KR20230049286A - A novel compound derived from Paucibacter aquatile and uses thereof - Google Patents

Abstract

The present invention relates to a novel compound derived from a Paucibacter aquatile strain and uses thereof. More specifically, the novel compound has an excellent algicidal effect on Microcystis, a species that causes green algae, and has very low cytotoxicity, thereby being able to be usefully used to control Microcystis while minimizing disturbance to the aquatic ecosystem.

Description

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

The present invention relates to novel compounds derived from Paucibacter aquatile strains and uses thereof.

Eutrophication of water resources due to the accumulation of nutrients has recently been raised as a major problem in water quality. Eutrophication of water resources acts as a factor of mass proliferation of phytoplankton, that is, floating algae, causing algae blooms. Algal blooms generally occur only in fresh water. Various terrestrial pollutants such as factory wastewater, domestic sewage, fertilizers, pesticides, and livestock and human excreta flow into rivers or lakes, and organic matter decomposed by microorganisms present in the lower part of the water body It produces a large amount of nitrogen and phosphorus, which are food for plankton, and green algae occur. These algae reduce dissolved oxygen in water, produce toxic substances to kill fish and aquatic organisms, and furthermore, cause many problems such as destruction of the water ecological environment, introduction of advanced purification facilities for water sources, and damage to natural aesthetics. In particular, toxins produced by these toxic blue-green algae have been found to have a toxic effect on the liver of vertebrates. It has been found to inhibit the function of protein phosphatase like the compound of , and the possibility of acting as an accelerator of carcinogenesis has also been reported. Furthermore, damage to animals due to hydration of toxic substance-producing blue-green algae has been reported in many countries such as the United States, Canada, the United Kingdom, and Japan.

It is known that cyanobacteria, green algae, and diatoms act as causative bacteria for green algae in lakes in Korea. However, in reality, the proportion of green algae caused by green algae and diatoms is not small, and blue-green algae called cyanobacteria are found to be the biggest cause. Like plants, these blue-green algae proliferate through an oxygen-generating photosynthesis process, and require a large amount of nutrient elements such as nitrogen (N) and phosphorus (P). Therefore, in order to suppress the occurrence of algae, overall control such as cutting off the supply of nitrogen, phosphorus and organic and inorganic nutrients in the stagnant lake, suppressing the increase in water temperature suitable for the growth of blue-green algae, and blocking the supply of light is required to effectively suppress the occurrence of algae. Currently, methods for controlling algae generally include physical control such as underwater aeration, electromagnetic waves, and light shielding, chemical control using oxidizing agents, coagulants, and natural algicides, and biological control using bacteria and natural enemy organisms. The chemical treatment method is widely used at home and abroad as a way to control algae in a short period of time, and materials such as ocher, coagulant, copper sulfate, chlorine, and ozone are mainly used.

On the other hand, Korean Patent Publication No. 2007-0016137 relates to a macrocyclic compound as a viral replication inhibitor, and discloses a composition for treating Polavivirus infection including hepatitis C virus infection and liver fibrosis containing the compound as an active ingredient. Korean Patent Publication No. 2020-0077541 relates to a novel macrocyclic derivative, a method for preparing the same, and a pharmaceutical composition containing the same, and discloses the use of the compound for cancer treatment, and Korean Patent Registration No. 1842803 Disclosed is a CR182 strain of the genus Pausibacter that inhibits the growth of methacillin-resistant Staphylococcus aureus.

However, the novel compound derived from Faucibacter aquatyl 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 Registered Patent No. 1842803

The present invention has been derived from the above needs, and the present inventors have found that the new compound produced by the Paucibacter aquatile strain, which has been reported as an algicidal microorganism, has a very excellent algicidal effect on Microcystis, the causative species of algae, The present invention was completed by confirming that the cytotoxicity was very low.

In order to solve the above problems, the present invention provides a compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[Formula 1]

In addition, the present invention provides a method for preparing the 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 are blue-green algae, preferably the blue-green algae are Microcystis sp., more preferably the Microcystis sp. aeruginosa ).

As another example, the present invention provides a harmful algae control method comprising the step of treating harmful algae-generated water with the composition for controlling harmful algae.

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

The new compound produced by Paucibacter aquatile strain reported as an algicidal microorganism of the present invention has an excellent algicidal effect on Microcystis, which is a causative species of algae, and has very low cytotoxicity, thereby minimizing the impact on the ecosystem and algae can be efficiently controlled, so it can be usefully used as an algicide.

1 illustrates a process of separating and purifying a sample 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.
5 shows the ¹ H- ¹ H COSY spectrum of the active ingredient DH153-1 according to one embodiment of the present invention.
Figure 6 shows the HSQC spectrum of the active ingredient DH153-1 according to one 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.
8 shows the chemical structure of the active ingredient DH153-1 identified by a two-dimensional NMR experiment according to an embodiment of the present invention.
9 shows the assignment of ¹ H (red) and ¹³ C (blue) peaks of the active ingredient DH153-1 according to an embodiment of the present invention.
10 shows the ESI-mass spectrum of the active ingredient DH153-1 according to one embodiment of the present invention.
Figure 11 shows the algicidal effect of the active ingredient DH153-1 on Microcystis aeruginosa (A) and well plate culture photos (B) of the isolated compound according to an embodiment of the present invention.
Figure 12 shows the comparison results of the algicidal effect by concentration of the active ingredient DH153-1 according to an embodiment of the present invention.
13 shows the cytotoxicity test results 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, which are provided to help a more general understanding of the present invention, and it is common 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 of And, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter 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 represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[Formula 1]

In addition, the present invention provides a method for preparing the 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 having 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, Afanizomenone ( Aphanizomenon ) genus, Nodularia genus or Cochlodinium genus, more preferably Microcystis aeruginosa ( Microcystis aeruginosa ), Microcystis viridis ( M. viridis ), Microcystis It may be Stys polos aqui ( 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 according to the method, and the extract can be used by mixing with water or an organic solvent, and to improve the stability of the effect and the attachment of the drug to the target organism, the non- Ionic or ionic surfactants may be used together. In addition, suitable carriers and additives may be further included.

The composition for controlling harmful algae of the present invention may be formulated into emulsions, wettable powders, granules, powders, calceps, and gel formulations, and is provided as a contact agent through a donut-type formulation for buoyancy of the formulation. desirable.

As another example, the present invention provides a harmful algae control method comprising the step of treating harmful algae-generated water with the composition for controlling harmful algae.

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

The advantages and features of the present invention, and how to achieve them, will become clear with reference to the detailed description of the following embodiments. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various different forms, and only the present embodiments will complete the disclosure of the present invention and allow common knowledge in the art to which the present invention belongs. It is provided to fully inform the holder of 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 (Accession No. 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 the fraction was analyzed by TLC and divided into 5 groups (FIG. 1).

Example 2. Isolation and Identification of Active Material Components

As a result of the activity assay, activity was shown in the DH15-2, DH15-3, and DH15-4 fractions. Among them, DH15-2 and DH15-3, which have similar HPLC profiles, were combined and subjected to silica gel column chromatography (CHCl ₃ :MeOH=100:1-> 10:1, v/v), followed by three fractions (DH153-1, DH153-2, DH153-3). Of these, DH153-1 showed algicidal activity, and DH153-1 was finally purified by performing preparative HPLC as necessary for chemical structure analysis.

The separation and purification process of the sample is as shown in FIG. 1, and the chemical structure analysis of DH153-1 was performed.

In order to identify the chemical structure of the active ingredient DH153-1 _, after dissolving it in DMSO- d6 , 1-dimensional NMR such as ¹ H NMR, ¹³ C NMR, and DEPT and 2-dimensional NMR spectrum such as ¹ H- ¹ H COSY, HSQC, and HMBC was measured and analyzed.

1) Measurement and interpretation of ^1H NMR spectrum: _As a result of measuring ^1H NMR spectrum after dissolving it in DMSO- d6 , protons derived from -OH or -NH at 12.78, 11.10, and 9.81 ppm, 7.73, 6.80, 6.44, 6.00 , 6 protons attributed to aromatic and double bonds at 5.44 and 5.24 ppm, methoxy proton at 3.92 ppm, nitrogen bound methylene proton at 3.66/3.63 ppm, one methine proton at 2.82 ppm, and 9 protons at 0.8–2.4 ppm Methylene proton, methyl proton was observed at 1.23 ppm (Fig. 2).

2) Measurement and interpretation of ¹³ C NMR and DEPT spectrum: As a result of measuring and interpreting the ¹³ C NMR spectrum and DEPT spectrum, one oxygenated sp ² quaternary carbon at 163.5 ppm, four sp at 143.5, 141.6, 120.8, and 109.0 ppm ² quaternary carbon, 141.8, 133.8, 124.4, 113.8, 108.2, 6 sp ² methine carbon at 92.2 ppm, methoxy carbon at 58.5 ppm, 10 methylene carbon between 23-50 ppm, 1 methine at 32.7 ppm and 20.9 ppm One methyl carbon was observed in (Figs. 3 and 4).

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

4) Measurement and interpretation of HSQC spectrum : To investigate the hydrogen-carbon correlation ( ¹ J _CH ) of the active ingredient DH153-1 The HSQC spectrum was measured and analyzed. As a result, the correlation between hydrogen and carbon constituting this active ingredient was identified (FIG. 6).

5) Measurement and interpretation of HMBC spectrum : To identify the chemical structure of the active ingredient DH153-1, a hydrogen-carbon 2-bond or 3-bond bonding relationship ( ² J _CH , ³ J _CH ) can be identified. The HMBC spectrum was measured and analyzed. As a result, from 6.80 and 6.00 ppm of methine proton to 143.5 and 120.8 ppm of sp ² carbon, from 6.44 ppm of methine proton to 141.6, 120.8 and 109.0 ppm of sp ² carbon, and from 7.73 ppm of methine proton to 163.5, 109.0 and 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 they were bound to the 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 mutually coupled from the process of elimination, proton, carbon chemical shift, and HMBC correlation. Therefore, the chemical structure of this compound was determined as shown in the figure below, and as a result of the search, the same compound was not found, so it is judged to be a new substance. The assignment of each proton and carbon peak is shown in FIGS. 7 to 9.

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

From the NMR measurement, the active ingredient DH153-1 was identified as a novel compound. To confirm the chemical structure, ESI-mass spectrum was measured in positive mode. As a result, M ⁺ peak was observed at m / z 382.2, and the molecular weight was confirmed as 382 (FIG. 10). This compound has a 4-bond nitrogen, so the compound has a positive charge, which is well matched in molecular weight and chemical structure.

Example 3. Comparison of algicidal effects of novel compounds

In order to compare the algicidal effects on DH153-1, DH153-2, and DH153-3 isolated from silica gel column , microcystis aeruginosa , a causative agent of green algae, was treated with a culture medium of 20 ppm, respectively. The number of cells in the 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 a microscopic counting method. 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 was confirmed by more than 99% in DH153-1 (Fig. 11A), and 12 well plate culture medium was compared. As a result, as shown in the picture, it was confirmed that all microcystis cells died and the green color disappeared. As a result, a high algicidal effect was confirmed in DH153-1 (FIG. 11B).

Example 4. Comparison of algicidal effect by concentration of the novel compound (DH153-1)

The finally selected DH153-1 was compared with the algicidal effect over time at concentrations of 0.1, 1, 5, 10, and 15 ppm. After 12 hours of treatment with the new compound by concentration in Microcystis culture medium (cell count: 2.0×10 ⁶ cells/ml), 0.1ppm showed 49.3% algicidal effect, and after 24 hours, 62.2% showed higher algicidal effect. . 1ppm showed high algicidal effect up to 66.2% after 12 hours, 81.6% after 24 hours, and 95% after 36 hours. When treated with 5, 10, or 15 ppm, it was confirmed that the algicidal effect appeared rapidly up to 90% in 12 hours (FIG. 12).

Example 5. Comparison of cytotoxicity

The CCK-8 method was performed to evaluate the toxicity of the isolated compound single-compound 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 form 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 in a 6 well plate at 1×10 ⁵ cells/ml. Cells cultured for one day were treated with DH153-1 at each concentration, 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, and after incubation for 2 hours, 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 by the isolated and purified novel compound DH153-1 up to a concentration of 1 ppm.

In the above, the present invention has been described as an 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 intended to explain, not limit, the present invention, and the spirit and scope of the present invention are not limited by these embodiments. The protection scope of the present invention should be construed by the following claims, and all techniques within the equivalent range should be construed as being included in the scope of the present invention.

Claims (8)

A compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
[Formula 1]

A method for producing the compound of formula 1 of claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, comprising culturing a Paucibacter aquatile strain. 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 is 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 . Harmful algae control method comprising the step of treating harmful algae-generated water with the composition for controlling harmful algae of claim 3 The method for controlling harmful algae according to claim 7, wherein the water is seawater or fresh water.

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