KR102589629B1 - A novel compound derived from Paucibacter aquatile and uses thereof - Google Patents

Abstract

The present invention relates to a new compound derived from the Paucibacter aquatile strain and its use. More specifically, the new compound has an excellent algicidal effect against Microcystis, a species that causes green algae, and is highly cytotoxic. Because it is low, it can be useful for killing algae while minimizing disturbance to the aquatic ecosystem.

Description

{A novel compound derived from Paucibacter aquatile and uses thereof}

The present invention relates to novel compounds derived from Paucibacter aquatile strain and their uses.

Eutrophication of water resources due to accumulation of nutrients has recently been raised as a major problem in water quality. Eutrophication of water resources acts as a factor in the mass proliferation of plankton, or plankton, which causes green algae. Green algae generally occurs only in fresh water. Various terrestrial pollutants such as factory wastewater, domestic sewage, fertilizers, pesticides, livestock and human waste flow into rivers and lakes, and organic matter decomposed by microorganisms present in the lower part of the water body Green algae occurs by producing large amounts of nitrogen and phosphorus, which serve as food for plankton. These green algae reduce dissolved oxygen in the water, produce toxic substances that kill fish and aquatic life, and further cause many problems such as destruction of the water ecology, introduction of advanced purification facilities for water sources, and damage to natural beauty. In particular, the toxins produced by these toxic blue-green algae have been found to have a toxic effect on the liver of vertebrates. In particular, microcystin is a member of the okadaic acid family in addition to causing acute toxicity such as liver bleeding and liver dysfunction. Like the compounds in , it has been found to inhibit protein phosphatase function, and the possibility of acting as an accelerator of carcinogenesis has also been reported. Furthermore, damage to animals caused by hydration of toxic substance-producing blue-green algae has been reported in many countries, including the United States, Canada, the United Kingdom, and Japan.

It is known that green algae phenomenon in lakes in Korea is mainly caused by blue-green algae, green algae, and diatoms. However, in reality, the proportion of green algae phenomenon caused by green algae and diatoms is small, and blue-green algae called cyanobacteria are found to be the biggest cause. Like plants, blue-green algae proliferate through the process of oxygen-generating photosynthesis and require large amounts of nutrients such as nitrogen (N) and phosphorus (P). Therefore, in order to suppress the occurrence of green algae, comprehensive control such as blocking the supply of nitrogen, phosphorus and organic and inorganic nutrients in stagnant lakes, suppressing the increase in water temperature suitable for the growth of blue-green algae, and blocking the supply of light must be implemented to effectively suppress the occurrence. Currently, general methods for controlling algae include physical control such as underwater aeration, electromagnetic waves, and light shielding, chemical control using oxidizing agents, coagulants, and natural algaecides, and biological control using bacteria and natural enemies. Chemical treatment is a method that can control algae in a short period of time and is widely used at home and abroad. Substances such as red clay, coagulants, copper sulfate, chlorine, and ozone are mainly used.

Meanwhile, Korean Patent Publication No. 2007-0016137 relates to a macrocyclic compound as a viral replication inhibitor, and discloses a composition for treating liver fibrosis and polarivirus infection, including hepatitis C virus infection, containing the compound as an active ingredient. and Korean Patent Publication No. 2020-0077541 relates to a new macrocyclic derivative, a manufacturing method thereof, and a pharmaceutical composition containing the same, and discloses the use of the compound for cancer treatment, and Korean Patent No. 1842803 We are disclosing the CR182 strain of the Faucibacter genus, a novel microorganism that inhibits the growth of methacillin-resistant Staphylococcus aureus.

However, the new compound derived from Faucibacter aquatil of the present invention and its algicidal use have not yet been disclosed.

Korean Patent Publication No. 2007-0016137 Korean Patent Publication No. 2020-0077541 Korean Patent No. 1842803

The present invention was developed in response to the above-mentioned needs, and the present inventors have found that a new compound produced by a strain of Paucibacter aquatile , which has been reported as an algaic microorganism, has an excellent algicidal effect on Microcystis, the species that causes green algae, The present invention was completed by confirming that cytotoxicity was very low.

In order to solve the above problems, the present invention provides a compound of the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[Formula 1]

Additionally, the present invention provides a method for producing a compound of Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, comprising culturing a Paucibacter aquatile DH15 strain.

As another example, the present invention provides a composition for controlling harmful algae comprising the compound of Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

In the present invention, the harmful algae is blue-green algae, preferably the blue-green algae is Microcystis sp., and more preferably the Microcystis sp. is Microcystis aeruginosa. aeruginosa ).

As another example, the present invention provides a method for controlling harmful algae, including the step of treating water in which harmful algae occurs with the composition for controlling harmful algae.

In the present invention, the water is seawater or fresh water.

The new compound produced by the Paucibacter aquatile strain, reported as an algaic microorganism of the present invention, has an excellent algicidal effect on Microcystis, the species that causes green algae, and has very low cytotoxicity, minimizing the impact on the ecosystem while producing green algae. Since it can be controlled efficiently, it can be usefully used as an algicide.

Figure 1 shows a sample separation and purification process according to an embodiment of the present invention.
Figure 2 shows the ¹ H NMR spectrum of the active ingredient DH153-1 in one embodiment of the present invention.
Figure 3 shows the ¹³ C NMR spectrum of the active ingredient DH153-1 in one embodiment of the present invention.
Figure 4 shows the DEPT (135) spectrum of the active ingredient DH153-1 in one embodiment of the present invention.
Figure 5 shows the ¹ H- ¹ H COZY spectrum of the active ingredient DH153-1 according to an embodiment of the present invention.
Figure 6 shows the HSQC spectrum of the active ingredient DH153-1 according to an embodiment of the present invention.
Figure 7 shows the HMBC spectrum of the active ingredient DH153-1 according to one embodiment of the present invention.
Figure 8 shows the chemical structure of the active ingredient DH153-1 identified by two-dimensional NMR experiment according to an embodiment of the present invention.
Figure 9 shows the assignment of ^{the 1} H (red) and ¹³ C (blue) peaks of the active ingredient DH153-1 according to an embodiment of the present invention.
Figure 10 shows the ESI-mass spectrum of the active ingredient DH153-1 according to an embodiment of the present invention.
Figure 11 shows the algicidal effect of the isolated compound of the active ingredient DH153-1 on Microcystis aeruginosa according to an embodiment of the present invention (A) and a photograph of well plate culture (B).
Figure 12 shows the results of comparing the algaecide effect by concentration of the active ingredient DH153-1 according to an embodiment of the present invention.
Figure 13 shows the results of a cytotoxicity test of the active ingredient DH153-1 according to an embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific details, such as specific components, are shown, but this is provided to facilitate a more general understanding of the present invention, and it is known in the art that the present invention can be practiced without these specific details. It will be self-evident to those who have the knowledge. Additionally, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description will be omitted.

In order to achieve the object of the present invention, the present invention provides a compound of the following formula (1), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[Formula 1]

Additionally, the present invention provides a method for producing a compound of Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, comprising culturing a Paucibacter aquatile strain.

In the present invention, the Paucibacter aquatile strain may be a Paucibacter aquatile DH15 strain with the accession number KCTC18831P.

As another example, the present invention provides a composition for controlling harmful algae comprising the compound of Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

In the composition for controlling harmful algae according to the present invention, the harmful algae may be blue-green algae, preferably Microcystis genus, Anabaena genus, Oscillatoria genus, Afanizomenon ( It may be the genus Aphanizomenon , Nodularia , or Cochlodinium , and more preferably Microcystis aeruginosa , M. viridis , or Microcystis. It may be M. flos-aquae or Microcystis wesenbergii ( M. wesenbergii ), most preferably Microcystis aeruginosa, but is not limited thereto.

The composition for controlling harmful algae of the present invention can be prepared in various forms depending on the method, and the extract can be mixed with water or an organic solvent for use. In order to improve the stability of the effect and the adhesion of the drug to the target organism, non- Ionic or ionic surfactants can be used together. Additionally, appropriate carriers and additives may be additionally included.

The composition for controlling harmful algae of the present invention can preferably be formulated into emulsion, wettable powder, granule, powder, calcep-type and gel-like formulations, and is provided as a contact agent through a donut-shaped formulation for buoyancy of the formulation. desirable.

As another example, the present invention provides a method for controlling harmful algae, including the step of treating water in which harmful algae occurs with the composition for controlling harmful algae.

In the method for controlling harmful algae according to an embodiment of the present invention, the water may be seawater or fresh water, and preferably fresh water, but is not limited thereto.

The advantages and features of the present invention and methods for achieving them will become clear with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various different forms. The present embodiments are merely intended to ensure that the disclosure of the present invention is complete and that common knowledge in the technical field to which the present invention pertains is not limited. It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.

Example 1. Extraction of active substances from algicidal microorganisms

In the present invention, Paucibacter aquatile DH15 ( Paucibacter aquatile ), accession number KCTC18831P, was cultured at 25 degrees in R2A medium. The cultured bacteria were extracted using an acetone solvent, partitioned and extracted with ethyl acetate, and then concentrated. The concentrate was subjected to Sephadex LH-20 column chromatography (CHCl ₃ :MeOH=1:1, v/v), and then the fractions were analyzed by TLC and divided into 5 groups (Figure 1).

Example 2. Separation and identification of active substance components

The activity assay results showed activity in DH15-2, DH15-3, and DH15-4 fractions. Among them, DH15-2 and DH15-3, which showed similar HPLC profiles, were combined and silica gel column chromatography (CHCl ₃ :MeOH=100:1-> 10:1, v/v) was performed, and then three fractions (DH153-1, Divided into DH153-2 and DH153-3). Among these, DH153-1 showed algicidal activity, and preparative HPLC was performed as necessary for chemical structure analysis, ultimately purifying DH153-1.

The sample separation and purification process is shown in Figure 1, and chemical structure analysis was performed on DH153-1.

To determine the chemical structure of the active ingredient DH153-1 _{, after dissolving it in DMSO-d6} , 1D NMR spectrum such as ^1H NMR, ^13C NMR, DEPT and 2D NMR spectrum such as ^1H - ^1H COZY, HSQC, HMBC etc. was measured and interpreted.

1) Measurement and interpretation of ¹ H NMR spectrum: As a result of measuring ¹ H NMR spectrum dissolved in DMSO- d ₆ , protons derived from -OH or -NH at 12.78, 11.10, 9.81 ppm, 7.73, 6.80, 6.44, 6.00 , 6 protons attributed to aromatics and double bonds at 5.44 and 5.24 ppm, a methoxy proton at 3.92 ppm, a methylene proton bonded to nitrogen at 3.66/3.63 ppm, one methine proton at 2.82 ppm, and 9 protons between 0.8 and 2.4 ppm. Methylene proton, methyl proton was observed at 1.23 ppm (Figure 2).

2) Measurement and interpretation of ¹³ C NMR and DEPT spectrum: As a result of measuring and interpreting ¹³ C NMR spectrum and DEPT spectrum, one oxygenated sp ² quaternary carbon at 163.5 ppm, 4 sp at 143.5, 141.6, 120.8, 109.0 ppm. ² quaternary carbons, 6 sp ² methine carbons at 141.8, 133.8, 124.4, 113.8, 108.2, 92.2 ppm, methoxy carbon at 58.5 ppm, 10 methylene carbons between 23-50 ppm, one methine at 32.7 ppm and 20.9 ppm One methyl carbon was observed (Figures 3 and 4).

3) Measurement and interpretation of ¹ H- ¹ H COZY spectrum: ¹ H- ¹ H COZY spectrum that can identify the correlation between neighboring hydrogens ( ³ J _HH ) to determine the chemical structure of the active ingredient DH153-1. was measured and interpreted. As a result, four partial structures shown in the figure below, including allylic coupling, were identified (Figure 5).

4) Measurement and interpretation of HSQC spectrum : To determine the correlation between hydrogen and carbon ( ¹ J _CH ) of the active ingredient DH153-1 The HSQC spectrum was measured and interpreted. As a result, the correlation between hydrogen and carbon, which constitutes this active ingredient, was identified (Figure 6).

5) Measurement and interpretation of HMBC spectrum : To determine the chemical structure of the active ingredient DH153-1, the 2-bond or 3-bond bond relationship between hydrogen and carbon ( ² J _CH , ³ J _CH ) can be identified. The HMBC spectrum was measured and interpreted. As a result, from 6.80 and 6.00 ppm of methine protons, 143.5 and 120.8 ppm of sp ² carbon, from 6.44 ppm of methine protons to 141.6, 120.8, and 109.0 ppm of sp ² carbon, and from 7.73 ppm of methine protons, 163.5, 109.0, 49.6. A long-range correlation was observed for ppm of carbon. In addition, a long-range correlation was observed from 1.23 ppm of methyl proton to 143.5, 34.3, and 32.7 ppm of carbon, confirming that it was bound to a pyrrole group. Therefore, the chemical structure was identified except for several methylene groups with overlapping proton signals between 0.8 and 1.2 ppm. It was confirmed that these overlapping parts are coupled to each other through the removal process, proton and carbon chemical shift, and HMBC correlation. Therefore, the chemical structure of this compound was determined as shown in the picture below, and as the same compound was not found as a result of the search, it was judged to be a new substance. The assignment of each proton and carbon peak is shown in Figures 7 to 9.

6) ESI-mass analysis of the active ingredient DH153-1

From NMR measurements, the active ingredient DH153-1 was identified as a new compound. To confirm the chemical structure, the ESI-mass spectrum was measured in positive mode. As a result, the M ⁺ peak was observed at m/z 382.2, confirming the molecular weight as 382 (Figure 10). This compound has a 4-bond nitrogen, so the compound has a positive charge, which means that its molecular weight and chemical structure match well.

Example 3. Comparison of algicidal effects of new compounds

To compare the algicidal effects of DH153-1, DH153-2, and DH153-3 isolated on a silica gel column, they were each treated to 20ppm in the culture medium of Microcystis aeruginosa , a species that causes green algae. The cell number of Microcystis culture medium was 2.0×10 ⁶ cells/ml. After 48 hours of culture, changes in the number of Microcystis cells were observed using microscopic counting. Compared to the control group, the algicidal effect of DH153-2 and DH153-3 was confirmed to be 54% and 81%, respectively, but the highest algicidal effect of over 99% was confirmed in DH153-1 (Figure 11A), comparing 12 well plate cultures. As a result, as seen in the photo, it was confirmed that all Microcystis cells died and the green color disappeared. As a result, high algicidal effect was confirmed in DH153-1 (Figure 11B).

Example 4. Comparison of algicidal effects by concentration of the new compound (DH153-1)

Finally, the algicidal effect of the selected DH153-1 was compared over time at concentrations of 0.1, 1, 5, 10, and 15 ppm. After 12 hours of treating Microcystis culture medium (cell count ^2.0 . 1ppm showed a high algicidal effect of 66.2% after 12 hours, 81.6% after 24 hours, and 95% after 36 hours. When treated at 5, 10, and 15 ppm, it was confirmed that the algaecide effect rapidly increased to 90% in 12 hours (Figure 12).

Example 5. Comparison of cytotoxicity

The CCK-8 method was performed to evaluate the toxicity of single-compound isolated compounds to cells. The cells used were B16F10 melanoma and were cultured under conditions of 37°C, 5% CO ₂ , and 95% humidity for cell culture. The medium was cultured in an adherent manner in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and Penicillin/Streptomycin solution (100 U/ml). DH153-1 was treated with B16F10 cells at 0.1, 0.5, and 1 ppm for one day. Cells were cultured at 1×10 ⁵ cells/ml in a 6 well plate. Cells cultured for one day were treated with DH153-1 at various concentrations, and cell viability was measured using Cell counting kit-8 (CCK-8, Dojindo). Cells were treated with CCK-8 according to the manufacturer's method, incubated for 2 hours, and absorbance was measured at 450 nm using a microplate reader (TECAN).

It was normalized based on the control group, and it was confirmed that more than 95% of the cells were alive up to a concentration of 1 ppm (FIG. 13). It was confirmed that there was no cytotoxicity caused by the isolated and purified new compound DH153-1 up to a concentration of 1ppm.

Above, the present invention has been described by way of example, and those skilled in the art will be able to make various modifications without departing from the essential characteristics of the present invention. Accordingly, the embodiments disclosed in this specification are for illustrative purposes rather than limiting the present invention, and the spirit and scope of the present invention are not limited by these embodiments. The scope of protection of the present invention should be interpreted in accordance with the claims below, and all technologies within the equivalent scope should be interpreted as being included in the scope of rights of the present invention.

Claims (8)

A compound of formula 1 below, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
[Formula 1]

delete A composition for controlling harmful algae comprising the compound of claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient. The composition for controlling harmful algae according to claim 3, wherein the harmful algae are blue-green algae. The composition for controlling harmful algae according to claim 4, wherein the blue-green algae is Microcystis sp. The composition for controlling harmful algae according to claim 5, wherein the Microcystis sp. is Microcystis aeruginosa . A method for controlling harmful algae comprising the step of treating water containing harmful algae with the composition for controlling harmful algae of paragraph 3. The method of claim 7, wherein the water is seawater or fresh water.