KR101268170B1 - Novel cyclic peptide-based compound having anitmicrobial activity and uses thereof - Google Patents

Abstract

The present invention relates to a cyclic depsipeptide compound separated from and purified from the fungus Alternaria SF-5016 isolated from marine sediments and an antimicrobial composition comprising the same as an active ingredient. The novel cyclic peptide-based compound has antimicrobial activity against Bacillus subtilis or Staphylococcus aureus.

Description

Novel cyclic peptide-based compound having antimicrobial activity and use thereof

The present invention is an antimicrobial active peptide-based compound derived from a fungus isolated from the ocean, an antimicrobial pharmaceutical composition or cosmetic composition comprising the compound as an active ingredient, a method for separating the compound and the genus Alternaria sp. .) SF-5016 strain.

The classical meaning of antibiotics is a secondary metabolite produced by microorganisms, which means that they kill or inhibit growth at very low concentrations. The term antibiotic was first used by Waksman in the 1940's and initially used penicillins produced by the synthetic sulfonamides and penicillin sp. Used to distinguish. Since the discovery and synthesis of many substances that exhibit antimicrobial activity, it has become difficult to uniquely define the meaning of antibiotics. Currently, antibiotics generally have a natural, semisynthetic or It is taken to mean a compound of synthesis.

Research on the exploration and use of natural products is the exploration and utilization of the biosynthetic ability of the organism, so far, mainly plants and microorganisms have been the target species. In particular, microorganisms, along with species diversity, produce many unique and interesting bioactive substances, have short generation periods, are easy to mass-produce, and have high industrial applicability.

The exploration of useful substances produced by microorganisms has been actively conducted mainly on terrestrial microorganisms, and many of them have found usefulness in fermentation industry and medicine. Compounds that have been substantially applied to the medicinal field so far are produced by microorganisms isolated from the land, and with the progress of many studies, the probability of obtaining new compounds is significantly reduced.

On the other hand, the ocean is a larger area than the land, and due to abundant ecological changes, it is believed that many times the microorganisms of the land are growing in an unknown state. Therefore, these marine microorganisms, unlike their terrestrial microorganisms, have also been suggested to produce new compounds. Recently, marine microorganisms, which have been left behind due to difficulty in isolation and culture, are attracting attention as new microbial resources, and thus, new drug lead compounds are expected to be discovered by exploration using cultures of marine microorganisms. Moreover, the use of cosmetic materials and health foods is expected to increase in the future.

The first report of antibiotic production in marine microorganisms was made in 1947, but it was also the first report of segregation of practical compounds in 1966 (Fenical, W. Chem . Rev. , 93 , 1753 (1993)). Research on marine bacteria has been successful since the early 1990s, and since 1994, when marine fungi have been successful. Therefore, marine microbial resources can be regarded as important as a new source in the search for new bioactive substances. According to the current technology, about 1% or less of cultured marine microorganisms are known and less than 0.1% are according to the researchers. In particular, in the symbiotic microorganisms, the cultureable microorganisms are extremely limited. Although the research field of the use of biosynthetic genes of cultivable bacteria in marine microorganisms has been progressed, resource development of oocyte cultured microorganisms through the development of culture technology such as medium and culture conditions is also one of the important research fields. Among the new compounds isolated from marine microorganisms, most of them are those whose biological activity is not revealed, which is the biggest reason for the limited range of physiological activity assay. In addition, the specific activity that is different from the biological activity set up for the separation of the compound is increasingly found after separation. Therefore, it is considered that a wide range of pharmacological activity studies are necessary because compounds having unique structures exist among the compounds for which no physiological activity is reported.

In addition, antibiotic-resistant bacteria that are resistant to conventional antibiotics and appear to be ineffective are emerging, and as new diseases such as AIDS are increasing, the development of new drugs that can replace existing antibiotics is urgently required. It is becoming. Previously developed antibiotics are classified as beta-lactam class of antibiotics, which are recognized to be superior to penicillin, methicillin (methicillin) or bacteria that have become resistant to the methicillin, Specifically, vancomycin and the like have been developed to have an antimicrobial effect on methicillin resistance staphyllococus aureus (MRSA). However, with the recent increase in resistance to existing antibiotics, such as vancomycin resistance staphyllococus aureus (VRSA), called superbacteria, the need for development of substances or drugs that are resistant to previously reported antibiotics is increasing. It is being emphasized.

Accordingly, recently, attention has been focused on the development of a medicament with a new mechanism of action that can replace an existing antibiotic. Therefore, in order to develop new active substances, target-specific search methods and securing new resources are required.

The inventors of the present invention, while searching for the metabolite of marine microorganisms for the excellent antimicrobial activity, selected a fungal strain that produces an excellent antimicrobial activity from marine sediment, from which the cyclic peptide compound of the new structure is purely The cyclic peptide-based compound was isolated and purified, and Staphylococcus aureus ) and Bacillus The present invention was confirmed to exhibit antimicrobial activity against Gram-positive bacteria such as subtilis ). It is therefore an object of the present invention to provide a cyclic peptide-based compound of novel structure.

In order to solve the above problems, the present invention provides an antimicrobial active peptide-based compound derived from the fungus isolated from the ocean represented by the formula (1) or a pharmaceutically acceptable salt thereof.

The present invention also provides an antimicrobial pharmaceutical composition or cosmetic composition comprising the compound as an active ingredient.

The present invention also provides a method for separating the compound.

The present invention also provides an Alternaria sp. SF-5016 strain producing the compound.

As described above, a composition containing a novel alternaramid compound, which is a cyclic peptide-based compound of the present invention, as an active ingredient may be useful as an antimicrobial composition for the treatment of bacterial infectious diseases by exhibiting inhibitory activity against Gram-positive bacteria.

Figure 1 shows a ¹ H NMR spectrum (400MHz, CDCl ₃ ) of the alternaramid compound isolated in the present invention.
Figure 2 shows a ¹³ C NMR spectrum (100MHz, CDCl ₃ ) of the alternaramid compound isolated in the present invention.

In order to achieve the object of the present invention, the present invention provides a novel cyclic peptide-based compound represented by the formula (1) or a pharmaceutically acceptable salt thereof:

The novel cyclic depsipeptide-based compound of the present invention is a compound isolated from marine fungi, preferably Alternaria sp., More preferably Alternaria sp. It is characterized by having an unknown chemical structure.

The cyclic depsipeptide compounds according to the present invention can be used in the form of pharmaceutically acceptable salts and include all salts, hydrates, solvates and isomers prepared by conventional methods.

The present invention also provides an antimicrobial pharmaceutical composition comprising the cyclic peptide-based compound as an active ingredient. The antimicrobial pharmaceutical composition containing the novel cyclic depsipeptide compound of the present invention showed antimicrobial activity against Gram-positive bacteria without having cytotoxicity. The Gram-positive bacteria is preferably Bacillus sp. Or Staphylococcus sp.) may be bacteria. In addition, the genus Bacillus sp. Or Staphylococcus genus sp.) bacteria are preferably each Bacillus subtilis ) or Staphylococcus aureus , but is not limited thereto.

The compounds of the present invention are generally administered in the form of pharmaceutical compositions comprising the active compound of the invention and comprising a pharmaceutically acceptable carrier or carrier suitable for use in pharmaceutical preparations.

In general, the compounds of the present invention are administered in a pharmaceutically effective amount. The amount of the compound actually administered will usually be determined by the physician taking into account relevant conditions, including the subject disease, the route of administration chosen, the compound actually administered, age, weight and individual patient's response, the severity of symptoms, and the like. Dosages range from 10 mg to 500 mg once or several times daily.

The pharmaceutical compositions of the present invention can be administered by various routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular and nasal. The pharmaceutical compositions of the invention are preferably formulated in one of the injectable or oral compositions depending on the intended route of delivery.

Compositions for oral administration may take the form of bulk liquid dilutions or suspensions, or bulk powders. More generally, however, it is provided in unit dosage form to facilitate accurate dosing. The term "unit dosage forms" refers to completely separate units suitable for one dose to be administered to patients and other mammals, each of which is calculated and determined in advance to produce the desired therapeutic effect. Amounts of the active substance are in combined form with appropriate pharmaceutical excipients. Common unit dosage forms include ampules or syringes in which the liquid composition is prefilled and measured, or in the case of solid compositions, pills, tablets, capsules and the like.

Liquid forms suitable for oral administration may include suitable aqueous or non-aqueous carriers with buffers, suspensions and dispersants, colorants, spices, and the like. Solid preparations may include, for example, the following components or compounds of similar nature: binders such as microcrystalline cellulose, gum tragacanth or gelatin; Excipients such as starch or lactose; Disintegrating agents such as alginic acid, Primogel or corn starch; Lubricants such as magnesium stearate; Glidants, such as colloidal silicon dioxide; Sweeteners such as sucrose or saccharin; Or a flavoring agent such as mint, methyl salicylate or orange flavoring.

Injectable compositions are generally based on sterile saline for injection or phosphate-buffered saline (PBS) or other injectable carriers known in the art. As above, the present compounds in such compositions are generally a minor component and usually comprise about 0.05-10% of the combined weight of the residue, such as an injectable carrier.

The above ingredients for oral or injectable compositions are only representative. Other materials and treatment methods and the like are disclosed in Remington's Pharmaceutical Sciences (Part 8 of Remington's Pharmaceutical Sciences, 17th edition, 1985, Mack Publishing Company, Easton, Pa.).

The compounds of the present invention can also be administered in a sustained release form or a sustained release drug delivery system. Representative sustained release materials are described in the Remingtons Pharmaceutical Sciences Additives column.

The present invention also provides a cosmetic composition comprising the compound of Formula 1. The cosmetic composition of the present invention includes components commonly used in cosmetic compositions in addition to the compound of Formula 1, and includes conventional auxiliaries such as stabilizers, solubilizers, vitamins, pigments and flavors, and carriers.

Cosmetic compositions according to the invention can be prepared in any formulations conventionally prepared in the art and include, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing It may be formulated as cleansing, oil, powder foundation, emulsion foundation, wax foundation, spray, and the like, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters.

The present invention also relates to

1) culturing the SF-5016 strain of Alternaria sp.,

2) extracting the strain culture obtained in step 1) with ethyl acetate, and

3) It provides a separation method of the cyclic peptide-based compound of the present invention comprising the step of separating the ethyl acetate extract obtained in step 2) by column chromatography.

Step 1) is a step of culturing the fungus Alternaria sp. SF-5016 strain, strain culture is cultured in a medium containing nutrients that can be used by conventional microorganisms. As a nutrient source, the well-known nutrient source currently used for the cultivation of a mold is used. For example, as a carbon source, glucose, starch syrup, dextrin, starch, molasses, animal oil, vegetable oil, etc. may be used, and as nitrogen sources, bran, soybean meal, wheat, malt, cottonseed gourd, fishmeal, corn steep liquor, gravy, yeast Extracts, ammonium sulfate, sodium nitrate, urea and the like can be used. If necessary, it is very effective to add salts, potassium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other inorganic salts that promote ion generation. As a culture method, shaking culture or stationary culture is possible under aerobic conditions.

The culture temperature is slightly different depending on the conditions when incubated in each of the above conditions, it is usually appropriate to incubate at 20 ~ 37 ℃, in most cases incubated at 25 ~ 30 ℃.

Step 2) is a step of extracting the strain culture obtained in step 1) with ethyl acetate, the cyclic depsi peptide-based compound is present in the mycelia portion as well as the culture medium of the strain. Therefore, an organic solvent such as ethyl acetate is added to the culture medium and the mycelium of the strain to extract the active ingredient from the culture medium and the mycelium, and the obtained extract is concentrated by vacuum evaporation.

Step 3) is a step of separating the compound of the present invention, the ethyl acetate concentrate obtained in step 2) is subjected to flash column chromatography using a methanol: water mixed solvent, and then purified by high-performance liquid chromatography Compound 1 To separate.

The present invention also provides an Alternaria sp. SF-5016 strain (Accession No .: KCTC 18187P) that produces the compound represented by Chemical Formula 1.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

One. Alternaria  Genus ( Alternaria sp .) SF Isolation of -5016 Strains

This strain was isolated from marine sediments near the coast of Masan Bay in January 2006. The collected sediments were placed in sterilized plastic bags immediately after collection and stored frozen until used in the experiment. Sediment samples were diluted 10-fold with sterile seawater. 1 ml of the diluted solution was plated in a potato dextrose agar medium and cultured at 25 ° C. for 14 days to separate pure colonies.

2. Identification and naming of strains

GenBank search results of nucleotide sequences obtained through rRNA sequencing of the strain showed alternaria alternate ) (DQ678082) at 99.51%, Alternaria homology 99.51% to thalictrigena ) (EU040211) and 99.14% to Alternaria malorum (AY251081). This strain was identified as genus Alternaria and named as Alternaria sp. SF-5016 (Accession No .: KCTC 18187P).

3. Alternaria  Genus ( Alternaria sp .) SF Culture of -5016 Strains

In order to culture this fungal strain, a medium containing a nutrient source normally used by microorganisms was prepared. Coarse medium and production medium for fungal strains containing 0.4% (w / v) potato starch, 2% (w / v) dextrose, 3% (w / v) sodium chloride, 1.5% (w / v) agar Potato dextrose agar medium was used.

After sterilizing a 100 ml Erlenmeyer flask containing 20 ml of the seed medium at 121 ° C. for 20 minutes, platinum was inoculated from a slope culture tube of Alternaria sp. It was made. Ten plates of 20 ml of medium were inoculated and seeded with 2 ml of the seed culture solution.

4. Alternaria  Genus ( Alternaria sp .) SF Cyclic from -5016 strain Peptides  Isolation and Purification of the Compound

The culture of the Alternaria sp. SF-5016 strain cultured in 3 above was extracted with ethyl acetate (1 L), and the extract was concentrated using a reduced pressure dryer in a sensitization method. The concentrated solution was adsorbed to ODS Al 18 and subjected to ODS RP-18 flash column chromatography, where methanol / water (8: 2-10 / 0, v / v) was added. The mixture was eluted with increasing methanol concentration step by step as a mixed solvent. At this time, the active fraction containing Compound 1 eluted in 80% methanol and 100% methanol. After the two active fractions were concentrated and concentrated under reduced pressure, the fraction containing Compound 1 was acetonite containing 0.1% formic acid as a solvent using high performance liquid chromatography (column: Shiseido Capcell Pak C18, length 25 mm, diameter 1 mm). The reel and water were eluted at a 65:35-80:20 concentration gradient to elute flow rate of 2 ml / min to separate Compound 1 showing a 210 nm UV absorption peak at 35 minutes.

5. Alternaria  Genus ( Alternaria sp .) SF Cyclic from -5016 strain Peptides  Structural analysis of compounds

The fungus Alternaria sp. Compound 1 isolated from the culture medium of SF-5016 was determined molecular weight and molecular formula using an ESI mass spectrometer (Electrospray Ionization mass spectrometer). In addition, ¹ H NMR, ¹³ C NMR, Correlation Spectroscopy (COSY), 1H-Detected heteronuclear Multiple-Quantum Coherence (HMQC), and Heteronuclear Multiple-Bond Coherence (NMR) analysis (JEOL JNM ECP-400) ), DEPT (Distortionless Enhancement by Polarization), NOESY (Nuclear Overhauser effect spectroscopy) spectra were obtained, and the molecular structure of the compound was determined.

The measurement results are as follows, and the substance isolated from the culture medium of the Alternaria sp. SF-5016 strain was identified as a novel cyclic depsipeptide compound having the following formula, and alternaamide (Alternaramide) ( Named compound 1).

Alternaramide (Compound 1): white powder; [α] _D- 6 ( c 0.53, MeOH); UV (MeOH) λ _max (log ε) 208 (4.1); IR (neat) ν _max 3298, 3008, 2930, 1742, 1674, 1634, 1525, 1450, 1236, 750 cm ⁻¹ ; ¹ H, ¹³ C NMR data is shown in Table 1 below; HRESIMS m / z 589.3024 [M + H] ⁺ (calcd for C ₃₃ H ₄₁ N ₄ O ₆ , 589.3026).

[Formula 1]

Experimental Example  1: Alternaramide  Determination of Antimicrobial Activity of Compounds

The following strains from the accumulation culture collection of the Korea Gene Bank (KCTC) were used for the antimicrobial activity evaluation study: Candida albicans (KCTC 7965), Filobasidiella neoformans (KCTC 7003), Staphylococcus aureus (KCTC 1928), Bacillus subtilis (KCTC 1021), Proteus vulgaris (KACC 12674). Gentamicin was used as a control of activity evaluation, and its activity was Staphylococcus aureus (KCTC 1928) and Bacillus subtilis The antimicrobial activity of (KCTC1021) was 13 mm (concentration of 50 µg / disk) and 16 mm (concentration of 5 µg / disk).

The antimicrobial activity test was performed using the paper disc method. Agar medium containing each test bacteria solution was poured into a petri dish to make a flat medium, and then, Compound 1 was absorbed by 400 µg into a sterile paper disc, and then placed on the prepared activity evaluation flat medium, and 25 ° C. 48 hours of incubation in the incubator to determine the clear zone (mm) around the disc.

List of strains and media used for antimicrobial experiments Test microorganism Used badge Candida albicans KCTC 7965 YMA, MEA Filobasidiella neoformans KCTC 7003 YMA, MEA Staphylococcus aureus KCTC 1928 NA Bacillus subtilis KCTC 1021 NA Proteus vulgaris KACC 12674 (-) NA

-Nutrient Agar: [0.3% (w / v) beef extract, 0.5% (w / v) peptone, 1.5% (w / v) agar]

YMA (Yeast Mold Agar): [0.3% (w / v) yeast extract, 0.3% (w / v) malt extract, 0.5% (w / v) peptone, 1% (w / v) dextrose, 2% ( w / v) agar]

Malt Extract Agar (MEA): [1.275% (w / v) maltose, 0.275% (w / v) dextrin, 0.235% (w / v) glycerol, 0.078% (w / v) peptone, 1.5% (w / v) agar]

Antimicrobial Activity of Compound 1 Test microorganism Clear zone (mm) Candida albicans KCTC 7965 - Filobasidiella neoformans KCTC 7003 - Staphylococcus aureus KCTC 1928 13 Bacillus subtilis KCTC 1021 8 Proteus vulgaris KACC 12674 (-) -

Examples of formulations for the composition of the present invention are illustrated below.

Formulation Example 1: Preparation of powder

0.1 g of the cyclic peptide-based compound of the present invention

Lactose 1.5 g

Talc 0.5 g

The above ingredients were mixed and filled in an airtight container to prepare powders.

Formulation Example 2: Preparation of tablets

0.1 g of the cyclic peptide-based compound of the present invention

1.5 g of crystalline cellulose

0.5 g magnesium stearate

After mixing the above ingredients, tablets were prepared by direct tableting method.

Formulation Example 3 Preparation of Capsule

0.1 g of the cyclic peptide-based compound of the present invention

5 g of corn starch

4.9 g of carboxy cellulose

After mixing the above components to prepare a powder, the powder was filled in a hard capsule according to a conventional preparation method of a capsule to prepare a capsule.

Formulation Example 4: Preparation of injection

0.1 g of the cyclic peptide-based compound of the present invention

Sterile sterilized water for injection

pH adjuster

According to the conventional method for preparing an injection, the amount of the above-mentioned ingredient was prepared per ampoule (2 ml).

Formulation Example 5: Preparation of a liquid preparation

0.1 g of the cyclic peptide-based compound of the present invention

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the conventional method for preparing a liquid, and lemon flavor was added appropriately, followed by mixing the above components. Then, purified water was added thereto to adjust the total volume to 100 ml, and then filled in a brown bottle to sterilize to prepare a liquid.

Korea Biotechnology Research Institute KCTC18187P 20100208

Claims (9)

The cyclic peptide-based compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof:
[Formula 1]

The compound according to claim 1, wherein the cyclic peptide-based compound is isolated from Alternaria sp. delete delete The cyclic peptide-based compound of claim 1, or a pharmaceutically acceptable compound thereof, which exhibits antimicrobial activity against at least one Gram-positive bacterium selected from the group consisting of Bacillus sp. And Staphylococcus sp. Bacteria. Pharmaceutical composition for antimicrobial, characterized in that it comprises a salt. The method of claim 5, wherein the Bacillus sp. Or Staphylococcus genus sp.) bacteria are Bacillus subtilis ) or Staphylococcus aureus ) antimicrobial pharmaceutical composition, characterized in that. delete 1) culturing the SF-5016 strain of Alternaria sp.,
2) extracting the strain culture obtained in step 1) with ethyl acetate, and
3) separating the ethyl acetate extract obtained in step 2) by column chromatography, wherein the cyclic peptide-based compound of claim 1. Alternaria sp. SF-5016 strain producing the compound of claim 1 (Accession No .: KCTC 18187P).

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