A novel indole compound and its applications

Abstract

This invention discloses a novel indole compound with the following chemical structural formula: This invention utilizes fungal co-culture fermentation to obtain a new natural compound that exhibits good inhibitory activity on the growth of young roots of barnyard grass and has a significant damaging effect on its root tip cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of natural products, in particular to an indole compound. BACKGROUND

[0002] Plant endophytic fungi refers to a kind of fungi that live in plant tissues throughout or part of the life cycle without causing any harmful effects to the host. Microorganisms are one of the main sources of natural secondary metabolites. Due to the gene silencing phenomenon existing in biosynthetic gene clusters (BGCs) in microorganisms, especially in fungi, the biosynthetic gene clusters do not express or express at low levels under laboratory culture conditions, and the content of secondary metabolites produced by microorganisms is low, and the skeleton type is single.

[0003] Echinochloa crus-galli (L.) P. Beauv. is an annual grass weed that has strong adaptability and tolerance to adverse environments, and is one of the malignant weeds in rice fields nationwide. The main measure for controlling Echinochloa crus-galli (L.) P. Beauv. at present is to use chemical control, but the long-term use of chemical herbicides will cause environmental pollution, and the problem of weed resistance is increasingly prominent. Therefore, it is urgent to find a new herbicide to solve the problems faced by the existing Echinochloa crus-galli (L.) P. Beauv. harm. SUMMARY

[0004] The purpose of the present application is to solve the defects in the prior art and provide a natural product with herbicidal effect.

[0005] In order to achieve the above purpose, the technical scheme provided by the present application is as follows:

[0006] An indole compound, the chemical structural formula of which is as follows:

[0007]

[0008] The present application obtains a new indole compound (hereinafter referred to as Colletotriauxin G), which is obtained by activating the microbial silent gene cluster by co-culturing fungi, and is a hidden natural product. The indole compound of the present application has a natural source, does not cause environmental pollution and potential risks to humans, has good Echinochloa crus-galli (L.) P. Beauv. growth inhibition effect, and expands the application of natural products in weed control.

[0009] The above-mentioned co-culture of fungi uses the following fungi: endophytic fungus A. destruens CIY4 isolated from chicory leaves and C. acutatum.

[0010] The present application can stimulate the silent genes of microorganisms, produce secondary metabolites, obtain hidden natural products and extract new indole compounds by co-culturing fermentation of fungi, and the new indole compounds can be used for preparing natural herbicides.

[0011] The present application also provides the application of the above-mentioned new indole compounds in weeding, especially in preparing natural herbicides.

[0012] In some embodiments, as preferred, the above-mentioned new indole compounds are used for preparing natural herbicides, and the concentration of the new indole compounds is preferably 50-100 mu g / mL.

[0013] The present application has the following advantages compared with the prior art:

[0014] The present application activates silent gene clusters by co-culturing fungi to obtain hidden natural products, and screens out compounds with excellent weeding effects, thereby expanding the source and application of natural herbicides.

[0015] The obtained compound is a new indole compound, which not only expands the natural source of indole compounds, but also provides a new lead compound for the research and development of herbicide biological pesticides due to its growth inhibition effect on the young roots of barnyard grass. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The chemical structural formula of the new indole compound Colletotriauxin G of the present application is shown in the following formula (I);

[0017] Figure 2 The key data of the new indole compound Colletotriauxin G of the present application are shown in the following table (I); 1 H- 1 H COSY, HMBC and NOESY correlation diagrams;

[0018] Figure 3 The mass spectrum of the new indole compound Colletotriauxin G of the present application is shown in the following figure (I);

[0019] Figure 4 The IR spectrum of the new indole compound Colletotriauxin G of the present application is shown in the following figure (I);

[0020] Figure 5 The UV spectrum of the new indole compound Colletotriauxin G of the present application is shown in the following figure (I);

[0021] Figure 6 The hydrogen spectrum of the new indole compound Colletotriauxin G of the present application is shown in the following figure (I);

[0022] Figure 7 Carbon spectrum of the novel indole compound Colletotriauxin G of the present application;

[0023] Figure 8 Carbon spectrum of the novel indole compound Colletotriauxin G of the present application; 1 H- 1 H COSY diagram;

[0024] Figure 9 HMBC diagram of the novel indole compound Colletotriauxin G of the present application;

[0025] Figure 10 HSQC diagram of the novel indole compound Colletotriauxin G of the present application;

[0026] Figure 11 NOESY diagram of the novel indole compound Colletotriauxin G of the present application;

[0027] Figure 12 Comparison diagram of the inhibitory activity of the novel indole compound Colletotriauxin G of the present application on the growth of the young roots of barnyard grass under different concentrations;

[0028] Figure 13 Comparison diagram of the influence of the novel indole compound Colletotriauxin G of the present application on the growth of the young roots of barnyard grass under different concentrations;

[0029] Figure 14 Comparison diagram of the cell damage of the young roots of barnyard grass treated by the novel indole compound Colletotriauxin G of the present application under different concentrations. DETAILED DESCRIPTION

[0030] The present application will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0031] Embodiment 1

[0032] Preparation example of the novel indole compound Colletotriauxin G of the present application

[0033] The A. destruens CIY4 and C. acutatum isolated from the leaves of chicory are activated and cultured. The activation and culture conditions are as follows: the A. destruens CIY4 and C. acutatum are inoculated in potato dextrose agar medium (PDA), 27±1 ℃, and cultured for 5 days. Then the mycelial blocks are inoculated in potato dextrose broth (PDB) and cultured at 27-28 ℃ with 160 r / min shaking for 7 days to obtain seed liquid. The seed liquid of A. destruens CIY4 and C. acutatum is inoculated in sterilized and cooled rice medium at a ratio of 1:1, and fermented at room temperature of about 25 ℃ for 30 days. Then the rice and fermentation products are completely immersed in 3 times the volume of analytical pure methanol (CH3OH) for 1 day each time, and the three times of extraction liquid are combined and concentrated to no methanol smell by rotary evaporator under reduced pressure to obtain total extract 1.6 kg. Add appropriate amount of ultrapure water to suspend, extract with the same volume of ethyl acetate (EtOAc), a total of 3 times, to obtain ethyl acetate extract 41.6 g. The ethyl acetate extract is preliminarily separated by silica gel column chromatography, the sample is mixed with 100-200 mesh silica gel, the silica gel column is packed with 100-200 mesh silica gel, and the solvent system of petroleum ether (PE) / ethyl acetate (EtOAc) (100:0-100:100) and dichloromethane (CH2Cl2) / methanol (CH3OH) (100:4-0:100) is used for gradient elution, and the same components are combined by HPLC detection, to obtain components Fr.1-Fr.5. Component Fr.5 is gradient eluted by silica gel column (CH2Cl2:CH3OH=100:4-100:16) to obtain sub-components Fr 5.1-Fr 5.5, and Fr 5.5 is purified by semi-preparative high performance liquid chromatograph (40% CH3CN). The obtained monomeric compound is subjected to activity screening, and the compound Colletotriauxin G with barnyard grass growth inhibition activity is obtained, and the structure is identified by the following spectral techniques (such as MS, NMR, UV, IR, etc.):

[0034] Compound Colletotriauxin G, light white powder. The spectral data are as follows (combined with Figures 2-11 ): [α] 25 D= +31.579° (c 0.038, CH3OH); UV (CH3OH) λ max 218.6, 279.3 nm; IR (KBr) v max 3356, 2924, 2853, 1717, 1457, 1178, 1078, 1034 cm -1 ; from HR-ESI-MS data, m / z 336.1463 [M+H] + (Calcd. for C 17 H 22 NO6 336.1442), the molecular formula of the compound was deduced as C 17 H 21 NO6, with 8 degrees of unsaturation. According to the δ 1 H-NMR data (shown in Table 1 below), the presence of typical 3-substituted indole structure was deduced from the signals of δ H 7.18 (1H, s, H-2), 7.62 (1H, d, J = 7.9, H-4), 7.02 (1H, t, J = 7.5, H-5), 7.10 (1H, t, J = 6.9, H-6), 7.34 (1H, d, J = 8.1, H-7), one methine δ H 1.61 (3H, d, J = 7.2, H-3'), one methine attached with a methyl group δ H 4.10 (1H, m, H-2'), four oxygen methine signals at 4.98 (1H, ddd, J = 8.3, 4.8, 3.5, H-2"), 4.10 (1H, m, H-3"), 3.41 (1H, dd, J = 8.4, 1.2, H-4"), 3.68 (1H, ddd, J = 8.5, 6.0, 3.4, H-5"), and two oxygen methylene signals 3.76 (1H, d, J = 4.2, H-1"), 3.83 (1H, dd, J = 12.0, 3.6, H-1"), 3.53 (1H, dd, J = 11.3, 6.1, H-6") and 3.73 (1H, d, J = 3.4, H-6").

[0035] Table 1 NMR spectral data of compound Colletotriauxin G

[0036]

[0037] As shown in the above table, 13 C NMR showed the presence of 17 carbon signals, including one ester carbonyl carbon δ C 176.8 (C-1'), one methyl carbon δ C 18.2 (C-3'), two oxygen methylene carbons δ C62.0 (C-1"), δ C 65.1 (C-6"), 1 methine carbon δ C 38.5 (C-2'), 4 oxygen methine carbons δ C 75.5 (C-2"), δ C 69.2 (C-3"), δ C 71.2 (C-4"), δ C 72.6 (C-5"), 8 indole carbons δ C 123.2 (C-2), δ C 115.3 (C-3), δ C 127.6 (C-3a), δ C 119.7 (C-4), δ C 119.8 (C-5), δ C 122.5 (C-6), 112.4 (C-7), δ C 138.0 (C-7a). HMBC correlations of H-5 / C-3a, H-6 / C-4, C-7a, H-7 / C-3a, H-2 / C-3a, C-7a showed the presence of the indole, HMBC spectrum showed the presence of the methylation of the indole 3-acetoxy moiety, δ C 127.6 aromatic carbon C-3a with δ C 38.5 methine carbon C-2' with correlations, demonstrating the C-3 substitution of the indole moiety, HMBC correlations of H-3' / C-3, C-1' indicated the presence of the methylated indole 3-acetoxy moiety. Further, according to COSY correlations, the presence of a hexose fragment -CH2-CH-CH-CH-CH2- was deduced, which was further confirmed by the HMBC correlations of H-3" / C-1", C-5", H-4" / C-2", H-1" / C-5", and H-4" / C-6".

[0038] The HMBC correlation of H-2" / C-1' demonstrated the attachment of the hexose fragment 2" position to the indole end through the ethyl ester linkage. The above NMR data analysis indicated that the compound was a 3-(2'-methyl)-ethyl ester-1,2-deoxyglucose substituted indole, in addition, according to the NOESY spectrum Figure 2 and Figure 11 the correlations of 2" / 5" and 3" / 5", it was deduced that 2", 3", 5" were on the same side of the six-membered ring, thus, its structural formula and relative configuration as Figure 1 shown, was a new compound, named Colletotriauxin G.

[0039] Example 2

[0040] Test of the growth inhibition activity of compound Colletotriauxin G on the young roots of E. crus-galli

[0041] The present application adopts the plate method to determine the growth inhibition activity of compound Colletotriauxin G on the young roots of E. crus-galli, and glyphosate is used as a positive control drug. First, 3 mg of the compound is dissolved in a small amount of DMSO, so that the final content of DMSO in the test solution is less than 0.1%, 0.1% Tween 20 is added to 3 mL, and a stock solution of 1 mg / mL is prepared. Then, the test solution with the corresponding concentration is prepared according to Table 2. The E. crus-galli seeds are soaked in sterile deionized water for 1 day in advance, sterilized with 75% alcohol for 5 min, washed with sterile deionized water for 3 times, each time for 30 s, two filter papers are placed in each culture dish, 5 mL of the above test solution is added, and a water solution containing 0.1% (v / v) dimethyl sulfoxide and 0.1% (v / v) Tween 20 is used as a solvent control Figure 13 indicated as the solvent group), and sterile deionized water is used as a blank control Figure 14 indicated as the CK group), 10 seeds are placed in each dish, the dish is sealed with an extension film, and the culture is carried out at 23℃, humidity 70%, light intensity 14000, light / dark (16h / 8h) for 4 days, and each concentration is repeated 3 times. After 4 days, the photograph is taken, the length of the young roots of E. crus-galli is calculated using Image J, and the data is analyzed using GraphPad Prism 8.0.2. The results (as shown in Figure 12 , 13) show that compound Colletotriauxin G can significantly inhibit the growth of the young roots of E. crus-galli at a concentration of 50 μg / mL, and there is no significant difference between the inhibition of the young roots of E. crus-galli by Colletotriauxin G and glyphosate.

[0042] Table 2: Preparation method of test solution with gradient concentration

[0043]

[0044] Example 3

[0045] Effect of compound Colletotriauxin G on the root tip cells of E. crus-galli

[0046] The present application adopts the propidium iodide (PI) staining method to detect the effect of the compound on the root tip cells of E. crus-galli, and the young roots of E. crus-galli treated with the compound for 4 days in Example 2 are dyed with 10 μg / mL of PI staining agent for 30 min, washed with 0.01M PBS buffer, and then placed under a laser scanning confocal microscope to observe the above treated root tips at a wavelength of 561 nm. The test is repeated 3 times, and the images are recorded. The results (as shown in Figure 14As shown in the figure, compared with the blank control group (CK group), the root tip cells of the Echinochloa crus-galli seedlings treated with compound Colletotriauxin G were damaged, and with the increase of the concentration, the damage to the root tip cells of the Echinochloa crus-galli seedlings was obvious at 50 μg / mL, the effect was significant, and there was no obvious difference in the damage effect compared with the glyphosate treatment group, indicating that the compound has obvious damage effect on the root tip cells of the Echinochloa crus-galli seedlings.

Claims

1. An indole compound, having a chemical structure as follows:

2. Use of the indole compound of claim 1 in weed control.

3. Use of the indole compound according to claim 1 for the production of a natural herbicide, characterized in that, The indole compound is used for preparing a natural source herbicide, wherein the concentration of the indole compound is 50 μg / mL-100 μg / mL.

Images