KR100315098B1 - A New Peptibol Antibiotic KGT14-3 Produced by Apiocrea sp. 14T and Production Method and Use thereof - Google Patents

Abstract

The present invention is a peptibol antibiotic (KGT14-3) having a novel antiviral, antitumor and antimicrobial activity produced by 14T strain of genus Apiocrea sp. ) And a method of manufacturing and use thereof.

An object of the present invention is to culture the culture of the strain ( Sedonium sp ., Also known as 14T) in a rice medium to suppress the plant virus infection and to prepare a peptibol antibiotic KGT14-3 having antitumor and antimicrobial effect. The strain used in the present invention was incubated for 3-7 days at 25-28 ° C. in potato-glucose-based medium containing 0.1% lactic acid spores isolated from the net mushrooms ( Boletus sp.,) Collected from Gyeryongsan Donghaksa Valley. To separate. Sections of bacteria grown in potato agar medium for 7 days are inoculated in each rice medium and grown at 25-28 ° C. for 20-30 days. The rice medium is dried in a shade, pulverized, extracted with ethanol, concentrated in gam-am, fractionated using a separatory funnel in a butanol-water solvent system, and the butanol layer is collected and concentrated. The concentrated solution is purified by chromatography to remove KGT14-3, a novel compound. This material was identified as a novel substance by hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum, carbon nuclear magnetic resonance ( ¹³ C-NMR) spectrum.

This KGT14-3 compound has anti-viral activity against tobacco mosaic, anti-cancer activity with cytotoxicity against pancreatic cancer cells and melanoma cells, and anti-bacterial activity against Gram-positive bacteria. It is expected to use.

Description

Novel Peptib Ball Antibiotic from Apiocria 14T Strains of Aspergillus spp., Production Method and Uses thereof {A New Peptibol Antibiotic KGT14-3 Produced by Apiocrea sp. 14T and Production Method and Use approximately}

The present invention provides a novel antiviral, anti-tumor and antimicrobial activity of Peptide Ball Antibiotic (KGT14-3) produced by 14T strain of genus Apiocrea sp. It relates to a manufacturing method and use.

Of Bahia oak Liao (Apiocrea sp.) Genus 14T strain Ascomycetes (Ascomycetes) is the imperfection-family name as parasites isolated from mushrooms is sepe Doni umgyun (Sepedonium sp.).

Animal and animal diseases and human cancers remain unresolved tasks for humans. The development of drugs to prevent and treat them continues after World War II, but antimicrobial antibiotics have not been developed. In particular, the development of anti-phytoviral agents has been relatively neglected compared to animal or human viruses, and little has been found except for polymer materials such as complex polysaccharides and proteins, which have not been developed as drugs. Most of the plant viruses are genes with ribonucleic acid (RNA) genes and envelope proteins, which are the type of virus such as influenza virus, pandemic hemorrhagic fever virus (Hantan virus), HIV virus (HIV) and hepatitis C virus in animals. Given this, the discovery of antiviral agents for plant viruses could lead to the development of therapeutic agents for human viral diseases. In addition, antibiotics that can act on viruses are also effective in abnormal cells, and thus, are highly likely to be toxic to tumors and cancer cells. In this respect, the antiviral material search method for plant viruses can be used as a preliminary search method for anti-phytoviral agents as well as animal or human virus diseases and cancer treatments. Until now, it is very difficult to find new substances from organic biomaterials and actinomycetes or antibiotics producing soil microorganisms.

Microbial antimicrobial research on plant viruses reported that before 1950 several bacteria and fungi inhibited viral infections (Mulvania, 1926; Johnson & Hoggan, 1937). In 1950, Gupta & Price screened 49 fungal cultures, and Polyporus arcularius inhibited 88% infection of southern bean mosaic virus. There is a report. In 1952, Baden and Freeman also discovered polysaccharides that inhibit viral infection from basidiomycetes. In 1984, Shimomura et al. Isolated peptide glucan, a virus inhibitor, from shiitake mushrooms. In particular, shiitake mushrooms have been studied for their antiviral properties. In 1993, Aoki et al. Examined the antiviral activity of 125 strains of biliary culture filtrates and isolated polysaccharides, a virus-inhibiting substance. Peptiboball antibiotics isolated from the same genus or similar fungus as the strain used in the present invention have been studied as a model of the ion channel (ion channel) and the activity was reported to show mainly antifungal and antibacterial activity. An example of isolates of peptiball antibiotics using the bacteria most similar to the ones used by the present inventors is Chrysospermin A isolated from mycelia of Apiocrea chrysosperma by Donberger et al. In 1995. , B, C, D (chrysospermin a, B, C, D) and sepe belonging to the genera such as incomplete generation sepe Doni Titanium (Sepedonium) of the bacteria or the like 1997 Li Wu (Ritzau) Doni help ampoule Los Forum (Sepedonium ampullosporum ) is an ampullosporin. In particular, Apiochria chrysosperma is very similar to the strain used in the present invention, and the material to be produced is very similar to the strain used in the present invention because it has a common feature of being a peptabol material consisting of 19 amino acids. However, the product is different from KGT14-3 in the present invention, it is thought that the difference in species (species) or biotype (biotype) level. Also all were isolated from mycelium in liquid culture and did not use rice culture as in the present invention. Chrysospermin A, B, C, D and ampoulosporin produced by the above strains have all been reported to exhibit a narrow range of antimicrobial and antibacterial activity and have not been reported for antiviral and anticancer activity. The physiological activities of Tybol antibiotics are of little interest.

The present inventors have completed the present invention in view of the fact that forest fungi have relatively little research in the search for physiologically active substances, and thus the possibility of developing new useful active substances is high. Fungus inhabiting the forest was collected and 500 strains were isolated. Inhibition of the tobacco mosaic virus infection of these cultures was investigated through the inoculation test of the tobacco variety Xanthi-nc. A strain was selected and the compound KGT14-3 was isolated and purified from the rice culture of this strain and the chemical structure was revealed. This compound is a peptibol antibiotic with antibacterial virus activity against tobacco mosaic, anti-cancer activity cytotoxic to pancreatic cancer cells and melanoma cells, and antibacterial activity against Gram-positive bacteria. Therefore, the present invention provides a method for producing the above-mentioned compounds from rice cultures of peptibol novel compounds (KGT14-3) and apiocria 14T strains showing antiviral activity, anticancer activity and antibacterial activity.

1 is an electron micrograph of a 14T strain of Apiocrea sp.

Figure 2 is a separation process of the novel compound KGT14-3 from 14T strain rice culture.

3 is a hydrogen nuclear magnetic resonance ( ¹ H-NMR) spectrum of the KGT14-3 compound.

4 is a carbon nuclear magnetic resonance ( ¹³ C-NMR) spectrum of the KGT14-3 compound.

Aspergillus genus 14T strain used in the present invention is a potato agar medium containing 0.1% of lactic acid (lactic acid) that is parasitic in Boletus sp. Separated. For the primary screening of the antibacterial virus of the isolated strains and various other strains, each strain was grown in potato agar medium and the cultures were extracted with ethanol to examine the tobacco mosaic virus infection inhibitory activity. In this study, Apiocria 14T strain had the highest anti-plant virus activity. Separation and purification to obtain the compound of KGT14-3 from the 14T strain was carried out by the method of screening the tobacco mosaic virus infection inhibitory activity until the initial partial purification, and after partial purification, Bacillus subtilis, a gram-positive bacterium, in thin layer chromatography and high-performance liquid chromatography. Growth inhibition against Bacillus subtilis ) was used as an index. Finally, the purified active substance was used to re-inhibit tobacco mosaic infection inhibitory activity and to investigate cytotoxicity and antibacterial activity against human tumor cell lines.

Meanwhile, Apiocrea sp. 14T used in the present invention was deposited with Accession No. KCTC 8921P at Biotechnology Research Institute on January 11, 1999.

Hereinafter, the present invention will be described by the following examples and test examples. However, these do not limit the technical scope of the present invention.

<Example 1> Isolation and Identification of Strains

The strains used in the present invention is to separate that existed as a tan spore state on the surface of a mushroom (Boletus sp., Net mushrooms) collected in the valley near Kyeryongsan donghaksa that visitors to Chungcheongnam Gongju was named Bahia oak Ria in 14T. The yellow spores were incubated at 25-28 ° C. for 3-7 days in a potato-glucose-based medium containing 0.1% lactic acid (PDA) to isolate bacteria. After culturing the isolated strain for 7 days in pure PDA, the culture was extracted with ethanol to examine the tobacco mosaic virus (hereinafter referred to as TMV) infection inhibitory activity of the extract showed an 85% effect was superior to the other strains examined. This strain is parasitic to the fruiting body of mushrooms in nature, and powdered spores are observed on the surface of the fruiting body. Growth was good on Malt Extract Agar medium and PDA medium, but not on Czapek dox agar medium, water agar or low nutrient medium. In the medium, the white mycelium grows in the shape of fleece, and in the latter, it becomes yellow with spores forming and becomes yellowish brown or brown as old. In the agar medium, the hyphae layer is thin and the diameter of aerial hyphae is 3.2-1.9 ㎛ (average 2.4 ㎛), and there are two types of spores, conidia and phiallospores. The conidia are spherical in shape and have a rough surface with projections. The size of the conidia is 11.5 to 13.8 μm (average 12.8 μm), and the length of the conidia is 8.9 to 22.2 μm (average 15.1 μm), and the phiallospore ) Is elliptical and the size is 12.6 × 4.0 to 7.4 × 2.6 μm (average 10.7 × 3.4 μm) and the surface is smooth (see FIG. 1). This is similar to the full generation of Bahia oak Ria Cri source peoma (Apiocrea chrysosperma) of sepe Doni help Cri source pemum (Sededonium chrysospermum) as compared to the gyundeul recorded as the same type as those reported in the prior art (see Table 1). However, this 14T strain showed a slight difference in the diameter of the aerial hyphae and the size of the Pyroconidia spores, which may be homologous to the originally recorded strain, but due to the morphological differences, it was identified as Apiocrea sp. It was named 14T strain of genus Apiocrea sp.

Table 1.Cultural and Morphological Characteristics of 14T of Genus Apiocrea sp.

division Growth status and morphological characteristics 14T Apiocrea chrysosperma Used badge Potato grapes Growth Growth Growth Culture characteristics Early White Mycelial Yellow or Tan Spore Formation Early White Mycelial Yellow or Tan Spore Formation Malt Extract Agar Growth Growth Chapex Dogs Agar Poor growth Poor growth Water agar Poor growth Poor growth Agency Aerial hyphae 2.1-4.4 μm in diameter 3.5-6.0 μm in diameter Conidia Spherical, surface diameter 11.5-13.8 ㎛ Spherical, surface diameter 11.5-16.0 μm Conidia Erection length 8.9-22.2 ㎛ for aerial hyphae Upright about aerial hyphae Piaro Consortium Oval 2.6 × 7.4-4.0 × 12.6 μm Oval 5 × 8-9 × 12 μm ecology Parasitic on Net Mushrooms Parasitic on Net Mushrooms

<Example 2> Cultivation of 14T strain for obtaining KGT14-3 compound

In order to obtain the KGT14-3 compound of the present invention, when cultured for 15 days at 27 ° C. in a potato glucose liquid medium (potato dextrose broth medium), the cells grew completely and showed TMV infection inhibitory activity. A rice incubator was used. A rice incubator is prepared by putting 60 ml of distilled water into a 500 ml Erlenmeyer flask in 50 cc of white rice and autoclaving at 15 at 121 ° C. for 20 minutes. The prepared rice medium was inoculated with a piece of bacteria grown on potato agar medium for 7 days in each rice medium and grown at 25-28 ° C. for 20-30 days, and then the whole rice culture was used as a material for material extraction. The addition of carbon sources such as glucose and lactose, nitrogen sources such as peptone and soy flour, and inorganic salts to rice media helped to speed up the growth, but did not significantly affect the production of the material.

<Example 3> Separation and Purification of the KGT14-3 Compound

After culturing this strain in rice medium for 20-30 days, the culture was dried in the shade, pulverized with an electronic millstone, extracted with ethanol, and concentrated under reduced pressure to remove ethanol. Subsequent separation of KGT14-3 is shown in FIG. That is, the concentrate of the ethanol extract of the rice culture was fractionated using a separating funnel in a butanol-water solvent system, and the butanol layer was collected and concentrated. The concentrated solution was sequentially purified by changing the ratio of chloroform and methanol in silica gel column chromatography, and then eluted with chloroform: methanol = 4: 1 and 2: 1. The mixture was concentrated again, and then reversed-phase silica gel column chromatography. After collecting 90% methanol fractions from silica gel (ODS) column chromatography, the fractions were subjected to thin layer chromatography (reverse phase) in a solvent system of 90% methanol, and then fractions of Rf = 0.38 were collected. Purified by high performance liquid chromatography, a novel compound KGT14-3 was isolated purely.

<Test Example 1> Physical and chemical properties of KGT14-3 compound

The physicochemical properties of the KGT14-3 compound obtained in the present invention were tested. The results are as follows.

· Appearance of substance: White powder.

Melting point: collapses above 250 ° C.

Molecular weight: 1,913

Molecular Formula: C ₉₁ H ₁₄₄ N ₂₂ O ₂₃

UV maximum absorption spectrum (UV lambda max, methanol; nm): 214, 224, 282

ㆍ Infrared Absorption Spectrum (IR ν _max ^KBr cm ^-1 ): 3290, 1660, 1520

Solubility: Soluble in dimethylsulfuroxide, methanol, ethanol, insoluble in hexane, water.

Rf position of thin layer chromatography (reverse phase, 90% methanol): 0.38

Nuclear Magnetic Resonance (NMR) Spectrum: Hydrogen magnetic resonance ( ¹ H-NMR) spectrum and carbon nuclear magnetic resonance ( ¹³ C-NMR) spectrum measured using deuterated methanol as a solvent are shown in FIGS. 3 and 4, respectively. same.

ㆍ chemical structure

AcPhe-Aib-Ala-Aib-Iva-Leu-Gln-Gly-Aib-Aib-Ala-Ala-Aib-Pro-Iva-Aib-Aib-Gln-Trpol

The abbreviation for chemical structure of KGT 14-3 material is as follows.

AcPhe: acetylated phenylalanine, Aib: aminoisobutyric acid, Iva: isovaline, Leu: leucin, Gln: glutamine, Gly: glycine, Ala: alanine, Pro: proline, Trpol: tryptophanol

<Test Example 2> Tobacco virus infection inhibitory activity of the KGT14-3 compound

To measure the antiviral activity of KGT14-3, the most virulent and representative tobacco mosaic virus (TMV) among plant viruses was used. TMV inoculum was used to dry the tobacco leaves of the diseased plants in the shade, and then added the juice of 0.02M phosphate buffer of 50 times the weight of the leaves, and then pulverized with jujitsu and filtered with filter paper. To inoculate the virus, sprinkle carborundum on the leaf surface of 8-10 leaf tobacco plants (Xanthi-nc>) and rub the juice with cotton. 3-4 days after the inoculation, the leaves show many spots of viral infection of about 1mm in diameter. To confirm the antiviral activity of KGT14-3, the purified active substance was dissolved in 10% ethanol to make dilutions sequentially, and the dilutions were inoculated on one leaf by mixing the same amount with the TMV inoculation solution. % Ethanol was inoculated with the same amount of inoculum and inoculated, and 3-4 days later, the number of spots on the leaves was compared to determine the inhibitory activity. As a result of examining the antiviral activity of KGT14-3 in this way, as shown in Table 2, it showed 96.2% infection inhibition activity at 100 ppm, 80.3% at 10 ppm, and showed strong antiviral activity at 1 ppm. Did.

Table 2.Antiviral Activity of KGT14-3 Compounds

KGT14-3 concentration (ppm) Infection Control Effect (%) 100 96.2 10 80.3 One 9.1 0 0.0

<Test Example 3> Antibacterial activity of KGT14-3 compound

In order to measure the antimicrobial activity of KGT14-3 compounds, gram-positive and negative bacteria, yeasts, and fungi were used. Assay, 30 ㎍ of purified active substance was dissolved in methanol, absorbed by paper disc, and air-dried. After placing the bacteria on the mixed medium, the size of the inhibition zone formed around the paper disk was examined. As a result, as shown in Table 3, it showed antimicrobial activity forming 9-20mm low-lying in yeast, Gram-positive bacteria, muco ramanias fungus and Saccharomyces cerevisiae, except for Streptococcus sp. There was no antimicrobial activity.

Table 3. Antimicrobial Activity of KGT14-3 Compounds

Black microorganism Diameter of lowlands (mm) Bacillus subtilis 10 Staphylococcus aureus 10 Escherichia coli 10 Streptococcus SP 0 Corynebacterium Lilium 20 Candida Albicans 0 Aspergillus Niger 0 Magna Forte Gricia 0 Colletotricum Lagenerium 0 Huzium Solani 0 Muco Ramanaanus 9 Alternaria Mali 0 Saccharomyces cerevije 9

<Test Example 4> Cytotoxicity of KGT14-3 Compound

In order to measure the cytotoxicity of KGT14-3 compounds, purely purified active substances were diluted in dimethylsulfur oxide (DMSO) by concentration, followed by incubation of pancreatic cancer cells (PC-3) and melanoma cells (UACC 62), which are cancer cell lines. (37% incubation in 10% Petalcap serum culture) and then examined by quantifying the viable cells. The results for pancreatic cancer cell lines was 50% growth inhibitory concentration (GI ₅₀₎ 0.46ppm, melanoma cells showed strong toxicity to cancer cells 0.99ppm. (See Table 4)

Table 4. Cytotoxicity of KGT14-3 Compounds against Human Tumor Cells

Tumor cell line 50% Growth Inhibitory Concentration (GI ₅₀ ) (ppm) Pancreatic cancer cell line 0.46 Melanoma cell line 0.99

The peptiball antibiotic KGT14-3 compound of the present invention exhibits anti-viral activity against tobacco mosaic, anti-cancer activity with cytotoxicity against pancreatic cancer cells and melanoma cells, and antibacterial activity against gram-positive bacteria. It is expected to be used as antitumor and antibacterial agent.

Claims (9)

A salt comprising, as an active ingredient, a peptiball antibiotic KGT14-3 or a peptiball antibiotic KGT14-3 represented by the following structural formula (I): AcPhe-Aib-Ala-Aib-Iva-Leu-Gln-Gly-Aib-Aib-Ala-Ala-Aib-Pro-Iva-Aib-Aib-Gln-Trpol Structural Formula (I) Abbreviations in the chemical formula are as follows. AcPhe: acetylated phenylalanine, Aib: aminoisobutyric acid, Iva: isovaline, Leu: leucin, Gln: glutamine, Gly: glycine, Ala: alanine, Pro: proline, Trpol: tryptophanol Apiocrea sp. 14T strain (No. KCTC 8921P, 1999. 1) isolated from a fungus isolated from mushrooms producing the structural formula (I) of the peptide peptibball antibiotic KGT14-3 of claim 1 11) Separating the bacteria by incubating the Apiocria 14T (Accession No. KCTC 8921P) isolated from the mushrooms ( Boletus sp.,) At 25-28 ° C. for 3-7 days in a potato-glued cloth medium containing 0.1% lactic acid And, inoculating each rice medium with the sections of the bacteria grown on potato agar medium and growing them at 25 to 28 ° C. for 20-30 days, drying and pulverizing the aforementioned culture in a cow, extracting with ethanol, and gam Concentrate to remove the organic solvent and recover the concentrated solution by eluting with silica gel column chromatography with a mixed solvent of chloroform and methanol. Separation of fractions by reverse phase thin layer chromatography in the system, purification with high performance liquid chromatography, and pure separation of a novel compound KGT14-3 Method of producing a novel antibiotic peptides view from Bahia oak Ria in 14T strains of Ascomycetes which is characterized in that it comprises. According to claim 3, Rice cultivator Apiocria 14T strain of asymptomatic fungus, characterized in that the rice medium prepared by 45g white rice and 60g of distilled water in a 500ml Erlenmeyer flask container and autoclaved at 15 at 121 ° C for 20 minutes. Method for producing a novel peptibol antibiotic. The method of claim 3, wherein the concentrated solution is recovered from silica gel column chromatography, chloroform: methanol, 4: 1 and 2: 1, and sequentially eluted to recover and concentrate. Method for preparing novel peptiball antibiotics. The method of claim 3, wherein the reverse phase chromatography is fractionated using 90% methanol and developed by thin layer chromatography using 90% methanol. The fraction of Rf = 0.38 is collected and purified. A method for producing a novel peptibol antibiotic from the Apiocria 14T strain of Aspergillus fungus. An anticancer agent comprising as an active ingredient a structural formula (I) or a pharmaceutically acceptable salt thereof, which is a peptiball antibiotic prepared by the method of any one of claims 3 to 6. An antiviral agent comprising as an active ingredient a structural formula (I) or a pharmaceutically acceptable salt thereof, which is a peptiball antibiotic prepared by the method of any one of claims 3 to 6. An antimicrobial agent comprising the structural formula (I) or a pharmaceutically acceptable salt thereof as a peptibol antibiotic prepared by the method of any one of claims 3 to 6 as an active ingredient.