Compounds and their uses

Abstract

The present invention provides a novel compound having nitrification-inhibiting activity, a soil conditioner and fertilizer containing the compound, and a method for producing the compound. [Solution] The compound represented by formula (1) below or the compound represented by formula (2) below shall be used. [C1] TIFF0007864320000010.tif59170

Description

[Technical Field]

[0001] This invention relates to soil conditioners and their use. More specifically, it relates to compounds, soil conditioners, fertilizers, methods for producing the active ingredients of soil conditioners, and methods for inhibiting nitrification. [Background technology]

[0002] A considerable amount of ammonia nitrogen fertilizer applied to farmland is not absorbed and utilized by plants, but is converted into nitrate nitrogen through nitrification by nitrifying bacteria. This nitrate nitrogen not only infiltrates groundwater and causes water pollution, but is also converted to nitrous oxide through denitrification in the soil and released into the atmosphere. Nitrous oxide is known to act as a powerful greenhouse gas and is a factor in environmental burden (e.g., Non-Patent Document 1).

[0003] A phenomenon has been observed in which crops secrete natural substances from their root systems that inhibit nitrification; this phenomenon is called "Biological Nitrification Inhibition (BNI)." By utilizing BNI, nitrogen loss and environmental burden caused by nitrification can be effectively reduced. Therefore, the identification of compounds exhibiting BNI activity (hereinafter sometimes referred to as BNI substances) and the establishment of methods for producing such substances are essential requirements for the development of crops with enhanced BNI activity (hereinafter sometimes referred to as BNI crops). For example, Patent Document 1 discloses a cyclic diterpene compound having BNI activity. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2010-248088 [Non-patent literature]

[0005] [Non-Patent Document 1] Subbarao and Searchinger PNAS. Vol. 118,No. 22, e2107576118, 2021 [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to provide a novel compound having nitrification-inhibiting activity, a soil conditioner and fertilizer containing the compound, and a method for producing the compound. [Means for solving the problem]

[0007] The inventors focused on the fact that finger millet, which can grow under low input conditions, exhibits tolerance to nutrient-poor soils, and hypothesized that BNI is involved in nitrogen retention in the rhizosphere.

[0008] Finger millet, a type of grain, has the advantage of being able to be cultivated even in harsh environments such as drought and high temperatures compared to major crops (wheat, corn, and rice), and it also has superior nutritional value. Much of the chemical composition of finger millet is unknown compared to major grains, and most research has been limited to nutrients useful to humans, such as calcium, flavonoids, and unsaturated fatty acids. In particular, information on secondary metabolites is overwhelmingly scarce compared to other grass crops.

[0009] Therefore, the present inventors isolated and purified compounds having BNI activity from the root secretions of finger millet, determined their structures, confirmed the BNI activity of the identified compounds, and thus completed the present invention.

[0010] The present invention includes the following embodiments. A first aspect of the present invention is a compound represented by the following formula (1) or a compound represented by the following formula (2), or a solvate thereof.

[0011] [ka]

[0012] The second aspect of the present invention is a soil conditioner containing, as an active ingredient, the compound according to the first aspect or a solvate thereof.

[0013] The third aspect of the present invention is a fertilizer containing the soil conditioner according to the second aspect.

[0014] The fourth aspect of the present invention is a method for producing the compound according to the first aspect, including immersing the plant body of Echinochloa crus-galli in an organic solvent to obtain an extract, and purifying the compound according to claim 1 from the extract by chromatography.

Effect of the Invention

[0015] According to the present invention, it is possible to provide a novel compound having nitrification inhibitory activity, a soil conditioner containing the compound, a fertilizer, and a method for producing an active ingredient of the soil conditioner.

Brief Description of the Drawings

[0016] [Figure 1A] It is a diagram showing the results of separation by chromatography in Experimental Example 1. [Figure 1B] It is a diagram showing the results of separation by chromatography in Experimental Example! [Figure 1C] It is a diagram showing the results of separation by chromatography in Experimental Example 1. [Figure 2A] It is a diagram showing the results of ultraviolet absorption spectrum analysis and circular dichroism dispersion spectrum analysis of colacanol A in Experimental Example 2. [Figure 2B] It is a diagram showing the results of ultraviolet absorption spectrum analysis and circular dichroism dispersion spectrum analysis of colacanol B in Experimental Example [Figure 3A] It is a diagram showing the results of high-resolution electrospray ionization mass spectrometry of colacanol A in Experimental Example 1. [Figure 3B]This figure shows the results of high-resolution electrospray ionization mass spectrometry for Kolacanol B in Experimental Example 1. [Figure 4A] This figure shows the results of the NMR analysis of Coracanol A in Experimental Example 1. [Figure 4B] This figure shows the results of the NMR analysis of Coracanol A in Experimental Example 1. [Figure 4C] This figure shows the results of the NMR analysis of Kolacanol B in Experimental Example 1. [Figure 4D] This figure shows the results of the NMR analysis of Kolacanol B in Experimental Example 1. [Figure 5A] This figure shows the measurement results of the nitrification inhibitory activity of Coracanol A in Experimental Example 2. [Figure 5B] This figure shows the measurement results of the nitrification inhibitory activity of Coracanol B in Experimental Example 2. [Figure 5C] This figure shows the measurement results of the nitrite production inhibitory ability of Kolacanol A and Kolacanol B in Experimental Example 3. [Modes for carrying out the invention]

[0017] [Compound] In one embodiment, the present invention provides a compound represented by the following formula (1) or a compound represented by the following formula (2), or a solvate thereof. In this specification, the compound represented by the following formula (1) may be referred to as Coracanol A. The compound represented by the following formula (2) may be referred to as Coracanol B.

[0018] Kolacanol A and Kolacanol B are unprecedented 6-6-6-6 carbon four-membered ring diterpenoids having a bicyclo[2.2.2]octane skeleton and an octaline skeleton, respectively. As will be described later in the examples, these compounds have nitrification inhibitory activity.

[0019] [ka]

[0020] The solvates in this embodiment are not particularly limited as long as they exert their effects, and examples include hydrates, organic solvent hydrates, and the like.

[0021] This embodiment may be a compound represented by formula (1) or a compound represented by formula (2).

[0022] [Soil conditioner] In one embodiment, the present invention provides a soil conditioner containing as an active ingredient a compound represented by formula (1) or a compound represented by formula (2) according to the above embodiment, or a solvate thereof. The soil conditioner according to this embodiment may contain the compound represented by formula (1) or the compound represented by formula (2) according to the embodiment as an active ingredient.

[0023] The soil conditioner according to this embodiment may contain components other than the compound represented by formula (1) or the compound represented by formula (2) according to the embodiment. In the soil conditioner according to this embodiment, the total content of the compound represented by formula (1) or the compound represented by formula (2) according to the embodiment may be 10 to 100% by mass, 30 to 100% by mass, 50 to 100% by mass, 70 to 100% by mass, or 90 to 100% by mass. The soil conditioner according to this embodiment may consist of a compound represented by formula (1) or a compound represented by formula (2).

[0024] By adding soil conditioners to the soil, the nitrification of nitrogen components in the soil can be suppressed, thereby preventing the deterioration of the soil environment. The amount of active ingredient added to the soil is not particularly limited as long as the effects of the present invention are achieved, but for example, it is in the range of 100 to 500 μg per gram of soil.

[0025] [fertilizer] In one embodiment, the present invention provides a fertilizer containing a soil conditioner according to the above embodiment.

[0026] Because the fertilizer contains the aforementioned soil conditioner, it has a nitrification-inhibiting effect. This suppresses the nitrification of nitrogen components and prevents the deterioration of the soil environment.

[0027] The fertilizer according to this embodiment can be manufactured by adding a soil conditioner to a known fertilizer. Examples of known fertilizers include inorganic fertilizers and organic fertilizers, and mixtures thereof may also be used. Examples of inorganic fertilizers include nitrogenous fertilizers such as urea, ammonium sulfate, and ammonium chloride; phosphate fertilizers such as superphosphate; and potassium fertilizers such as potassium sulfate and potassium chloride. Examples of organic fertilizers include bone meal and compost.

[0028] [Manufacturing method] In one embodiment, the present invention provides a method for producing a compound represented by formula (1) or a compound represented by formula (2) according to the above embodiment.

[0029] The manufacturing method according to this embodiment includes the steps of (A) immersing finger millet plants in an organic solvent to obtain an extract, and (B) purifying the extract from the extract by chromatography for the compound represented by formula (1) or the compound represented by formula (2) according to the embodiment.

[0030] As described later in the examples, the inventors isolated colacanol A and colacanol B, which have nitrification inhibitory effects, from the roots of finger millet. The inventors also confirmed that colacanol A and colacanol B are produced in the leaves of finger millet.

[0031] In step (A), finger millet plants are immersed in an organic solvent to obtain an extract. In this specification, the term "plant body" includes not only the individual plant but also plant organs and tissues such as leaves, stems, and roots, which are parts of the plant body. Roots are preferred as the plant body. The plant material to be immersed in the organic solvent may, for example, be pre-cut into small pieces or pulverized into a powder.

[0032] The organic solvent used to obtain the leachate is not particularly limited as long as the present invention is effective, and examples include alcohol, acetonitrile, diethyl ether, dichloromethane, chloroform, ethyl acetate, or mixtures thereof.

[0033] In step (B), the compound represented by formula (1) or the compound represented by formula (2) according to the embodiment is purified from the leachate by chromatography. The chromatography method is not particularly limited as long as the effects of the present invention are achieved, and examples include partition chromatography, normal-phase chromatography, reverse-phase chromatography, etc. [Examples]

[0034] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0035] [Experimental Example 1] (Separation and purification of Coracanol A and Coracanol B) Compounds exhibiting nitrification-inhibiting activity were isolated and purified from an extract obtained from the roots of finger millet, and their structures were determined.

[0036] In a constant temperature chamber with a room temperature of 25 degrees Celsius, humidity of 50%, and 12 hours of light and dark exposure, 100 seeds of finger millet (ICRISAT stock IE2872, origin: Zambia) were sown in pots (9 kg of black volcanic soil) and cultivated in the pots for 60 days. No fertilizer was added during cultivation, and water was replenished twice a week to compensate for the amount of moisture lost.

[0037] <Root exudate> The roots of cultivated finger millet were immersed in dichloromethane to obtain a root extract. The root extract was concentrated using a rotary evaporator, and the resulting root extract (10 mg) was dissolved in methanol.

[0038] <Separation by chromatography> The obtained methanol solution was separated using a reversed-phase column (TSKgel® Super-ODS, 50 mm × 4.6 mm id, flow rate 1.2 ml). Linear gradient elution was performed using a mixed solvent of acetonitrile and 0.1% formic acid aqueous solution as the mobile phase under the following conditions: At the start of elution, the mixture was 1% acetonitrile / 99% water (0.1% formic acid), which was increased to 30% acetonitrile / 70% water (0.1% formic acid) after 6 minutes, and then the ratio was continuously changed to 100% acetonitrile after 9 minutes. As a result, three fractions (Fr.1, Fr.2, Fr.3) were obtained, as shown in Figure 1A.

[0039] Next, Fr.1 was fractionated using a reversed-phase column (TSKgel® Super-ODS, 100 mm × 4.6 mm id, flow rate 1.0 ml) with 40% acetonitrile / 60% water (0.1% formic acid) as the mobile phase. The results are shown in Figure 1B. As shown in Figure 1B, three fractions were identified: Fr.1-1, Fr.1-2, and Fr.1-3.

[0040] Next, Fr.1-2 were further separated using a reversed-phase column (COSMOSIL® piNAP column, 100 mm × 4.6 mm id, flow rate 1.0 ml). Elution was performed using 40% acetonitrile / 60% water (0.1% formic acid) as the mobile phase, and fraction B (0.05 mg) with a retention time of 9.2 to 10.2 minutes was isolated. The results are shown in Figure 1B. As shown in Figure 1B, fraction B, which has a single peak, was observed with a retention time of 9.2 to 10.2 minutes.

[0041] Furthermore, Fr.2 was fractionated using a reversed-phase column (COSMOSIL® 5C18 column, 250 mm × 4.6 mm id, flow rate 1.0 ml) with 80% acetonitrile / 20% water (0.1% formic acid) as the mobile phase. The results are shown in Figure 1C. As shown in Figure 1C, two fractions, Fr.2-1 and Fr.2-2, were identified.

[0042] Next, Fr.2-1 was fractionated using a reversed-phase column (TSKgel® Super-ODS, 100 mm × 4.6 mm id, flow rate 1.0 ml) with 50% acetonitrile / 50% water (0.1% formic acid) as the mobile phase. The results are shown in Figure 1C. As shown in Figure 1C, three fractions were identified: Fr.2-1-1, Fr.2-1-2, and Fr.2-1-3.

[0043] Next, Fr.2-1-2 was further separated using a reversed-phase column (COSMOSIL® piNAP column, 150 mm × 4.6 mm id, flow rate 1.0 ml). Elution was performed using 50% acetonitrile / 50% water (0.1% formic acid) as the mobile phase, and fraction A (0.1 mg) with a retention time of 15.0 to 16.0 minutes was isolated. The results are shown in Figure 1B. As shown in Figure 1B, fraction A, which has a single peak, was observed with a retention time of 15.0 to 16.0 minutes.

[0044] <Ultraviolet spectrum, circular dichroic dispersion spectrum> The ultraviolet (UV) spectra and circular dichroic dispersion (ECD) spectra of the compounds in fractions A and B were analyzed. The UV spectra of the compounds were measured using a Shimadzu UV-1600, and the ECD spectra were measured using a JASCO J-820.

[0045] The analysis results of the compounds contained in fraction A were as follows: UV (methanol) λ max (logε): 229nm (3.19) ECD (methanol) λ ext (Δε):195nm(-6.26),221nm(+0.58),248nm(+3.64) nm

[0046] The analysis results of the compounds contained in fraction B were as follows: UV (methanol) λ max (logε):202nm(3.43), 279nm(1.52) ECD (methanol) λ ext(Δε): 197 nm (+0.58), 305 nm (-0.70)

[0047] <Mass spectrometry> For the compounds contained in fraction A and fraction B, mass spectrometry was performed. The mass spectrometry was carried out by high-resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (HR-ESI-FT-ICR-MS), and Orbitrap Velos Pro (manufactured by Thermo Fisher) was used as the analytical instrument. The analysis results of the compounds contained in fraction A are shown in Figure 3A, and the analysis results of the compounds contained in fraction B are shown in Figure 3B.

[0048] The analysis results of the compounds contained in fraction A were as follows. m / z 331.2268 [M + H] + Calculated value C 21 H 31 O3, 331.2273

[0049] The analysis results of the compounds contained in fraction B were as follows. m / z 303.2315 [M + H] + Calculated value C 20 H 31 O2, 303.2318)

[0050] <NMR analysis> For the compounds contained in fraction A and fraction B, nuclear magnetic resonance (NMR) analysis was performed. As the analytical instrument, AVANCE III HD 800 MHz manufactured by Bruker was used.

[0051] The analysis results of the compounds contained in fraction A are shown in Figures 4A and 4B. The analysis results of the compounds contained in fraction B are shown in Figures 4C and 4D.

[0052] The peak assignment results of the compounds contained in fraction A are shown in Table 1. The assignment and stereochemistry of each peak shown in Table 1 were determined based on the measurement results of two-dimensional NMR (HSQC, HMBC, DQF-COSY, NOESY) shown in Table 2.

[0053] [Table 1]

[0054] [Table 2]

[0055] Table 3 shows the peak assignments for the compounds contained in fraction B. The assignment and stereochemistry of each peak shown in Table 3 were determined based on the two-dimensional NMR (HSQC, HMBC, DQF-COSY, NOESY) measurement results shown in Table 4.

[0056] [Table 3]

[0057] [Table 4]

[0058] Based on the above analysis results, the compound contained in fraction A was identified as the compound represented by the following formula (1). This compound is sometimes referred to as coracanol A.

[0059] [ka]

[0060] Based on the above analysis results, the compound contained in fraction B was identified as the compound represented by the following formula (2). This compound is sometimes referred to as coracanol B.

[0061] [ka]

[0062] [Experimental Example 2] (Measurement of nitrification inhibitory activity) The nitrification inhibitory activity of coracanol A and coracanol B was measured using bioluminescent nitrifying bacteria. The measurement method was described in Japanese Patent Publication No. 2022-016389.

[0063] Nitrifying bacteria (Nitrosomonas europaea IFO14298) into which the luciferase gene (luxAB) derived from the bacterium (Vibrio harveyi) was introduced were cultured in 200 mL of P medium (in a 500 mL baffled Erlenmeyer flask) at 30°C and 150 rpm for 4 days with shaking. The composition of the P medium (1.0 L) was as follows: (NH4)2SO4 (final concentration: 18.9 mM), KH2PO4 (final concentration: 5.1 mM), Na2HPO4 (final concentration: 95.0 mM), NaHCO3 (final concentration: 6.0 μM), MgSO4·7H2O (final concentration: 405 μM), CaCl2·2H2O (final concentration: 340 μM), Fe-EDTA (final concentration: 2.7 μM), and kanamycin·nH2SO4 (0.25 mg) was added.

[0064] The obtained nitrifying bacteria were transferred to 50 mL plastic tubes as needed, concentrated at the bottom using a centrifuge (9000 rpm, 5°C, 10 minutes), the liquid portion was removed, and the tubes were washed twice with 20 mL of fresh P medium. After concentrating the bacteria at the bottom of the tubes again, fresh P medium was added to adjust the turbidity to OD. 660 The ratio was adjusted to 1.0. Next, the nitrifying bacterial suspension was left to stand at 25°C for 10 minutes in the dark before the experiment.

[0065] To pre-confirm the luminescence state of the nitrifying bacteria to be used in Experimental Example 2, allylthiourea and 6-methoxy-2(3H)-benzoxazolone (MBOA) were used as positive controls. Bacterial suspensions that showed an 80% inhibition rate of luminescence with allylthiourea (final concentration: 0.22 μM) and a 70-80% inhibition rate with MBOA (final concentration: 50 μM) were used in Experimental Example 2.

[0066] In Experimental Example 1, Coracanol A and Coracanol B were each dissolved in DMSO, and 1.0 μL of each solution was added to 199 μL of water. 250 μL of nitrifying bacterial suspension was added to this sample aqueous solution (200 μL), and the mixture (total 450 μL) was then incubated at 15°C for 20 minutes. Next, a portion of the reaction medium (100 μL) was added to a luminescent substrate, a 10% n-decylaldehyde / 90% ethanol mixture, and the bioluminescence of the sample was measured using a Pro-mega Corp. GLOMAX20 / 20 luminometer.

[0067] The bioluminescence associated with nitrification decreases if a nitrification-inhibiting active substance is present in the test material. The luminescence of a nitrifying bacterial suspension without the addition of Coracanol A and Coracanol B was set as 100%, and the relative luminescence values ​​when various concentrations of Coracanol A or Coracanol B were added to the aqueous solution of the nitrifying bacterial suspension were defined as the nitrification inhibition rate (%). The luminescence of each sample was measured at least three times, and the average value was used to calculate the nitrification inhibition rate. The results are shown in Figures 5A and 5B. In Figures 5A and 5B, the concentrations of Coracanol A and Coracanol B refer to the concentrations in the mixture. (ED of Coracanol A) 50 It is 12 μM, and the ED of Coracanol B 50 The concentration was 21 μM.

[0068] [Experimental Example 3] (Measurement of the ability of nitrifying bacteria to suppress nitrite production) The inhibitory effects of colacanol A and colacanol B on nitrite production by nitrifying bacteria were measured. The measurement method followed a known method (Otaka et al., Plant Soil. Vol. 489, No. 1, 341-359, 2023).

[0069] In Experimental Example 1, Coracanol A and Coracanol B were each dissolved in DMSO, and 5.0 μL of each solution was added to 250 μL of water to obtain sample aqueous solutions. Next, a suspension of nitrifying bacteria cultured for 4 days in the same manner as in Experimental Example 2 was added to the sample aqueous solution (255 μL) (however, the final concentration of (NH4)2SO4, which is the nitrogen source, was set to 2.0 mM, and OD 660 2.0 mL (in a 15 ml plastic tube) of the solution (adjusted to 1.0) was added to obtain the mixture. The final concentrations of Coracanol A in each mixture were set to 30 μM and 100 μM, and the final concentrations of Coracanol B were set to 100 μM and 250 μM. Next, the mixture (total 2255 μL) was cultured in the dark at 30°C and 150 rpm with shaking. Reaction cultures (100 μL each) were collected at 0 minutes, 6 hours, 24 hours, and 72 hours after incubation. The nitrite concentration in each culture was quantified using the Gries method (using the Cayman Chemical Company Nitrate / Nitrite Colorimetric Assay Kit). The results are shown in Figure 5C. The inhibitory activity of kolacanol A against nitrite production by nitrifying bacteria was over 99% at 100 μM, and the inhibitory activity of kolacanol B was over 99% at 250 μM. [Industrial applicability]

[0070] The compound of the present invention can be suitably used as a naturally derived nitrification inhibitor. Furthermore, this compound is an unprecedented 6-6-6-6 carbon four-membered ring diterpenoid, and is expected to have applications in drug discovery, for example.

Claims

1. A compound represented by the following formula (1) or a compound represented by the following formula (2). 【Chemistry 1】

2. A soil conditioner comprising the compound described in claim 1 as an active ingredient.

3. A fertilizer containing the soil conditioner described in claim 2.

4. A method for producing the compound described in claim 1, Obtaining an extract by immersing finger millet plants in an organic solvent, A method for producing a compound according to claim 1, comprising purifying the leachate by chromatography.

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