of the culture medium, the diameter of the antimicrobial activity was assayed.

Example 3

Isolation and Purification of the Compounds (I) and (II)

In order to separate and purify the structural formula (I) and the structural formula (II) compound, 30 L of the culture supernatant of the manure mushroom strain was adsorbed on a DIION HP-20 column, washed with 30% methanol and eluted with 70% methanol. Methanol was removed and concentrated. The concentrate was extracted three times with ethyl acetate, and the ethyl acetate layer was concentrated again, and dissolved in a small amount of a mixed solvent having a ratio of chloroform: methanol of 100: 1.

Adsorption chromatography was then performed on a silica gel column (70-230 mesh, manufactured by Merck) using a mixed solvent having a ratio of chloroform: methanol of 100: 1, 50: 1 and 10: 1, respectively, and the active fraction was concentrated. It was.

The concentrated active fraction was dissolved in a small amount of 40% methanol and ODS column chromatography was performed on a solvent of 40% methanol to obtain two active fractions A, B, which were each divided and concentrated. Preparative TLC chromatography was performed on each of the concentrated active fractions using a mixed solvent having a chloroform: methanol ratio of 10: 1 to recover the band at fraction RF of 0.35 and fraction B of RF of 0.4. And 10 mg of new material were obtained.

During the purification of the active material, the sample was dissolved in a certain amount of methanol and then dried by absorbing 100 μl into a paper disk, and the dried paper disk was placed on a Staphylococcus aureus (KCTC 1928) plate and cultured. At this time, the antimicrobial activity of the strain culture solution was assayed whether growth resistant ring was formed.

Example 4

Physicochemical Characteristics of Compounds (I) and (II)

The physicochemical characteristics of the compounds of Structural Formula (I) and Structural Formula (II) isolated and purified from the culture of manure myrtle mushroom are as follows.

(4-1) Compound of structural formula (I): (4R ^* , 7S ^* )-2, 10-dihydro-4,14-dihydroxyyl-5 (9) -en-1-wan ((4R ^＊ , 7S ^＊ ) -2, 10-dehydro-4, 14-dehydroxyillud-5 (9) -en-1-one)

[Formula I]

1) Appearance of substance: Colorless oily

2) Molecular Weight: 248

3) Molecular formula: C ₁₅ H ₂₀ O ₃

4) High resolution mass spectrometry (m / z): found (M ^＊ ) 248.1419

Calculated ^Value (M ^＊ ) 248.1412

5) Intrinsic Light Rotational Speed ([α] _d : +20 in methanol (C = 0.3)

6) UV absorption spectrum (UV max MeOH nm (ε)):

205 (9,700), 264 (5,800)

7) infrared absorption spectrum (IR ν _max KBR cm ^-1 ):

3380, 1660, 1600, 1040

8) nuclear magnetic resonance (NMR) spectra

Hydrogen nuclear magnetic resonance ( ¹ H NMR) spectra and carbon nuclear magnetic resonance ( ¹³ C NMR) spectra measured using chloroform (CDCl ₃ ) as a solvent and tetramethyl silane (TMS) as reference materials are shown in FIGS. Same as

(4-2) Compound of formula (II): (4R ^* , 7S ^* )-2, 10-dihydro-4,13-dihydroxyyl-5 (9) -en-1-wan ((4R ^＊ , 7S ^＊ ) -2, 10-dehydro-4, 13-dehydroxyillud-5 (9) -en-1-one)

[Formula II]

1) Appearance of substance: Colorless oily

2) Molecular Weight: 248

3) Molecular formula: C ₁₅ H ₂₀ O ₃

4) High resolution mass spectrometry (m / z): found (M ^＊ )

Calculated ^Value (M ^＊ ) 248.1413

5) Intrinsic Light Rotational Speed ([m] _D in methanol: -15.4 (C = 0.26)

6) UV absorption spectrum (UV max MeOH nm (ε)):

205 (11,500), 270 (7,300)

7) infrared absorption spectrum (IR ν _max KBR cm ^-1 ):

3380, 1660, 1600, 1040

8) nuclear magnetic resonance (NMR) spectra

Hydrogen nuclear magnetic resonance ( ¹ H NMR) spectra and carbon nuclear magnetic resonance ( ¹³ C NMR) spectra measured using chloroform (CDCl ₃ ) as a solvent and tetramethyl silane (TMS) as reference materials are shown in FIGS. Same as

As described above, the structural formula (I) and the structural formula (II) compound showed the same structure through the results of nuclear magnetic resonance spectra (NMR), but the intrinsic optical rotation ([α] _D ) values were +20 and -15.4, respectively. These compounds could be identified as being structural isomers with each other.

Example 5

Determination of Antimicrobial Activity

Agar dilution method was used to measure the growth inhibitory concentration of microorganisms of the structural formula (I) and the structural formula (II) compound obtained by purification in Example 3.

As a result, as shown in Table 1, the Structural Formula (I) and Structural Formula (II) compounds were 12-25 ppm in the Staphylococcus aureus KCTC 1916 strain and the KCTC 1928 strain, 1-2 ppm in the E. coli BE 1186 strain. The minimum inhibitory concentration of growth was about 25ppm in the red pepper bacterium, the vegetable vegetable pathogen, and 25ppm in the vegetable gray fungus.

In this case, the Staphylococcus aureus KCTC 1928 strain used as the disclosed strain is a strain made to be resistant to penicillin, kanamycin, chromamphenicol, tetracycline and macrolide antibiotics, and thus, the structural formula (Ⅰ) and structural formula (Ⅱ) ), The compound was compared to the antimicrobial activity in the Staphylococcus aureus KCTC 1916 strain used as a comparative group, it was confirmed that the higher activity to the strain resistant to the antibiotic.

As described above, the structural formula (I) and the structural formula (II) compound of the present invention are Staphylococcus aureus KCTC 1916 strain, Staphylococcus aureus KCTC 1928 strain, Escherichia coli BE 1186 strain, red pepper bacterium bacterium, and vegetable gray The fungus showed excellent antibacterial effect even at low concentrations.

In addition, considering the antimicrobial activity of the Staphylococcus aureus KCTC 1928 strain uniquely, it can be seen that the structural formula (I) and the structural formula (II) compounds of the present invention exhibit higher antimicrobial activity against strains resistant to antibiotics. . Therefore, antibiotics extracted from the culture of manure mycelium mycelium can be useful for strains resistant.

Claims (7)

New sesquiterpenoid compounds of formula (I), their derivatives and salts thereof. [Formula I] New sesquiterpenoid compounds of formula (II), their derivatives and salts thereof. [Formula II] The culture solution of the manure mushroom (Coprinus atramentatius ASI 20013) was extracted with methanol, concentrated, extracted with ethyl acetate, concentrated, dissolved in chloroform and methanol mixed solvent, and subjected to adsorption chromatography, followed by ODS column chromatography and prep TLC. A method for producing a sesquiterpenoid compound of formula (I), characterized in that to perform chromatography. The culture solution of the manure mushroom (Coprinus atramentatius ASI 20013) was extracted with methanol, concentrated, extracted with ethyl acetate, concentrated, dissolved in chloroform and methanol mixed solvent, and subjected to adsorption chromatography, followed by ODS column chromatography and prep TLC. A method for producing a sesquiterpenoid compound of formula (II), characterized in that to perform chromatography. Structural mushrooms and their cultures were extracted with methanol, concentrated, extracted with ethyl acetate, concentrated, dissolved in a mixed solvent of chloroform and methanol, and subjected to adsorption chromatography to obtain structural formulas (I) and (II). Extract of manure myrtle mushroom containing the compound. The pharmaceutical composition for antibiotics according to claim 1 or 2, wherein the sesquiterpenoid compound and derivatives thereof are added as pharmaceutically acceptable substances. The pharmaceutical composition for antibiotics, characterized in that the pharmaceutically acceptable substance is added to the extract of the manure water mushroom of claim 5 as an active ingredient.